**Contents**


The Beneficial Health Effects of Vegetables and Wild Edible Greens: The Case of the Mediterranean Diet and Its Sustainability Reprinted from: *Appl. Sci.* **2020**, *10*, 9144, doi:10.3390/app10249144 . . . . . . . . . . . . . . . . . **127**

#### **Carlos Graci´an-Alcaide, Jose A. Maldonado-Lob ´on, Elisabeth Ortiz-Tikkakoski, Alejandro G ´omez-Vilchez, Juristo Fonoll ´a, Jose L. L ´opez-Larramendi, M ´onica Olivares and Ruth Blanco-Rojo**

Effects of a Combination of Elderberry and Reishi Extracts on the Duration and Severity of Respiratory Tract Infections in Elderly Subjects: A Randomized Controlled Trial Reprinted from: *Appl. Sci.* **2020**, *10*, 8259, doi:10.3390/app10228259 . . . . . . . . . . . . . . . . . **155**

#### **Ahmed Ezzat, Attila Heged ˝us, Szil´ard Szab ´o, Amin Ammar, Zolt ´an Szab ´o, J ´ozsef Ny ´eki, Bianka Moln ´ar and Imre J. Holb**

Temporal Changes and Correlations between Quality Loss Parameters, Antioxidant Properties and Enzyme Activities in Apricot Fruit Treated with Methyl Jasmonate and Salicylic Acid during Cold Storage and Shelf-Life Reprinted from: *Appl. Sci.* **2020**, *10*, 8071, doi:10.3390/app10228071 . . . . . . . . . . . . . . . . . **167**

#### **Jacek Gawro ´nski, Jadwiga Zebrowska, Marzena Pabich, Izabella Jackowska, Krzysztof ˙ Kowalczyk and Magdalena Dyduch-Siemi ´nska**

Phytochemical Characterization of Blue Honeysuckle in Relation to the Genotypic Diversity of *Lonicera* sp.

Reprinted from: *Appl. Sci.* **2020**, *10*, 6545, doi:10.3390/app10186545 . . . . . . . . . . . . . . . . . **189**

## *Editorial* **Potential Health Benefits of Fruits and Vegetables**

**Luca Mazzoni 1,\* , Maria Teresa Ariza Fernández 2,\* and Franco Capocasa 1,\***


In recent decades, the consciousness of consumers regarding the importance of a balanced diet to prevent the occurrence of chronic diseases has significantly increased. In particular, the consumption of plant-based foods, both vegetables and fruits, have been demonstrated to have a central role in the prevention of many chronic diseases, due to the high amount of bioactive compounds they contain. To date, many researchers and scientists in different fields of research have contributed with great efforts to the characterization of the phytochemical pattern of hundreds of fruits and vegetables, and have elucidated several mechanisms of actions and metabolic pathways through which fruits and vegetables exert their health-promoting and/or disease-preventing activities.

The aim of this special issue was to compile the most recent research on fruit and vegetable phytochemical composition, on the health-promoting effects and mechanisms of action of their application/assumptions in different models, such as in vitro cellular models, in vivo animal trials and in vivo human trials.

The first step of the evaluation of the potential health benefits of fruit and vegetables is the evaluation of the phytochemical composition. In this special issue, six studies were focused on the evaluation of the phytochemical composition and the antioxidant capacity of different fruit/vegetables species.

Li et al. characterized the phenolic composition of five different varieties of apples grown in Australia ('Royal Gala', 'Pink Lady', 'Red Delicious', 'Fuji' and 'Smitten') through liquid chromatography. The results underlined that different genotypes showed different amounts of total phenolic and total flavonoid content. Furthermore, a total of 97 different phenolic compounds were detected in the five apple varieties, highlighting the interest of Australian apple varieties as a rich source of bioactive compounds [1].

Among fruits, berries are raising interest for their proven high nutritional quality, due to the high amount of several phytochemicals. Two berries were analyzed in this special issue: in the first study, Kruger et al. evaluated the effect of cultivar, environmental variations and their interaction on anthocyanin composition of six strawberry cultivars grown in five locations from the North to South of Europe, for two different years. As a general trend, fruits grown in southern locations were richer in total anthocyanins and pelargonidin-3-glucoside content. Principal component analysis revealed that anthocyanin content of cultivars is influenced by environmental factors; in particular, the minor anthocyanins (cyanidin-3-glucoside, cyanidin-3-(6-*O*-malonyl)-glucoside, pelargonidin-3-rutinoside and pelargonidin-3-(6-*O*-malonoyl)-glucoside) were sensitive to the maximum temperature value. However, different cultivars changed their anthocyanin pattern in relation to the environmental conditions to varying extent, with 'Gariguette' and 'Clery' cultivars remaining unaffected [2].

The second study on a berry species involved the phytochemical characterization of 30 haskap berry genotypes. In this manuscript, different spectrophotometric and spectrofluorimetric methods were used to evaluate the antioxidant capacity and the amount of

**Citation:** Mazzoni, L.; Ariza Fernández, M.T.; Capocasa, F. Potential Health Benefits of Fruits and Vegetables. *Appl. Sci.* **2021**, *11*, 8951. https://doi.org/10.3390/app11198951

Received: 3 September 2021 Accepted: 13 September 2021 Published: 26 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

different phenolic compounds and vitamin C. This study allowed for the identification of those genotypes, which were more interesting for a high content of the studied phytochemicals, and that could be recommended to consumers for a healthy diet. Furthermore, the heritability and genetic process analyses allowed for the indication of the effectiveness of breeding for the transmission of the analyzed traits to the progeny, suggesting the most suitable genotypes for the implementation of future breeding programs aimed at obtaining healthier fruits [3].

Together with fruits, fresh vegetables are also interesting for their content of specific bioactive compounds. In particular, vegetables belonging to the *Brassicaceae* family have attracted increasing attention in recent years for the quantity and quality of their bioactive compounds, and this attention has also been confirmed in this special issue, where two studies (one review and one research) were published on this argument.

The research study presented an investigation on the amount of nine inorganic elements (Cd, Co, Cr, Cu, Fe, Ni, Mn, Pb and Zn) in genotypes from three different species belonging to the *Brassicaceae* family (*Brassica rapa*, *Eruca vesicaria* and *Sinapis alba*), grown according to both conventional and organic cultivation techniques, during two agricultural seasons on two different experimental farms. The results underlined that the inorganic elements amount is influenced mainly by many factors other than the cultivation technique, comprising the soil characteristics. The organic cultivation technique did not decrease the heavy metal content or increase the nutritional quality of *Brassicaceae*, as commonly believed. As a final result, it was predicted that the consumption of 150–200 g of these vegetables, both from organic and conventional agriculture, fulfill the same percentage of Dietary Reference Intakes for Co, Cr, Cu, Fe, Mn and Zn. Regarding the heavy metal (Cd, Ni and Pb) tolerable intakes, only slight differences (mainly for Pb) have been found between both cropping systems [4].

The second manuscript regarding the quality of *Brassicaceae* is a review article, which aimed to highlight the main phytochemical compounds present in brassicas used as a food vegetable that confer nutritional and sensorial quality to the final product, and to investigate the main factors that affect the phytochemical concentration and the overall quality of Brassica vegetables. In summary, the bioactive molecules responsible for the nutritional quality of *Brassicaceae* can be divided in antioxidant compounds (e.g., phenols, vitamin C) and non-antioxidant compounds (e.g., minerals, glucosinolates). The amount of these compounds in Brassica vegetables could be influenced by many factors, including the genetic source, the environmental conditions and the cultivation techniques adopted for the vegetable production [5].

As mentioned above, the nutritional quality of fresh fruits and vegetables depends on many factors, which could affect the final quality of the products. However, if the product is not stored properly after its harvest, the loss or degradation of phytochemical compounds is a tangible possibility. In this regard, a study in this special issue evaluated different options for prolonging the apricot fruit quality during cold storage and shelf life, decreasing the postharvest losses of apricots. The quality parameters (quality losses, antioxidant properties and enzyme activities) were evaluated at different time periods (from 7 to 21 days) at cold storage (1 ◦C) and shelf life (25 ◦C), comparing post-harvest treatments with methyl jasmonate and salicylic acid. As a general trend, both post-harvest treatments significantly decreased the quality loss of chilling injury and fruit decay on all dates. The antioxidant capacity and the phenolic patterns increased for both treatments at all dates, and almost all the antioxidant enzyme activities increased significantly on all dates for both treatments (except catalase activities, which decreased with the methyl jasmonate treatment). In conclusion, both methyl jasmonate and salicylic acid are useful and inexpensive techniques to maintain the apricot fruit quality in both cold storage and shelf life conditions [6].

The second step for the evaluation of the health potential of fruit vegetables is testing the product in an in vitro model. Usually, in this step the capacity of the fruit/vegetable extract is evaluated to limit the viability of pathological cells, or to protect the healthy cells

from an induced external stress. In this special issue, a study evaluating the effect of olive (*Olea europaea* L.) vegetation water on human cells regarding its antioxidant properties and radical scavenger bioactivities was published. The study involved the treatment of two cell lines, human hepatocellular carcinoma and human keratinocytes, with two food supplements containing concentrated olive water in combination with 6% lemon juice or 70% grape juice, respectively. The first analysis of the extracts revealed that hydroxytyrosol was the most abundant polyphenol in both formulations, followed by tyrosol and oleuropein (for the olive-derived concentrate with lemon juice), and by proanthocyanidins and tyrosol (for the olive concentrate with grape juice). Both extracts were demonstrated to be effective antioxidants, also preventing the advanced glycation end product formation. In addition, preliminary data indicate that the administration of hydroxytyrosol through these hydrophilic matrices is better absorbed into the human body [7].

After the demonstration of the beneficial effects of fruit and vegetable consumption in in vitro models, the following step is the evaluation of the positive effects also in in vivo animal models. A trial on morbidity and mortality in the context of sepsis and septic shock on male Sprague Dawley® rats was presented in this special issue. In this study, a thiosulfinate-enriched *Allium sativum* extract was used as adjuvant in the management of sepsis induced by intraperitoneal *Escherichia coli* ATCC 25922 inoculation. To evaluate the efficacy in the sepsis-induced management, clinical, analytical, microbiological and histopathological parameters were evaluated in the control group, in the group treated with antibiotic, and in the group treated with antibiotic plus *Allium sativum* extract. The results confirmed that the utilization of *Allium sativum* extract as an adjuvant to antibiotic treatment in the management of sepsis could improve the sepsis attenuation, ameliorating clinical parameters of rats as weight, ocular secretions, whiskers separation and physical activity level, inhibiting *Escherichia coli* proliferation and thus, reducing overall mortality after an animal peritonitis model [8].

The final step for the valorization of the potential health benefits of fruit and vegetable consumption is the introgression of these products in the human diet. With this aim, two studies were published in this special issue. The first study involved a particular group of people affected by prediabetes mellitus, whose glucose levels did not meet the criteria for diabetes but were higher than those considered normal. These people were fed with two servings per day of *Gynura bicolor*, a red purple-colored vegetable, and the effect on glycemic control and antioxidant ability was evaluated. People were divided into control group and *Gynura bicolor*-fed group, and data on anthropometry and biochemical analysis were collected at 0, 8 and 12 weeks. The results clearly showed that *Gynura bicolor* consumption improved both the glycemic control and the antioxidant activity, mainly because of its high content of polyphenols [9].

The second interventional study was focused on the vulnerable category of elderly people, in particular, regarding the respiratory tract infections. The objective of this study was to evaluate the efficacy of the consumption of a combination of elderberry and reishi extracts on the incidence, severity and duration of respiratory tract infections in a group of healthy elderly volunteers. A group of 60 nursing home residents ≥65 years of age randomly received a combination of 1.5 g of elderberry + 0.5 g of reishi or a placebo daily for 14 weeks. If the incidence of respiratory infections was similar in both groups, the berryfed group presented a significant reduction of common cold event duration and of high severity influenza-like illness events. Moreover, the sleep disturbances were significantly reduced in the berry-fed group. Thus, the suitability of the elderberry + reishi extract in reducing the respiratory tract infections was confirmed [10].

To summarize, in this special issue we have published several works demonstrating that fruit and vegetables contain several bioactive compounds, which give high potentiality to these foods in the prevention of many chronic human diseases. Furthermore, we have published some studies showing that fruit and vegetables demonstrated their positive activities both in in vitro and in vivo models. The last study of the special issue that we want to present is the correct conclusion of this editorial because it translates all the previous suggested findings for a healthy life; in fact, the broad recognition of the positive effects of the Mediterranean Diet, the dietary patterns that were followed in specific regions of the area in the 1950s and 1960s on the longevity of Mediterranean populations led to the adoption of this diet in other regions of the world. This study reviewed the scientific knowledge regarding the beneficial health effects of adherence to this diet, underlying that it is not only linked to the consumption of specific food products but also to social, religious, environmental and cultural aspects. Therefore, the Mediterranean Diet represents a healthy lifestyle in general that can allow to optimize the positive effect of fruit and vegetable consumption [11].

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Effects of** *Gynura bicolor* **on Glycemic Control and Antioxidant Ability in Prediabetes**

**Chu-Hsuan Hsia <sup>1</sup> , Yu-Tang Tung <sup>2</sup> , Yu-Sheng Yeh <sup>3</sup> and Yi-Wen Chien 1,4,5,6,\***


**Abstract:** There exists an intermediate group of individuals whose glucose levels do not meet the criteria for diabetes yet are higher than those considered normal (prediabetes mellitus (preDM)). Those people have a higher risk of developing diabetes in the future. *Gynura bicolor* (GB) is a redpurple-colored vegetable, which is common in Taiwan. GB has shown antioxidant, anti-inflammatory and anti-hyperglycemic effects in previous studies. The aim of this study was to assess the effects of serving two serving sizes of GB every day on the glycemic control and antioxidant ability of preDM subjects. According to the age and anthropometry data of the participates, we assigned them into a control or GB group for the 8-week intervention and 4-week washout period. Data of anthropometry and biochemical analysis were collected at 0, 8 and 12 weeks. Oral glucose tolerance tests were performed, and we collected dietary records on the baseline and Week 8. Both groups received nutrition education and a diet plan individually. After intervention, the fasting glucose and malondialdehyde (MDA) values were significantly decreased in the GB group. HOMA-IR and QUICKI values were improved, and antioxidant activity was increased in the GB group. GB could improve glycemic control and decrease oxidative stress because of its large amounts of polyphenols.

**Keywords:** *Gynura bicolor*; prediabetes; phytochemical; blood glucose; oxidative stress

#### **1. Introduction**

According to The American Diabetes Association (ADA), people whose fasting plasma glucose (FPG) level is 100 to 125 mg/dL were defined as impaired fasting glucose (IFG), or whose 2 h value in the oral glucose tolerance test (OGTT) is 140 to 199 mg/dL were defined as impaired glucose tolerance (IGT), or individuals with glycated hemoglobin (HbA1c) of 5.7–6.4%. People who meet one of three diagnosis criteria were called "prediabetes (preDM)" [1]. Individuals with both IFG and IGT have more severe dysglycemic condition and are especially at high-risk for type 2 diabetes [2]. According to the International Diabetes Federation (IDF), in 2019, there were 463 million people with preDM, and this number is expected to reach 700 million by 2045 [3]. In total, 5–10% of individuals with preDM develop diabetes annually, with up to 70% eventually developing diabetes. Several trials have reported on the risk of diabetes development in preDM individuals after lifestyle and drug-based interventions. PreDM can convert back to normoglycaemia [4,5].

Under hyperglycemia or IR condition, oxidative stress will be increased and endothelial dysfunction will occur [6]. The endothelium is a key regulator of vascular function [7], when dysfunction will cause inflammation, vasoconstriction, thrombosis and platelet activation, which are linked with atherosclerosis [8]. Evidence from prospective studies

**Citation:** Hsia, C.-H.; Tung, Y.-T.; Yeh, Y.-S.; Chien, Y.-W. Effects of *Gynura bicolor* on Glycemic Control and Antioxidant Ability in Prediabetes. *Appl. Sci.* **2021**, *11*, 5066. https:// doi.org/10.3390/app11115066

Academic Editors: Luca Mazzoni, Maria Teresa Ariza Fernández and Franco Capocasa

Received: 21 April 2021 Accepted: 27 May 2021 Published: 30 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

suggest that cardiovascular disease (CVD) may be associated with preDM [9]. Lifestyle intervention includes diet modification, weight reduction or moderate physical activity. Some studies reported that intensive lifestyle interventions such as achieving and maintaining ideal body weight may prevent the progression to type 2 diabetes in IGT or IFG subjects [10]. In the Da Qing trial in China, 577 individuals with IGT were randomized to dietary counseling, increased exercise, diet plus exercise or control (general recommendations). The cumulative 6-year incidence of diabetes was significantly lower in the diet group (43.8%), the exercise group (41.1%) and diet plus exercise group (46.0%) than the control group (67.7%). In a proportional hazards analysis adjusted for differences in baseline BMI and fasting glucose, the diet, exercise, and diet-plus-exercise interventions were associated with 31% (*p* < 0.03), 46% (*p* < 0.0005), and 42% (*p* < 0.005) reductions in risk of developing diabetes, respectively [11]. In a meta-analysis, it was reported that consuming large amounts of vegetables and fruits may reduce the risk of diabetes and associate to inflammation and oxidative stress [12]. Giugliano reported that diets containing vegetables and fruits, n-3 fatty acids and fiber attenuate the inflammation [13]. Phytochemicals such as vitamin C, vitamin E, carotenoids, phytosterols, anthocyanins and alkaloids showed anti-inflammation and antioxidant effects [14].

Anthocyanins belong to the widespread class of phenolic compounds collectively named flavonoids. Mechanic studies support the beneficial effects of flavonoids, including anthocyanins, on the biomarkers of CVD risk such as NO, inflammation and endothelial dysfunction. The role of anthocyanins in CVD prevention is strongly related to against oxidative damage [15]. Red-purple vegetables are rich in anthocyanins, the common vegetables in Taiwan such as purple-leaved sweet potato, *Gynura bicolor* (GB) and purpleleaved celosia. GB is widely distributed in Asia and common cuisine in Taiwan. The leaves of GB distinctively show a reddish purple color on the abxial side and a green color on the adaxial side. *Gynura* is used as traditional Chinese medicine for treatment of inflammation, fever, hypertension and diabetes [16,17]. GB has been shown to have antioxidant, anti-inflammatory and anti-hyperglycemic effects [18–20]. However, limited clinical human studies have examined the effects on glycemic control and antioxidant ability in preDM subjects.

The aim of this study was to assess the effects of serving two exchanges of GB per day on glycemic control and antioxidant ability on preDM subjects.

#### **2. Materials and Methods**

#### *2.1. GB Extract Preparation*

Fresh GB was obtained from Taiwan Seed Improvement and Propagation Station, COA, Taiwan. Leaves of GB were removed, washed, dried and ground to fine powder. The extract was produced from 100 mg of the lyophilized vegetable powder with a 10-fold volume of 70% methanol (0.1% of HCl) in the shaker for 30 min at room temperature, then centrifuged at 1400× *g* for 10 min at 4 ◦C, adjusted the volume of supernant and repeated extraction for 3 times. The collected extracts were kept at −20 ◦C until used for total polyphenol and total anthocyanin analysis.

#### *2.2. Total Polyphenol and Total Anthocyanin Analysis of GB Extracts*

Total polyphenol contents were determined by the colorimetric method using Folin– Ciocalteau reagent [21]. Briefly, the extracts, blank or standard (galic acid), were added to 2 mL of 2% Na2CO3. After 2 min, 100 µL of 50% Folin–Ciocalteau reagent was added. The mixture was left at room temperature in the absence of light for 30 min. The absorbance of the colored product was measured at 750 nm. The standard curve of gallic acid was used for calculating the polyphenol contents.

Total anthocyanins were determined by using the pH differential method [22]. Before analysis, two dilutions of the sample were prepared, one for pH 1.0 using potassium hydroxide buffer (0.025 M, KOH) and the other for pH 4.5 using sodium acetate buffer (0.4 M, 54.4 g CH3COONa). Samples were diluted 100 times and waited 15 min. The ab-

sorbance was measured at 520 and 700 nm. The concentration (mg/L) of each anthocyanin was calculated according to the following formula and expressed as cyanidin-3-glucoside equivalent (CGE):

$$\text{Total enthalpyans} = \frac{(\text{A} \times \text{MW} \times \text{DF} \times 100)}{\varepsilon \times \text{L}}$$

where A is the absorbance =(A520nm − A700nm)pH1.0 − (A520nm − A700nm)pH4.5, MW is the molecular weight (g/mol) = 449.2 g/mol for cyanidin-3-glucoside, DF is the dilution factor = 100, and ε is the extinction coefficient = 26,900, where L (path length in cm) was 1.

#### *2.3. Subjects*

Subjects were recruited from the neighborhood near by Taipei Medical University. Inclusion criteria include whose blood glucose data meet the preDM diagnosis: FPG 100–125 mg/dL or 2 h values in the OGTT 140–190 mg/dL or HbA1c 5.7–6.4% and 20–70 years old. Exclusion criteria were individuals with a history of diabetes or use hypoglycemic agents/insulin in past 3 months, hepatic or renal disease, history of cardiovascular disease or cancer, pregnancy, breast feeding, or intending to become pregnant during the study period, thyroid or pituitary disease, gastrointestinal disease, hematological disorders or neurological disease, drinking alcohol or smoker. Written informed consent was obtained from all subjects, and the study was approved by the Joint Institutional Review Board at Taipei Medical University in Taiwan (JIRB number: 201307030).

#### *2.4. Study Design*

The 12-week study consisted of an 8-week intervention period (Week 0 to Week 8) and a 4-week washout period (Week 8 to Week 12). Subjects were assigned to the control group (prohibit eating red-purple food) or GB group (diet including 2 serving sizes of GB, equivalent to 200 g of edible portion (E.P)). The experimenter weighed GB, and asked the participants to take it home every week and cook it it by themselves. Both groups received dietary counseling based on the guideline of the Taiwanese Association of Diabetes Educators. Subjects were asked to do a 3-day dietary record (2 weekdays and 1 weekend) at Week 0 and 8, and the compliance was checked by dietary record. Every 4 weeks of the intervention period (Week 0, 4 and 8) and 4 weeks washout period (Week 12), anthropometric data and blood pressure were measured. Anthropometric data included height, weight, body composition, body mass index (BMI), waist and hip circumference and waist to hip ratio. Body composition was measured by Inbody 3.0 (Biospace, Seoul, Korea) according to the principle of biochemical impedance analysis (BIA). At Week 0 and Week 8, blood samples were collected after they had fasted overnight.

#### *2.5. Blood Sample Analysis*

Plasma collected by centrifugation at 1500× *g* for 10 min at 4 ◦C was stored at −80 ◦C until further analysis. Fasting glucose, fasting insulin and glycated hemoglobin (HbA1c), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), aspartate transaminase (AST) and alanine transaminase (ALT) levels were analyzed at the laboratory of Taipei Medical University Hospital. Homeostatic model assessment-insulin resistance (HOMA-IR) was calculated according to the formula insulin (µU/mL) × glucose (mmol/L)/22.5, and the quantitative insulin sensitivity check index (QUICKI) was calculated according to the formula 1/[log (fasting insulin) + log (fasting glucose)]. Total polyphenol levels in plasma were measured by the method described by Serafini et al. [23] using Folin–Ciocalteau reagent. Absorption at 750 nm was measured spectrophotometrically. The other blood measurements included the ferric reducing ability of plasma (FRAP), vitamin C and malondialdehyde (MDA), which involved standardized methods. Glutahione peroxidase (GSH-Px) and IL-6 were measured using Cayman standard glutathione assay kit and R&D human IL-6 high sensitivity ELISA kit, respectively. The total polyphenol content was expressed as gallic acid equivalent (GAE).

#### *2.6. Dietary Nutrient Intake Analysis*

Daily nutrient intake was calculated as the mean daily intake from a 3-day dietary record using the nutrient analysis software (E-kitchen, Taichung, Taiwan). Nutrient assessment included total caloric intake, dietary macronutrient intake (protein, fat and carbohydrate), dietary fiber, vitamin C and vitamin E.

#### *2.7. Statistical Analysis*

All data were expressed as the mean ± SEM. The data were analyzed using one-way ANOVA followed by Duncan's multiple range test, Mann–Whitney U test and paired *t*-test. A value of *p* < 0.05 was used to indicate statistical significance.

#### **3. Results**

#### *3.1. Total Polyphenols and Total Anthocyanins of GB*

The total polyphenol of GB extract was 25.54 ± 1.77 mg GAE/g DW. The total anthocyanin of GB extract was 1.53 ± 0.15 mg CGE/g DW.

#### *3.2. Baseline Characteristics and Dietary Intake of Subjects*

Nine subjects (two male and seven female) assigned to the control group and nine subjects (two male and seven female) assigned to the GB group completed the whole study period. General baseline characteristics were shown in Table 1. There were no differences in age or height. Although the dietary fiber intake of both groups did not show significant differences at baseline, the intake in the GB group was significantly increased (36%) after intervention (Table 2).

**Table 1.** Baseline subject characteristics.


Values are mean ± SEM (n = 9), § *p* < 0.05 between groups by Student's *t*-test, GB, *Gynura bicolor*; BMI, Body mass index; HbA1c, Glycated hemoglobin.



Values are mean ± SEM (n = 9), § *p* < 0.05 between groups by Mann–Whitney U test, GB, *Gynura bicolor*; E, energy.

#### *3.3. Glycemic-Control-Related Markers*

Glycemic-control-related markers were shown in Table 3. At baseline, there was no difference in FPG, fasting insulin, HOMA-IR and QUICKI between groups. After an 8-week intervention, FPG and HOMA-IR were significantly decreased in the GB group when compared with baseline. In addition, the results remain maintain after the 4-week washout period (Week 12). The changes of fasting insulin, HOMA-IR and QUICKI in the GB group were significant when compared with the control group (∆0–8 week).

**Table 3.** Effects of *Gynura bicolor* on glycemic control profile.


Values are mean ± SEM (n = 9), § *p* < 0.05 between groups by Mann–Whitney U test; \* *p* < 0.05 compared with Week 0 by Mann–Whitney U test, HOMA-IR, homeostasis model assessment-insulin resistance; QUICKI, quantitative insulin sensitivity check index.

#### *3.4. Anthropometric Measures, Blood Pressure, Lipid Profile and Liver Function*

Anthropometric measures included weight, BMI, body fat, fat-free mass, waist circumference, hip circumference and waist:hip ratio. There were no differences at baseline, Week 4, Week 8 and Week 12 between groups or within groups (Table 4). Blood pressure, TC, HDL-C, LDL-C, AST, and ALT levels did not differ between groups or within groups at baseline, Week 4, Week 8 and Week 12 (Tables 5 and 6).


**Table 4.** Effects of *Gynura bicolor* on anthropometric and body composition measures.

Values are mean ± SEM (n = 9).

**Table 5.** Effects of *Gynura bicolor* on blood pressures.


Values are mean ± SEM (n = 9).


**Table 6.** Effects of GB on blood lipid profile and liver function 1–3 .

<sup>1</sup> Values are mean ± SEM, 2,\* *p* < 0.05 compared with Week 0 by Mann–Whitney U test, <sup>3</sup> GB, *Gynura bicolor*; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; AST, aspartate transaminase; ALT, alanine transaminase.

#### *3.5. Oxidative-Stress-Related Markers*

Oxidative-stress-related markers were shown in Figure 1. At baseline, vitamin C (Figure 1a), total polyphenol (Figure 1b) and MDA (Figure 1e) in the GB group were significantly different when compared with the control group. The baseline total polyphenol level in the GB group was significantly lower when compared to the control group (0.39 ± 0.01 vs. 0.43 ± 0.01). After asking clients to avoid red-purple food for 8 weeks, the total polyphenol level in the control group was significantly decreased when comparing Week 8 to Week 0, though neither group showed any differences at Week 8. Although FRAP was significantly increased in both control and GB groups after 8 weeks, the delta value from Week 0 to Week 8 in the GB group was significantly higher than the control group (3.92 ± 0.56 vs. 1.80 ± 0.19) (Figure 1c). At Week 8, GSH-Px activity in the GB group was significantly higher than baseline (Figure 1d). At baseline, the levels of MDA in the GB group were significantly higher than in the control group (9.32 ± 0.62 vs. 5.88 ± 0.65). After the intervention, the MDA levels of the GB group were significantly decreased when comparing Week 8 to Week 0, and there were no significant differences between Week 0 and Week 8 of the control group (Figure 1e). Moreover, the differences of MDA levels between Week 8 and Week 0 were significantly higher in the GB group when compared to the control group (−5.23 ± 0.66 vs. −0.81 ± 0.43). There were no differences in IL-6 between groups or within groups (Figure 1f).

− −

μ μ ρ **Figure 1.** Effects of *Gynura bicolor* on antioxidants, antioxidant capacity and inflammation marker. (**a**) Vitamin C (µg/mL), (**b**) total polyphenols (mg/mL), (**c**) FRAP (mM), (**d**) GSH-Px (nmol/min/mL), (**e**) MDA (µM), and (**f**) IL-6 (ρg/mL). Values are mean ± SEM (n = 9). § *p* < 0.05 between groups by Mann–Whitney U test; \* *p* < 0.05 compared with Week 0 by Mann– Whitney U test; # the difference value of Week 0 and Week 8 with a significant difference between groups by Mann–Whitney U test (*p* < 0.05) FRAP, ferric reducing ability of plasma; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; IL-6, interlukin-6.

#### **4. Discussion**

≥ ≥ ≥ β In 1988, Reaven described a cluster of risk factors for DM and CVD as "syndrome X", the former name of "metabolic syndrome (Mets)" [24]. Mets have several definitions. In Taiwan, Mets is defined as any three of the following five features: waist circumference >90 cm in men or >80 cm in women, HDL-C < 40 mg/dL in men or <50 mg/dL in women, FPG ≥ 100 mg/dL, TG ≥ 150 mg/dL and BP ≥ 130/85 mmHg. Insulin resistance (IR) is considered one of the main pathophysiology that caused Mets. IR causes abnormal nutrient metabolism such as hepatic gluconeogenesis, lipid peroxidation and TG synthesis. Moreover, vasoconstriction, oxidative stress and inflammation happened. Under this condition, it increases the risk of developing CVD [25]. In previous studies, it showed that IR and β-cell dysfunction were already happening before developing to diabetes [26]. Ford et al. performed a meta-analysis; it concluded that IFG and IGT are associated with modest increases in the risk of cardiovascular disease. Therefore, preDM is related to Mets [27]. In our study, all subjects' FPG levels were over 100 mg/dL and have a large waist circumference. We considered the subjects in this study to be at a higher risk of developing Mets and CVD than others.

HOMA-IR was proposed as a simple and inexpensive alternative to more sophisticated techniques by Matthew et al., and had been evaluated to be reliably used in large-scale or epidemiological studies [28,29]. QUICKI was first performed by Katz et al. QUICKI is a novel, simple, accurate and reproductive method for determining insulin sensitivity in humans [30]. HOMA-IR and QUICKI can only use FPG and fasting insulin to assess the levels of insulin resistance (IR) and insulin sensitivity. The higher the HOMA-IR, the higher the levels of IR. QUICKI is the reverse to HOMA-IR. Several studies showed the different cutoff point of HOMA-IR and QUICKI. Ascaso et al. observed 65 subjects aged 30–60 years in Spain, showing that subjects had IR when HOMA-IR > 2.6 and QUICKI < 0.33 [31]. Keskin et al. described that the cutoff point for diagnosis of IR is >2.5 for adults

and >3.15 for adolescents [32]. In China, a total of 2217 subjects were observed. It increases the risks of developing Mets and diabetes when the quartile1 (Q1) of HOMA-IR > 2.8. No matter which cutoff point they are based on, HOMA-IR and QUICKI in both groups were abnormal before the intervention period. After an 8-week intervention of GB and a 4-week washout period, HOMA-IR in the GB group were significantly decreased when compared with baseline. The difference of QUICKI between baseline and the 8-week intervention was significantly decreased when compared to the control group. Therefore, we speculated that an intake of GB may improve the insulin resistance and elevate the insulin sensitivity. Furthermore, the efforts of improving insulin resistance can be maintained.

Increased consumption of vegetables, whole grains, and soluble and insoluble fiber is recommended to treat preDM and diabetes individuals. Wolfram and Ismail-Beigi collected 14 randomized clinical trials on diabetes from the past decade. They concluded that improving insulin sensitivity and glucose homeostasis on a plant-based diet is more effective than other commonly used diets, because plant-based diets contain fiber, micronutrients (potassium, folate and magnesium) and phytochemicals such as anthocyanins, flavonoids and chlorophyll [33–35]. The PREDIMED study (PREvencio'n con DIetaMEDiterra'nea) conducted a 3-month clinical trial. It showed increasing dietary fiber intake with natural food (22 g per day) on risk factors for CVD in subjects at high risk decreased FPG, TC and increased HDL-C, which is associated with CVD risk factors [36]. Another randomized clinical trial suggested that a moderate amount of dietary fiber intake (7 g per day) may be beneficial for managing the FPG but no effects on weight and BMI in Japanese men with hyperglycemia and visceral fat obesity [37]. According to "2005–2008 Nutrition and Health Survey in Taiwan (NAHSIT)", the dietary fiber intake in men was 13.7 g and 14 g in women, both of which were less than 25–35 g, the recommended intake from the Ministry of Health and Welfare (MHW). In our study, the dietary intake (13–14 g) in the control and GB groups was no different at baseline. After an 8-week intervention of GB, the dietary intake in the GB group was increased 6 g/day and FPG was decreased when compared with baseline. One exchange of GB contained dietary fiber (3.1 g). Although not reaching the recommended intake from the MHW, it did indeed raise the dietary intake. We speculated that decreased FPG was associated with an increasing intake of dietary fiber, which is from GB. In addition, total polyphenols and total anthocyanins of GB extract were 25.54 ± 1.77 mg GAE/g DW and 1.53 ± 0.15 mg CGE/g DW, respectively. A previous study showed that the main chemical components of GB are phenolic acids, flavonoids, carotenoids and anthocyanins [38]. Using UPLC-MS/MS analysis, 53 phenolics were identified [39]. Most polyphenols exist in the form of glycosylated derivatives in GB. These polyphenols must undergo digestive enzymes and intestinal microbiota metabolism to become bioactive in the human body [40]. Clinical study in obese healthy subjects did not support the use of dietary supplementation with dried purple carrot (259.2 mg/day of phenolic acids) to achieve weight loss, improvements in body composition, LDL-C and blood pressure [41]. The same results were also observed in our study, because the total polyphenol was much lower.

Several antioxidants exist in plasma, such as ascorbic acid, vitamin E, carotenoid and polyphenols. The ability of dietary polyphenols to reduce inflammation is related to acting as antioxidants, interfering with oxidative stress signaling, suppressing the proinflammatory signaling transductions. The biological significance of phenolic compounds is not only in direct reaction with ROS, but also in activation of cell signaling pathways [42]. FRAP is presented as a method for assessing total antioxidant power. Ferric ferrous ion (Fe3+) reduction at low pH causes a navy blue ferrous-tripyridyltriazine (Fe2+-TPTZ) complex form. The deeper the color, the better the antioxidant power [43]. We observed that FRAP both in the GB group and control group were increased after an 8-week experimental period. It meant that total antioxidant power was both enhanced. Moreover, the change between baseline and Week 8 in the GB group was more significant than in the control group. MDA is an intermediate product of lipid peroxidation, which usually uses thiobarbituric acid-reactive substances (TBARS) to quantify. One study showed that smoking and alcohol

consumption were confounding factors of MDA [44]. GSH-Px is the important enzyme of the cell defense system. GSH-Px could breakdown peroxide to oxygen and water. The activity of GSH-Px would decrease when peroxidant increases [45]. A previous study observed that Aronia extract results from the influence of anthocyanins and possibly other flavonoids on decreasing MDA and catalase and increasing GSH-Px and superoxide dismutase activities [46]. In our study, we excluded smoker and alcohol consumption subjects. MDA and GSH-Px in the GB group were decreased/increased in Week 8 when compared with baseline, respectively. It showed that, after an 8-week intervention, the oxidative stress in subjects was decreased.

Because of increasing oxidative stress, the pathophysiology of preDM and T2D were considered as a chronic inflammation. IL-6 is an important mediator of inflammatory response [47]. Lucas et al. observed that TNF-α and IL-6 were significantly higher in overweight/obese young subjects with preDM (BMI > 33 kg/m<sup>2</sup> ) than healthy overweight/obese ones [48]. It is not consistent with our study. We speculated that this is because the study population consisted of all overweight individuals (BMI > 25 kg/m<sup>2</sup> ). The inflammation generated from adipocytes was fewer than obesity.

The bioavailability of polyphenols was influenced by the molecular weight, conjugated form (methyl, glucuronide or sulphate) and cooking method. For most flavonoids absorbed in the small intestine, the plasma concentration then rapidly decreases (elimination half-life period of 1–2 h). The maintenance of a high concentration in plasma thus requires a repeated ingestion of the polyphenols over time. Anthocyanins are quite rapidly absorbed, but their bioavailability seems to be the lowest of all flavonoids [40]. Nielsen et al. concluded that urinary flavonoids may be useful as a new biomarker for vegetables and fruits [49]. In the GB group, there were no differences in total polyphenols of plasma. It could be associated with the fact that the blood sample collection of subjects was not conducted all at the same time. When all were finished to collect, it already passed the half-life time of polyphenols. In the control group, total polyphenols of plasma were significantly decreased. It might be associated with prohibiting eating red-purple food for 8 weeks. We suggested that future studies must use urine tests to assess total polyphenol intake.

Our study was the first interventional trial using GB for preDM, by using natural ingredients and at least eating two exchanges of vegetables every day. GB is not only rich in fiber; it also contains higher polyphenols than other green-leafy vegetables. This might have resulted in improvement of glycemic control and reducing oxidative stress in preDM subjects. Although the beneficial effects of GB could be observed, some limitations needed be addressed. (1) PreDM without medication subjects are difficult to find, because people pay less attention to their own blood report. (2) The intervention period was 8 weeks. We did not consider that the half-life of HbA1c is about 3 months. (3) The baseline characteristics had biases between the control group and GB group. This is because we did not assign randomly, but based on subjects' dietary habit. Further studies, we suggest, should consider expanding the intervention time and the allocations of groups.

In conclusion, the subjects in our study were overweight and preDM subjects. After intervention of two serving sizes of GB for 8 weeks, FPG and MDA were decreased, FRAP and GSH-Px were increased in the intervention group and HOMA-IR and QUICKI were improved. We concluded that GB could improve glycemic control and decrease oxidative stress because of its large amounts of polyphenols. In addition, GB not only has health-care functions, but its pigments also show potential uses as natural food colorings. Therefore, GB extract can be used to make functional foods, such as adding to fruit mousse or yogurt.

**Author Contributions:** C.-H.H. and Y.-W.C. were involved in the conceptualization and the design of this study. C.-H.H. conducted this study and biochemical analyses. C.-H.H. wrote the original draft preparation. C.-H.H. and Y.-T.T. reviewed and edited this study. Y.-S.Y. supervised this study. Y.-W.C. visualized and supervised this study. All authors provided critical inputs to data analyses and the interpretation of the data. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Taipei Medical University in Taiwan.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** No new data were created or analyzed in this study. Data sharing is not applicable to this article.

**Acknowledgments:** We thank all subjects for participating in this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Thiosulfinate-Enriched** *Allium sativum* **Extract as an Adjunct to Antibiotic Treatment of Sepsis in a Rat Peritonitis Model**

**Francisco Javier Redondo-Calvo 1,2,3,†, Omar Montenegro 1,†, David Padilla-Valverde 3,4, Pedro Villarejo <sup>4</sup> , Víctor Baladrón 1 , Natalia Bejarano-Ramírez 3,5, Rocío Galán 1 , Luis Antonio Gómez <sup>6</sup> , Natalia Villasanti 3,7 , Soledad Illescas 3,8, Vicente Morales <sup>9</sup> , Lucía Medina-Prado <sup>2</sup> , José Ramón Muñoz-Rodríguez 2,3 and José Manuel Pérez-Ortiz 2,3,\***


**Featured Application: Thiosulfinate-enriched** *Allium sativum* **extract used as an adjuvant to antibiotic treatment and to sepsis management could improve the response profile and attenuate the outcome of the sepsis shock.**

**Abstract:** Up to now, there are no studies that have shown a decrease in morbidity and mortality in the context of sepsis and septic shock, except for antibiotic therapy and the objective-guided resuscitation strategy. The goal was to evaluate the use of thiosulfinate-enriched *Allium sativum* extract (TASE) as an adjuvant in the management of sepsis. An experimental in vivo study was carried out with male Sprague Dawley® rats. Animals were randomized in three treatment groups: the control group (I), antibiotic (ceftriaxone) treatment group (II) and ceftriaxone plus TASE treatment group (III). All animals were housed and inoculated with 1 × 10<sup>10</sup> CFU/15 mL of intraperitoneal *Escherichia coli* ATCC 25922. Subsequently, they received a daily treatment according to each group for 7 days. Clinical, analytical, microbiological, and histopathological parameters were evaluated. Statistically significant clinical improvement was observed in the ceftriaxone plus TASE vs. ceftriaxone group in weight, ocular secretions, whiskers separation and physical activity level (*p* ≤ 0.05). When comparing interleukins on the third day of treatment between II and III, we found statistically significant differences in IL-1 levels (*p* < 0.05). Blood and peritoneal liquid cultures of group I were positive for multisensitive *E. coli*. Group II and III cultures were negative for *E. coli*, although an overgrowth of *Enterococcus faecalis* was found. In conclusion, TASE used as an adjuvant to antibiotic treatment in the management of sepsis could improve response profiles with sepsis attenuation, thus reducing overall mortality after an animal peritonitis model.

**Keywords:** garlic; *Allium sativum*; thiosulfinate; allicin; sepsis; immunomodulation; interleukins; rats

**Citation:** Redondo-Calvo, F.J.; Montenegro, O.; Padilla-Valverde, D.; Villarejo, P.; Baladrón, V.; Bejarano-Ramírez, N.; Galán, R.; Gómez, L.A.; Villasanti, N.; Illescas, S.; et al. Thiosulfinate-Enriched *Allium sativum* Extract as an Adjunct to Antibiotic Treatment of Sepsis in a Rat Peritonitis Model. *Appl. Sci.* **2021**, *11*, 4760. https://doi.org/10.3390/ app11114760

Academic Editor: Luca Mazzoni

Received: 22 April 2021 Accepted: 19 May 2021 Published: 22 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

There are few areas in critical medicine that generate as much interest and research as sepsis. Despite diagnostic and therapeutic advances, sepsis morbidity, mortality, and incidence remain very high. Sepsis is an altered host response to an infectious pathogen, causing potentially life-threatening organ dysfunction, and septic shock is a subset of sepsis in which the underlying circulatory and metabolic abnormalities are deep enough to substantially increase mortality [1]. Despite critical care progress in recent years in critical care, sepsis and septic shock account for more than 50% of deaths in critical care units.

Sepsis is now recognized as a multifactorial host response to an infectious pathogen that can be significantly amplified by endogenous factors involving the early activation of both pro- and anti-inflammatory responses along with major modifications in nonimmunological pathways such as cardiovascular, neuronal, autonomic, hormonal, bioenergetic, metabolic and coagulative [2,3], all of which are of prognostic importance. Additionally, the biological and clinical heterogeneity of affected individuals is important, as well as age, underlying comorbidities, concurrent injuries (including surgery), medications, and source of infection [1].

What differentiates sepsis from infection is an aberrant or poorly regulated host response with the presence of organ dysfunction. The severity of organ dysfunction has been evaluated with various scoring systems that quantify abnormalities based on clinical findings, laboratory data, or therapeutic interventions. Differences in these scoring systems have also resulted in inconsistent information. The predominant score currently in use is the Sequential Organ Failure Assessment Score (SOFA), which has been simplified in quick SOFA [1,3].

Garlic (*Allium sativum*) has long been a medicinal ingredient used as an antineoplastic and antimicrobial agent. Sulfur compounds (i.e., thiosulfinates) appear to be the active components in the root bulb of the garlic plant [4,5]. Allicin is the main thiosulfinate of *Allium sativum* and could act on four points of the inflammatory cascade. The ability of allicin to inhibit the activation of the nuclear factor NF-κβ [6], prevent the adhesion of T cells to endothelial cells and reduce transendothelial migration [7] have been described. Allicin can also reduce the activity of induced nitric oxide synthase [8] and decrease the amount of nitric oxide and the vasodilatation that may lead to shock. Additionally, it could act by preventing the activation of the coagulation cascade by acting as an antiplatelet [9].

Up to now, there are no studies that have shown a decrease in morbidity and mortality in the context of sepsis and septic shock, except for antibiotic therapy and the objectiveguided resuscitation strategy proposed in the 2016 sepsis campaign. This strategy was made by a group of international experts who established a series of based-on-evidence recommendations for the management of acute sepsis and septic shock. It is also the basis for the better outcome of high-mortality critically ill patients [10]. However, many drugs have been used unsuccessfully in both animal models and clinical trials [11–15], so there is still a need of new therapeutics that can overcome the antibiotic resistance.

As allicin is not stable [16], here, we decided to explore whether intraperitoneal applications of thiosulfinate-enriched *Allium sativum* extract (TASE) could be an adjuvant to specific antibiotic treatment in sepsis and septic shock and to evaluate its possible immunomodulatory role.

#### **2. Materials and Methods**

#### *2.1. Animals and Sepsis Model*

Male 5-week-old Sprague Dawley® rats (Harlan Laboratories Models SL) were used. The study was conducted at the Translational Research Unit of the University General Hospital, Ciudad Real. The procedures were carried out at the same time of day to avoid the possible influence of the circadian cycle on the results of the work.

Rats were kept with food and water ad libitum, in a cycle of 12 h of light and 12 h of darkness, and a room temperature of 22 ± 2 ◦C with a relative humidity of 50–70% and 15–20 air renewals per hour without recirculation. They were housed according to RD

53/2013 and no rat was caged alone to favor their group behavior. In addition, they were maintained in these environmental conditions to allow acclimatization for a week before the study started. Animals were randomized in three groups. Group I: physiological saline (*n* = 6); group II: ceftriaxone (*n* = 9); and group III: ceftriaxone + TASE (*n* = 9). A model of peritonitis was generated in all groups. Rats from different groups were never housed in the same cage.

To create the peritonitis model, each rat was administered with an intraperitoneal injection of bacteria after anesthesia with ketamine/xylacin (75/10 mg/kg), also directly into the abdominal cavity. Prior to this experiment, we conducted an experimental study to determine the most optimal inoculum dose to generate the sepsis and septic shock model. We determined that it was necessary to use a concentration of *Escherichia coli* ATCC 25922 of 1 × 10<sup>10</sup> colony forming units (CFU) in 15 mL of distilled water [17].

#### *2.2. Thiosulfinate-Enriched Allium sativum Extract*

Lyophilized *Allium sativum* extract was obtained from the purple garlic ecotype from Las Pedroñeras (Ciudad Real, Spain), the only European region with protected geographical status for garlic (ES/PGI/005/0228/12.03.2002). A patented protocol (WO 2008/102036 A1. Method for obtaining a freeze-dried, stable extract from plants of the *Allium* genus) was employed for extraction to guarantee the stability and concentration of allicin and other thiosulfinates. The standardized composition and concentration of lyophilized *Allium sativum* extract are stable for over 10 months at 4 ◦C (Table 1, WO 2008/102036 A1). We employed diallyl thiosulfinate (allicin) concentration as the reference for the elaboration of the experimental treatment.


**Table 1.** Composition of organic and inorganic compounds of lyophilized *Allium sativum* extract from Las Pedroñeras (Ciudad Real, Spain) under optimized conditions (WO 2008/102036 A1).

#### *2.3. Experimental Design and Analytical Parameters*

In relation to the treatments used, group I received 4.4 mL of 0.9% physiological saline intraperitoneally, group II received the same intraperitoneal volume with the antibiotic ceftriaxone (100 mg/kg) and group III the same volume with ceftriaxone (100 mg/kg) + TASE (0.5 mg/kg; referred to allicin content). In Figure 1, we showed the experimental scheme of the study.

Vitamin E (α

**Figure 1.** Chronological scheme of the study. Day zero started with the arrival of the animals, which included their respective housing, marking and the beginning of an adaptation process to the animal facility that lasted seven days. On the seventh day, *E. coli* inoculation and first treatment dose were performed. Blood samples for interleukin determination were taken 72 h after. On day 14, animals were sacrificed by lethal doses of anesthesia, with subsequent sampling for microbiology (blood and peritoneal fluid), interleukins (blood) and pathological anatomy (peritoneum, liver).

The following clinical parameters were evaluated daily: weight, mobility, appearance (normal, ocular secretions, nasal secretions, whisker position, lack of grooming, piloerection and dehydration), clinical signs (abdominal distension, hardening distension, temperature) and behavior (normal, hypoactive, lethargy; response to stimuli).

Interleukin (IL) 1β/IL α) were determanufacturer's instructions. All Interleukin (IL) 1β/IL-1F2, IL-6 and tumor necrosis factor alpha (TNF-α) were determined in blood samples on treatment day 3 (T3) and 7 (T7) with the corresponding Quantikine ® Rat ELISA method (R&D Systems), following the manufacturer's instructions. All samples were diluted 1:3 in RD5Y diluent. Each diluted sample and standards were processed in duplicate. Internal quality control was performed with recombinant buffered IL control material of known concentration. The final reading was made at 450 nm and corrected to 550 nm in a microplate reader.

Peritoneal fluid was also sampled on T7 (last day of the experiment) directly from the peritoneal cavity. Peritoneal fluid study was performed to determine cellularity by Giemsa staining. During the exploratory laparotomy, the degree of peritoneal inflammation was evaluated macroscopically. Liver and peritoneum samples were taken for histopathological evaluation. Thus, samples were paraformaldehyde fixed, paraffin embedded, and 4 µm sections were made for hematoxylin/eosin staining to analyze the presence or absence of congestion and immune cells. A blinded expert pathologist evaluated the samples.

#### *2.4. Statistical Analysis*

A descriptive statistic of the quantitative variables was carried out to verify that the minimum and maximum values were in an adequate range. All data were expressed as mean ± standard error of the mean (x ± SEM).

To analyze the qualitative variables, proportions were compared with the chi-square test and Fisher's exact correlation. The means of the continuous variables were compared with U Mann–Whitney's test and normal distribution was verified by Shapiro Wilk's test.

In comparisons at different times within the same groups, tests were applied for paired variables (Student t for dependent variables or Wilcoxon test as the case may be). For the comparison between groups, the Kruskal–Wallis test was used as a function of normality. A significance level of 95% was used for statistical analysis. SPSS 25.0 for Windows (SPSS Inc., Chicago, IL, USA). ' – ' ' –

#### **3. Results**

Our model of abdominal sepsis is able to generate 100% lethality in rats if no antibiotic treatment is provided [17], so the control group could not finish the experimentation due to the fact that all members of the group died after 4–6 h post-inoculum. Unfortunately, one rat from group II also died after the inoculum despite the ceftriaxone administration, so we completed group II with eight rats.

In relation to clinical parameters, we found differences when comparing the body weights of group II and III on days 9, 10 and 11 (*p* < 0.05), corresponding to the third (T3), fourth (T4), and fifth (T5) dose of treatment (Figure 2). In relation to stress and suffering (nasal secretions, ocular secretions, whiskers position, lack of grooming, piloerection, lethargy, and diarrhea), we could only find differences in the level of activity of rats during the septic process 72 h post-inoculum (T3), showing greater hypoactivity in group II compared to group III (*p* ≤ 0.05). Statistically significant differences were also observed in the position of the whiskers (*p* ≤ 0.05) and in the presence of ocular secretions (*p* ≤ 0.05) at that time point. No statistically significant differences were found in the rest of the clinical signs studied (Table 2), and from T4 to T7 (end of the study; data not shown). –

In relation to the biochemical parameters studied (IL-1, IL-6, TNF- α), a comparison was made for the values of each interleukin between treated groups in T3 and T7 (Figure 3). Considering the levels of IL-1, in T3 we found statistically significant differences when comparing group II with respect to III (*p* < 0.05). It was also observed that IL-6 in T3 was lower in group III, although the values were not statistically significant. As for TNF-α, no differences between groups were assessed. *p ≤ p ≤ p ≤* 

Body weight

**Figure 2.** Weight monitoring during experiment: control group (8 days), group treated with ceftriaxone (CEF; 14 days) and group treated with ceftriaxone + thiosulfinate-enriched *Allium sativum* extract (CEF + TASE; 14 days). On day 7, the bacterial inoculum was introduced. Mean ± SEM. \* *p* < 0.05.


**Table 2.** Clinical parameters in relation to stress and suffering at T1, T2 and T3 (24, 48 and 72 h postinoculum, respectively) for nasal secretions, eye secretions, position of whiskers, lack of grooming, hair erection, lethargy, and diarrhea. \* *p* ≤ 0.05.

≤

**Figure 3.** Interleukin levels in T3 and T7 (treatment day 3 and 7, respectively). CEF = ceftriaxone. CEF + TASE = ceftriaxone + thiosulfinate-enriched *Allium sativum* extract. Mean ± SEM. \* *p* < 0.05.

> The peritoneal liquid and blood cultures of the control group were positive for multisensitive *E. coli* ATCC 25922 and identical to the inoculum (Table 3). Additionally, as mentioned before, all the rats from the control group did not recover from the inoculum and died after 4–6 h, showing that our sepsis model is lethal if left untreated. In group II, only one rat died after inoculation, and of the remaining eight rats, six showed *Enterococcus faecalis* in blood cultures, and two in peritoneal liquid. In the blood cultures of the two remaining rats, the multi-sensitive bacteria *E. coli* ATCC 25922 appeared. In group III, *E. coli* ATCC 25922 was not detected neither in blood nor in peritoneal liquid. In fact, eight out of nine rats showed *Enterococcus faecalis* in blood samples, and only one out of nine rats

showed *Enterococcus faecalis* in peritoneal liquid. There was also one rat that was negative for both bacteria.

**Table 3.** Results of blood and peritoneal fluid cultures stratified by treatment groups and their respective antibiogram.


\* TASE (0.5 mg/kg; referred to allicin content).

Regarding the histopathological analysis and organ evaluation, the inflammatory cell count, the presence of bacteria in the liver and on the peritoneal surface, as well as the congestion and hepatic vacuolization between treatment groups (group II and III), no statistically significant differences were found (Table 4).

**Table 4.** Histopathological analysis and organs evaluation in relation to inflammation, bacteria, congestion, and vacuolization.


#### **4. Discussion**

Until now, many models of sepsis have been described in animal experimentation, but most of them failed to replicate the human heterogeneous septic process, which is dependent on the genetic susceptibility of each individual and influenced by sex, age, comorbidities and drug consumption [18,19]. The murine model described here generates an efficient, controlled and easily reproducible intraperitoneal infection that could serve as a basis for future lines of research.

Scientific research based on the use of garlic derivatives has led to ambiguous conclusions on the beneficial effects of this plant, thus preventing the application of garlic products in the treatment of certain diseases [20]. This situation can be attributed to several factors, among which the following can be highlighted: the chemical instability of this type of compound [21], the great diversity of industrial processes for their production, the lack of coherence in terms of the medical properties investigated, and the chemical composition of the products used in these clinical investigations [22]. With the freeze-dried

garlic used in this experimental model, these deficiencies could be overcome by using stable and known concentrations of allicin and other thiosulfinates over time.

At present, there are no studies in the scientific literature that have demonstrated a decrease in morbidity and mortality in relation to sepsis, except for antibiotics and the goal-guided resuscitation strategy [23]. Some of the drugs that have been tested are, among others, corticoids [12], immunoglobulins [13], antithrombin III [14], vasopressin [24] or anti-TNF [25]. However, the clinical results obtained in our animal experimentation study are encouraging in this sense and confirm our working hypothesis showing earlier recovery of weight, less ocular secretions, separation of whiskers and decrease in hypoactivity in the group where TASE was administered. We also found lower levels of IL-1 on the third day of treatment, and a tendency to decrease the pro-inflammatory cytokine IL-6 in the TASE group. All these data would support the immunomodulatory role of the lyophilized garlic, thanks to its action in the inflammatory cascade [26], thus achieving the attenuation of sepsis and septic shock. Previous work has described the ability to suppress inflammatory signals of lipopolysaccharide (LPS) through the expression of anti-inflammatory genes, and the reduction in pro-inflammatory cytokines (IL-6 and MCP-1) [27]. The work of Lee et al. [28] showed the immunomodulatory activity of garlic in an experimental sepsis model where clamping and blind puncture were performed to induce peritonitis. The authors described how the administration of methyl 3-formyl-4-methylpentanoate (a natural compound derived from garlic) led to the inhibition of apoptosis of lymphocytes in the spleen and significantly inhibited the production of proinflammatory cytokines such as TNF-α, IL-1β and IL-6. The production of TNF-α and IL-6 stimulated by LPS was also strongly inhibited by the compound methyl 3-formyl-4 methylpentanoate sucrose in macrophages derived from mouse bone marrow [28]. In our study, we could not assess any significant difference on TNF-α levels in the TASE group.

The microbiological analysis showed how the blood cultures of the control group were positive for *E. coli*. This bacterium corresponded to the same inoculum with which sepsis was generated. However, no blood culture or peritoneal fluid were positive for *E. coli* in the TASE-treated group. This result highlights the fact that the inoculum was sensitive to the antibiotic and coadjuvant treatments used. In fact, it is known that there is a 90% sensitivity of *E. coli* to ceftriaxone and only 65% sensitivity of *Klebsiella pneumoniae*. Despite these moderately high percentages, we are currently in a global state of alarm due to the increased resistance of several microorganisms to this antibiotic compared to previous studies [29]. Moreover, most of the blood and peritoneal fluid cultures in our antibiotic treatment group showed the presence of *Enterococcus faecalis*. This result could be explained by considering the broad-spectrum efficacy of ceftriaxone on Gram-negative and some Gram-positive bacteria. This change of gastrointestinal microbiota would favor *Enterococcus faecalis*. One likely explanation for this selection is based on related studies in mice where LPS and flagellin from Gram-negative and anaerobic bacteria stimulated the production of RegIIIγ in Paneth cells by interactions with Toll-like receptors. Paneth cells have an important role in the defense mechanisms of the gastrointestinal tract in several animal species, thanks to their secretions of lysozyme, phospholipase A2 and defensins [30]. RegIIIγ is a C-type lectin receptor, capable of recognizing carbohydrates present on the surface of pathogens and is responsible of the internalization of the pathogen for antigen presentation and the induction of an immunological response. Thus, the level of RegIIIγ maintains the balance between the bacteria that compose the intestinal microbiota and the host [31]. When ceftriaxone used in our study killed the Gram-negative bacteria, it decreased the production of RegIIIγ and facilitated the growth of Gram-positive coccus (i.e., *Enterococcus faecalis*). Then, those Gram-positive could cross the intestinal barrier and reach the systemic circulation, liver and more. Therefore, if the antibiotic treatment persists for a long time, there could be a potential risk of bacterial endocarditis.

Few studies with animal models associate histological, clinical, and microbiological findings in intraperitoneal organs such as liver, peritoneum, and intestine after induced peritonitis. In this sense, our study tried to correlate those findings with the clinical response

to a treatment based on a thiosulfinate-enriched garlic extract. Only Lee et al. [28] examined the lung after peritonitis for these inflammatory changes, and also after therapy with a garlic derivative. Unfortunately, we could not obtain any histopathological difference between treatment groups because the tissue and organ evaluations were assessed at the end of the experiment and rats from both treatment groups were mostly recovered from the septic insult. Moreover, our study has several limitations that are inherent to the animal model of sepsis and septic shock that we use. First, the amount of blood collected was limited and did not allow the measurement of a greater number of inflammatory factors and biomarkers of endothelial damage. Secondly, no measurements were taken in relation to myocardial function and macrocirculation such as mean arterial pressure, contractility, peripheral vascular preload, and resistance to perform a target-guided therapy as it is usually performed in humans.

#### **5. Conclusions**

Thiosulfinate-enriched *Allium sativum* extract used as an adjuvant to antibiotic treatment and to sepsis management could improve the response profile and attenuate the outcome of the sepsis shock, mostly during the first days of the combined treatment. Further research would be necessary to clarify the immunomodulatory role of this plant extract.

#### **6. Patents**

Patent WO 2008/102036 A1. Method for obtaining a freeze-dried, stable extract from plants of the *Allium* genus.

National patent (Spanish Trademark number ES2675282A1). *Allium sativum* extract, its use for the manufacture of a medicinal product for the treatment of diseases, and its obtaining procedure.

**Author Contributions:** Conceptualization, F.J.R.-C., D.P.-V. and J.M.P.-O.; data curation, L.M.-P. and J.R.M.-R.; formal analysis, F.J.R.-C., O.M., V.B., N.V., S.I. and V.M.; investigation, O.M., N.B.-R., R.G. and L.M.-P.; methodology, D.P.-V., P.V. and S.I.; project administration, F.J.R.-C., D.P.-V. and J.M.P.-O.; resources, F.J.R.-C., D.P.-V. and L.A.G.; supervision, F.J.R.-C., D.P.-V. and J.M.P.-O.; visualization, J.R.M.-R.; writing—original draft, F.J.R.-C., O.M. and S.I.; writing—review and editing, F.J.R.-C., P.V., V.B., N.B.-R., R.G. and J.M.P.-O. All authors have read and agreed to the published version of the manuscript.

**Funding:** There are no sources of funding for the work.

**Institutional Review Board Statement:** The study (ref. PI-HGUCR 1/2014) was approved by the Animal Experimentation Committee of the University General Hospital, Ciudad Real. It was authorized by the Office of Agriculture of Castilla-La Mancha (Spain) and this experiment followed the ARRIVE guidelines developed by the National Center for the Replacement, Refinement and Reduction of Animals in Research (nc3rs).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are grateful to the Pathology, Microbiology and Clinical Analysis Departments of the Hospital General Universitario de Ciudad Real. We also thank Clara Villar-Rodríguez for her technical support (Translational Research Unit, Hospital General Universitario de Ciudad Real).

**Conflicts of Interest:** L.A.G. is part of the authors of the registered brand Aliben© (European Trade mark number 10543429) which entitles the lyophilized *Allium sativum* extract employed in this study (patent WO 2008/102036 A1. Method for obtaining a freeze-dried, stable extract from plants of the *Allium* genus). D.P., P.V., J.M.P-O., J.R.M-R., L.A.G. and F.J.R-C. are co-contributors of a national registered patent (Spanish Trade mark number ES2675282A1), which employs the lyophilized *Allium sativum* extract, its use for the manufacture of a medicinal product for the treatment of diseases, and its obtaining procedure.

#### **References**


## *Article* **Cellular Antioxidant Effects and Bioavailability of Food Supplements Rich in Hydroxytyrosol**

**Cecilia Bender \* , Sarah Straßmann and Pola Heidrich**

Institut Kurz GmbH, Stöckheimer Weg 1, 50829 Köln, Germany; s.strassmann@institut-kurz.de (S.S.); p.heidrich@institut-kurz.de (P.H.)

**\*** Correspondence: c.bender@kurz-italia.com

**Abstract:** The present study evaluates the effect of olive (*Olea europaea* L.) vegetation water on human cells regarding its antioxidant properties and radical scavenger bioactivities. To this aim, two food supplements containing concentrated olive water in combination with 6% lemon juice or 70% grape juice, respectively, were assessed in different oxidation assays. From the investigated polyphenols, hydroxytyrosol, present in olives and in a lesser extent in grapes, was found to be the most abundant in both formulations, followed by tyrosol and oleuropein for the olive-derived concentrate with lemon juice, and by proanthocyanidins and tyrosol for the olive concentrate with grape juice. Cellular studies suggest that both formulations are effective antioxidants. In particular, the combination of olive and grape extracts showed a remarkable superoxides-, hydroxyl radicals-, and hydrogen peroxides-scavenging activity, while the formulation containing 94% olive concentrate wasmore potent in protecting the cells against lipoxidation. Both products showed a significant and similar effect in preventing advanced glycation end products' (AGEs) formation. In addition, preliminary data indicate that hydroxytyrosol is absorbed into the human body when administered via these hydrophilic matrices, as confirmed by the urinary excretion of free hydroxytyrosol. Since the availability of phytochemicals largely depends on the vehicle in which they are solved, these findings are of relevance and contribute to supporting the healthful effects here assessed in a cellular environment.

**Keywords:** hydroxytyrosol; olive extract; olive polyphenols; grape extract; oleuropein; antioxidant capacity; F2-isoprostanes; AGEs

#### **1. Introduction**

Many consumers associate the fruit of the olive tree (*Olea europaea* L.) mainly with the resulting oil, the precious olive oil that is considered particularly healthy compared to other oils. The raw olive fruit contains several types of phenols, thecontents of which vary with the olive cultivar, mainly tyrosol and its derivatives, phenolic acids, and flavonoids [1]. However, many of these substances that the olive has to offer from a health perspective are water-soluble, and thus remain largely in the residue of the olive pressing and the oil contains only a small part. This is the case of hydroxytyrosol, a polar phenol slightly soluble in fats, which can be found in olives as a simple phenol, or either esterified with elenolic acid to form oleuropein aglycone, and which is naturally present in significantly higher concentrations in the olive fruit's aqueous fraction.

The vegetation water, resulting from the pressing of the olive fruits duringthe production of the olive oil, is rich in bioactive compounds, particularly polar phenols, and typically contains 98% of the total phenols of the olive fruit [2].

The positive health effects of olive polyphenols are already known; in particular, hydroxytyrosol has potential antioxidant, anti-inflammatory, and health benefits mainly related with cardiovascular diseases [3–6].

Bioavailability and pharmacokinetic analyses, which were mainly reported with pure hydroxytyrosol and with olive oil, suggest that hydroxytyrosol can be rapidly absorbed

**Citation:** Bender, C.; Straßmann, S.; Heidrich, P. Cellular Antioxidant Effects and Bioavailability of Food Supplements Rich in Hydroxytyrosol. *Appl. Sci.* **2021**, *11*, 4763. https:// doi.org/10.3390/app11114763

Academic Editor: Luca Mazzoni

Received: 30 April 2021 Accepted: 20 May 2021 Published: 22 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

from blood and distributed in the human body [7], metabolized, and quickly eliminated in urine mainly as glucuronide and sulfate [8]. Currently, hydroxytyrosol from different sources is available on the market. Its absorption and subsequent urine excretion may be dependent on the vehicle of administration [9]. Thus, the bioavailability of hydroxytyrosol and its precursors (oleuropein and tyrosol) from those specific sources would be a prerequisite for its health effects in humans.

The present study addresses the bioactivity of hydroxytyrosol-rich extracts, obtained from the vegetation water resulting from olive oil production. Herein, hydroxytyrosol is present as both a simple phenol and as oleuropein aglycone.

For the study, olive-derived concentrates combined with 6% lemon juice or 70% grape juice and marketed as liquid supplements were characterized. Bioactivities, mainly related to the antioxidant potential, were evaluated in cultured cells by means of the antioxidant capacity (cellular antioxidant activity assay, superoxide dismutase and catalase activities), the protection against lipoxidation (inhibition of F2-isoprostanes formation) and glycation (inhibition of AGEs formation). In addition, preliminary data on the bioavailability and urinary recovery of free hydroxytyrosol through acute administration of the food supplements are presented from an open-label cross-over study with four volunteers.

Despite the difference in the composition of both formulations, the main phytochemical in the ones that were investigated was hydroxytyrosol, present in both olive fruit and to a lesser extent in grapes. The treatment of the cells with the supplements gave positive results, although these were slightly different in magnitude, through antioxidant actions. The high bioactivity observed suggests a possible application in the maintenance of the cellular redox state and for related health benefits.

#### **2. Materials and Methods**

#### *2.1. Standards and Reagents*

2,2′ -azobis [2-methylpropionamide] dihydrochloride (AAPH), quercetin dihydrate, and 2′ -7′ -dichlorodihydrofluorescin diacetate (DCFH2-DA) were purchased from Sigma-Aldrich (Milan, Italy). Hydroxytyrosol and oleuropein were procured from Cayman Chemical (Ann Arbor, MI, USA). Resveratrol was purchased from Sigma-Aldrich (Steinheim, Germany). Dulbecco's modified Eagle's medium (DMEM) high-glucose culture media, L-glutamine, trypan blue solution, and trypsin-EDTA solution 10X were culture grade and purchased from Merck (Milan, Italy). Fetal bovine serum (FBS), Dulbecco's phosphate buffered saline (PBS) without Mg2+ and Ca2+, and Hank's balanced salts solution (HBSS) were culture grade and purchased from Euroclone SpA (Milan, Italy). Water, acetonitrile, formic acid, and methanol (all LC-MS-grade) were purchased from VWR Chemicals (Darmstadt, Germany). All other chemicals were analytical grade and purchased from common sources.

#### *2.2. Sample Material*

The food supplements analyzed are derived from olive fruit (*Olea europaea* L.) vegetation water subjected to filtration and concentration, and were supplied by Fattoria La Vialla (Castiglion Fibocchi, Arezzo, Italy). The commercial brands are Oliphenolia bitter™ and Oliphenolia™, hereinafter referred to as P-1 and P-2, respectively. P-1 consists of 94% concentrated olive aqueous fraction and 6% lemon juice (*Citrus limon* L. fructus); while P-2 is characterized by 30% concentrated olive extract and 70% grape juice (*Vitis vinifera* L. fructus).

#### *2.3. Analysis of Polyphenols*

The samples were diluted with methanol (50:50 *v*/*v*), ultrasonicated, centrifuged, and filtrated through 0.45 µm regenerated cellulose filters prior to measurement by UHPLC-MS, with an Acquity UPLC I-Class system coupled to a XEVO-TQS micro mass spectrometer (bothWaters, Milford, MA, USA). The instrument consisted of a sample manager cooled at 10 ◦C, a binary pump, a column oven, and a diode array detector measuring at 280 nm. The column oven temperature was set at 40 ◦C. The gradient started with 2% A and raised linearly to 15% within 5.5 min, then to 100% A within 1 min before holding for 1.5 min as a washing step; it then decreased back to 2% B within 1 min and was equilibrated for 2 min. Eluent B was water with 0.1% formic acid, eluent A was acetonitrile with 0.1% formic acid, the flow was 0.4 mL/min on an HSS T3 RP column (150 mm x 2.1 mm, 1.7 µm particle size) combined with a precolumn (Acquity UPLC HSS T3 VanGuard, 100 Å, 2.1 mm × 5 mm, 1.8 µm), both from Waters (Milford, MA, USA). The injection volume was 2 µL.

The peaks were identified by MS/MS (MRM 153 > 123 for hydroxytyrosol, 539 > 377 for oleuropein and SIR 137 for tyrosol in negative mode and MRM 229 > 135 for resveratrol operating in positive ion mode). The source voltage was kept at 1.5 kV, and the cone voltage was 20 V. The source temperature was set at 150 ◦C and the desolvation temperature at 350 ◦C with a desolvation gas flow of 650 L/h and a cone gas flow of 50 L/h. Standard substances were used as reference.

Proanthocyanidin monomers were determined according to Kelm et al. [10].

Data were acquired and processed using MassLynx (Waters, Milford, MA, USA).

#### *2.4. Cell Cultures*

Human hepatocellular carcinoma (HepG2) and human keratinocytes (HaCat) cell lines were obtained from CLS (Cell Lines Service GmbH, Germany) and cultured at 37 ◦C under a humidified atmosphere of 5% CO<sup>2</sup> in DMEM containing 2 mM L-Glutamine, 4.5 g/L glucose, and 10% of heat-inactivated FBS. Experiments were performed with DMEM low-glucose (Lonza Ltd., Morristown, NJ, USA) supplemented with 2 mM L-Glutamine without FBS in either, 6-well culture plates for AGEs, catalase, and superoxide dismutase (SOD), 12-well plates for F2-isoprostanes, or 96-well black plates for the cellular antioxidant activity (CAA) and vitality assays. For each cell-based test, P-1 and P-2 samples were centrifuged, sterile-filtered and directly diluted into culture media before testing.

#### *2.5. Cellular Viability Assay*

To determine the optimal growth conditions of cells following 4 h treatment with the sample material, five serial dilutions were evaluated for P-1 and P-2 (range 1:150 to 1:750 and 1:250 to 1:1250 for HaCat and HepG2 cells, respectively), and the metabolic activity was monitored using the resazurin toxicity assay according to the manufacturer's instructions (Tox-8 kit, Sigma-Aldrich, Italy). Fluorescence was read at 37 ◦C (Em. 590 nm/Ex. 540 nm) in a multiwell fluorescence reader (Fluostar Optima, BMG LabTech, Offenburg, Germany). Data were processed using Mars 2.0 Optima Data Analysis software (BMG LabTech GmbH, Germany).

#### *2.6. Cellular Antixidant Activity (CAA)*

The intracellular reactive oxygen species (ROS) formation was detected with the CAA method by spectrofluorimetry using the cell-permeable probe DCFH2-DA, as previously described [11,12]. Briefly, HepG2 cells were cultured until confluence and pre-incubated for an hour with DCFH2-DA and increasing concentrations of the sample dilutions (1:750 to 1:250 *v*/*v*) or the quercetin standard. After the addition of AAPH, the absorbed probe was oxidized to a high fluorescent molecule within the cytoplasm, which was measured at 37 ◦C for an hour at excitation 485 nm and emission 540 nm (Fluostar Optima, BMG LabTech, Germany). Raw data were analyzed using MARS 2.0 Optima Data Analysis software (BMG LabTech, Germany). Results are expressed as µmol of quercetin equivalency (QE) per mL of product and as the mean of five independent measurements ± standard deviation.

#### *2.7. Cellular Extract Preparation*

At the indicated time points, the cells were harvested in ice-cold PBS and collected in 2 mL centrifuge tubes before homogenization for the AGEs, catalase and SOD experiments. For whole lysates preparation, samples were homogenized using the Cell Disruptor Genie® (Scientific Industries Inc., Bohemia, NY, USA) with 0.5 mm glass beads, according to the

manufacturer's instructions. Whole protein lysates were obtained by centrifugation for 10 min at 10,000× *g*, clear supernatants were transferred to clean tubes and their total protein content was determined according to the Bradford method [13], and they were then preservedat −80 ◦C for further analysis.

#### *2.8. Catalase Activity Assay*

HepG2 cells were cultured without (untreated control) or with the specific sample material (dilution 1:750 *v/v*) for 72 h. After treatment cells were harvested and lysed, the catalase activity was immediately measured by fluorescence using a commercial assay (Arbor Assays Ltd., Ann Arbor, MI, USA, Cat. No: K033-F1) according to manufacturer's instructions. Raw data were analyzed using Mars 2.0 Optima Data Analysis software (BMG LabTech GmbH, Germany), and the results were normalized with the total protein content, expressed as the mean of two experiments and as units of catalase activity per mg of protein ± standard deviation, and then compared with the untreated control.

#### *2.9. Superoxide Dismutase (SOD)*

HepG2 cells were incubated for 72 h with 1:750 *v/v* dilution of the samples.After treatment cells were harvested and lysed for further absolute quantification of SOD activity using a commercial kit (Sigma-Aldrich, Italy; SOD assay, Cat. No: 19160) following the manufacturer's recommendations. In the presence of oxygen, xanthine oxidase generates O<sup>2</sup> •−, which in turn converts a colorless substrate into a yellow product. Samples with increasing levels of SOD cause a decrease in the O<sup>2</sup> •− concentration, reducing the yellow color, which is read at 450 nm. Raw data were analyzed using Mars 2.0 Optima Data Analysis software (BMG LabTech GmbH, Germany), and the results were normalized, expressed as mean of three experiments in terms of units of SOD activity per mg of protein ± standard deviation, and compared with the untreated cells.

#### *2.10. Endogenous F2-Isoprostanes Measurement*

HaCat cells were seeded in 12-well plates (500,000 cells/mL) and pre-incubated overnight without (untreated control) or with diluted samples (1:750 *v*/*v*). After replacing the culture media, lipoxidation was provoked by incubating with AAPH 1 mM for 2.5 h. Supernatants were then removed, centrifuged and immediately investigated for 8-epi PGF2α concentrations using a commercial ELISA kit (item n. 516360, Cayman Chemical, USA) following manufacturer's protocol. Results, expressed as the mean of three experiments ± standard deviation, were determined using Mars 2.0 Optima Data Analysis software (BMG LabTech GmbH, Germany).

#### *2.11. Endogenous AGEs Measurement*

HaCat cells were plated and incubated overnight in complete culture medium. For the experiments the culture medium was replaced with serum-free medium without (untreated control) or with diluted samples (1:750 *v*/*v*). After an hour of incubation, the medium was replaced with an AGEs-inducer solution containing glyoxal and/or S-pbromobenzylglutathione cyclopentyl diester at increasing concentrations and incubated for 4 h. Whole protein lysates were processed for quantitative determination of AGEs with a commercial ELISA kit (Cusabio Ltd., Wilmington, DE, USA, Cat. No: CSB-E09412h) according to the manufacturer's recommendations. Spectrophotometric measurements were recorded with a multiwell reader (Fluostar Optima, BMG Labtech, Germany), and the raw data were analyzed using Mars 2.0 Optima Data Analysis software (BMG LabTech GmbH, Germany) and expressed as the mean of two experiments ± standard deviation and as AGEs concentrations relative to protein content.

#### *2.12. Bioavailability*

A pilot, open-label, single-dose, two-period, cross-over design study was conducted in our laboratory to test the urinary excretion of free hydroxytyrosol and trans-resveratrol in

self-reported healthy volunteers. In the investigation, two males and two females received, after an overnight fast, a single dose (50 mL) of one food supplement with 200 mL of water separated by one week wash-out period before administration of a single dose of the second food supplement. Urine samples were collected immediately before intake (baseline) and after 30 min of intake. The samples were centrifuged and filtered before being measured by LC-MS/MS, as described above. Freshly prepared urine-blank samples spiked with standards were used for the hydroxytyrosol calibration.

#### **3. Results**

#### *3.1. Characterization of the Food Supplements*

Table 1 shows the chemical characterization of a representative batch of P-1 and P-2. Of the selected phytochemicals identified, hydroxytyrosol is the main phenolic compound in both samples, as shown by the LC-MS analysis.

**Table 1.** Chemical analysis of selected phytochemicals of olive-derived food supplements. Values are expressed as mean ± standard deviation of 2 determinations. <sup>a</sup> single determination. \* manufacture's data; – not determined.


A comparison of the average amount of total phenolics gives similar results for both samples. Despite the ratio of olive water being lower in P-2, this compound contains higher amounts of hydroxytyrosol and derivatives. This result is consistent with P-2′ s combination of grape juice and further concentrated olive vegetation water, as both would contribute to the hydroxytyrosol content. In addition, P-2 comprises trans-resveratrol and proanthocyanidin monomers from grapes, phytonutrients that have considerable antioxidant properties, and that are also said to have positive effects on health [14–18]. It is very likely that P-2 contains additional polyphenols from grapes, and that the two products may also differ in the content of vitamins, or the phytocomplex they contain, though they will not be described in detail here, as hydroxytyrosol is the main polyphenol from the olive on which we are focused.

#### *3.2. Bioassays*

Since the aqueous olive extracts show a high content of natural phenols, we examined their possible connection with the prevention of oxidative stress. To this end, sample dilutions found to be non-toxic to the HepG2 and HaCat cell lines were further investigated for their antioxidant effects.

#### 3.2.1. CAA

The CAA measures the ability of an antioxidant sample to inhibit the formation of induced reactive oxygen species (ROS) within cells [11]. While antioxidant samples inhibit the formation of free radicals in a dose-dependent manner, an increment in intracellular fluorescence denotes an increment in ROS formation [19]. Both purified extracts exert a strong antioxidant activity in the CAA assay (Table 2 and Figure 1) by inhibiting the production of peroxides at the intracellular level.


**Table 2.** CAA results. lts. **<sup>Ŧ</sup>** HepG2 HepG2 cells' viability > 90% in the dilution range tested; \*\* QE = quercetin equivalency; \* *p* < 0.05 according to *t* test. **Ŧ**

**Ŧ**

± ±

**Figure 1.** CAA of P-1 and P-2. Data are represented as box plots, showing median, 25–75% quartiles, standard error, and total range of values from 5 experiments. QE = quercetin equivalency.

We found that P-2, containing grape and olive concentrates, displayed a more potent cellular antioxidant potential compared to P-1 containing 94% of the olive extract (*p* < 0.05). In a preventive treatment, the CAA values were 9.20 and 6.77 µmoles quercetin equivalents per mL for P-2 and P-1, respectively.

Interestingly, the results for both purified extracts of olive water are even superior to those of extracts known to be highly antioxidant, such as pure chokeberry juice, which in the CAA test yields an average of 5.27 µmol QE/mL (unpublished results).

Similarly, data reported elsewhere have showed that polyphenols extracted from olive vegetation water are able to inhibit ROS production in human neutrophils and in endothelial cells exposed in vitro [20,21].

#### 3.2.2. SOD and Catalase Activities

To minimize the harmful effects of excess ROS, aerobic organisms have developed several lines of antioxidant defense, which are employed in addition to the direct action of antioxidant molecules [22]. Among these, the enzymatic defense plays a key role in oxidative damage prevention [23]. Catalase and superoxide dismutase (SOD) directly scavenge hydrogen peroxide and superoxide radicals, respectively, converting them into less reactive species.

Given the performance of P-1 and P-2 samples in the CAA test, we evaluated the endogenous SOD and catalase defense in HepG2 human cells after treatment with both formulations, and then compared these to the untreated cells (baseline activity). Under the conditions tested, both supplements showed an increased SOD activity (*p* < 0.01) in liver cells compared to the untreated control (Table 3). A similar effect on catalase activity was found with the P-2 sample, but not with the P-1 sample.


**Table 3.** Antioxidant enzyme activity measured in cell-lysates after 72 h of treatment with the sample material. Average results are expressed in terms of activity units per mg of protein ± standard deviation. UTC: untreated control cells. \* *p* < 0.01 according to *t*-test.

Similar results supporting the increase in catalase and SOD after treatment with phenolic-rich olive water were obtained by others through a series of cellular tests [20], and in vivo in rats' liver [24].

In accordance with the results obtained in the CAA dosage, the P-2 formulation containing grape and olive extracts exerts a stronger antioxidant effect than the formulation without grape extract. This suggests a potent synergism of olive water polyphenols in the presence of grape extract, and could be explained by the different phenolic compositions, which include, among other things, grape-derived phenolic compounds, such as transresveratrol, anthocyanins and proanthocyanidins, as well as a further concentration of the olive vegetation water, which results in a higher content of hydroxytyrosol and tyrosol.

#### 3.2.3. Cellular Peroxidation

Isoprostanes are a family of eicosanoids produced by the random oxidation of tissue phospholipids by oxygen radicals. Several studies have shown that the content of F2 isoprostanes in the human body (i.e., measured in vivo) is directly related to oxidative damage to lipids. In particular, the 8-epi-prostaglandin F2-alpha isomer (8-isoprostane), an end product of the lipid peroxidation chain reaction, has been proposed as a reliable signaling molecule for antioxidant deficiency and oxidative stress [25–28]. The degradation of lipids by lipoxidation occurs as a result of oxidative damage, and consequently the levels of isoprostanes increase, contributing in turn to the development of many diseases related to oxidative stress.

To evaluate the potential of olive-derived supplements in protecting cellular lipids from oxidative damage, we measured the levels of free 8-isoprostanes in cultured cells. Modest amounts of 8-isoprostanes were present in the culture media under normal culture conditions (untreated control), which were increased by oxidative stress after induction by AAPH treatment, but these decreased after incubation with olive-derived extracts or quercetin treatments (Figure 2 and Table 4); compared with untreated cells (baseline level), treatment with P-1 caused a significantly (*p* < 0.05) greater reduction in the content of free 8-isoprostanes (~29% reduction), while P-2 showed a moderate yet not significant effect on lipid oxidation protection (~6% reduction).

α **Figure 2.** Box plots represent the free isoprostanes (8-epi PGF2α) released in culture, showing median, 25–75% quartiles, standard error, and total range of values from 3 experiments. UTC: untreated control cells; +: lipid peroxidation initiator; Q: quercetin control. \* *p* < 0.05 versus UTC.

**α**

**Table 4.** Free isoprostanes released in culture after treatments. The relative values (third column) were normalized to the UTC, which represents the basal amount of isoprostanes under normal culture conditions, and multiplied by 100. UTC: untreated control cells; AAPH: lipid peroxidation initiator; Q: quercetin 100 µM. \* *p* < 0.05 vs. UTC.


The products derived from olives appear to cut down the number of oxidative attacks on the cell lipid membrane; in fact, the basal oxidative stress is effectively quenched compared to the untreated control cells, thus promoting cellular health. Similarly, in humans, oils rich in olive polyphenols have been shown to reduce the urinary excretion of F2-isoprostanes [29].

#### 3.2.4. Antiglycation Activity

Advanced glycation end products (AGEs) are a heterogeneous group of substances that are formed in the human body during non-enzymatic glycosylation between the carbonyl group of a reducing sugar and a free amino group of a protein [30,31]. AGEs play an important, albeit complicated, role in cellular aging processes, in which they are produced in large quantities, causing oxidative stress, inflammatory reactions, and chronic diseases such as diabetes and cardiovascular disease [32,33]. Hyperglycemia, the accumulation of triosephosphates and ketone bodies, lipid peroxidation, and oxidative stress can increase AGEs formation [34]. The irreversible glycation of proteins in turn results in structural alterations and the accumulation of defective proteins in cells, which impactnormal physiological functions [35].

Since increased intracellular and extracellular stress is a source of AGEs accumulation in vivo, and the antioxidant activities of natural phenolics may inhibit the AGEs production [36,37], we investigated cellular AGEs to further support the potential of P-1 and P-2 in preventing the cellular stress.

We have found that both products reduce the formation of new AGEs (Table 5).

**Table 5.** AGEs content in cell lysates after treatment with the extract products and an AGEs-inducer mix. UTC: untreated control cells; +: AGEs inducer solution. Relative AGEs values are normalized to the UTC, which represents the basal amount of AGEs in normal culture conditions, multiplied by 100.


Relative values greater than 100 indicate cellular accumulation of AGEs (e.g., treatment with the AGE-inducer alone), while lower values indicate a potential for the sample to reduce the AGEs formation. Pre-incubation of the cells with each product, followed by the induction of AGEs formation, effectively cleanses the cellular AGEs, with respect to both the AGEs-inducer treatment alone and with the cells under normal culture conditions. The strong positive influence is similar for both pre-treatments with each olive-derived concentrates, and both show a nearly 50% reduction in AGEs formation in vitro.

Our results are consistent with data from a recent study conducted with an olive leaf extract concentrated in hydroxytyrosol (54.5 mg/g), in which a reduction in AGEs production was demonstrated in HepG2 cells subjected to carbonyls-induced stress [38].These authors reported that, likely due to a synergistic effect of hydroxytyrosol and other minor compounds with similar polarities, the olive leaf extract exerts a wide antiglycative activity.

#### *3.3. Bioavailability*

Several cellular effects have been evaluated in vitro to test the antioxidant potential of olive water concentrates. Although, the bioavailability of such components is a prerequisite to any health claim made based on cellular tests. To investigate whether the hydroxytyrosol and trans-resveratrol consumed with the food supplements here studied are bioavailable, we conducted a pilot internal study with four volunteers (two male, two female). Volunteers in a fasted state received a single dose of 50 mL of P-2 in period 1 and 50 mL of P-1 in period 2, separated by a one-week wash-out period. Trans-resveratrol and hydroxytyrosol in its free form were undetectable in urinary samples collected immediately before intake. Free hydroxytyrosol, but not free trans-resveratrol, was detectable in urinary samples collected 30 min after the intake of both supplements and in all the volunteers (Figure 3).

**Figure 3.** UHPLC-MS/MS chromatograms of the MRM 153 > 123. (**A**) hydroxytyrosol standard in urine; (**B**) representative urine sample taken 30 min after ingestion; (**C**) representative urine sample before intake of the supplement/product.

Similarly, previous human studies have showed that plasma and/or urinary hydroxytyrosol increase following the oral administration of hydroxytyrosol, consumed with olive oil [39–41], liquid or encapsulated olive leaf extract [42], and encapsulated extract from oil mill wastewater [8]. In these experiments, the absorption and excretion of orally administered hydroxytyrosol, collected mainly in conjugated forms and in a dose-dependent manner, have been shown. Furthermore, it has been shown that human absorption may differ depending on the composition of the food matrix through which the hydroxytyrosol is dispensed. Besides this, the bioavailability is likely influenced by wide interindividual variability in the absorption and metabolism [43].

Our preliminary results show that hydroxytyrosol consumed together with a hydrophilic vehicle is bioavailable in the human body, independently of the interactions of the combined fruit extracts used. Moreover, hydroxytyrosol can be detected in urine in its free form. This is relevant as the absorption of hydroxytyrosol is dependent on the vehicle of administration [9]. This is likely because the interaction between olive water and grape or lemon juices influences the hydroxytyrosol absorption and recovery yield. Further studies should be done to verify this hypothesis.

#### **4. Discussion**

Our study explores the antioxidant potential of two food supplements derived from olive vegetation water, mainly characterized by a high content of hydroxytyrosol. The antioxidant activity was determined by measuring the cellular antioxidant activity, the catalase and SOD activities in the HepG2 cell line, as well as the inhibition of lipid peroxidation and glycoxidation in the HaCat cell line. The tests carried out have shown that both olive-derived products have a strong positive influence on cells; this influence is complex and not one-dimensional. This reflects the complex nature of the sample materials and suggests a powerful synergy of hydroxytyrosol with other olive phenols, which is further potentiated in the presence of the grape phytocomplex. In particular, both supplements are able to reduce oxidative parameters in vitro.

On one hand, P-1, containing 94% olive water concentrate, showed a good capacity in the CAA and SOD assays, but a better performance in preventing isoprostanes formation in vitro when compared to P-2 (Table 6). On the other hand, P-2, containing olive and grape concentrates, showed a greater antioxidant potential for scavenging reactive species, as indicated by its greater potential in removing the hydroxyl radicals and by its higher SOD and catalase activities. Regarding the prevention of AGEs accumulation, both products showed an excellent capacity in vitro. The overall better antioxidant performance of P-2 in vitro could be explained by the higher concentration of olive-derived polyphenols, as well as the presence of grape-derived antioxidants, which include, among others, the trans-resveratrol, anthocyanins and proanthocyanidin monomers.

Table 6 describes the cellular effects measured, and their links to the attributed in vivo effects.

The mechanism of this positive influence needs to be better understood and has led us to further investigations on the mechanisms and dynamics of the effects of food supplements derived from olive vegetation water on human cells. However, understanding the absorption and bioavailability of these key molecules after oral administration remains a prerequisite before any potential health effect can be derived. In this sense, the pilot trial shows that the hydroxytyrosol supplied with ahydrophilic matrix combining olive fruit concentrate and lemon or grape juices is effectively absorbed, and then urinarily excreted as hydroxytyrosol in its free form.

In subsequent studies, the exact excreted fraction will be determined, and further focus will be placed on the metabolites to obtain a broader picture of the entire ADME properties.

Overall, preliminary data obtained in vitro indicate that the aqueous extracts of olives can actually improve the cellular redox status and related markers, and that their main active ingredient is bioavailable to the human body. Aqueous olive concentrates, with or without grape concentrate, are valid candidates for the prevention of cellular oxidative damage, and thus merit further attention.

**Table 6.** Results of bioassays on human cell lines and the effects' connections in vivo. Direction of in vitro effect: increased (↑), decreased (↓), or no effect (~). Effect magnitude compares P-1 to P-2.


**Author Contributions:** Conceptualization, investigation, writing—original draft preparation, methodology, formal analysis: C.B.; writing—review and editing, formal analysis: S.S.; visualization, methodology: P.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the German Federal Ministry for Economy and Energy under the program ZIM (Zentrales Innovationsprogramm Mittelstand), grant numbers 16KN038023 and ZF4226901SK6.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study, as only urine was taken, there was no medical intervention and there was no danger to the health of the test persons.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Phenolic Profiling of Five Different Australian Grown Apples**

**Heng Li <sup>1</sup> , Vigasini Subbiah <sup>1</sup> , Colin J. Barrow <sup>2</sup> , Frank R. Dunshea 1,3 and Hafiz A. R. Suleria 1,2,\***


**Abstract:** Apples (*Malus domestica*) are one of the most widely grown and consumed fruits in the world that contain abundant phenolic compounds that possess remarkable antioxidant potential. The current study characterised phenolic compounds from five different varieties of Australian grown apples (Royal Gala, Pink Lady, Red Delicious, Fuji and Smitten) using LC-ESI-QTOF-MS/MS and quantified through HPLC-PDA. The phenolic content and antioxidant potential were determined using various assays. Red Delicious had the highest total phenolic (121.78 ± 3.45 mg/g fw) and total flavonoid content (101.23 ± 3.75 mg/g fw) among the five apple samples. In LC-ESI-QTOF-MS/MS analysis, a total of 97 different phenolic compounds were characterised in five apple samples, including Royal Gala (37), Pink Lady (54), Red Delicious (17), Fuji (67) and Smitten (46). In the HPLC quantification, phenolic acid (chlorogenic acid, 15.69 ± 0.09 mg/g fw) and flavonoid (quercetin, 18.96 ± 0.08 mg/g fw) were most abundant in Royal Gala. The obtained results highlight the importance of Australian apple varieties as a rich source of functional compounds with potential bioactivity.

**Keywords:** apple; royal gala; pink lady; red delicious; smitten; fuji; phenolic compounds; antioxidant activity; LC-ESI-QTOF-MS/MS; HPLC

#### **1. Introduction**

Apples (*Malus domestica*) are widely grown and consumed fruits. In 2018, apple production across the globe was 86 million tonnes, mainly from China, America and New Zealand, whereas the apple production in Australia was over 2.6 million tonnes [1]. Apples are usually supplied to the market in the form of fresh fruit or processed products, including dried apples, apple cider, apple juice and sauce [2]. Apples are enriched with bioactives compounds [3], vitamins (water and fat soluble) and minerals like calcium, potassium and phosphorus [4]. These compounds are required by the human body to perform various functions like strengthening of the bones, building muscles, filtering out waste [3], and have positive health benefits against several chronic diseases, including type 2 diabetes, asthma and rheumatoid arthritis [5].

The varieties of apples are due to the difference of agroclimatic regions and zones, cultivation practices, nutritional composition and sensory characteristics [6]. Royal Gala, one of the variety of apples having bright shiny red colour, with stripes ranging from straw yellow to amber orange, has a sensory profile that is sweet, soft, crunchy and slightly acidic [7,8]. Pink Lady is a variety that has been originated from a cross between 'Golden Delicious' and 'Lady Williams', known for its sweet taste, firmness and possesses a scald-free surface [6]. A consumer panel in New Zealand appreciated the Pink Lady variety for its dense flesh, excellent crispness, juiciness, good sugar-acid balance and sweet flavour [9]. The Red Delicious variety when compared to the previous two varieties has a darker crimson red surface with traces of yellow and orange [10]. The physical

**Citation:** Li, H.; Subbiah, V.; Barrow, C.J.; Dunshea, F.R.; Suleria, H.A.R. Phenolic Profiling of Five Different Australian Grown Apples. *Appl. Sci.* **2021**, *11*, 2421. https://doi.org/ 10.3390/app11052421

Academic Editor: Luca Mazzoni

Received: 19 February 2021 Accepted: 4 March 2021 Published: 9 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

characteristics of Red Delicious is an elongated form with a thick peel, grainy and tender with a melting texture, usually exhibiting small but evident humps on the skin surface [11]. While different varieties exhibit different appearances, taste and shapes, apples have one common characteristics, which are the high concentrations of phenolic compounds that exhibit high antioxidant potential [12].

Phenolic compounds are important plant secondary metabolites which exhibit excellent abilities to reduce and eliminate free radicals thereby providing antioxidant and anti-lipid peroxidation properties [13,14]. The phenolic compounds exhibiting antioxidation potential have made the food and nutrition market interested in phenolic compounds, thus replacing the existing chemical anti-oxidation ingredients in food to increase the nutritional value and health benefits [14]. One of the polyphenol mechanisms is the removal of free radicals by supplying hydrogen atoms or separate electrons from the phenol group and eliminating related enzymes, thereby preventing the production of free radicals and their intermediate products [15]. Additionally, phenolic compounds can react with metal ions to inactivate the Fenton reaction [16]. The antioxidant potential are often determined by using a series of different in vitro spectrophotometric-based assays including the total antioxidant capacity (TAC), 2,2′ -azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay, the ferric reducing ability of plasma (FRAP) and 2,2′ -diphenyl-1-picrylhydrazyl (DPPH) [17].

Liquid chromatography coupled with mass spectrometry (LC-ESI-QTOF-MS/MS) is an effective tool used for the identification and characterisation of phenolic compounds. High pressure liquid chromatography (HPLC) combined with photodiode array detector (HPLC-PDA) is used for the quantification of the phenolics [18,19]. According to a previous study, few phenolic compounds have been identified in apples through HPLC and LC-ESI-QTOF-MS analysis including flavanols (catechin), dihydrochalcones (chlorogenic acid), phenolic acids and anthocyanins [20].

Although there are many studies that have isolated and identified phenolic compounds in different apples, only a few have focused on Australian grown apples. The novelty of this study will encourage the Australian producers to utilise the low-grade produce of the apples to a better use as it is rich in phenolics, since premature or overripe fruits compromise the quality and do not meet the standards of the supermarkets. Therefore, in the current research we extracted phenolics from five popular varieties of Australian grown apples (Royal Gala, Pink Lady, Red Delicious, Fuji and Smitten) and estimated their antioxidant potential. The outcome of the current research will add adequate information on the phenolics and antioxidant potential of Australian grown apples for their further application in the food, nutraceutical and pharmaceutical industries.

#### **2. Materials and Methods**

#### *2.1. Chemicals and Reagents*

The chemicals used for the extraction and characterisation were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA). The chemicals used for phenolic estimation and antioxidant assays were procured from Sigma-Aldrich (St. Louis, MO, USA) including ferric (III) chloride anhydrous, 50% acetic acid, 2,4,6-tripyridyl-s-triazine (TPTZ), acetonitrile, catechin, ascorbic acid, vanillin, aluminium chloride hexahydrate, 2,2′ diphenyl-1-picrylhydrazyl, 2,2′ -azino-bis(3-ethylbenz-thiazoline-6-sulphonate), potassium persulfate and Folin-Ciocalteu´s phenol. The standards for HPLC including protocatechuic acid, epicatechin, gallic acid, epicatechin gallate, caffeic acid, quercetin, chlorogenic acid, *p*-hydroxybenzoic acid and kaempferol were procured from Sigma-Aldrich (Castle Hill, NSW, Australia). Ammonium molybdate and sodium acetate hydrated were procured from Sigma-Aldrich (Castle Hill, NSW, Australia). Moreover, 99% ethanol was procured from Thermo Fisher (Waltham, MA, USA), and 98% sulfuric acid was purchased from RCI Labscan Ltd. (Rongmuang, Thailand).

#### *2.2. Sample Preparation and Extraction*

Australian grown apple varieties (Royal Gala, Pink Lady, Red Delicious, Fuji and Smitten) were bought from a local market in Melbourne, VIC, Australia. All the samples were fully matured and ripen before harvested, transported and distributed to the local retailers within 2–3 days using refrigerated trucks. The apple peels were removed by a peeler and the core was separated to obtain the pulp. Subsequently, the pulps were blended into a slurry using a blender. 5 g of slurry samples were macerated in 20 mL of 70% ethanol (*w*/*v*) by slightly modifying the protocol of our earlier published study of Gu et al. [21]. The slurry samples were homogenised to prepare the sample extracts of the apples in a homogeniser at 10,000 rpm for 30 s. The homogenised extract samples were incubated in a shaking incubator at 120 rpm, 4 ◦C for 12 h. The samples were centrifuged for 15 min at 5000 rpm (4 ◦C). A syringe filter was used to filter the extracts used for LC-ESI-QTOF-MS/MS and HPLC-PDA studies and the samples were stored at −20 ◦C for further analysis.

#### *2.3. Estimation of Phenolic Compounds and Antioxidant Assays*

The estimation of phenolic compounds present in the samples and their potential antioxidant activities were analysed following our previously published protocols of Tang et al. [22] and Wang et al. [23].

#### 2.3.1. Determination of Total Phenolic Content (TPC)

The spectrophotometric method of Yunfeng et al. [24] was used for the determination of TPC with some modifications. For this, 25 µL of the apple extract with 200 µL water and 25 µL Folin–Ciocalteu reagent solution were added to 96-well plates. The reaction mixture was incubated for 5 min (25 ◦C). Then, 5 µL of 10% sodium carbonate was added to the reaction mixture and incubated for 60 min in the dark at room temperature. The absorbance of the reaction mixture was measured at 765 nm using spectrophotometer. The standard used was gallic acid (0–200 µg/mL) to construct the standard curve and the values of TPC was expressed in mg of gallic acid equivalent per gram of sample (mg GAE/g of sample) (fw).

#### 2.3.2. Determination of Total Flavonoids Content (TFC)

The Total Flavonoids Content (TFC) was determined by improvising the aluminium protocol described in Rajurkar and Hande [25]. For this, 80 µL of the apple extract with 120 µL of 50 g/L sodium acetate solution and 80 µL of 2% aluminium chloride were added into the 96-well plate subsequently incubate the reaction mixture at 25 ◦C for 2.5 h. The absorbance was measured at 440 nm. Quercetin calibration curve (0–50 µg/mL) was constructed and TFC was expressed in quercetin equivalent (mg QE/g fw).

#### 2.3.3. Determination of Total Tannin Content (TTC)

The vanillin-sulfuric acid method with some modifications of Mesfin and Won Hee [26] was used to determine TTC. 25 µL of the apple extract was added to 25 µL of 32% sulfuric acid and 150 µL of 4% vanillin solution in the 96-well plate. The reaction mixture was incubated for 15 min at 25 ◦C. The absorbance was measured at 500 nm and expressed in mg of catechin equivalent per g of sample weight (mg CE/g fw) based on a calibration curve with concentration from 0–1000 µg/mL.

#### 2.3.4. 2,2′ -Diphenyl-1-picrylhydrazyl (DPPH) Assay

The DPPH method was used to determine the free radical scavenging activity [27]. For this, 40 µL of DPPH methanolic solution (0.1 mM) and 40 µL of extract were added into the 96-well plate. The reaction mixture was shaken vigorously and incubated for 30 min at 25 ◦C. The absorbance was measured at 517 nm. The standard used was ascorbic acid to construct the standard curve (0 to 50 µg/mL). The obtained values were expressed in mg of ascorbic acid equivalent per gram (mg AAE/g) (fw).

#### 2.3.5. Ferric Reducing Antioxidant Power (FRAP) Assay

The ferric reducing ability was assessed by modifying the FRAP method of Faiza et al. [28]. The FRAP solution was prepared at the ratio of 10:1:1, 300 mM sodium acetate solution, 20 mM Fe [III] solution and 10 mM TRTZ. 20 µL of the apple extract and 280 µL of FRAP dye solution added to the 96-well plate. The reaction mixture was incubated for 10 min at 37 ◦C. The absorbance was measured at 593 nm. The ascorbic acid standard curve (0–150 µg/mL) was constructed and the values obtained were expressed in mg of ascorbic acid equivalent per gram of sample (mg AAE/g fw).

#### 2.3.6. 2,2′ -Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) Assay

In ABTS assay, the free radical scavenging activity of the apple samples were determined by following the protocol as in Rajurkar and Hande [25]. First, 88 µL of 140 mM potassium persulfate and 5 mL of 7 mM ABTS solution were mixed to form the ABTS<sup>+</sup> stock solution and incubated for 16 h in a dark area. 290 µL of prepared diluted ABTS solution was mixed with 10 µL of extract. Subsequently, incubation of the reaction mixture in the dark area for 6 min (25 ◦C). The absorbance was measured at 734 nm. The standard curve used to calculate the antioxidant potential was of ascorbic acid (0 to 150 µg/mL). The values were expressed in ascorbic acid equivalents (mg AAE/g) of sample.

#### 2.3.7. Total Antioxidant Capacity (TAC)

The phosphomolybdate [29] method was used to determine the TAC. The formulation for phosphomolybdate reagent was 0.6 M sulphuric acid, 0.004 M ammonium molybdate and 0.028 M sodium phosphate. Then, 260 µL phosphomolybdate reagent was mixed with 40 µL extracts in the 96-well plate. The incubation of the reaction mixture was at 95 ◦C for 10 min. The absorbance was read at 695 nm after the reaction mixture cools down to room temperature. Ascorbic acid standard curve (0–200 µg/mL) constructed to determine the values of TAC and expressed in mg ascorbic acid equivalents (AAE) per gram (fw).

#### *2.4. LC-ESI-QTOF-MS/MS Analysis of Phenolic Compounds*

The identification and characterisation of phenolics in five varieties of apples were conducted using LC-ESI-QTOF-MS/MS and following the protocol described in Suleria et al. [18]. The separation of compounds was carried out through LC column 250 × 4.6 mm, 4 µm with column temperature at 25 ◦C. The HPLC buffers were sonicated at room temperature for 10 min. The binary solvent delivery system was used as follows: Mobile phase A: 2% acetic acid and 98% water; Mobile phase B: acetonitrile, water and acetic acid (50:49.5:0.5, *v*/*v*/*v*). The injected sample volume was 6 µL and the flow rate was at 0.8 mL/min. The program set was carried out as following: 0 min (10% B), 20 min (25% B), 30 min (35% B), 40 min (40% B), 70 min (55% B), 75 min (80% B), 77 min (100% B), 79 min (100% B), 82–85 min (isocratic 10% B). Negative and positive modes were performed for peak identification. Nitrogen gas was used as a nebulizer and drying gas at 45 psi, temperature at 300 ◦C with the flow rate of 5 L/min. The range of mass spectra were 50– 1300 amu. Agilent LC-ESI-QTOF-MS/MS Mass Hunter workstation software (Qualitative Analysis, version B.03.01, Agilent, Santa Clara, CA, USA) was used for data acquisition and analysis.

#### *2.5. HPLC-PDA Analysis*

The HPLC-PDA analysis of polyphenols in apples was carried out using Agilent 1200 series HPLC [30,31]. The volume of the injected sample was 20 µL. 280 nm, 320 nm and 370 nm were the wavelengths used for detection. The column and the conditions used were as followed in LC-ESI-QTOF-MS/MS analysis. The wavelengths were used for the identification of hydroxybenzoic acids, hydroxycinnamic acids and flavanol group, respectively. The acquisition of the data and analysis were carried out using Agilent LC-ESI-QTOF-MS/MS Mass Hunter workstation software (Qualitative Analysis, version B.03.01, Agilent, Santa Clara, CA, USA).

#### *2.6. Statistical Analysis*

The experiments were performed in triplicates (n = 3) and the data was expressed in mean ± standard deviation. One-way analysis of variance (ANOVA) followed by Tukey's honestly significant differences (HSD) multiple rank test were performed to see the significant difference between the phenolic compounds and antioxidant activities at *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Phenolic Compound Estimation (TPC, TFC and TTC)*

The Folin–Ciocalteu's reagent method determined the total phenolic content in the apple extracts and were expressed as gallic acid equivalents (GAE/g fw) as shown in Table 1. Red Delicious apple showed the highest TPC with 121.78 ± 3.45 mg GAE/g and significantly higher than other samples (*p* < 0.05). The total polyphenol content of five different varieties of apples were in the order of Red Delicious > Royal Gala > Fuji > Pink Lady > Smitten. According to the study of Ting et al. [32], Praveen et al. [33] and Almeida et al. [34], Red Delicious had more phenolic content than Gala, Fuji and Pink Lady, which is consistent to the result of our study. Almeida et al. [34] reported that Fuji apple contains 14.7 ± 0.4 mg (GAE)/g and Ting et al. [32] study showed that Fuji has 489.59 ± 4.21 mg (GAE)/g, the difference in the phenolic content might be due to the geographical location, soil nutrients, growth period and harvest season [35]. Additionally, due to the lack of research on Smitten apple variety, there is no valid data for Smitten for comparison.

Flavonoids have attracted a lot of attention due to their strong antioxidant activity [36]. In TFC, Red Delicious apple had the highest flavonoid content of 101.23 ± 3.75 mg QE/g and the lowest flavonoid content was present in Smitten. In a previous study, TFC of Red Delicious (98 mg QE/g) and Royal Gala (89 mg QE/g) were similar to that of our apple samples [37]. In another study, the values of total flavonoid content of Fuji apple (108 mg QE/g) was reported more than our value which may be due to the difference of varieties or solvent extraction ratio [38]. The TTC in our selected apples ranged between 4.65 ± 0.03 to 2.17 ± 0.05 mg CE/g. Fuji apple showed higher level of tannin content followed by Pink Lady, Smitten, Royal Gala and Red Delicious. Previously, the total tannin content of different varieties ranged from 0.75 mg CE/g to 14.79 mg CE/g, which is consistent with our results [39]. Overall, the variety of Red Delicious had the highest content of TPC and TFC and Fuji variety had a high content of TTC.


**Table 1.** Phenolic content and antioxidant potential in five varieties of apples.

All values are expressed as the mean ± SD and performed in triplicates. Different letters (a, b, c, d, e) within the same column are significantly different (*p* < 0.05) from each other. The five varieties of apples are reported based on fresh weight. CE (catechin equivalents), QE (quercetin equivalents), GAE (gallic acid equivalents), AAE (ascorbic acid equivalents). TFC (total flavonoids content), TPC (total phenolic content), TTC (total tannins content), FRAP (ferric reducing ability of plasma), DPPH (2,2′ -diphenyl-1-picrylhydrazyl), TAC (total antioxidant capacity), ABTS (2,2′ -azino-bis-3-ethylbenzothiazoline-6-sulfonic acid).

#### *3.2. Antioxidant Activities (DPPH, FRAP, ABTS and TAC)*

The antioxidant potential of five varieties of apple samples were estimated by four assays including DPPH, FRAP, ABTS and TAC assays, and the antioxidant activities were expressed in ascorbic acid (AAE) per gram (fw) as mentioned in Table 1.

In the DPPH assay, the free radical scavenging activity is determined which is attributed to the phenolic compounds [40]. The apple varieties in the current study varied from 1.17 to 3.53 mg AAE/g. Red Delicious had the highest antioxidant potential followed by Royal Gala, Pink Lady, Fuji and Smitten. Previous studies reported that antioxidant potential for over ten varieties of apples ranged from 0.26 to 9.30 mg AAE/g [41,42]. The values of Fuji and Red Delicious apples are slightly higher than ours which might be because of the cultivar, location, maturity and storage of apples which may change the concentration of antioxidant potential [43].

FRAP assay can provide comprehensive information about the antioxidant activities of five varieties of apples since various antioxidant assays can help us to understand the antioxidant properties of apples better [44]. In FRAP assay, the electron transfer method was used to measure the capacity to reduce Fe3+ to Fe2+ [20]. The FRAP values were significantly different (*p* < 0.05) from 2.12 ± 0.04 mg AAE/g to 4.42 ± 0.01 mg AAE/g among the apple varieties. The highest FRAP capacity was recorded in Red Delicious, followed by Royal Gala, Pink Lady, Fuji, and Smitten.

In the ABTS assay, the antiradical scavenging activities were determined based of the hydrogen atom donating tendency of polyphenols [40]. The highest antioxidant ability was demonstrated in the order of Red Delicious > Royal Gala > Pink Lady > Fuji > Smitten. Upon comparison with the previous studies' Royal Gala and Fuji showed higher antioxidant ability than the previous reported values [41,42]. The reason might be because of the cultivar, location, maturity and storage of apples which may change the concentration of antioxidant potential [43]. In the TAC assay, the mechanism very similar to FRAP where reduction of molybdenum (VI) to molybdenum (V) in the presence of phenolics. In the current study, Red Delicious had the highest total antioxidant followed by Royal Gala, Pink Lady, Fuji and Smitten. Previously Khanizadeh et al.'s [35] study showed the values ranging from 0.323 to 1.246 mg AAE/g and the values were lower than our study. A difference in the concentration might be because of the difference between cultivars, location, harvesting time and maturity of samples [6].

#### *3.3. Correlation between Phenolic Compounds and Antioxidant Activities*

The correlation between the polyphenols and antioxidant activities was performed with a Pearson's correlation test (Table 2). TPC shows a strong positive correlation with TFC with *r* <sup>2</sup> = 0.975, *p* ≤ 0.01, this indicates that TFC contributes largely to the total phenolic content. Additionally, TPC was strongly correlated with TAC with *r* <sup>2</sup> value of 0.920 (*p* ≤ 0.05). A previous study by Vasantha Rupasinghe and Clegg [45] reported a similar correlation between TPC and TAC.


**Table 2.** Correlation coefficients (*r* 2 ) between phenolic contents and antioxidant assays.

\*\* Significant correlation with *p* ≤ 0.01; \* Significant correlation with *p* ≤ 0.05.

TFC had a significantly strong correlation with DPPH and TAC with *r* <sup>2</sup> value of 0.903 (*p* ≤ 0.01) and 0.952 (*p* ≤ 0.05) respectively indicating that flavonoids were one of the significant contributors for the antioxidant activities. The results confirm with the previous studies of Maleeha et al. [46] and Ruiz-Torralba et al. [47], on phenolic compounds contributing towards antioxidant potential. A non-significant correlation were observed between TTC and antioxidant assays indicating the contribution of tannins to antioxidant activity is limited, which confirms with Kam et al. [48] study.

The correlation among the antioxidant assays had strong correlation with each other. Significant positive correlation was observed between DPPH with ABTS, FRAP and TAC (*r* <sup>2</sup> = 0.952, *r* <sup>2</sup> = 0.961, and *r* <sup>2</sup> = 0.980, *p* ≤ 0.01). The correlation displayed in our study was similar to Kriengsak et al. [49], where a high correlation was observed between the four assays. Similarly, ABTS was observed to have high significant correlation with FRAP and TAC with *r* <sup>2</sup> = 0.938, *p* ≤ 0.01 and *r* <sup>2</sup> = 0.931 (*p* ≤ 0.05), respectively. On the other hand, FRAP was correlated with TAC with *r* <sup>2</sup> = 0. 912 (*p* ≤ 0.05).

Overall, phenolic compounds were highly correlated with antioxidant assays, which indicated that both classes of phenolic compounds including phenolic acids and flavonoids have strong antioxidant potential. The four antioxidants' assays were strongly correlated with each other.

#### *3.4. Phenolic Compounds Profile by LC-MS/MS Analysis*

LC- MS/MS has been a useful and reliable tool for identification and characterisation of phenolics in several plant samples. Qualitative analyses of phenolics from five varieties of apples (Royal Gala, Pink Lady, Red Delicious, Fuji and Smitten) were achieved using mass spectrometry in both negative and positive modes of ionisation (ESI−/ESI<sup>+</sup> ). The compounds in the apples were identified based on their precursor ions and MS spectra. The basis for the compounds to be further analysed were the PCDL library score more than 80 and mass error < 5 ppm (Table 3). In our current study, 97 different phenolic compounds were characterised in five apple samples, including 27 phenolic acids, 52 flavonoids, 5 lignans and 13 other polyphenols.


**Table 3.** Identification and characterisation of polyphenols in apples by using LC-ESI-QTOF-MS/MS.


**Table 3.***Cont.*


**Table3.***Cont.*


**Table3.***Cont.*


**Table 3.***Cont.*

\* Data presented in the table are from the sample indicated with an asterisk; \*\* Compounds were detected in both negative [M-H]− and positive [M+H]+ mode of ionization while only single mode data was presented. Apple samples mentioned in abbreviations are Royal Gala "RG"; Red Delicious "RD"; Fuji "F"; Smitten "S"; Pink Lady "PL".

#### 3.4.1. Phenolic Acids

In our research, 27 phenolic acids including hydroxyphenylacetic acids (2), hydroxycinnamic acids (18), hydroxybenzoic acids (5), and hydroxyphenylpropanoic acids (2) were identified and characterised in five varieties of apples.

Compound **1** was tentatively characterised as protocatechuic acid 4*-O-*glucoside present in negative mode of ionisation and identified in Royal Gala, Red Delicious and Fuji apples. The compound had precursor ion at *m/z* 315.0718 and on further MS/MS analysis showed product ions at *m/z* 125 (loss of CO2, 44 Da) and *m/z* 169 (loss of hexosyl moiety, 162 Da) [50]. In previous study of Gu et al. [21] reported tentatively characterised protocatechuic acid 4*-O-*glucoside from fresh apples. Compound **12** (([M-H]<sup>−</sup> *m/z* at 325.0925) was tentatively characterised as *p*-Coumaric acid 4*-O-*glucoside based on the product ions at *m/z* 163, due to the loss of hexosyl moiety (162 Da) from the precursor ions [50]. Identified in Pink Lady, Royal Gala and Fuji apples.

Compound **7** was tentatively characterised as caffeic acid in Smitten variety based on the precursor ion at [M+H]<sup>+</sup> at *m/z* 181.0494 and confirmed based on the MS<sup>2</sup> fragmentation with product ions at *m*/*z* 143 (loss of two water molecules, 36 Da) and *m*/*z* 133 (loss of HCOOH, 46 Da) [51]. Compound 15 was observed in Smitten, Pink Lady and Fuji and tentatively characterised as ferulic acid based on the precursor ion at ([M-H]<sup>−</sup> at *m*/*z* 193.0505. Upon further MS/MS analysis, the product ions at *m*/*z* 178 (loss of CH3, 15 Da), *m*/*z* 149 (loss of CO2, 44 Da) and *m*/*z* 134 (loss of CH3-CO2, 59 Da) confirmed the compound [52]. Compounds **19** (([M-H]<sup>−</sup> *m*/*z* at 223.0603) identified in Fuji, Pink Lady and Smitten apples. MS/MS analysis confirmed the compound as sinapic acid by fragments at *m*/*z* 205 and *m*/*z* 163 due to the consecutive loss of H2O and 2CHO from the precursor ion respectively [53]. Previously, Lee et al. [54] reported the presence of caffeic acid, ferulic acid and sinapic acid in apples. Caffeic acid abundantly present in both pulp and peel [54]. Other phenolic compounds to our best knowledge were first time detected in Australian grown apples.

#### 3.4.2. Flavonoids

A total of 52 Flavonoids were identified in the five apple samples including anthocyanins (8), dihydrochalcones (3), dihydroflavonols (3), flavanols (9), flavones (4), flavanones (7), flavonols (10), and Isoflavonoids (8).

Compound **31** (Cyanidin 3,5*-O-*diglucoside) and compound **33** (Delphinidin 3*-O*glucosyl-glucoside) were both detected in the positive mode of ionization with the precursor ions at *m*/*z* 612.1693 and *m*/*z* 628.1648, respectively. The MS/MS experiment allowed the further identification of these compounds based on the peaks after removal of the sugar moieties for both compounds [55].

Compound **36** and compound **37** were tentatively characterised as 3-hydroxyphloretin 2 ′ *-O-*glucoside and 3-hydroxyphloretin 2′ *-O-*xylosyl-glucoside present in negative mode of ionisation with precursor ions at *m*/*z* 451.1249 and *m*/*z* 583.1665, respectively. 3 hydroxyphloretin 2′ *-O-*glucoside was confirmed by fragment ions at *m*/*z* 289 [M-Hglucoside] and *m*/*z* 273 [M-H-phloretin aglycon] [56] identified in Pink Lady, Royal Gala, Fuji and Smitten apples. Whereas, 3-Hydroxyphloretin 2′ *-O-*xylosyl-glucoside was identified by fragment ions at *m*/*z* 289, due to the loss of xylosyl-glucoside disaccharide (132 + 162 Da) [57] observed in Royal Gala apples. Phloridzin (compound **38)** with precursor ion at [([M-H]−, *m*/*z* 435.1284], and confirmed by product ions at *m*/*z* 273 due to the loss of glucoside (162 Da) [58] identified in Pink Lady, Royal Gala, Fuji and Smitten apples. Kelebek et al. [58] reported the presence of phloridzin in apples.

Three flavanols derivatives (Compound **44**, **46**, **48**) were all detected in four samples including Pink Lady, Royal Gala, Fuji and Smitten apples. Compound **44**, **46**, **48** with negative mode of ionisation with precursor ions at *m*/*z* 577.1333, *m*/*z* 289.0706 and *m*/*z* 865.1961 were tentatively characterised as procyanidin dimer B1, (+)-catechin and procyanidin trimer C1 respectively. The compound procyanidin trimer C1 was confirmed by product ions at *m*/*z* 739, *m*/*z* 713 and *m*/*z* 695, due to the loss of heterocyclic ring fission

(HRF) reaction (126 Da), loss of retro-Diels-Alder (RDA) (152 Da) and loss of H2O [59]. While the loss of phloroglucinol (126 Da) from the precursor ion confirmed the presence of procyanidin dimer B1 [60]. Whereas, (+)-catechin compound confirmed based on the fragment ions at *m*/*z* 245, *m*/*z* 205 and *m*/*z* 179, due to corresponding loss of CO<sup>2</sup> (44 Da), flavonoid A ring (84 Da) and flavonoid B ring (110 Da) from the precursor ion, respectively [50]. Previously Nicoli et al. [61] reported the presence of (+)-catechin in apple varieties. (+)-catechin has a positive health benefit including scavenging free radicals, delaying aging and benefitting the intestinal microbes [62].

Compound **51** (hesperetin 3′ ,7*-O-*diglucuronide) and compound **53** (narirutin) were found both in negative ionization modes based on the precusor ions at *m*/*z* 653.1361 and *m*/*z* 579.1710, respectively. Compound **51** was confirmed by the product ion at *m*/*z* 477 [M-H-glucuronide, loss of 176 Da], *m*/*z* 301 [M-H-2 glucuronide, loss of 352 Da], *m*/*z* 286 [M-H-2glucuronide-CH3, loss of 367 Da] and *m*/*z* 242 [M-H-2glucuronide-OCH2-CHO] [63], while compound **53** was confirmed by loss of neohesperidose moiety (308 Da) [64] from the precursor ion. In our study compound **51** was identified in Smitten and Pink Lady whereas compound **53** was identified in Royal Gala and Red Delicious. To our best knowledge it was first time detected in Australian grown apples.

Apigenin 7*-O-*glucuronide (Compound **56**) and cirsilineol (compound **58**) were tentatively characterised in negative mode of ionisation at *m*/*z* 447.0930 and *m*/*z* 345.0962, respectively. The MS/MS analysis confirmed the compound **56** at product ions *m*/*z* 271 due to the corresponding loss of glucuronide (176 Da) and loss of glucuronide and *m*/*z* 253 due to the loss of H2O-CH2O (194 Da) from the precursor ion [65]. The presence of cirsilineol was confirmed by the product ions at *m*/*z* 330 [M+H-CH3], *m*/*z* 312 [M+H-CH3- H2O], *m*/*z* 297 [M+H-2CH3-H2O] and *m*/*z* 284 [M+H-CH3-H2O-CO] [66]. According to previous reports, compounds have been characterised in several plants including Ocimum species [66].

Compound **62** (Myricetin 3*-O-*galactoside with ([M-H]<sup>−</sup> *m*/*z* at 479.081) identified in Red Delicious and compound **63** (Quercetin 3*-O-*glucosyl-xyloside with ([M-H]<sup>−</sup> *m*/*z* at 595.1291) identified in Pink Lady were only detected in the negative ionization mode, and identified according to the fragment peaks at *m*/*z* 317 [M-H-glucoside, loss of 162 Da] [67] and *m*/*z* 265 [M-H-glucose-xylose, loss of 330 Da] [51], respectively. Compound **65, 66** and **68** present in the negative mode of ionisation were identified as kaempferol 3*-O-*glucosylrhamnosyl-galactoside, kaempferol 3*-O-*(2"-rhamnosyl-galactoside) 7*-O-*rhamnoside and kaempferol 3,7*-O-*diglucoside according to the ([M-H]<sup>−</sup> at *m*/*z* 755.2068, *m*/*z* 739.2115 and *m*/*z* 609.1451, respectively Kaempferol 3*-O-*glucosyl-rhamnosyl-galactoside exhibited the product ions at *m*/*z* 285, corresponding to the loss of the sugar units from the precursor ion [68]. The presence of kaempferol 3*-O-*(2"-rhamnosyl-galactoside) 7*-O-*rhamnoside was confirmed by the product ions at *m*/*z* 593 [M-H-C6H10O4], *m*/*z* 447 [M-H-2C6H10O4], and *m*/*z* 285 [M-H-2C6H10O4-C6H10O5] [69]. Whereas, kaempferol 3,7*-O-*diglucoside exhibited the product ions at *m*/*z* 447 and *m*/*z* 285, corresponding to the loss of glucoside and consecutive loss of glucoside from the parent ion [70]. It worth noted that these compounds were first time detected in Australian grown apple samples to the best of our knowledge.

Compound **73** and **75** detected in positive mode were identified as 6"*-O-*Malonyldaidzin and violanone with precursor ion at *m*/*z* 503.1200 and *m*/*z* 317.1016, respectively. 6"*-O-*Malonyldaidzin was confirmed by the product ion at *m*/*z* 255 [71], corresponding to the loss of malonyl-glucoside from precursor, while the compound violanone was confirmed by the intensive peaks at *m*/*z* 300 [M+H-CH3, loss of 15 Da], *m*/*z* 285 [M+H-2CH3, loss of 30 Da] and *m*/*z* 135 [M+H-C10H12O3] [72]. Previously, several studies had discovered the existence of the above isoflavonoids in fruits [71,73–76].

#### 3.4.3. Lignans

Compound **82** (Schisandrin C) was detected only in the positive ionization mode with precursor ions at *m*/*z* 385.1663. The fragmentation peaks confirmed the compound schisantherin C based on product ions at *m*/*z* 370 [M+H-CH3OH], *m*/*z* 315 [M+H-C5H10] and *m*/*z* 300 [M+H-CH3-C5H10] [77].

#### 3.4.4. Other Polyphenols

In other polyphenols, curcuminoids (1), furanocoumarins (1), hydroxybenzaldehydes (2), hydroxycoumarins (1), hydroxyphenylpropenes (1), phenolic terpenes (2), tyrosols (3) and other polyphenols (2), while tyrosols was the dominant subclass were identified in apple samples.

Compound **88** was tentatively characterised as 4-hydroxybenzaldehyde based on the precursor ion at ([M-H]<sup>−</sup> at *m*/*z* 121.0301 and confirmed based on the MS<sup>2</sup> fragmentation, which exhibited the loss of CO<sup>2</sup> (44 Da) from the precursor, resulting in the product ion at *m*/*z* 77 [78]. Rosmanol (compound **93**) was found in positive modes, and tentatively characterised according to the precursors [M+H]<sup>+</sup> at *m*/*z* 347.1844. In the MS<sup>2</sup> experiment, peaks at *m*/*z* 301 (loss of H2O) and *m*/*z* 231(loss of CO2) achieved the identification of coumarin [79]. Meanwhile, compound **94** (carnosic acid with ([M-H]<sup>−</sup> at *m*/*z* 331.1905) was confirmed by the fragments at *m*/*z* 287 and *m*/*z* 296, resulting from the loss of CO<sup>2</sup> and further loss of H2O from the precursor [80]. To best of our knowledge, this is the first time it has been detected in apple samples.

Compounds **95** and **96** detected in negative mode were detected as hydroxytyrosol 4*-O-*glucoside and 3,4-DHPEA-AC, precursor ion at *m*/*z* 315.1090 and *m*/*z* 195.0658, respectively. On further analysis, hydroxytyrosol 4*-O-*glucoside was confirmed by the product ions at *m*/*z* 153 and *m*/*z* 123, corresponding to the loss of glucoside (162 Da) and glucoside-CH2O (192 Da) from the precursor ion, respectively [78] and 3,4-DHPEA-AC was confirmed by the product ions at *m*/*z* 135 [M-H-C2H4O2] [81].

Compounds **91** and **92** were found in negative ionization mode and identified as salvianolic acid C and salvianolic acid B with precursor ions at *m*/*z* 491.0963 and *m*/*z* 717.1436, respectively. Salvianolic acid C was confirmed by the product ion at *m*/*z* 311 [M-H-caffeic acid], *m*/*z* 267 [M-H-caffeic-CO2] and *m*/*z* 249 [M-H-CO2-H2O][82], while salvianolic acid B was confirmed by the intensive peaks at *m*/*z* 519 [M-H-Danshensu, loss of 198 Da], *m*/*z* 339 [M-H-Danshesu-caffeic acid, loss of 378], *m*/*z* 321 [M-H-2 × Danshensu, loss of 396 Da] and *m*/*z* 295 [M-H-Danshensu-caffeic acid-CO2, loss of 422 Da][82]. Previously, both compounds were detected in *Salvia miltiorrhiza* [83]. Salvianolic acid, known for its antioxidant potential, can effectively remove oxygen free radicals in the human body. This compound is one of the natural products with the strongest antioxidant effect [84]. However, these compounds have been discovered for the first time in apple varieties to the best of our knowledge.

#### *3.5. Quantitative Analysis of Phenolic Compounds by HPLC-PDA*

The most effective way of quantification of phenolic compounds is by HPLC-PDA analysis [85]. In our study, 10 phenolic compounds (mainly phenolic acids and flavonoids) were chosen to be quantified since it is difficult to complete the qualification of all the identified compounds. Since a few compounds have too low UV absorption to be detected, the content of phenolic compounds in five apple samples are shown in Table 4.

In phenolic acids, chlorogenic acid, *p*-hydroxybenzoic acid and caffeic acid were the major phenolic acids in Royal Gala, while Pink Lady contained high content in chlorogenic acid, *p*-hydroxybenzoic acid and protocatechuic acid. It was observed that Red Delicious had highest content in caffeic acid when compared to other samples. Caffeic acid, chlorogenic acid and protocatechuic acid were detected in Fuji. Whereas Smitten apples had gallic acid and *p*-hydroxybenzoic acid, these compounds were not observed in Fuji.

According to previous studies, chlorogenic acid and caffeic acid have been identified and quantified in several apple cultivars [86,87]. While Soares et al.'s [88] study indicated that apples, including gala, showed a low concentration of gallic acid and *p*-hydroxybenzoic acid, only few studies focused on identification of Fuji. Hence, further studies are required to analyse the quantitation of Fuji and Smitten.


**Table 4.**Quantitative analysis in phenolic compounds of five kinds of apple samples.

 Experiments performed in triplicates are expressed as the mean ± SD. Means followed by different letters (a, b, c, d, e) within the same column are significantly different (*p* < 0.05) from each other. Data of five kinds of apples are reported (fw).

In flavonoids, a total of four flavonoids (catechin, epicatechin, quercetin, kaempferol) were detected among five apple samples. In general, Fuji was detected the highest catechin content while Red Delicious was the lowest. In contrast, the highest quercetin was detected in Red Delicious while Fuji contained the lowest quercetin. Epicatechin was detected in Royal Gala and Smitten the compounds were 7.13 ± 0.08 mg/g and 7.59 ± 0.09 mg/g respectively. Smitten contained the highest Kaempferol (14.25 ± 0.09 mg/g) among five samples. Compound epicatechin gallate was negligible in all the samples.

Previous studies showed that catechin and quercetin are main flavonoids that contribute to the antioxidant potential of apples [61,89]. Previously reported that epicatechin and kaempferol have been successfully synthesised and characterised [90,91]. However, to the best of our knowledge epicatechin gallate was not detected in apples hence more further studies are needed to verify the detection of this flavonoids.

In conclusion, Royal Gala, Red Delicious and Smitten had abundant quercetin content. Pink Lady had a high concentration of compounds including chlorogenic acid and catechin. Fuji had most abundant amount kaempferol and catechin content among five samples. Finally, phenolic acids were more abundant in Pink Lady and Royal Gala while flavonoids were more abundant in Royal Gala, which is consistent with the previous study.

#### **4. Conclusions**

In conclusion, various methods have been successfully utilized for the determination, characterisation, and quantitation of phenolic compounds among five different varieties of Australian grown apples. In phenolic compound estimation, Red Delicious showed higher TPC, TFC, DPPH, FRAP, ABTS and TAC values than other apple samples while Fuji exhibited the highest TTC value. The correlation between flavonoids and phenolic acids exhibited a major contribution towards the antioxidant activities of apples. The LC-ESI-QTOF-MS/MS qualification identified a total of 97 different phenolic compounds in five apple samples, including phenolic acids, flavonoids, lignans, other polyphenols and stilbenes. 10 phenolic compounds were quantification through HPLC-PDA based on the difference of UV spectra and retention times. The analysis showed that phenolic acids were more abundant in Pink Lady and Royal Gala whereas flavonoids were more abundant in Royal Gala.

**Author Contributions:** Conceptualization, methodology, formal analysis, validation and investigation, H.L. and H.A.R.S.; resources, H.A.R.S., C.J.B. and F.R.D.; writing—original draft preparation, H.L. and H.A.R.S.; writing—review and editing, H.L., V.S., C.J.B., H.A.R.S. and F.R.D.; supervision, H.A.R.S. and F.R.D.; ideas sharing, H.A.R.S.; C.J.B. and F.R.D.; funding acquisition, H.A.R.S., F.R.D. and C.J.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the University of Melbourne under the "McKenzie Fellowship Scheme" (Grant No. UoM-18/21), the "Richard WS Nicholas Agricultural Science Scholarship" and the "Faculty Research Initiative Funds" funded by the Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Australia and "The Alfred Deakin Research Fellowship" funded by Deakin University, Australia.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** We would like to thank Nicholas Williamson, Shuai Nie and Michael Leeming from the Mass Spectrometry and Proteomics Facility, Bio21 Molecular Science and Biotechnology Institute, the University of Melbourne, VIC, Australia for providing access and support for the use of HPLC-PDA and LC-ESI-QTOF-MS/MS and data analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Review* **Environmental Conditions and Agronomical Factors Influencing the Levels of Phytochemicals in** *Brassica* **Vegetables Responsible for Nutritional and Sensorial Properties**

**Francesca Biondi 1,2, Francesca Balducci <sup>1</sup> , Franco Capocasa <sup>1</sup> , Marino Visciglio <sup>2</sup> , Elena Mei <sup>2</sup> , Massimo Vagnoni <sup>2</sup> , Bruno Mezzetti <sup>1</sup> and Luca Mazzoni 1,\***


**Abstract:** Recently, the consumption of healthy foods has been related to the prevention of cardiovascular, degenerative diseases and different forms of cancers, underlying the importance of the diet for the consumer's health. Fruits and vegetables contain phytochemicals that act as protective factors for the human body, through different mechanisms of action. Among vegetables, *Brassica* received a lot of attention in the last years for the phytochemical compounds content and antioxidant capacity that confer nutraceutical value to the product. The amount of healthy bioactive compounds present in the *Brassica* defines the nutritional quality. These molecules could belong to the class of antioxidant compounds (e.g., phenols, vitamin C, etc.), or to non-antioxidant compounds (e.g., minerals, glucosinolates, etc.). The amount of these compounds in *Brassica* vegetables could be influenced by several factors, depending on the genotypes, the environmental conditions and the cultivation techniques adopted. The aim of this study is to highlight the main phytochemical compounds present in brassicas used as a food vegetable that confer nutritional and sensorial quality to the final product, and to investigate the main factors that affect the phytochemical concentration and the overall quality of *Brassica* vegetables.

**Keywords:** phytochemical compounds; antioxidant capacity; *Brassica* spp.; vegetables; cultivation techniques; glucosinolates

#### **1. Introduction**

In recent years, the increasing incidence of cardiovascular, degenerative diseases and different forms of cancers has stimulated the interest of consumers in distinguishing healthy from unhealthy foods, as a consequence of the abandonment of Mediterranean diet which, in contrast to other eating regimes, was considered a model of healthy eating for years. The interest for consuming healthy food led to coining the new definition "functional food" for several foodstuffs. This term defines a food product that, in addition to carrying out the traditional alimentary function, also performs preventive and/or therapeutic effects against various human diseases, in particular chronic-degenerative diseases [1,2]. Fruits and vegetables contain phytochemicals that are responsible for these positive effects on human body.

Among vegetables, brassicas received a lot of attention in the last few years. They comprise a large and diverse group of widely consumed vegetables. *Brassica* is the Latin name of a genus that is taxonomically placed within the *Brassicaceae* (*Cruciferae*). The main cultivated and most consumed as food vegetables *Brassica* species in the world are indicated in Table 1. Other closely related vegetables within the *Brassicaceae* family are also reported.

**Citation:** Biondi, F.; Balducci, F.; Capocasa, F.; Visciglio, M.; Mei, E.; Vagnoni, M.; Mezzetti, B.; Mazzoni, L. Environmental Conditions and Agronomical Factors Influencing the Levels of Phytochemicals in *Brassica* Vegetables Responsible for Nutritional and Sensorial Properties. *Appl. Sci.* **2021**, *11*, 1927. https:// doi.org/10.3390/app11041927

Academic Editors: Alessandro Genovese and Carmela Spagnuolo

Received: 14 December 2020 Accepted: 19 February 2021 Published: 22 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The healthy potential of *Brassica* is bound to their phytochemical compounds. The main compounds responsible for healthy function are phenolic compounds, vitamins (C, B9, K), provitamin A (β-carotene), lutein and different types of glucosinolates [3]. The increasing interest for *Brassica* vegetables has been underlined by their economic importance (among the top 10 economic crops in the world [4]) and by the fact that there was an increase of about 11.5% of both the cultivated area and the production quantity from 2009 to 2019, with a slight increase in yield of about 1.5%. The Organization for Food and Agriculture of the United Nations (FAO) also reported that, in 2019, the global production of cauliflower, broccoli, cabbage and other Brassica crops was about 97 million tonnes, occupying a cultivated area of almost 4 million hectares. Asia accounts for more than 75% of the global *Brassica* vegetable production, with China producing almost a half of all of these vegetables (45 million tonnes). India, with more than 18 million tonnes, then Korea, Russia, and USA with more than 2 million tonnes, are also the biggest producers of cauliflower, broccoli, cabbage and other Brassica crops [5].

**Table 1.** Main species, subspecies (ssp.) and varieties (var.) of *Brassicaceae* family crops consumed as food vegetable in the world [4,6–8].


**Table 1.** *Cont.*


The present review summarizes the main chemical compounds responsible for the sensorial and nutritional quality of *Brassica* spp., with particular emphasis on the factors affecting their level.

#### **2. Nutritional Quality**

Nutritional quality could be defined as the value of the product for the consumer's physical, psychological, or emotional well-being. The first term of this extended definition concerns the effects of food determined by its phytochemicals, i.e., the sum of all beneficial and harmful compounds and their nutritional (or biological) aspects [9]. In the case of *Brassica* spp., these molecules could belong to the class of antioxidant compounds (e.g., phenols, vitamin C, etc.), exerting their health effects through the ability to scavenge free radicals, or to non-antioxidant compounds (e.g., minerals, glucosinolates, etc.) that exert their function through direct mechanisms in the human metabolism, different from the scavenger activity.

#### *2.1. Antioxidant Compounds*

Total antioxidant capacity (TAC) is the ability of food to preserve an oxidizable substrate, inactivate the radical species or reduce an oxidized antioxidant. TAC is considered a fundamental parameter for the description of fruits and vegetables nutritional quality; it is an indicator of the presence of bioactive substances belonging to the antioxidants group. Each antioxidant compound performs its protecting activity through different mechanisms and with different efficiency, depending on its chemical structure and the matrix it acts on. For this reason, TAC analysis is usually preferred to the measurement of the single concentration of each antioxidant, mainly if the objective of the study is a general screening of the health effects of different fruit and vegetables.

*Brassica* vegetables, i.e., broccoli and kale, showed higher antioxidant potential than other vegetable crops, such as spinach, carrots, potatoes, beans and onions. In general, among *Brassica* vegetables, Brussels sprouts, broccoli, and red cabbage belong to the group that has the highest antioxidant capacity. Common cabbage possesses the lowest antioxidant capacity [10,11]. Contrasting results were reported in cauliflower by Azuma et al. [12] and Wu et al. [13]. The analysis of TAC is influenced by the extraction method and the type of reactive species in the reaction mixture [12].

Many researchers studied and identified the main antioxidant molecules present in *Brassicaceae* [13–16]. These antioxidant compounds belong to two main groups: watersoluble antioxidants and lipo-soluble antioxidants [14,15]. Kurlich et al. [16] and Wu et al. [13] reported that hydrophilic antioxidants are responsible for 80–95% of TAC in *Brassicaceae*, while lipo-soluble antioxidants account for only 5–20%.


Phenolic compounds are the most widespread antioxidant family present in vegetables. This large group of compounds is particularly present in *Brassica* vegetables and constitutes the main source of antioxidants in these plants [14,17]. These plants produce them as secondary metabolites for protection from pest and insect attack.

Their importance in human health is related to antioxidant and anti-inflammatory properties that could have preventive and/or therapeutic effects against obesity, cancer, and neurodegenerative and cardiovascular diseases [18]. Among *Brassica* species, kale and broccoli have the highest quantity of total polyphenols with about 13 mg gallic acid/g of dry weight [19,20].

Flavonoids represent common phenolic compounds in *Brassica*; they possess a lot of biological properties, e.g., antioxidant activity, a capillary protective effect, and an inhibitory effect elicited in various stages of tumours [14,21]. They are characterized by numerous subclasses, but the most important in *Brassica* are the following:


Among phenolic compounds, even if they are not hydro-soluble, it is worth mentioning the lignans, diphenolic compounds that possess several biological activities, through their antioxidant and oestrogenic properties. Lignans may reduce the risk of certain cancers and cardiovascular diseases [16]. Some studies reported that lignans are mainly present in the kale family, broccoli and Brussel sprouts with lariciresinol (972, 599 and 493 µg/100 g fresh edible weight of Broccoli, Curly kale and Brussel sprouts, respectively) and pinoresinol (315, 1691 and 220 µg/100 g fresh edible weight of Broccoli, Curly kale and Brussel sprouts, respectively) being the most abundant [15,32].

• Vitamin C and vitamin B9 (Folic Acid)

Vitamin C, or ascorbic acid, is a powerful antioxidant, widely present and studied in fruits; however, many recent works have been focused on the importance of vitamin C in vegetables, mostly in *Brassicaceae* family. In *Brassica* vegetables vitamin C concentration varies a lot among species and subspecies, and it is strictly genotype- and environmentdependent [33,34]. Vitamin C performs countless biological activities in the human body and represents a nutritional compound fundamental for health. Ascorbic acid is a radical scavenger, an enzyme cofactor and a donator/acceptor in electrons transport at the plasma membrane level; its role is fundamental in the regeneration of α-tocopherol, and in the prevention and treatment of malignant and degenerative diseases [33,35].

Among *Brassica* genotypes, Brussel sprouts (76–192 mg/100 g edible portion) and kale (92–186 mg/100 g edible portion) seem to possess the highest content of vitamin C, followed by broccoli (34–146 mg/100 g edible portion) and cauliflower (17–81 mg/100 g edible portion), while white cabbage (19–47 mg/100 g edible portion) possesses the lowest amount [14].

Vitamin B9 (Folic acid) is an important vitamin present in *Brassica*, mainly in raw broccoli (63 µg/100 g of edible portion), Brussel sprouts (61 µg/100 g of edible portion) and kale (141 µg/100 g of edible portion) [36], that act as a coenzyme in many single carbon transfer reactions, in the synthesis of DNA and RNA and of protein components. Furthermore, it reduces the level of homocysteine in the blood, a risk factor for cardiovascular diseases. Among the several health activities that folic acid performs, it is strongly important in the prevention of megaloblastic anaemia, neuropsychiatric disorders and various forms of cancer in the foetus during pregnancy, also reducing the risk of neural tube defects [33,37]. These beneficial effects of folic acid, in particular on the pathogenesis of cancer, and neurological, haematological, and cardiovascular diseases may, in part, be due to its antioxidant activity, via its electron-accepting capacity [38,39].

#### 2.1.2. Lipo-Soluble Antioxidants

Despite the low incidence of lipo-soluble antioxidants on the TAC of *Brassica*, several studies confirm the high content of lipo-soluble antioxidant in kale and broccoli, moderate in Brussels sprouts, and low amount in cauliflower and cabbage [33]. Among lipo-soluble antioxidants, carotenoids and vitamin E are the most important found in *Brassica* vegetables.

• Carotenoids

Carotenoids are responsible for the orange, yellow and red pigmentation of several fruits and vegetables, mainly carotenes and xanthophylls. The most represented carotenoids in *Brassica* vegetables are β-carotene, which the organism transforms to vitamin A, and lutein and zeaxanthin [14]. β-carotene prevents the insurgence of cancer and cardiovascular diseases, and decreases the risk of myocardial infarction, of immune dysfunction and age-related macular degeneration among smokers [33,40]. Muller [41] analysed the total carotenoid content of several *Brassica* species and reported them in decreasing order: Brussel sprouts (6.1 mg/100 g), broccoli (1.6 mg/100 g), red cabbage (0.43 mg/100 g) and finally white cabbage (0.26 mg/100 g). In the *Brassica oleracea* genus, kale possesses the highest content of carotenoids with over 10 mg/100 g of the edible portion [41].

The *Brassica* vegetable with the highest content of lutein and zeaxanthin is kale (3.04– 39.55 mg/100 g); interesting contents were also found in broccoli and Brussels sprouts [14]. In *B. rapa* species, 16 carotenoids were identified by Wills and Rangga [42]; in *B. chinensis, parachinensis* and *pekinensis,* lutein and β-carotene are the most abundant carotenoids [15].

• Vitamin E

Vitamin E is formed by groups of compounds known as tocopherols and tocotrienols; in detail, α-tocopherol is the main compound found in *Brassica* vegetables, with the exception of cauliflower, that contains mainly γ-tocopherol [14,43]. Vitamin E performs a protective activity against coronary heart disease through the inhibition of LDL oxidation [44]. A high intake of vitamin E helps in the prevention of cancers, cardiovascular diseases, neurological disorders, and inflammatory diseases [33]. The content of vitamin E in *Brassica* species has been studied in the literature, as reported here in decreasing order: broccoli (0.82 mg/100 g), Brussels sprouts (0.40 mg/100 g), cauliflower (0.35 mg/100 g),

Chinese cabbage (0.24 mg/100 g), Red cabbage (0.05 mg/100 g), and white cabbage (0.04 mg/100 g) [43].

#### *2.2. Micro- and Macro-Elements*

Macro-elements, also called macronutrients, are those nutrients that the plants need in greater quantities for essential structural and energetic role. They are indispensable elements for the growth and development of the metabolic functions of plants. The fundamental nutrients are represented by nitrogen (N), phosphorus (P) and potassium (K).

Minerals, such as Boron (B), Copper (Cu), Cobalt (Co), Iron (Fe), Manganese (Mn), Zinc (Zn), and Selenium (Se), are required by plants in very small quantities and are known as microelements. Although trace elements are present in small quantities in plants, they play key roles in plant life; this is also demonstrated by the symptoms associated with deficiency phenomena. Their availability depends on the conditions of the soil. The high capacity of *Brassicaceae* to accumulate the metals present in the soil led this family to be considered a good heavy metal hyperaccumulator, giving the significant number of genera (11) and species (90) of those kinds of plants belong to *Brassicaceae* family [45].

However, micro- and macro-elements also play important roles in the human body. The elements K, Ca, Mg, Fe, Zn, Se, and Mn are fundamental in the regulation of many metabolic activities, in bones and teeth health, in cancer prevention, in the production of red blood cells, and participating as enzyme co-factors. Among *Brassicaceae*, kale is the richest in almost all the main macro and micro elements, with a particularly high amount of calcium (95–539 mg/100 g of edible portion), magnesium (20–67 mg/100 g of edible portion), phosphorus (13–92 mg/100 g of edible portion), potassium (20–491 mg/100 g of edible portion), zinc (0.3–0.9 mg/100 g of edible portion), iron (0.4–3.1 mg/100 g of edible portion), manganese (0.4–1.9 mg/100 g of edible portion), copper (0.02–1.03 mg/100 g of edible portion), and selenium (0–0.94 mg/100 g of edible portion). Mustard green is the richest in iron (1.64 mg/100 g of edible portion), Turnip in sodium (67 mg/100 g of edible portion) and Broccoli in selenium (2.5 mg/100 g of edible portion) [36,46].

#### *2.3. Glucosinolates (GLS) and Isothiocyanates (ITCS)*

Glucosinolates (GLS) are one of the most important secondary metabolites in *Brassicaceae* derived from amino acid biosynthesis [14,47]. GLS are glucosidic compounds containing sulphur, present in *Brassica* leaves, compartmentalized in the vacuole, at concentrations that are able to prevent the development of pathogens, diseases and pests [48]. Their concentrations vary among *Brassica* species [49], according to the developmental stage, tissue type, exposure to salt stress, environmental factors, or plant signalling molecules, including treatment with salicylic acid (SA), jasmonic acid (JA) and methyl-jasmonic acid (MeJA) [50–52]. However, their amount generally ranges from the 4.7–32.2 mg/100 g of Mustard spinach, 8.7–12.8 mg/100 g of Rocket, 9.7–33.7 mg/100 g of Chinese cabbage, up to the 65.4–151.1 mg/100 g of Kale, 149.4 mg/100 g of Chinese broccoli and 87.6–332.8 mg/100 g of Radish [53,54]. GLS can be divided into three chemical classes: arylaliphatic, indole and aliphatic, based on their amino acid precursor (aromatic amino acid, tryptophan and methionine, respectively) [55], as reported in Table 2. In *Brassica* vegetables, the most important GLS belong to the methionine-derived ones [56]. Some authors declared that the most popular food processing methods, such as boiling, blanching, and steaming, can significantly affect the final content of GLS. A mild-processing technique, such as blanching, is recommended in order to minimize the loss of GLS or their derivatives [57].



GIB: glucoiberin (3-methylsulfinylpropyl GSL); PRO: progoitrin ((R)−2-hydroxybut−3-enyl GSL); SIN: sinigrin (prop−2-enyl GSL); GAL: glucoalysiin (5-methylsulphinylpentyl GSL); GRA: glucoraphanin (4-methylsulphinylbutyl GSL); GNA: gluconapin (but−3-enyl GSL); GBN: glucobrassicanapin (pent−4-enyl GSL); GIV: glucoiberverin (3-methylsulfanylpropyl GSL); GER: glucoerucin (4-methylsulfanylbutyl GSL); GNL: gluconapoleiferin (2-hydroxypent−4-enyl GSL); GBS: glucobrassicin (indol−3-lylmethyl GSL); NGBS: neoglucobrassicin (1-methoxyindol−3-ylmethyl GSL); 4HGBS: 4-hydroxyglucobrassicin (4-hydroxyindol−3-ylmethyl GSL); 4MGBS: 4-methoxyglucobrassicin (4-methoxyindol−3-ylmethyl GSL); GST: gluconasturtiin (2-phenylethyl GSL).

GLS have no direct functions to human health: the health effects are exerted by their hydrolysis breakdown products, the isothiocyanates (ITCs). These are aromatic volatile compounds containing sulphur, derived from the hydrolytic action of the enzyme myrosinase on GLS. The plant myrosinase acts in the human gut and hydrolyses GLS in ITCs during human ingestion. However, during the cooking of the vegetables, the exposure to heat treatment can inactivate the plant myrosinase, so the ITCs are obtained thanks to the action of myrosinase produced by the human gut flora. Unfortunately, its activity and efficiency are lower than plant myrosinase [68,69]. It is possible to obtain many ITCs, and their production strictly depends on the original GLS, the substrate, the pH conditions, the availability of ferrous ions, and the level of activity of the ESP (epithiospecifier protein), a specific protein factor [52,70].

ITCs are mainly responsible for the bitterness, and spicy and typical aroma and smell of *Brassica* vegetables [63]. They possess protective and preventing effects against several kinds of cancer e.g., prostate, intestinal, liver, lung, breast, and bladder, chronic inflammation and neurodegeneration, acting on the apoptotic phase of cell developmental cycle; they are also effective in the reduction in cholesterol [19,71,72].

The most studied ITCs in medical research is sulforaphane [73,74], mainly represented in broccoli and Brussel sprout. It is the most important ITCs considering its health benefits, it derives from the glucoraphanin [75]. Sulforaphane is an indirect antioxidant, because it acts as a catalyst in the stimulation of cellular antioxidant system. In particular, sulforaphane stimulates some enzymes active against tumoral cell proliferation [65,66].

#### **3. Sensorial Quality**

The quality of vegetables for the consumer not only concerns the nutritional aspects, but also includes the sensorial parameters that can be defined by several indicators.

The principal sensorial parameters are:


Considering *Brassica* vegetable quality, fundamental sensorial parameters are those related to aroma and taste. All of these parameters can be investigated through analytical measurements or the implementation of a panel test.

#### *3.1. Brassica Aroma*

The typical aroma is one of the main reasons for the consumers' rejection against *Brassica* vegetables [77]. Raw vegetables are rich in aroma compounds, which are usually produced because of enzymatic reactions. The typical sulphurous and pungent odour of *Brassicaceae* crops are often attributed to GSL/ITC content. These traits predominantly stem from sulphur-compound degradation products, such as from S-methyl-L-cysteine sulfoxide (SMCSO) [78], and formation can be facilitated by factors, such as bacterial metabolism, plant senescence, cooking, and enzymatic breakdown because of tissue damage (e.g., cutting) [79–81]. Sulphides are generally undesirable odour attributes [82], and compounds such as methanethiol, dimethyl sulphide (DMS), dimethyl trisulphide (DMTS), and dimethyl disulphide (DMDS) are regularly linked with sulphurous aromas and overcooked off-flavours. The main responsible of the fresh cabbage odour is the allyl isothiocyanate, a hydrolysis product of sinigrin thanks to the action of myrosinase [83,84]. Additionally, green note is a particularly important characteristic to recognize in *Brassica* and is conferred by alcohols and aldehydes formed by the enzymatic degradation of free fatty acids [83].

Cooking is the main adopted form to eat *Brassica*, because makes those vegetables more easily digestible and causes a flavour change in them, increasing the consumers' acceptance [85]. Reductions in alcohols, aldehydes and nitriles concentration were reported in cooked *Brassica*, as well as of the sulphides amount (except in broccoli) [85]. The concentration of isothiocyanate was found to increase after cooking [85]. Additionally, the storage of vegetables in frozen form could impact their volatile profile, in particular, influencing the alcohol, aldehydes, and isothiocyanates content [85].

#### *3.2. Brassica Taste*

As previously stated, *Brassica* vegetables contain health-related compounds that possess undesirable sensory characteristics. Bitterness is particularly accentuated in *Brassica*; this sensation is caused by ITCs that derive from sinigrin, gluconapin, progoitrin, glucobrassicin, neoglucobrassicin at different intensities [86,87]. Many studies identified the relation between the bitter taste and sinigrin and goitrin in cooked Brussels sprout, and between bitterness and sinigrin and neoglucobrassicin in cooked cauliflower [88]. Several studies affirm that the GLS and their breakdown products are not the only ones responsible for the bitter taste and *Brassica* aroma, but these resulted from a synergistic activity of various phytochemicals (indole hydrolysis products, flavonoids, etc.) [65,89].

The overall taste of *Brassica* vegetables is not only linked to bitter compounds but derives from the interaction between the bitter and the sweet tastes [88]. Some evidence demonstrated how the taste is the main driver of liking a food product [90,91], and that there is an innate preference for sweet taste in respect to bitter and sour taste [92,93]. This explains why a bitter taste in vegetables could deter most consumers from buying them. Some studies demonstrated that consumers prefer *Brassica* with low amounts of bitter GLS and higher concentrations of sucrose and, more generally, that the sweet taste is a favourable characteristic for the consumer's appreciation of *Brassica* [87,91].

#### **4. Factors Influencing the Phytochemical Compounds of** *Brassica* **Vegetables**

The quality of the final product can be influenced by several factors such as genetic, environmental, and agricultural (Figure 1).

**Figure 1.** Factors that influence the quality of the final product.

#### *4.1. Genetic Factors*

As for all other crops, *Brassica* quality is influenced by several factors, but the principal is represented by the genotype characteristics. Many breeding programs are working towards the creation and selection of new better productive and qualitative genotypes. These programs are particularly implemented in the Mediterranean Area, where many spontaneous and wild *Brassica* provide genetic diversity and variability, allowing for the development of new pre-breeding and advanced breeding materials. Particularly in Italy and Spain, it is possible to find countless ecotypes and populations of *Brassica oleracea* and *Brassica rapa* species, handed down by generations of farmers [49].

These two species have been widely studied and showed a wide diversity in terms of nutritional quality.

#### 4.1.1. Brassica Oleracea Species

Kale could be considered the ancestor of several *B. oleracea* vegetable crops because it has been found to be very similar to the *B. oleracea* wild type and to several wild *Brassica* species (*n* = 9) [94].

Several differences among varieties within this species were reported, e.g., the highest content of total phenolic was found in curly kale that showed a concentration 10-times higher than cauliflower and white cabbage [95]. Although the methodologies of analysis used in many studies were different, all of them agree on the lower content of phytochemicals in white cabbage, in respect to broccoli, Brussel sprouts, curly kale and red cabbage. There are controversial results regarding cauliflower as it showed high activity in liposomal phospholipid suspension system, but low activity in oxygen radical absorption capacity (ORAC method) [14,15].

As mentioned above, the variability is also expressed among genotypes of the same species in broccoli [96], cauliflower [97], cabbage [26] depending on their characteristics; in general, the higher content of antioxidants is detected in the varieties with red or purple pigmentation. Broccoli is important for its cancer-protective compounds; in particular, for its content of glucoraphanin, and its active form sulforaphane. Sicilian landraces of violet cauliflower could be considered an environmentally friendly crop, being characterized by high plant rusticity and adaptability to the Mediterranean climatic condition that allows one to limit the use of pesticides and fertilizers for its cultivation [98].

#### 4.1.2. *Brassica rapa* Species and Other Cruciferous Crops

*Brassica rapa* species include turnip tops and leaves ("cima di rapa" and "friariello"), turnip, pak choi, Chinese cabbage, choy-sum and mizuna, evidencing a wide variability among close species [99] and varieties of the same species [100]. Phenolic compounds are mainly affected by the interaction between environment and genotype; this means that their variability strictly depends on the environmental conditions, hence they possess low heritability.

Choy sum, a *Brassica rapa* variety, showed the highest antioxidant potential compared to broccoli, cabbage, and cauliflower [101]. Some studies found that watercress showed a higher antioxidant potential in comparison to salad rocket; however, these two varieties, together with wild rocket and mizuna, are good sources of antioxidants [102,103]. In *B. rapa,* the aliphatic glucosinolates (GLS) is the predominant form, with gluconapin as the most abundant, followed by glucobrassicanapin [104]. *B. rapa* varieties have shown a high concentration of isorhamnetin, irrespective of the plant organs considered [105].

#### 4.1.3. Plant Portion and Plant Developmental Stage

The variation in nutritional and phytochemical content does not differ only among species and varieties of the same species, but also can change during the growth period [6] and based on plant portion [50], as reported below.

For most of these crops, the more interesting parts of the plant for the nutritional quality are not consumed. For example, seeds seem to possess the highest content of phytochemical compounds but are not usually consumed and appreciated by consumers; this aspect is confirmed in kale, where seeds possess higher antioxidant capacity than leaves [22]. In turnip, flower buds registered the highest antioxidant content, in respect to leaves, stems and roots [106].

GLS content also differs based on plant portion. Seeds possess the highest concentration of GLS, followed by inflorescences, siliques, leaves, roots, stems and petioles [107]. Indeed, the concentration of aliphatic GLS in kale (*B. oleracea acephala*) leaves increases over time, from seedling to early flowering stages. At that stage, the aliphatic GLS content in leaves of *B. oleracea* declined drastically over time as the content in the flower buds increased [50].

A comparison study on turnip tops and turnip greens also reported several and appreciated differences in phytochemicals compounds. Turnip tops gave a higher GLS value (26.02 µmol/g dw) than turnip greens (17.78 µmol/g dw). The opposite trend was reported for total phenolic, whereby turnip greens showed a higher content (43.81 µmol/g dw) than turnip tops (37.53 µmol/g dw) [104].

Several studies confirmed the possibility to detect differences within the same portion of plant. In tronchuda cabbage, the mainly consumed portion are the internal leaves, utilised for salad or cooking; nonetheless, these have an antioxidant capacity lower than the external ones, which are usually discarded [22]. The same results were observed in Chinese cabbage, whereby the variation in bioactive compounds was also evident among different layers of the same head cabbage; phenolic acids and flavonoids were higher in the outer leaves, followed by the mid- and inner leaves. This result could be explained by the higher exposure of outer leaves to sunlight, which stimulates the production of antioxidants [108].

The stage of growth can influence the content and concentration of phytochemical compounds in *Brassica*, and the knowledge of this aspect is fundamental in choosing the proper harvesting moment for obtaining products with the highest quality. Indeed, the juvenile cabbage possesses more flavonols than the mature one [26].

Total GLS content also varies in the function of the stage of growth and increases from vegetative to reproductive stages and maturity. Consequently, the highest content is found either in flower buds, or in leaves harvested at the optimum consumption stage, 180 days after the sowing of kale [50]. In broccoli heads, the highest glucoraphanin content was also observed 180 days after sowing, with a following decline during flowering [109].

Vallejo et al. [110] found an increase in ascorbic acid and phenol compounds during the development of the inflorescence in three broccoli cultivars.

Carotenoids are also affected by the plant developmental stage. In kale, the highest content of lutein was registered in 1- to 2-week-old leaves, and the highest content of β-carotene was found in 2- to 3-week-old leaves [111].

Some of the health-promoting factors may be present 10-times higher in sprouts than in mature vegetables. Sprouting resulted in an overall increase in the total phenolic content and antioxidant capacity and, although germination time was not a discriminating factor, longer germination times resulted in the lower antioxidant capacity of the sprouts [112].

#### *4.2. Environmental and Agricultural Factors*

Seasonal variation, light exposure, temperature, water availability [113], phytosanitary measures, sowing date and harvesting period [114] are all factors linked to environmental conditions that can influence the quality, in particular nutritional content and profile, of *Brassica* vegetables [6]. Different responses to seasonal variations were reported in several *Brassica* crops, such as broccoli, kale, and turnip [115]; this effect is determined mainly by temperatures and day length during the period before harvest.

Countless studies agree that spring season crops, growing at intermediate temperatures, high light intensity, during longer days and in dry conditions (or low average of rainfall) during their vegetative period, contain an increased total GLS and phytochemicals concentration [50,104,114]. For example, in canola (*Brassica napus*), it was found that GLS

concentration increased when a temperature of 40 ◦C was maintained for 4 h on five successive days, giving a total of 15-degree days of stress (15 DD/40 ◦C) [116]. Some authors reported that higher and lower temperatures, rather than intermediate temperatures, brought about an increase in GLS concentration, e.g., growing temperatures between 7 ◦C and 13 ◦C brought about an increase in glucoraphanin and lutein in broccoli; furthermore, they acted as a trigger for biosynthetic pathways [117]. Moreover, broccoli sprouts grown at constant high (29–33 ◦C) or low (11–16 ◦C) temperatures had higher antioxidant content than sprouts grown at intermediate temperature (21.5 ◦C) [118]. The same authors confirm that the main antioxidant content is observed in sprouts that grow with a strong temperature range of 30/15 ◦C day/night.

Autumn/winter season crops, grown at lower temperature, lower light intensity, shorter days, and higher water availability, tend to have the lowest total GLS and other phytochemicals concentration [119,120]. An exception is represented by a turnip that produces higher flavonoids and vitamin C content in the autumn/winter season; this crop accumulates and produces the main phytochemicals with low/moderate temperature and considerable radiation, mainly in turnip tops [121]. More precisely, in *Brassica rapa*, the correlation with temperature is also bound to the plant portion; indeed, the number of days with a minimum temperature below 0 ◦C was negatively correlated with total GLS content in turnip greens. In turnip tops, GLS content was positively correlated with the number of days with a maximum temperature above 20 ◦C. In the case of phenolics, no correlation was found between climatic factors and turnip greens, while in turnip tops, total flavonoids and total phenolics content seemed to be correlated with the number of days with a minimum temperature below 0 and 10 ◦C, respectively [104]. In broccoli, freezing temperature can positively influence the concentration of sulforaphane [122].

The biotic and abiotic factors that characterize the surrounding environment can influence the quality of *Brassicaceae*. With respect to biotic sphere, aphid infestation brought about an increased production of primary metabolites, including amino acids, as well as some secondary metabolites, as a plant defence mechanism against these pathogens. Concerning abiotic factors, the water stress condition and metal exposure produce an initial increase in photosynthetic pigments, proteins, free amino acids and sugar content, followed by a subsequent decrease [123]. In detail, a relation between copper stress and the production of amino acids was found as free amino acid production takes part in the detoxification from excess copper [124]. In *Brassica juncea,* the accumulation of metals produces a 35% increase in oil content [123]. Moderate salinity in water or soil affects the myrosinase-GLS system in broccoli, inducing the production of GLS; also, phenolic compounds increase in this stressful condition, but in the case of strong salinity both GLS and phenolics decrease [125]. Seedlings of *Brassica oleracea* L. var. *italica* subjected to water shortage (applied by increasing the time between two irrigation events) showed a decrease in inflorescence chlorophylls, carotenoids, ascorbic acid, total phenols and total soluble carbohydrates [126].

Ragusa and co-author [127] investigated the effect of different germination temperatures (10, 20 and 30 ◦C) on the phytochemical content as well as on reducing and antioxidant capacity of broccoli and rocket sprouts. In both seeds and sprouts, the total GLS and ascorbic acid contents did not differ between vegetables, while broccoli exhibited exceptionally higher polyphenols and a greater reduction in antioxidant capacity compared to rocket. In both species, an increase in germination temperature positively affected the glucosinolate content. Ascorbic acid increased during germination without a difference among the three tested temperatures. The phenol content increased in broccoli sprouts when grown at 30 ◦C, while the reverse was true in rocket. The antioxidant capacities increased with germination, and higher indexes were detected at 10 ◦C, particularly in rocket.

#### 4.2.1. Cultivation System and Soil Composition

The cultivation system influences the quality of vegetable product, in particular the concentration of primary and secondary metabolites in *Brassica* vegetables.

Some authors reported a higher antioxidant (phenolic compounds, in particular flavonoids) and GLS concentration in *Brassica* growth in organic cultivation system than in conventional systems [128], as demonstrated in early harvested tronchuda cabbage [129]. This result could be linked to the fact that, under organic cultivation, crops are subjected to more biotic and abiotic stress; these stressing conditions lead to an increase in the production of secondary metabolites as a defence mechanism, and consequently obtaining vegetables with higher nutritional and antioxidant potential than in a conventional system.

Several studies described an opposite situation and contrasting evidence about phytochemical enhancement in organic vegetables [130,131]. In fact, Conversa et al. [132] reported that the choice of cultivation systems does not modify the antioxidant properties of raw and processed products, but differences can be found in the chlorophyll and carotenoid contents of organic "cima di rapa" landraces. The lipophilic antioxidant content was improved in organic product while the hydrophilic component, which constituted 99% of the total antioxidant capacity, was not affected by the different crop management in "cima di rapa". However, the organic system influenced the quality of products during storage: after 7 days of storage at 5 ◦C, the organic "cima di rapa" maintained the best colour with high chlorophyll levels, probably due to a higher availability of nitrogen in organic management; on the contrary, the quality declined with a higher production of strong off-odour after 14 days of storage, in comparison to the conventional products.

Regarding the soil composition effect on *Brassica* quality, it was reported that the highest GLS and phenolic compounds content were detected in locations with the highest soil pH and available potassium; the content can be also influenced by nitrogen and sulphur applications in turnip [104]. On the contrary, in *B. rapa L. Subsp. Sylvestris,* flavonols (kaempferol and quercetin derivatives) were reduced by sulphur availability [113].

#### 4.2.2. Water Stress

It was reported that a moderate water stress increases the concentration of bioactive compounds in *Brassica*, partly due to an increased concentration per unit of dry weight; if the stress becomes intensive, the secondary metabolite production should decrease [133]. Phenolic compounds and GLS content increase in the absence of irrigation, because of a reduction in vegetative growth, mainly in turnip, cabbage and broccoli [6]. The association between low availability of water in the soil during plant growth and postharvest cold storage brought about the best maintenance of antioxidant activity in *Brassica*. Water stress conditions also affect sugar content, as it is increased in cabbage [134].

#### 4.2.3. Plant Density, Intercropping and Trap Cropping

Plant density seems to affect the plant morphology and phytochemical compound content: a higher density decreases the head size but increases the GLS content, because the competition for nutrients in high density conditions causes stress on plants which, in turn, stimulates the production of secondary metabolites [120].

Intercropping and trap cropping are strategies utilised for weed and pest control [135]; however, the presence of another crop can generate stressful conditions, such as plant competition for light, nutrients, and water, decreasing their availability and, hence, affecting the accumulation of phytochemicals in *Brassica* plant tissue.

#### 4.2.4. Fertilization Practices

A correct fertilization plan is fundamental for obtaining high quality, healthy and safe vegetables. The nutritional and sensorial profile of *Brassica* is conditioned by the availability of fertilizers and nutrients as they determine the biosynthesis of secondary metabolites.

Countless studies have been conducted on the effect of sulphur fertilization on phytochemical concentration, mainly on GLS production, considering their sulphurous nature [136,137]. There is a correlation between the increase in sulphur supply and higher levels of total GLS [138], in turnip [136], kale [137] and broccoli, mainly when associated with a reduction in water, at the expense of yield [139]. Vallejo and co-authors [119] sug-

gested that the effect of sulphur application on GLS varies with the development stage of broccoli plants and differs for each kind of GLS; in fact, they found an increase in total GLS content at the start of the inflorescence development, followed by a rapid decrease thereafter. Increasing sulphur fertilization brought about a positive impact in the synthesis of polyphenols, such as flavonols and phenolic acids, increasing the total antioxidant capacity in turnip top (*B. rapa ssp. Sylvestis*) [113], and broccoli [110]. Sulphur fertilization in pre-harvest (from 2.6 mmol/L to 6.5 mmol/L) increases the lipophilic and hydrophilic antioxidant capacity but does not affect the nitrate and chlorophyll contents in ready-to-eat "friariello" product [140]. Sulphur deficiency induced an increased vulnerability of *Brassica* crops to diseases and fungal pathogens [141]. Sulphur fertilization, besides improving the antioxidant activity, it is also associated with a genotype-dependent significant reduction in leaf nitrate content, since it enhances the incorporation of nitrogen into organic compounds and consequently reducing the leaf nitrate concentration [113].

Nitrogen is the main constituent of chlorophyll structure: for this reason, its availability influences the content of carotenoids such as lutein and β-carotene, indeed high NO3-N:NH4-N ratio led to a higher content of both [142]. Consequently, the colour and pigmentation of leafy vegetables are also improved [132]. Nitrogen fertilisation led to a decrease in the total GLS content [136]; nonetheless, it acts differently according to the type of GLS; in fact, abundant nitrogen applications increase progoitrin and decrease sinigrin concentration in *Brassica napus* [143]. A reduced nitrogen fertilisation generated an increase in the bioactive compound content, mainly phenolics, as nitrogen stress triggers the gene expression of flavonoid pathway enzymes [128]. Combined fertilisation with NO<sup>3</sup> <sup>−</sup>:NH<sup>4</sup> + is the optimal solution to maintain plant growth and increase the total GLS content [144].

An optimal balance between nitrogen and sulphur fertilisation influences the biosynthesis of secondary metabolites [21]. GLS, for example, can be enhanced by the presence of low nitrogen and high sulphur fertilizers: this balance influences the quantity and the quality of GLS produced, according to the corresponding amino acids synthetized. Some authors reported the effect of different nitrogen/sulphur combinations on GLS content in *Brassica*, with an increasing amount of nitrogen (80–320 kg/ha) applications. When enough sulphur was available (60 kg/ha), there were no effects on total GLS content, but their production moved to indolics; when the combination was with a low concentration of sulphur supply (10–20 kg/ha), the arylaliphatic and aliphatic GLS decreased [138]. Increased nitrogen/sulphur ratio pushes the plants towards the vegetative growth, at the expense of GLS production [136]. Fabek et al. [145] showed that the type of fertilisation may influence mineral composition in plants: nitrogen fertilisation was negatively associated with potassium (K) and calcium (Ca) content in broccoli, while sulphur fertilisation increased manganese (Mn) and zinc (Zn), and decreased copper (Cu). Applications of sodium selenate (Na2SeO4) produced an increase in GLS [144].

Similarly, microelements availability can influence the phytochemical concentration in *Brassica*, with salts stress increasing GLS content. In detail, selenium seems to increase the GLS content (in particular sulphuraphane), when applied up to a certain dose; above this level, it decreases the GLS production [51].

Furthermore, some *Brassica* species are used as metal hyperaccumulator, and the type and amount of metal in the soil affects the concentration of glucosinolates in plant tissues. In particular, it was reported that glucosinolate concentrations in roots and shoots of *Thlaspi caerulescens* responded in different way to enhanced Zn accumulation: decreased glucosinolate levels were observed in leaves of plants accumulating high Zn concentrations, while increased levels were detected in roots, with Zn accumulation [146]. Similarly, the content of total glucosinolates, mostly due to indolic glucosinolates as glucobrassicin, was increased only in the roots of Chinese cabbage, when subjected to high soil copper stress [147]. In two *B. juncea* cultivars subjected to high arsenic levels, the increased levels of thiol related proteins, sulphur content and phytochemicals (phenolics and ascorbic acid) in leaves allow us to better tolerate the oxidative stress induced in the plant; different

response pattern of total and individual GSLs content was observed in both cultivars under arsenic stress [148].

Besides the classical fertilizers, in the last years, new proposed products that are beneficial on crops, such as improving safety, enhancing growth and production, improving the defence against weeds and pests and nutritional quality, were developed. Among these, signalling molecules, biocontrol agents, and biostimulants are now gaining high interest for improving plant resilience and quality. Leaves and cotyledons of *B. napus*, *B. rapa* and *B. juncea* showed an up to 20-fold increase in glucobrassicin content after treatment with JA (Jasmonic Acid), or MeJA (Methyl Jasmonate) [149]. In contrast, treatment with ABA (Abscisic Acid) reduced the accumulation of indole GLS in *B. napus* [150].

In summary, the levels of hormones, such as JA, SA (Salicylic Acid) and ABA, seem to be related to the regulation of GLS and of other bioactive compound content [151]. Consequently, hormonal elicitation can be a useful tool to induce the synthesis of bioactive compounds interesting for human health.

Concerning the application of biocontrol agents, Gallo et al. [152] affirmed that the use of *Trichoderma* and its metabolites led to an increase in GLS in plants. This could probably be due to their capability of inducing resistance mechanisms, stimulating the synthesis of salicylic and jasmonic acids and the cascade of events leading to the production of various metabolites; only ascorbic acid was lower compared to control plants.

Additionally, in *Brassica* spp. cultivation is increasing the use of biocontrol agents, there is also the utilisation of seaweeds extract, mycorrhizae, nematodes [153], humic acids such as vermicompost foliar sprayed [154], and protein hydrolysates, all compounds now classified as "biostimulants", useful to increase plant yield and the accumulation of bioactive compounds [155].

*Brassica* species can contrast the main soil-borne agents thanks to their secondary metabolites that act as biofumigants. A study reported the effectiveness of the flour of dry plants of *Brassica juncea, Eruca sativa, Raphanus sativus* and *Brassica macrocarpa* in nematodes control (*Meloidogyne* spp.) on tomatoes. Minced flour was distributed before planting (60 and 90 g m−<sup>2</sup> ) and was successful for the sinigrin presence [156].

#### **5. Conclusions**

*Brassica* vegetables are a good source of many phytochemical compounds that exert positive effects on the final consumer's health. This study presented an investigation on the presence of these bioactive compounds, analysing how they affect the sensorial and nutritional quality, and on the factors that can modify their concentration in *Brassica* food vegetables, such as genetic, environmental and agronomic factors.

There is a large possibility to improve the nutritional and sensorial quality of *Brassica* vegetables through the implementation of appropriate agronomic practices; nevertheless, the effects of the treatments are strictly genotype-dependent, and a good selection of the genotype before the start of cultivation is required. Furthermore, the environmental factors could influence to different extents the quality of *Brassica* genotypes, and they should be considered in the evaluation of the phytochemical compounds amount.

All this information is useful for developing new fresh and processed products with increased nutritional and sensorial quality, according to the final users' needs and the final purpose of consumption. If the consumer will be informed and made conscious of the healthy potential of the phytochemical compounds present in *Brassica*, they may be willing to accept these products despite the bitter taste and the intense aroma, which are often responsible for a low consumer acceptance.

**Author Contributions:** Conceptualization, M.V. (Marino Visciglio), B.M., F.C., and M.V. (Massimo Vagnoni); Methodology, F.B. (Francesca Biondi), F.C., E.M., and L.M.; Investigation, F.B. (Francesca Biondi), F.B. (Francesca Balducci), and L.M.; Resources, M.V. (Marino Visciglio), and B.M.; Data Curation, F.B. (Francesca Biondi), F.C., and L.M.; Writing—Original Draft Preparation, F.B. (Francesca Biondi), and L.M.; Writing—Review & Editing, B.M., and L.M.; Visualization, L.M.; Supervision, M.V. (Marino Visciglio), F.C., and B.M.; Project Administration, M.V. (Marino Visciglio), M.V. (Massimo Vagnoni), E.M., and B.M.; Funding Acquisition, M.V. (Marino Visciglio), and B.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The authors thank Cecilia Limera and Maria Teresa Ariza Fernandez for extensively revise the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Influence of Post-Flowering Climate Conditions on Anthocyanin Profile of Strawberry Cultivars Grown from North to South Europe**

**Erika Krüger 1,\*, Frank Will <sup>2</sup> , Keshav Kumar <sup>2</sup> , Karolina Celejewska <sup>3</sup> , Philippe Chartier <sup>4</sup> , Agnieszka Masny <sup>3</sup> , Daniela Mott <sup>5</sup> , Aurélie Petit <sup>4</sup> , Gianluca Savini <sup>5</sup> and Anita Sønsteby <sup>6</sup>**


**Abstract:** The effect of cultivar and environmental variations and their interaction on anthocyanin components of strawberry were assessed for six cultivars grown in five locations from North to South of Europe in two different years. To evaluate the impact of latitude- and altitude-related factors, daily mean (Tmean), maximum (Tmax) and minimum (Tmin) temperature and global radiation accumulated for 3, 5, 10 and 15 days before fruit sampling, was analyzed. In general, fruits grown in the south were more enriched in total anthocyanin and pelargonidin-3-glucoside (pel-3-glc), the most abundant anthocyanin in strawberry. Principal component analysis (PCA) provided a separation of the growing locations within a cultivar due to latitudinal climatic differences, temporary weather changes before fruit collection and cultivation technique. PCA also depicted different patterns for anthocyanin distribution indicating a cultivar specific reaction on the environmental factors. The linear regression analysis showed that pel-3-glc was relatively less affected by these factors, while the minor anthocyanins cyanidin-3-glucoside, cyanidin-3-(6-O-malonyl)-glucoside, pelargonidin-3-rutinoside and pelargonidin-3-(6-O-malonoyl)-glucoside were sensitive to Tmax. The global radiation strongly increased cya-3-mal-glc in 'Frida' and pel-3-rut in 'Frida' and 'Florence'. 'Candonga' accumulated less pel-3-glc and total anthocyanin with increased global radiation. The anthocyanin profiles of 'Gariguette' and 'Clery' were unaffected by environmental conditions.

**Keywords:** anthocyanins; *Fragaria* × *ananassa*; latitude; temperature; global radiation; cultivar × environmental interaction

#### **1. Introduction**

Strawberry (*Fragaria* × *ananassa* Duch.) is the most important berry crop being cultivated from North to South of Europe. Beside its unique color, taste and aroma, strawberry fruits are enriched with several nutritious and bioactive compounds providing health benefits by reducing risk of diseases such as inflammation disorders and oxidative stress, obesity-related disorders and heart disease, and protection against various types of cancer [1–4]. Anthocyanins are a type of flavonoids that are commonly found in strawberries. The functional properties and the sensory qualities of the anthocyanins could easily be explained based on their chemical reactivity [5]. The antioxidative activity of anthocyanins could mainly be attributed to the presence of the flavylium cation moiety. Despite their low bioavailability [6], anthocyanins have been shown to exhibit a range of biological effects,

**Citation:** Krüger, E.; Will, F.; Kumar, K.; Celejewska, K.; Chartier, P.; Masny, A.; Mott, D.; Petit, A.; Savini, G.; Sønsteby, A. Influence of Post-Flowering Climate Conditions on Anthocyanin Profile of Strawberry Cultivars Grown from North to South Europe. *Appl. Sci.* **2021**, *11*, 1326. https://doi.org/10.3390/app11031326

Received: 29 December 2020 Accepted: 28 January 2021 Published: 2 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

including antioxidant activity, photoprotection, anti-carcinogenesis, induction of apoptosis, and prevention of DNA damage [3,7]. Anthocyanins also serve as visual attractants for pollinators and seed dispensers and play a crucial role in plant protection against biotic and abiotic stress, and hence, in adaptability to environmental conditions at site [8].

Thus, the total anthocyanin contents in strawberry are both qualitatively and quantitatively known to be strongly influenced by the genotype (among others [9–14]) and likewise, by external factors such as high or low temperature and light (photoperiod, quantity and wavelength including UV-light). Recent review articles have highlighted the influence of temperature and light on the synthesis and accumulation of plant secondary metabolites including anthocyanins [15–17]. For example, some studies reported positive correlation between anthocyanin contents and temperature in strawberries grown in controlled environment [18–20], as well as in ambient conditions [14,21]. The studies describing the influence of incident light are mostly related to protected cultivation systems and includes shading [22], UV-B radiation [23–26] and blue and red LED-light [27,28].

The synthesis and accumulation of anthocyanins in strawberries are primarily known to be influenced by the genotype, however, little is known about latitudinal effects on the anthocyanin content of strawberry causing quantitative or qualitative changes in the content of these compounds [21,23]. The ambient conditions, including temperature and photoperiod, varies with latitude, and may also influence the different anthocyanin components. In future, due to the climate changes, the northern regions of Europe will become more suitable for berry production [25]. Moreover, it is desirable that quality traits of a cultivar, including bioactive compounds such as anthocyanins, remain invariant to the changes caused by environmental conditions during the cultivation season in different years at all growing locations. Cultivars with such stable fruit qualities could serve as useful parents in breeding programs with regard to climate warming.

The objective of the present work was to study the cultivar and environmental impact and their interaction on the anthocyanin content of six strawberry cultivars: 'Candonga', 'Clery', 'Frida', 'Florence', 'Gariguette' and 'Sonata' grown at five geographical distant locations throughout Europe from South-East Norway to South-West France, covering a distance of more than 15.5 degree of latitudes or more than 2000 km. These cultivars are mainly selected because of their diversity and popularity in Europe. The present study would help in identifying the appropriate cultivar with desirable anthocyanin traits that could be cultivated in a given environmental condition.

#### **2. Materials and Methods**

#### *2.1. Experimental Sites, Plant Material and Cultivation*

The experiments were conducted during two consecutive cropping seasons (2017 and 2018) at five locations from North to South Europe at NIBIO (Norwegian Institute of Bioeconomy Research, Bergen, Norway) (NO, 60◦ N), INHORT (PL, 51◦ N), HGU (DE, 49◦ N), Sant'Orsola (IT, 46◦ N) and Invenio (FR, 44◦ N), hereinafter named as Norway, Poland, Germany, Italy and France. As common for the different regions, experiments in Norway, Poland and Germany were carried out in open field, whereas in Italy and France they were performed in polytunnels that were open-sided after anthesis. Details of the respective latitude, altitude, yearly mean temperature, soil type, soil pH and cultivation type, as well as start of flowering, harvest season and day length at start of harvest are given in Table 1. Air temperature (Tmean, Tmax and Tmin) and global radiation were recorded at each location. To describe the environmental conditions of the respective harvest season, weekly mean temperature and global radiation were calculated starting 2 weeks prior to start of harvest of the earliest cultivar 'Clery' (Figure 1).

**Figure 1.** Weekly mean temperature and global radiation courses during the harvest season for the five experimental **Figure 1.** Weekly mean temperature and global radiation courses during the harvest season for the five experimental locations in 2017 and 2018, starting two weeks before the first picking of the earliest cultivar 'Clery'.


**Table 1.** Geographical location, soil and climatic conditions, cultivation type and harvest season for the five experimental locations in Europe.

(a) For the period 1981–2010. (b) Earliest cultivar. (c) Latest cultivar. (d) Warm temperature in March enhanced start of flowering followed by extremely cold temperature delaying fruit development.

> The six short-day strawberry genotypes used in this study were selected for their diverse genetic background and adaptability to different environments: 'Candonga'® (E), 'Clery' (IT), 'Florence' (UK), 'Frida' (NO), 'Gariguette' (FR) and 'Sonata' (NL). All cultivars were cultivated in Norway, Poland, Germany and France, while Italy only grew 'Clery', 'Frida', 'Gariguette' and 'Sonata'. Each season, all locations propagated their own plants from mother-plants purchased from the same nursery to avoid epigenetic variation within a cultivar due to different origin. Thus, each year, plug-plants were planted in week 32 in Norway, Poland and Germany for the harvest season the next year. At all sites, three randomized replicated plots, each with 20 plants per genotype, were trialed. In Italy and France, where strawberry are commonly grown in tunnels, cold-stored plants were set in peat bags, as usual, in week 9 in 2017 and 2018 for harvesting in the respective year. Furthermore, in Italy and France, the experimental design consisted of three plots, and of three replications per genotype with 22–24 plants per plot for the harvest in the same year. Plant protection, fertilization and irrigation in open-field sites, and fertigation of the plants in peat bags were performed according to local guidelines.

> To evaluate the impact of latitude-related factors on the anthocyanin synthesis and accumulation, daily temperature (Tmean, Tmax and Tmin) and global radiation were accumulated for 3, 5, 10 and 15 days (3d, 5d, 10d and 15d) before sampling of the berries to be analyzed for each cultivar.

#### *2.2. Sample Preparation for Anthocyanin Analysis*

Three independent biological replications were collected twice in an approximately weekly interval at mid-harvest at each location per cultivar (Figure 1 and Table 1). Thus, the samples (500 g) consisted mainly of secondary and tertiary fruits, being fully ripe and undamaged. Each biological replicate of freshly harvested strawberries was promptly prepared for analytical purposes by first cutting the calyx and then slicing the fruits into 2–4 pieces. Slices were frozen in a liquid nitrogen bath for at least 10 s. For anthocyanin analysis, the frozen slices were ground either in a lab mill (e.g., Retsch GM 200) or crushed under liquid nitrogen with a precooled mortar and pistil to a fine powder. The frozen powders were filled into 50 mL plastic tubes with screw caps, and stored at −20◦ C. Anthocyanin analysis was carried out at Geisenheim. Suitable shipping conditions were chosen to prevent thawing of the samples during transportation until solvent extraction. The frozen

powders (5.0 g) were weighed into a 50 mL plastic tube and extracted twice in an ultrasonic bath (30 min, 120 W, Bandelin Sonorex RK 106, VWR, Darmstadt, Germany) and intermediate centrifugation (4500 upm 15 min, Hettich Rotanta 460, Tuttlingen, Germany) with a total of 2 × 10 mL acidified methanol/water/formic acid (80/20/1 *v*/*v*/*v*). Sonication and centrifugation were performed under cool conditions. After final centrifugation, the pooled supernatants were made up to 25 mL in a volumetric flask; aliquots were 0.45 µm filtered prior to HPLC analysis.

#### *2.3. HPLC Analysis of Anthocyanins*

HPLC analysis of the methanolic extracts was performed on an Accela HPLC system coupled with a PDA detector (Thermo Fisher, Dreieich, Germany) using a 125 × 2 mm i.d., 3 µm ODS-3 column (Dr. Maisch, Ammerbuch, Germany) protected with a guard column of the same material. Injection volume was 4 µL, elution conditions were: 250 µL/min flow rate at 40 ◦C; solvent A was 5% formic acid (ULC/MS grade, Promochem, Wesel, Germany); solvent B, methanol (gradient grade, Roth, Karlsruhe, Germany); 1 min isocratic conditions with 10% B, linear gradient from 10% to 40% B in 12 min, followed by washing with 100% B and re-equilibrating the column. Quantitation was carried out using peak areas (500 nm trace for pelargonidins, 515 nm for cyanidins) from external calibration via the reference substances pelargonidin-3-glucoside and cyanidin-3-glucoside, respectively. Anthocyanin analysis was carried out in duplicate.

#### *2.4. Electrospray Ionization (ESI)-MS Identification of Anthocyanins*

For mass detection, the Accela HPLC system was coupled to a ThermoFinnigan LXQ mass spectrometer (Thermo Fisher, Dreieich, Germany) equipped with an ESI source and an ion trap mass analyzer. The whole system was controlled by Xcalibur software. For anthocyanins, the mass spectrometer was operated in the positive mode under the following conditions: source voltage 4.5 kV; capillary voltage 32 V; capillary temperature 275 ◦C; collision energy 30% (MS<sup>2</sup> ) and 33% (MS<sup>3</sup> ).

#### *2.5. Software Used for Statistical Analysis*

All statistical analysis was conducted on the MATLAB (2016b) platform. Significant differences were calculated using post hoc analysis with Tukey's honestly significant difference criteria on the ANOVA (analysis of variance) results. Principal component analysis (PCA) [29] was performed using the PLS-Toolbox (Eigenvector Research, Manson, WA, USA). Linear regression analysis was used to study the correlation between different cultivar-specific anthocyanin components and the mean, minimum, maximum temperatures, and global radiation summarized 3, 5, 10 and 15 days before start of harvest. Coefficient of determinations (R<sup>2</sup> ) ≥ 0.2 at *p* ≤ 0.05 are presented.

#### *2.6. Data Arrangement for PCA*

The data for 'Clery', 'Frida', 'Gariguette' and 'Sonata' grown at the locations in France, Italy, Germany, Poland and Norway were arranged in a matrix of dimension 60 × 22; 60 represents the number of samples (five locations, two cropping seasons, two picking dates and three biological replicates) and 22, the number of variables (cya-3-glc, pel-3-glc, pel-3-rut, cya-3-mal-glc, pel-3-mal-glc, total anthocyanin as well as global radiation, mean, maximum and minimum temperatures summarized 3, 5, 10 and 15 days prior to harvest). The data for 'Candonga' and 'Florence' were arranged in matrices of dimension 48 × 22, where 48 is the number of samples (four location × two seasons × two picking dates × three biological replicates) and 22 is the same variables as specified above. The PCA analysis carried out on all the cultivars together clearly indicated (given in the Supplementary Figure S1) that the anthocyanin profiles of the strawberries are mainly influenced by the location. Thus it was important that each of the six cultivars were analyzed separately.

To ensure that each component had equal variance and comparable impact on the PCA modeling, the specific data for each cultivar were auto-scaled prior to PCA. Autosscaling [29] is a common pre-processing method that uses mean-centering followed by dividing each variable by the corresponding standard deviation. Each variable upon auto-scaling has a mean of zero and unit variance.

#### **3. Results and Discussion**

#### *3.1. Harvest Season and Environmental Characterization of the Growing Locations*

This study investigated the adaption of six strawberry genotypes to different environments related to anthocyanin accumulation in the fruits. As expected, the harvest period varied along the North-South axis (Table 1), due to the longitudinal difference between the growing sites (>15.5◦ of latitude). In addition, yearly differences in temperature and global radiation affected the harvest period.

For instance, the harvest period was much earlier in 2018 compared to 2017 in Norway, Poland and Germany, while there were no variations in Italy and France. To highlight the environmental differences between the five growing sites, ambient weekly mean temperature and global radiation during the harvest seasons are shown in Figure 1, starting two weeks prior to start of harvest of the earliest cultivar 'Clery'. Higher year-on-year variations were observed in Norway. Here, weekly mean temperature was, on average, 3 ◦C lower, and global radiation 1200 W m−<sup>2</sup> less in 2017 compared to 2018 (18 ◦C and 6412 W m−<sup>2</sup> ). In this year, weekly mean temperature and global radiation in Norway were similar to those in both years in Poland and Germany, and for temperature in France, only. Due to its high altitude (Table 1), Italy exhibited lower weekly mean temperature in both years, thus being similar to Norway in 2017. In Italy, large yearly variations occurred for global radiation in 2017 (5917 W m−<sup>2</sup> ) with similar values as for Norway, Poland and Germany, while in 2018, Italy had the same low values as for both years in France. The low values for global radiation at the southern locations are probably due to the earlier cropping season and shorter photoperiod compared to Norway, as the other extreme.

The temperature and global radiation may vary within the harvest season, therefore, the sum of temperature and global radiation 3, 5, 10 and 15 days before the two fruit samplings were calculated for each cultivar and location, and used for principal component and regression analyses. The complete list is provided in the Supplementary Materials (Table S1).

#### *3.2. Effect of Genotype on Total and Individual Anthocyanins*

Cyanidin-3-glucoside (cya-3-glc, m/z [M<sup>+</sup> ] 449, MS<sup>2</sup> 287), pelargonidin-3-glucoside (pel-3-glc, m/z [M<sup>+</sup> ] 433, MS<sup>2</sup> 271), pelargonidin-3-rutinoside (pel-3-rut, m/z [M<sup>+</sup> ] 579, MS<sup>2</sup> 433, 271), cyanidin-3-(6-O-malonyl)-glucoside (cya-3-mal-glc, m/z [M<sup>+</sup> ] 535, MS<sup>2</sup> 287), and pelargonidin-3-(6-O-malonoyl)-glucoside (pel-3-mal-glc, m/z [M<sup>+</sup> ] 519, MS<sup>2</sup> 271) were assigned by their mass spectra as the major strawberry anthocyanins (Tables 2–7). Over all, the pel-3-glc showed the highest concentrations (Table 4).

**Table 2.** Effect of growing location and year on total anthocyanin content as the mean of two picking dates in fruits of six strawberry cultivars.



**Table 2.** *Cont.*

Data are expressed as means ± SD (standard deviation) of two sampling dates per year. Before performing the statistical analysis, the homogeneity of the data were ensured using Bartlett's test. Mean values (*n = 3*) of different cultivars grown at a particular location followed by lower-case letters represent significant differences (*p* ≤ 0.05) between cultivars. Mean values of all the cultivars grown in a particular location followed by different upper-case letters represent significant difference between the two years 2017 and 2018 (*p* ≤ 0.05). Mean values of all cultivars grown at all the locations followed by different lower-case letters represent significant differences (*p* ≤ 0.05). \* = 0.05; \*\*\* = 0.001; ns = not significant. Can = 'Candonga'; Cle = 'Clery'; Flo = 'Florence'; Fri = 'Frida'; Gar = 'Gariguette'; Son = 'Sonata'.

**Table 3.** Effect of growing location and year on cyanidin 3-glucoside content as the mean of two picking dates in fruits of six strawberry cultivars.



Data are expressed as means ± SD (standard deviation) of two sampling dates per year. Before performing the statistical analysis, the homogeneity of the data were ensured using Bartlett's test. Mean values (*n =3*) of different cultivars grown at a particular location followed by lower-case letters represent significant differences (*p* ≤ 0.05) between cultivars. Mean values of all the cultivars grown in a particular location followed by different upper-case letters represent significant difference between the two years 2017 and 2018 (*p* ≤ 0.05). Mean values of all cultivars grown at all the locations followed by different lower-case letters represent significant differences (*p* ≤ 0.05). \*\*\* = 0.001; ns = not significant. Can = 'Candonga'; Cle = 'Clery'; Flo = 'Florence'; Fri = 'Frida'; Gar = 'Gariguette'; Son = 'Sonata'.

**Table 4.** Effect of growing location and year on pelargonidin-3-glucoside content as the mean of two picking dates in fruits of six strawberry cultivars.


Data are expressed as means ± SD (standard deviation) of two sampling dates per year. Before performing the statistical analysis, the homogeneity of the data were ensured using Bartlett's test. Mean values (*n =* 3) of different cultivars grown at a particular location followed by lower-case letters represent significant differences (*p* ≤ 0.05) between cultivars. Mean values of all the cultivars grown in a particular location followed by different upper-case letters represent significant difference between the two years 2017 and 2018 (*p* ≤ 0.05). Mean values of all cultivars grown at all the locations followed by different lower-case letters represent significant differences (*p* ≤ 0.05). \* = 0.05; \*\*\* = 0.001; ns = not significant. Can = 'Candonga'; Cle = 'Clery'; Flo = 'Florence'; Fri = 'Frida'; Gar = 'Gariguette'; Son = 'Sonata'.


**Table 5.** Effect of growing location and year on pelargonidin-3-rutinoside content as the mean of two picking dates in fruits of six strawberry cultivars.

Data are expressed as means ± SD (standard deviation) of two sampling dates per year. Before performing the statistical analysis, the homogeneity of the data were ensured using Bartlett's test. Mean values (*n* = 3) of different cultivars grown at a particular location followed by lower-case letters represent significant differences (*p* ≤ 0.05) between cultivars. Mean values of all the cultivars grown in a particular location followed by different upper-case letters represent significant difference between the two years 2017 and 2018 (*p* ≤ 0.05). Mean values of all cultivars grown at all the locations followed by different lower-case letters represent significant differences (*p* ≤ 0.05). \* = 0.05; \*\* = 0.01; \*\*\* = 0.001; ns = not significant. Can = 'Candonga'; Cle = 'Clery'; Flo = 'Florence'; Fri = 'Frida'; Gar = 'Gariguette'; Son = 'Sonata'.

**Table 6.** Effect of growing location and year on cyanidin-3-(6-O-malonyl)-glucoside content as the mean of two picking dates in fruits of six strawberry cultivars.



**Table 6.** *Cont.*

Data are expressed as means ± SD (standard deviation) of two sampling dates per year. Before performing the statistical analysis, the homogeneity of the data were insured using Bartlett's test. Mean values (*n* =3) of different cultivars grown at a particular location followed by lower-case letters represent significant differences (*p* ≤ 0.05) between cultivars. Mean values of all the cultivars grown in a particular location followed by different upper-case letters represent significant difference between the two years 2017 and 2018 (*p* ≤ 0.05). Mean values of all cultivars grown at all the locations followed by different lower-case letters represent significant differences (*p* ≤ 0.05). \* = 0.05; \*\*\* = 0.001; ns = not significant. Can = 'Candonga'; Cle = 'Clery'; Flo = 'Florence'; Fri = 'Frida'; Gar = 'Gariguette'; Son = 'Sonata'.

**Table 7.** Effect of growing location and year on pelargonidin-3-(6-O-malonyl)-glucoside content as the mean of two picking dates in fruits of six strawberry cultivars.


Data are expressed as means ± SD (standard deviation) of two sampling dates per year. Before performing the statistical analysis, the homogeneity of the data were ensured using Bartlett's test. Mean values (*n* =3) of different cultivars grown at a particular location followed by lower-case letters represent significant differences (*p* ≤ 0.05) between cultivars. Mean values of all the cultivars grown in a particular location followed by different upper-case letters represent significant difference between the two years 2017 and 2018 (*p* ≤ 0.05). Mean values of all cultivars grown at all the locations followed by different lower-case letters represent significant differences (*p* ≤ 0.05). \* = 0.05; \*\*\* = 0.001; ns = not significant. Can = 'Candonga'; Cle = 'Clery'; Flo = 'Florence'; Fri = 'Frida'; Gar = 'Gariguette'; Son = 'Sonata'.

The genotype influenced significantly (*p* ≤ 0.05) the abundance of total and individual anthocyanins in the fruits (Tables 2–7). When considering the cultivar means for all locations, 'Florence' and 'Frida' showed the highest total anthocyanin content (426.1 and 408.0 mg kg−<sup>1</sup> fresh weight (FW), respectively) whereas 'Sonata' had the lowest (193.2 mg kg−<sup>1</sup> FW) (Table 2). As expected, total anthocyanin content was strongly related to the predominant anthocyanin pel-3-glc (Table 4), resulting in the highest content for 'Florence' (357.4 mg kg−<sup>1</sup> FW) and, however, being significantly different from 'Florence', for 'Frida' (286.3 mg kg−<sup>1</sup> FW) and the lowest for 'Sonata' (139.3 mg kg−<sup>1</sup> FW) and also for 'Gariguette' (156.4 mg kg−<sup>1</sup> FW). Thereby, pel-3-glc contribution to the total anthocyanin content in the cultivars was in the range of 66–84%. 'Florence' (20.5 mg kg−<sup>1</sup> FW) also had, on average, a higher content of cya-3-glc compared to the other cultivars (Table 3). Pel-3-rut was highest in 'Candonga' and 'Frida' (21.8 and 21.3 mg kg−<sup>1</sup> FW) (Table 5), whereas cya-3-mal-glc was enriched in 'Florence' and 'Frida' (20.5 and 13.5 mg kg−<sup>1</sup> FW) (Table 6). The level of pel-3-mal-glc, the second abundant anthocyanin in strawberry, was again highest in 'Frida' (83.3 mg kg−<sup>1</sup> FW) and in 'Gariguette' (75.8 mg kg−<sup>1</sup> FW) (Table 7). The contribution of pel-3-mal-glc varied widely in a range of 9.9–31.7% of the total anthocyanin. In contrast to the other investigated cultivars, 'Candonga' contained only cya-3-glc, pel-3-glc, and pel-3-rut in large quantities, while cya-3-mal-glc and pel-3-mal-glc were only found in very small amounts in some locations and years (on average <0.4 and 0.7 mg kg−<sup>1</sup> FW). Each cultivar had an individual anthocyanin profile that will be discussed later (see Section 3.3). In general, the values for total and individual anthocyanin are in similar ranges as previously reported for these cultivars [11,12,14,21,30–32].

#### *3.3. Anthocyanins are Affected by Location*

To better characterize the influence of location and thus mainly latitude and climate, as well as yearly weather parameters on the anthocyanin profile of six strawberry cultivars, a cultivar-specific PCA was conducted. The score and loading plots comprised by the first two principal components (PC1 and PC2) explained ~70% of the total variance of the data set for each of the four cultivars ('Clery', 'Frida', 'Gariguette' and 'Sonata') grown in five locations (Figures 2 and 3), and of the two cultivars ('Candonga' and ''Florence') grown in four locations (Figure 4).

In addition, the PCA models also captured the effect of the two years and the two picking dates within each year. PC2 described 15.7–25.3% of the data variation and was mainly responsible for the separation of each cultivar by location, and thus by latitude and climatic factors, as well as local pre-harvest weather conditions on the anthocyanin synthesis and accumulation.

In general, the loading plots (Figures 2–4) of each cultivar indicates that pel-3-gluc are closely related to total anthocyanin, indicating a similar reaction on the different locations and their environmental conditions. Likewise, cya-3-glc and cya-3-mal-glc were grouped closely together. Thus, they were similar, but in an opposite way as pel-3-glc and total anthocyanins, influenced by locations and yearly weather variations. An exception was observed for 'Frida' where the total anthocyanin and pel-3-glc were clustered together with both cya-derivates, and hence, underlying the same location and environmental effects. Even though all pel-derivates have the same synthesis pathway, pel-3-rut and pel-3-mal-glc showed a cultivar and location-dependent distribution pattern. In the case of 'Clery', 'Gariguette' and 'Candonga', pel-3-rut was clustered more or less separately between both cya-derivates and total anthocyanin and pel-3-glc, while in 'Sonata', it was more related to the cya-derivates, in 'Frida', to cya-derivates, pel-3-glc, and total anthocyanin, while in 'Florence', pel-3-rut was linked to total anthocyanin and the other two pel-derivates. Pel-3 mal-glc was more or less related to pel-3-glc and total anthocyanin in the case of 'Clery', 'Gariguette' and 'Sonata'. In contrast, pel-3-mal-glc was clustered separately for 'Frida' and together with all other pel-derivates and total anthocyanins in the case of 'Florence'.

The score plots (Figures 2–4) indicated a similar clustering of location for each of the cultivars, showing a typical latitudinal division by the North-South axis. However, modification also occurred by cultivar, yearly variations and weather conditions during the harvest period.

The high amount of cya-3-glc and cya-3-mal-glc in 'Clery' samples from Poland and Norway (Tables 3 and 6), and the enriched levels of total anthocyanins, pel-3-glc, and pel-3-mal-glc and in fruits grown in France (Tables 2, 4 and 7), mainly contributed to the clustering of the different locations (Figure 2). The pel-3-rut content was high in 'Clery' fruits from Germany (Table 5), however, this was less important for the separation by location. Interesting to note is the effect of yearly variations combined with harvest period on the synthesis and accumulation of total anthocyanins and their individual compounds at the different locations. For example, 17A samples from France are overlapping with those from Italy in 2018. Another example is the clear separation of the Polish 18B samples from their other ones.

The loading plot for 'Frida' (Figure 2) separated the locations more clearly than for 'Clery'. However, the effects of yearly variations and harvest period are less pronounced. The main difference between locations were due to the high abundance of pel-3-glc in samples from Norway in both years (Table 4), and total anthocyanin both in Norway and Poland (Table 2). In addition, clustering was due to low levels of cya-3-glc in fruits from Italy, especially in 2017 (Table 3), and low values of pel-3-rut (Table 5) in both Italy and France in 2018. Moreover, pel-3-mal-glc (Table 7) was found to be relatively less abundant in samples from Norway.

**Figure 2.** Score and loading plots of PCA for 'Clery', and 'Frida' grown at five locations (F: France; G: Germany; N: Norway; P: Poland and I: Italy) characterized by total and individual anthocyanin and climatic data. The number and capital letters in the different panel are referring to the year (2017 and 2018, labeled as 17 and 18) and A or B to the harvest date, being one week apart.

**Figure 3.** Score and loading plots of PCA for 'Gariguette', and 'Sonata' grown at five locations (F: France; G: Germany; N: **Figure 3.** Score and loading plots of PCA for 'Gariguette', and 'Sonata' grown at five locations (F: France; G: Germany; N: Norway; P: Poland and I: Italy) characterized by total and individual anthocyanin and climatic data. The number and capital letters in the different panel are referring to the year (2017 and 2018, labeled as 17 and 18) and A or B to the harvest date, being one week apart.

Although the mean levels of total and individual anthocyanins in fruits of 'Gariguette' did not vary much between locations, as was the case for the other cultivars, the different locations were separated as well (see score plot in Figure 3). This was mainly due to the high level of cya-derivates in Poland in 2018 (Tables 3 and 6), and high amounts of pel-3-mal-glc in samples from France in both years, from Norway and Italy in 2017 and from Germany in 2018 (Table 7). Interestingly, total anthocyanin (Table 2) and its main component pel-3-glc (Table 4) did not contribute much to the separation of the locations because of contrasting year-on-year effects mainly in Norway, and even more pronounced, in Italy. In general, the anthocyanin profile of 'Gariguette' seemed to be less affected by the growing locations.

In the case of 'Sonata', total and individual anthocyanins did not vary much between the locations (Tables 2–7). Samples from France were clearly separated from the other locations (score plot in Figure 3), properly due to their relative low level of cya-derivates (Tables 3 and 6). Interestingly, Italian samples contained similar levels of these anthocyanins, but also less pel-3-glc (Table 4), and thus total anthocyanin (Table 2) in 2018, and this

distinguished these samples from those of France and partly from the other locations. Moreover, in 2018, Norwegian samples were enriched in pel-3-rut (Table 5), but contained, at the same time, low levels of pel-3-glc (Table 4), and total anthocyanin (Table 2) like samples from Italy. Both variables were of the factors responsible for the clustering of the Norwegian samples.

**Figure 4.** Score and loading plots of PCA for 'Candonga', and 'Florence' grown at four locations (F: France; G: Germany; N: Norway and P: Poland) characterized by total and individual anthocyanin and climatic data. The number and capital letters in the different panel are referring to the year (2017 and 2018, labeled as 17 and 18) and A or B to the harvest date, being one week apart.

Total anthocyanin and pel-3-glc were abundant in fruits of 'Candonga' grown in France but less enriched in those grown in Norway in 2018 (Tables 2 and 4). Thus, both compounds contribute to the separation of France from Norway, and both of these from the two other locations (Figure 4). Moreover, pel-3-rut (Table 5) was low in samples grown in Norway whereas samples grown in Poland were enriched in cya-3-glc (Table 3) and pel-3-mal-glc (Table 7) in 2018, and thus showing clear seasonal effects.

Fruits of 'Florence' grown in France were clearly separated from those grown in Norway (Figure 4). Moreover, PCA segregated both these locations from Germany and Poland. In both seasons, the level of pel-3-glc and anthocyanins were high in the French samples, whereas seasonal effects only enriched the levels of total anthocyanins in Poland and Germany, of pel-3-glc in Germany and of pel-3-mal-glc in all locations in 2018 (Tables 2, 4 and 7). In addition, fruits grown in Poland and Germany were enriched in cya-derivates in 2018, too (Tables 3 and 6).

In general, the PCA separated samples of all cultivars from France from those from the other locations. One reason might be the low values of global radiation for France in both years (Figure 1). However, more reasonable is the fact, that here, as in Italy, strawberries were cultivated in an open-sided tunnel covered with a standard plastic film that is well known to be non-transparent for UV radiation. Among others, flavonoid syntheses in plants is strongly induced by light and UV-B wavelength (280–315 nm). They are effective scavengers of reactive oxygen species (ROS) and absorb selectively UV radiation [17]. One of these flavonoids are anthocyanins being synthesized in higher amounts by excess UV-light. Previous studies reported a retarded coloring of the ripening fruit resulting in a decreased level of total anthocyanin in strawberries grown under UV opaque film compared to UV transparent film [26]. Moreover, no effect of UV radiation on total anthocyanin were observed for strawberry, raspberry and blueberry when grown under films varying from UV blocking to highly transparent [24]. In a study by Josuttis et al. [23], it was shown that the anthocyanin cya-3-glc decreased in strawberries when grown under a UV- blocking plastic film. Cya-3-glc is a minor anthocyanin in strawberry cultivars but abundant, for example, in red *Lettuce sativa* types [24,33]. Additionally, enhanced levels of the derivate cya-3-galactoside were found in the skin of apple [34,35] when exposed to low night temperature and light including UV wavelength. For peach, a different genetic background-dependent cya-3-glc level was detected in two peach cultivars after postharvest treatments with UVA or UVB light [36]. Accompanying transcriptomic studies identified different cultivar-specific expressed genes related to anthocyanin synthesis. In the current study, a cultivar-dependent reaction to UV exclusion was observed in the way that only fruits of 'Clery' and 'Sonata' showed a decreased cya-3-glc content under the UV-blocking tunnel production in France and Italy compared to those from open-field production in Norway, Poland and Germany.

The fact that the Italian samples of each cultivar were not clustered like the French samples but located between France and the other locations, may be due to the lower temperature during the harvest period (Table 1) in Italy, because of latitude and altitude. Thus, temperature effects probably modified the UV-reducing tunnel effect in samples from Italy.

#### *3.4. Impact of Temperature and Global Radiation on Cultivar-Specific Anthocyanin Profiles*

As shown by the PC analysis, the synthesis and accumulation of total and individual anthocyanin were influenced by temperature (Tmean, Tmax and Tmin) and global radiation and its interactions, being altogether affected by the latitude of the growing location. Environmental factors changing with latitude are mainly photoperiod, quantity and spectral composition of the solar radiation [17], as well as air temperature being indirectly dependent on solar radiation. However, weather conditions may vary between seasons at site and during the harvest period. Consequently, linear regression analyses were assessed to better explain the dependency of the cultivar-specific anthocyanin synthesis and accumulation on Tmean, Tmax, Tmin and global radiation. Moreover, to evaluate the effective time span of these factors, they were summarized 3, 5, 10 and 15 days prior to harvest. Overall, the percentage of variations (Figure 5), explained by these environmental factors, were rather low with some exceptions (highest R<sup>2</sup> = 0.62), highlighting again, the genotype specificbased anthocyanin syntheses. For example 'Gariguette' and 'Clery', with the exception of cya-3-mal-glc, were not affected by the environmental factors tested. For 'Clery', this result supports previous studies showing also no or little environmental effects on anthocyanin accumulation in fruits from this cultivar, when grown at three locations from North to Central Europe [21], and at different altitude in Switzerland [31]. In contrast, 'Florence', 'Frida' and 'Sonata' reacted most sensitively, but differently, to the pre-harvest temperature and global radiation conditions. For these cultivars, in general, (Tmax) had a higher impact on the syntheses of the less abundant anthocyanins cya-3-glc, pel-3-rut, cya-3-mal-glc, pel-3-mal-glc and on the total anthocyanin, than the minimum temperature. Thereby, the influence of Tmax often seemed to be stronger than the related Tmean itself (for example, for the cya-derivates in 'Florence' and 'Frida'). Only the synthesis of pel-3-glc, the main anthocyanin of strawberry, was slightly more influenced by Tmin than by Tmax. In the current study, global radiation was positive correlated with cyl-3-glc and pel-3-rut only in fruits of 'Frida'.

Noticeable is the contrasting behavior of 'Candonga' shown as the only cultivar with a negative relationship between global radiation and the main anthocyanin pel-3-glc (R <sup>2</sup> = 0.38 − 0.44), and thus also to the related total anthocyanin, and between cya-3-mal-glc and Tmin. 'Candonga' was bred and selected for tunnel production in Spain, which is the common production system for that area. Therefore, it is assumed that protection against UV-radiation was not an important growing factor for this genotype. In a Spanish study performed in tunnels, no correlation was found for 'Candonga' between total anthocyanin and Tmean, Tmax and solar radiation and, in contrast to this study, a positive relationship to Tmin [14]. In the same study, however, other cultivars reacted also on Tmean or Tmax.

Noteworthy also is the positive relationship between global radiation and the content of cya-derivates in the Scandinavian cultivar 'Frida'. Moreover, 'Frida', together with 'Florence'—bred in the United Kingdom—were both sensitive to Tmax with high R<sup>2</sup> values for cya-derivates and pel-3-rut, and in the case of 'Frida', also of pel-3-mal-glc. Thus, both cultivars showed a high adaption to their breeding locations to benefit best from Tmax in areas with, in general, lower temperature, and in case of 'Frida', also from the latitudinal long photoperiod in Norway.

Numerous studies evaluated the effect of temperature on the anthocyanin content of strawberry. In general, the content of total anthocyanin was enhanced with increasing temperature when plants were grown under controlled conditions [9,19,20]). However, the major anthocyanin pel-3-glc was less affected than the minor abundant pel-3-malglc [19]. Moreover, increased night temperature induced higher anthocyanin levels [9]. A contrasting result was obtained in a study with Japanese cultivars where high growing temperature (30/15 ◦C day/night) decreased the anthocyanin content compared to the control (20/15 ◦C), due to differently expressed genes involved in the anthocyanin synthesis [37].

There are only few studies evaluating latitudinal or altitudinal effects on the anthocyanin content of strawberry. In general, although pre-harvest weather conditions modified the data, fruits grown at higher latitudes had less anthocyanins than the southern ones [13,22,38]. In addition, quantitative changes in the anthocyanin profiles were found [20], giving northern fruits a higher percentage of minor anthocyanins like cya-3-glc and cya-3-mal-glc. These latitudinal depending results confirm our findings. In a study comparing different altitudes (differences ~600 m) with increased temperature but lower radiation in the period 10 days before harvest at the higher altitude, no influence on total anthocyanin and pel-3-glc were found, but cultivar specifically increased levels of cya-3-gluc and minor pel-derivates [31]. In contrast, Guerrero-Chavez et al. [39] found a decrease of total anthocyanin, mainly pel-3-glc and an unnamed pel-derivate, with increasing altitude (~600 m) in fruits of 'Elsanta'. Furthermore, in a recent study, temperature, UV radiation and sunshine duration were found to affect bioactive compounds of strawberry stronger than locations differing nearly by 800 m altitude. However, anthocyanin was the compound class that showed significant differences between locations in one cultivar only [32].

In this experiment, 'Florence' and 'Frida' showed a high adaptation to their breeding place, and in case of 'Candonga', to the tunnel cultivation technique used during selection. High adaption ability is well known for wild species. For example, wild populations of different *Vaccinium* species, grown from South to North Scandinavia, showed significant variations in the anthocyanin profile and total anthocyanin content giving northern populations a higher anthocyanin content in their berries [40–42]. It was explained by the

long photoperiod at northern sites and its intense radiation of UV, visible and far-red wavelength [42], but also by its lower mean temperature or by the interaction of both. The adaption was under strong genetic control and remained when cloned plants of the Nordic populations were grown in South Scandinavia [42].

**Figure 5.** Coefficient of determinations (R<sup>2</sup> ) between cya-3-glc, pel-3-glc, pel-3-rut, cya-3-mal-glc, ple-3-mal-glc and total **Figure 5.** Coefficient of determinations (R<sup>2</sup> ) between cya-3-glc, pel-3-glc, pel-3-rut, cya-3-mal-glc, ple-3-mal-glc and total anthocyanin (shown top to bottom) and the environmental factors' mean temperature (Tmean), maximum temperature (Tmax), minimum temperature (Tmin) and global radiation accumulated for 3, 5, 10 and 15 days (3d, 5d, 10d and 15d) before sampling of the berries to be analyzed for each cultivar 'Candonga' (Can), 'Clery' (Cle), 'Florence' (Flo), 'Frida' (Fri), 'Gariguette' (Gar) and 'Sonata' (Son), shown left to right. Cultivars with no data (blank) indicate the observed correlation between different anthocyanin components, and mean, maximum, minimum temperatures and global radiation were statistically insignificant.

−

The pigmentation of strawberry occurs relatively rapid at the end of the fruit development. Metabolomic studies indicate the first appearance of anthocyanins with rapidly increasing amounts until ripeness around 5–10 days after the fruit's white stage [38,43–45]. These previous studies have focused on fruit developmental stages and total anthocyanins only [38,44], or cya-hexose, pel-hexose and pel-rutinose [45]. The current study considered different time intervals up to 15 days prior to harvest and took into account not only the total anthocyanin but all individual anthocyanins evaluated. As expected, pel-3-glc were cultivar-specific, and presented only the last five days before harvest. However, in our study, it was surprising to find environmental effects occurring at an earlier fruit developmental stage affecting the different anthocyanins. For example, in 'Candonga' fruits, global radiation inhibited its synthesis up to 15 days pre-harvest. Furthermore, 'Candonga' was the exclusive cultivar where pel-3-rut, cya-3-mal-glc and pel-3-glc were not found for Tmax at d10 and d15 before harvest whereas they were partly present in 'Florence', 'Frida' and 'Sonata'. The synthesis of the other individual anthocyanins was affected by temperature and less by global radiation when taking the whole period into account. It is assumed that at this early stage, photosynthesis was enhanced by favorable temperature and radiation conditions and, in that way, precursors of the anthocyanins like carbon skeletons were accumulated. When evaluating primary and secondary metabolites during strawberry development [45], a decrease of diverse sugars was found over time, while anthocyanins increased at the late fruit developmental stages.

Herein, the impact of location on anthocyanin profiles of certain cultivars was systematically studied. The obtained results showed that the present work could serve as a useful starting point towards evaluating the latitudinal effects on plant performance and internal fruit quality. However, some further specific studies are required to validate this. In addition, certain optimizations are still required for accounting the seasonal variations in the global radiation and temperatures. These issues will be addressed in detail in our near future research work.

#### **4. Conclusions**

Our study indicated that the anthocyanin content of strawberry cultivars are, beside the well-known genetic origin, partly affected at site by the local environmental factors, namely temperature, global radiation and cultivation technique. While 'Clery' and 'Gariguette' displayed a very high stability in their anthocyanin content regardless of the growing location, the other cultivars partly reacted on the local conditions of the growing sites. Thus, a high cultivar x environment interaction was observed for the evaluated cultivars. Tmax, Tmin and global radiation, relatively less affected pel-3-glc. Cya-3-glc, cya-3-mal-glc, pel-3-rut, pel-3-mal-glc were found to be highly sensitive to Tmax. In addition, global radiation strongly increased cya-3-mal-glc and pel-3-rut in case of 'Frida', while in case of 'Candonga', the abundance of pel-3-glc decreased with global radiation. The anthocyanin profiles of 'Gariguette' and 'Clery' were unaffected by environmental conditions.

The minor strawberry anthocyanins cya-3-glc, pel-3-rut, cya-3-mal-glc and pel-3-malglc seemed to be cultivar-specific more sensitive to such environmental variations than the abundant pel-3-glc. Thereby, the minor anthocyanins might be useful to breed cultivars which are able to accumulate anthocyanins even under sub-optimal conditions, for instance, like 'Frida' which produced high content of anthocyanins in Norway being sensitive to Tmax and global radiation. In contrast, cultivars with high stability in their anthocyanin content like 'Clery' and 'Gariguette' may be valuable parents as well in breeding programs focusing on the challenge of increased temperature due to climate change and on weather instability between and within harvest periods. Thus, a better understanding of the cultivar x environmental interactions will be necessary. However, the cultivar-specific relationship between fruit anthocyanin content and the evaluated environmental factors in our study were rather low, indicating that other factors not considered were involved.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2076-341 7/11/3/1326/s1, Table S1: Sum of mean, maximum and minimum temperature as well as sum of global radiation, 3, 5, 10, and 15 days prior to sampling of fruits for analyses of 6 cultivars from North to South of Europe, Figure S1: PCA score plots for (a) the 6 cultivars (Clery (Cle), Candonga (Can), Frida (Fri), Florence (Flo), Gariguette (Gar) and Sonata (Son)) in 4 locations and (b) the 4 cultivars (Clery (Cle), Frida (Fri), Gariguette (Gar) and Sonata (Son)) in 5 locations clearly indicate that the location has major impact than the genetic variation. The '17' and '18' indicate years 2017 and 2018, respectively. The 'A' and 'B' indicate the two picking dates. In order to have better understanding, it is essential to analyze each cultivar separately.

**Author Contributions:** Conceptualization, E.K.; data curation, K.K.; investigation, E.K., F.W., P.C., A.M., D.M., A.P., G.S., K.C. and A.S.; writing—original draft, E.K., F.W. and K.K.; writing—review and editing, P.C., A.M., D.M., A.P., G.S., K.C. and A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by European Union's Horizon 2020 research and innovation programme, grant number 679303.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank all farm workers and laboratory stuff for skillful field management, sample collection and analyses, respectively.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


## *Article* **Comparative Effects of Organic and Conventional Cropping Systems on Trace Elements Contents in Vegetable Brassicaceae: Risk Assessment**

**Fernando Cámara-Martos <sup>1</sup> , Jesús Sevillano-Morales <sup>1</sup> , Luis Rubio-Pedraza <sup>2</sup> , Jesús Bonilla-Herrera <sup>2</sup> and Antonio de Haro-Bailón 3,\***


**Abstract:** Genotypes selected from 3 plant species (*Brassica rapa, Eruca vesicaria* and *Sinapis alba*) belonging to the *Brassicaceae* family were chosen to compare the concentrations of 9 inorganic elements (Cd, Co, Cr, Cu, Fe, Ni, Mn, Pb and Zn) in these varieties, that were grown under both conventional and organic conditions during two agricultural seasons (2018/2019 and 2019/2020) on two different experimental farms (Farm I and Farm II). We found that, together with agriculture practices, the inorganic element concentrations in Brassicas depended on many other factors, including soil characteristics. However, there were no conclusive results indicating a lower heavy metal content or a higher nutritionally beneficial trace elements content in vegetables grown under organic agriculture. Finally, a probabilistic assessment (@Risk) derived from the consumption of 150–200 g of these vegetables showed that organic Brassicas fulfill in comparison with the conventional ones, similar Dietary Reference Intakes (DRI) percentages for Co, Cr, Cu, Fe, Mn and Zn. Regarding heavy metals (Cd, Ni and Pb), we only found slight differences (mainly in the case of Pb) in the Tolerable Intakes (TI) between both cropping systems.

**Keywords:** organic farming; conventional farming; trace elements; heavy metals; risk assessment

#### **1. Introduction**

Nowadays, the maintenance of a good health status via appropriate dietary habits has become of great social concern, but we also have to bear in mind what has been called the "health trilemma", which tells us that food, health and the environment are closely linked, should also be borne in mind, in order to establish a balance between them for a healthier life on a more sustainable planet.

Currently, the consumption of plant-based foods with nutraceutical properties is one of the crucial factors for the welfare and promotion of health, preventing various pathologies like cancer, cardiovascular and neurodegenerative diseases [1,2]. Vegetable species belonging to the *Brassicaceae* (formerly *Cruciferae*) family are considered as being one of the first cultivated and domesticated plant groups and is appreciated for constituting a good source of minerals and trace elements [3] and for their health-promoting phytochemicals such as glucosinolates [4]. Plant species from this family include nutritionally important human and animal foodstuffs such as broccoli, turnip, cabbage, cauliflower, rapeseed, mustard, rocket, and is one of the ten most economically important plant families in the world [5,6].

**Citation:** Cámara-Martos, F.; Sevillano-Morales, J.; Rubio-Pedraza, L.; Bonilla-Herrera, J.; de Haro-Bailón, A. Comparative Effects of Organic and Conventional Cropping Systems on Trace Elements Contents in Vegetable Brassicaceae: Risk Assessment. *Appl. Sci.* **2021**, *11*, 707. https://doi.org/10.3390/app11020707

Received: 21 December 2020 Accepted: 10 January 2021 Published: 13 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In addition, in recent years, developed countries have been showing a greater interest in organic agriculture, with an increase of around 250% in the last 10 years [7]. This type of agriculture is based on the non-use of synthetic fertilizers or pesticides, and, instead, fertilization of the land with composted material, rich in organic matter, derived from the biodegradation of plant and animal sources [8].

Some authors have pointed out that organic foods contain higher concentrations of nutritionally beneficial trace elements and lower concentrations of harmful heavy metals [9]. In fact, vegetables can uptake and retain these inorganic elements from the surrounding environment through their roots and leaves [10]. However, the data existing in the bibliography on this topic are inconclusive, and it is difficult to make a valid comparison between both vegetable groups due to the limited availability of well-controlled or paired studies [11].

Therefore, for all the above reasons, the objectives of this research were (a) to compare the concentrations of nine inorganic elements (Co, Cr, Cu, Cd, Fe, Ni, Mn, Pb and Zn), well known both for their nutritional and toxicologic role in 3 species of *Brassicaceae (Brassica rapa, Eruca vesicaria* and *Sinapis alba*) grown under both conventional and organic conditions during two agricultural seasons (2018/2019 and 2019/2020) on two different experimental farms; (b) to make a probabilistic estimation with computer software (@Risk) of the contributions of the inorganic elements present in these vegetables to the recommended intakes or to the toxicologic limits established for them. This was to find out whether organicallygrown *Brassicas* have a greater nutritional value than the conventionally-grown ones.

The novelty of this work lies in the fact that few long-term studies have been made comparing vegetables grown under organic and conventional conditions during two agriculture seasons.

#### **2. Material and Methods**

#### *2.1. Plant Material*

Genotypes selected from 3 plant species belonging to the *Brassicaceae* family were chosen based on previous studies showing their differences in their glucosinolates profile and trace elements concentration: *Brassica rapa* L. (turnip greens and top greens), *Eruca vesicaria* L. (rocket) and *Sinapis alba* L. (white mustard). Turnip greens are the young leaves harvested in the vegetative growth period, and turnip tops are the fructiferous stems with flower buds and the surrounding leaves that are consumed before opening and while still green. These plant species are well adapted to Mediterranean environmental conditions and have been obtained by the Plant Breeding Group at the Institute for Sustainable Agriculture (IAS-CSIC) after several generations of breeding for seed yield and glucosinolate content.

This material was sown and cultivated during two seasons, 2018–2019 and 2019–2020, in two Farms (I and II) in Southern Spain (see Figure 1).

The Farm I land (37◦51′ N, 4◦48′ W) is located in Córdoba, next to the Guadalquivir River, in a position of the first terrace (altitude of 106 m), with a deep soil (Typic Xerofluvent) of sandy-loam texture with high pH (around 8), intermediate organic matter content (1.6%), and high carbonate content (17%). The experimental plot size for conventional cultivation of *Brassicaceae* species on Farm I was 25 × 25 m. The climate is typically continental Mediterranean (Csa in Köppen's climate classification), with relatively cold winters, intensely hot dry summers and mean annual precipitations of 650 mm. On this Farm, the three Brassicaceae species were only grown under conventional conditions with herbicides and mineral fertilization being applied. In pre-sowing, an herbicide with triflularin as its active matter was used at a dose of 1.5 L/ha. Moreover, before sowing, a basic dressing with 8-15-15 bottom fertilizer was applied at a rate of 600 kg/ha. A top dressing (cover fertilization) with 300 kg/ha of Ammonium Nitrate was applied after the winter stop at the resumption of vegetative growth.

**Figure 1.** Two Farms in the Southern Spain (A: Farm I. Alameda del Obispo (Córdoba). *Brassicaceae* grown in Conventional Cropping System; B: Farm II. Ribera Alta. Alcalá la Real (Jaén). *Brassicaceae* grown in Conventional and Organic Cropping System. The distance between both Farms is 121 km.

Farm II is located in the municipal district of Alcalá la Real (Jaén) (37◦27′ N 3◦55′ W, Spain) in the Sub-Baetic zone, next to the Velillos River (altitude 920 m) with a moderately stony structure and clay loam texture (Xerofluvent-Fluvisol calcareous) with high pH (8.2), high organic matter content (3%), and high carbonate content (16%). The experimental plot size for conventional and ecological cultivation of *Brassicaceae* species in Farm II were 25 × 25 m each. Both experimental plots were close together and separated only by a 2-mwide border. The climate is typically continental Mediterranean (Csa in Köppen's climate classification), with short summers, very hot, arid and mostly cloudless, winters are long, very cold and partially cloudy and mean annual precipitations of 650 mm. In this Farm, the three *Brassicaceae* species were grown both under conventional and organic conditions.

′ ′

The conventional cultivation conditions on Farm II were similar to those of the Farm I. In organic cultivation, neither herbicides nor mineral fertilizers were applied. Instead, only treatment with a mixture of goat and sheep manure was applied at a rate of 3 kg/m<sup>2</sup> .

When plants from the different species reached their optimal moment of consumption (from 3 to 5 months after sowing), leaf samples from individual plants of each species were harvested, pooled, and processed for chemical analysis. The number of analyzed plants throughout the two-years duration of the study were turnip greens (*Brassica rapa*) (n = 60), turnip tops (*Brassica rapa*) (n = 85), *Eruca vesicaria* (n = 18), and *Sinapis alba* (n = 12). The higher number of harvested *Brassica rapa* samples is due to turnip greens and turnip tops having good commercial prospects, and their consumption, both fresh and processed, has considerably increased in the last years. Furthermore, *Sinapis alba* and *Eruca vesicaria* are currently minority crops consumed only in salads, although their consumption may increase in the future due to their special composition in glucosinolates with medicinal properties (Sinalbine in *Sinapis alba* and Glucorafanine in *Eruca vesicaria*).

Plants were thoroughly washed with tap water to remove dirt and dust, and they were finally rinsed with deionized water. Then, they were stored at −80 ◦C until freezedrying, which was done in Telstar® model Cryodos-50 equipment (Telstar, Terrasa, Spain). The freeze-dried samples were ground in a Janke and Kunkel Model A10 mill (IKA-Labortechnik, Staufen, Germany) for about 20 s, and stored in a desiccator until their analysis.

#### *2.2. Materials and Reagents*

All the reagents were of an analytical-reagent grade. Ultrapure water (18 MΩ/SCF) prepared with a Milli-Q Reference Water Purification (Millipore, Madrid, Spain) was used throughout the experiments. All the glassware and plastic containers were soaked in 50% nitric acid overnight, then in 20% hydrochloric acid for an additional night and rinsed three times with de-ionized water prior to use. Hyperpure nitric acid (65%) and hydrochloric acid (35%) were obtained from Panreac (Barcelona, Spain). Hydrogen peroxide (33%) was acquired from Sigma Aldrich (St. Loius, MO, USA).

Standard solutions for measuring the elements Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn were prepared immediately before use by dilution with distilled deionized water of 1000 mg/L standard solutions (Certipur-Merck, Darmstad, Germany).

#### *2.3. Trace Element Determination*

To determine the trace element content (Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn) of *Brassicaceae* species, 0.5 g of freeze-dried sample was weighed in a porcelain crucible. Samples were incinerated in a muffle furnace at 460 ◦C for 15 h. The ash was bleached after cooling by adding 200 µL of hyperpure HNO<sup>3</sup> and 1 mL of deionized water, drying this on thermostatic hotplates, and maintaining it in a muffle furnace at 460 ◦C for 1 h more. Ash recovery was performed with 100 µL of hyperpure HNO3, making up to 10 mL with deionized water. To Cd analysis, in order to avoid Cd volatilization, 0.5 g of freeze-dried sample was placed in a Teflon vessel. Then, 3 mL of of hyperpure HNO<sup>3</sup> and 2 mL of H2O<sup>2</sup> were added to each vessel and kept for 10 min at room temperature. After sealing the vessels hermetically, they were placed in a microwave oven (Multiwave GO, Anton Paar, Germany) and digested following the instrumental parameters indicated by the manufacturer. Every sample was diluted up to a volume of 20 mL with ultrapure water

Elemental analyses for Fe (λ = 248.3 nm; Slit width = 0.2 nm), Mn (λ = 279.5 nm; Slit width = 0.2 nm) and Zn (λ = 213.9 nm; Slit width = 0.7 nm) were performed by flame absorption atomic spectroscopy (FAAS) with a Varian Spectra AA-50B model, equipped with standard air-acetylene flame, and single- element hollow cathode lamps. Finally, electrothermal atomic absorption spectroscopy (ET-AAS) was used for the determination of Cd, Co, Cr, Cu, Ni and Pb (Agilent Technologies model 240Z AA with a graphite furnace and autosampler). This equipment was certified by an equipment qualification report (EQR; Agilent Technologies). In this latter, analytical methodology was developed following the instrumental parameters indicated by the manufacturer with slight modifications (Table 1). For Cd and Pb analysis, a chemical modifier (200 mL solution) was prepared containing a mixture of 0.1% Paladium matrix modifier 10 g/L (Merck, Spain) plus 0.06% Magnesium nitrate hexahydrate in 10 mL HNO<sup>3</sup> hyperpure solution (69%). For each measurement, 15 µL of sample and 5 µL of modifier solution were injected. The accuracy and precision of the different analytical techniques used in determining trace element concentrations were validated by recovery experiments using Certified Reference Materials (Table 2).


**Table 1.** Instrumental conditions for Cd, Co, Cr, Ni and Pb analysis by ET-AAS in *Brassicaceae* samples.

**Table 2.** Analysis of certified references materials (mean ± standard deviation), limit of detection and limit of quantification.


\* Indicative value.

#### *2.4. Statistical Analyses and Risk Assessment*

The IBM SPSS 25 statistical software package was used for statistical analysis. The data were expressed as mean and standard deviation. Data were analyzed using ANOVA tests. Significant differences were considered when *p* < 0.05.

A probabilistic model was developed to estimate the intake level for Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn derived from feeding with Brassica vegetables. It should be pointed out that in developing the model, only the concentrations of trace elements for the *Brassica rapa* species were considered as it is the one most consumed in Spain, Portugal and Southern Italy, being part of very traditional recipes. Nowadays, turnip greens and turnip tops have good commercial prospects, and their consumption, both fresh and processed, has considerably increased in the last years. *Eruca vesicaria* and *Sinapis alba* have only eaten in small amounts in some salads, and they have a slightly spicy flavor like mustard greens.

This model followed a probabilistic approach in which variables were described by probability distributions, and they were fitted to concentration data obtained in this study for each element. Additionally, to estimate the intake level, serving size was considered assuming 15–20 g per day (around 150–200 g of fresh matter) of *Brassica rapa* (turnip greens or turnip tops). Daily intake was defined by a uniform distribution in the probabilistic model, meaning that all values in that range had the same probability to occur.

The probability distributions describing the trace element concentration data were fitted using @Risk v7.5 (Palisade, Newfield, NY, USA). The simulation ran with 10,000 iterations per element. The goodness of fit assessed how well the fitted distribution described the data; in this section, the Akaike Information Criterion (AIC) and Chi-square statistical tests were used. Additionally, the visual analysis was considered to assess the fit of the probability distributions to intake data. Data obtained through this probabilistic model was

compared to the Spanish DRI for adult population [12]. In the case of Cd, Ni and Pb [13–15] considering that they are heavy metals, tolerable intakes (µg/day) were considered.

#### **3. Results and Discussion**

*3.1. Trace Element Contents in Brassicaceae Species: Conventional Versus Organic*

The concentrations of Cu, Mn, and Zn for organic turnip greens (*Brassica rapa*) grown on Farm II were 7.4; 100.4 and 24.4 µg/g d.w (see Table 3). The concentrations of these trace elements in ones conventionally grown on the same farm were practically the same, with values of 7.5; 110.8 and 22.9 µg/g d.w. Conversely, there were statistically significant differences for Zn (*p* < 0.05) and Cu, Mn (*p* < 0.01) between the turnip greens grown on Farm I (conventional system) and those grown on Farm II (both conventional and organic systems). The highest concentrations of Cu and Zn (11.3; 38.9 µg/g d.w) and the lowest one Mn (56.0 µg/g d.w) found in turnip greens grown on Farm I compared to those analyzed on Farm II, demonstrate that, together with farming systems, the trace element concentrations in foodstuffs depend on many other factors, including soil characteristics, pollution from anthropogenic sources, genetic factors, seasonal influences and interactions between the elements [16].

**Table 3.** Total trace elements concentration (dry matter) in *Brassicaceae* species analyzed (mean ± standard deviation).


The Cu, Mn and Zn in turnip tops grown under the three experimental conditions were Farm I, conventional, 8.1; 24.2 and 29.9 µg/g d.w. respectively; Farm II, conventional 4.6; 25.5 and 25.1 µg/g d.w.; Farm II, organic, 9.0; 24.8 and 31.2 µg/g d.w. The concentrations in the turnip tops of the three trace elements were lower than those analyzed in the corresponding turnip greens. This could be explained by considering that turnip greens are the vegetative *Brassica rapa* leaves, whereas the turnip tops are the fructiferous stems with flower buds and the surrounding leaves. Previous studies have indicated that metals tend to accumulate preferentially in roots rather than in storage organs or fruits [17,18]. Moreover, unlike what happens to the turnip greens, statistically significant differences were found for Cu (*p* < 0.05) and Zn (*p* < 0.01) between the conventional and organic turnip tops grown on Farm II, the highest concentrations being found in the organic ones. These results are in agreement with those reported by Kelly and Bateman [9] for similar studies made with other vegetables species (tomatoes and lettuces)

Cu, Mn and Zn concentrations in *Sinapis alba* were 6.0; 17.8 and 23.8 µg/g d.w for the organic cropping system and 5.6; 18.7 and 25.9 µg/g d.w for the conventional one. Similarly, concentrations for these trace elements in *Eruca vesicaria* were 10.2; 48.1 and 33.0 µg/g d.w for the organic system and 9.3; 33.2 and 34.2 µg/g d.w for the conventional one. Zn

concentrations found in these *Brasicaceae* species are in agreement with those reported by Cámara–Martos et al. [3] in a previous study (*Sinapis alba* 20.8 µg/g d.w; *Eruca vesicaria* 23.5 µg/g d.w). However, our work again failed to find statistically significant differences in the concentrations of these trace elements between organic and conventional agriculture.

The results appearing in the bibliography do not show any clear trend for these trace elements either. Krejcova et al. [19], in conventionally-grown carrots, have shown a higher content of Mn and Cu but a lower content of Zn than in organic ones. Hadayat et al. [18], for organic lettuce, potato and carrot reported higher Cu contents than those in the same conventional vegetables. However, for conventional lettuce and carrot, higher Zn concentrations than those in the organic ones, were also found.

On the other hand, although in our study no differences were found in Mn concentrations between conventionally-grown *Brassicas* and organic ones, some authors have demonstrated that a lower mean concentration of Mn in organic crops is a common pattern. This could be due to the high concentrations of *arbuscular mycorrhizal fungi* in organic soils [9,20]. Although, this aspect has not been completely clarified. Other studies have shown that Mn is used as an additive to livestock feed supplements and, in turn, that this trace element would be present in the manure used in organic farming [9].

Co concentrations in turnip greens (*Brassica rapa*) were very similar in the three studied conditions (Farm I conventional 0.23 µg/g d.w; Farm II conventional 0.28 µg/g d.w and Farm II organic 0.22 µg/g d.w, with no significant differences between them (Table 3). Regarding Cr contents in turnip greens, in organic plants harvested on Farm II, lower concentrations (1.20 µg/g d.w) (*p <* 0.01) than in conventional ones of Farm I (2.41 µg/g d.w) and Farm II (2.06 µg/g d.w) were found. These results are in agreement with those reported by Krejcova et al. [19], who also found higher Cr concentration in conventional carrots (0.059 µg/g) than in organic carrots (0.046 µg/g). We have presumed that the main chemical form in which Cr is found in *Brassicaceae* vegetables, would be Cr (III). This chemical form is considered as being a beneficial element for human health, and according to several previous research works [21,22], it is the main chemical form (unlike Cr(VI) in which Cr is found in waters and foods. Therefore, according to their Co and Cr content, organic turnip greens would not have a higher nutritional value than conventional grown ones.

For the same reason as that already mentioned for the previous trace elements, Co and Cr concentrations in turnip tops (*Brassica rapa*) also decreased with respect to the corresponding turnip greens (*Brassica rapa*) (Farm I, conventional, 0.14 and 0.87 µg/g d.w.); (Farm II, conventional, 0.16 and 0.52 µg/g d.w.); (Farm II, organic, 0.19 and 0.48 µg/g d.w.) Again we find differences in Cr concentrations between turnip tops grown on the two Farms but not between the cropping systems (conventional versus organic). Thus, there were significant statistical differences for Cr contents (*p* < 0.05) between conventional turnip tops grown on Farm I and conventional turnip tops grown on Farm II. We also found statistically significant differences for Cr contents (*p* < 0.01) between conventional turnip tops grown on Farm I and organic ones grown on Farm II. Therefore, this again indicates that the total inorganic element content in vegetables does not only depend on the farming system [16].

*Sinapis alba* showed higher Cr concentrations (*p* < 0.01) for conventionally produced vegetables (0.51 µg/g d.w.) than organically ones (0.26 µg/g d.w.). Co contents for this *Brassicaceae* species were below the quantification limit (LOQ < 0.07 µg/g). On the other hand, Co and Cr contents in *Eruca vesicaria* were Farm I, conventional, 0.39 and 7.17 µg/g d.w; Farm II, conventional, 0.26 and 3.29 µg/g d.w, and, Farm II, organic 0.44 and 5.17 µg/g d.w. Cr contents in *Eruca vesicaria* are slightly higher with those reported in a previous study (2.59 µg/g) [3]. In addition, there were no statistically significant differences for both elements between organic and conventional *Eruca vesicaria*. Previous studies have reported that there is no clear trend in this matter. Thus, Hadayat et al. [18] gave higher Co contents in conventional potato, onion, tomato and carrot versus organic ones, whereas Cr contents were higher for organic onion, carrot and potato.

Regarding Fe, we found statistically significant differences (*p* < 0.01) between organic turnip greens (*Brassica rapa*) (0.10 mg/g d.w.) and conventional ones (0.19 µg/g d.w.) from Farm II; and between organic *Sinapis alba* (0.07 mg/g d.w.) and conventional one (0.15 mg/g d.w.) from Farm II (Table 3). Similarly, Krejcova et al. [19] have also reported higher Fe content for conventional carrots (5.24 µg/g) versus organic ones (4.96 µg/g). Nevertheless, for the rest of the vegetable *Brassicaceae studied*, the differences between conventional and organic cultivation were scant. In relation to this latter aspect, Kelly and Bateman [9] observed only minor variations in Fe concentrations between tomatoes and lettuces cultivated on organic and conventional farms.

While the above trace elements have a clear nutritional role, Pb and Cd are considered to be heavy metals that have harmful effects on the environment and human health. With respect to Ni, although the nutritional and/or toxicologic role of Ni in humans is unclear, in animal models, severe Ni deficiency can affect vision, Fe metabolism, and Na homeostasis [23]. However, high concentrations of this element can also affect vital processes in plants and induce toxic effects at morphologic, physiologic and biochemical levels [24].

For this latter element, we found statistically significant differences (*p* < 0.05) for Ni content in turnip tops (*Brassica rapa*) grown by organic agriculture (0.85 µg/g d.w.) on Farm II and turnip tops grown under conventional agriculture (1.02 µg/g d.w.) on the same Farm, with the lowest concentrations in organic ones (see Table 4). Similarly, Krejcova et al. [19] have also reported a lower Ni content in organic carrots (0.79 µg/g) than in conventional ones (1.58 µg/g). Other vegetables such as tomato and onion have also shown lower Ni contents when they are grown by organic systems. Conversely, other vegetable foodstuffs such as organic wheat (semolina samples) have shown higher Ni content than conventional wheat.


**Table 4.** Total heavy metals concentration (dry matter) in *Brassicaceae* species analyzed (mean ± standard deviation).

Ni values for conventional turnip tops grown on Farm I were (0.81 µg/g d.w.) and Farm II (1.02 µg/g d.w.) with statistically significant differences (*p* < 0.05) between them. Trace element content in organic *Sinapis alba* was below the quantification limit (LOQ < 0.18 µg/g) whereas Ni values for *Eruca vesicaria* ranged between 1.76–3.99 µg/g d.w without significant differences between conventional and organic system. These results are in agreement with those found in a previous study [3] for this latter *Brassica* specie (1.12 µg/g).

Regarding Pb, there were statistically significant differences (*p* < 0.05) between organic turnip greens (*Brassica rapa*) (0.33 µg/g d.w.) and conventional ones (0.62 µg/g d.w.) from Farm II (Table 4). Nevertheless, for the rest of the *Brassicaceae* studied, the differences between conventional and organic cultivation were scant. The influence of the soil in which the plants are grown has also been demonstrated. Thus, we found statistically significant differences (*p* < 0.01) for *Eruca vesicaria* between conventional plants grown on Farm I (3.40 µg/g d.w.) and plants grown on Farm II (0.55 µg/g d.w.). Furthermore, while for organic and conventional turnip tops grown on Farm II, Pb concentrations were below the quantification limit (LOQ < 0.160 µg/g), concentrations in conventional turnip tops grown on Farm I reached mean values of 0.90 µg/g d.w.

There are no conclusive results regarding a lower Pb content in vegetables grown through organic agriculture. Thus, Hadayat et al. [18] found lower Pb concentrations in organic tomato, lettuce, onion and carrot but not in potato. Krejcova et al. [19] reported higher Pb contents in conventional carrots (0.064 µg/g) versus organic ones (0.043 µg/g). However, Zaccone et al. [16] found higher contents of this heavy metal in organic wheat (94 µg/g) versus that in conventionally grown wheat (82 µg/g). Finally, Karavoltsos et al. [25] have indicated that, although the majority of organic vegetables may have lower Pb content, organic agriculture as such does not necessarily reduce the content of this heavy metal in organically cultivated products.

Another heavy metal whose consumption is aimed to reduce with the development of organic agriculture is Cd. Nevertheless, in the present study, we only found statistically significant differences for Cd levels (*p* < 0.01) between organic (0.07 µg/g d.w.) and conventional (0.23 µg/g d.w.) (Table 4) *Sinapis alba* grown on Farm II. For the rest of the *Brassicaceae* cultivated on Farm II, Cd values were very similar both in organic and conventional plants. Comparing our results with those in a previous study, Hadayat et al. (2018) reported lower Cd concentrations in organic tomato, onion, carrot and potato but not in lettuce. Cámara–Martos et al. [26] for infant foods, such as weaning jars, made with organic vegetable ingredients, it was also found that Cd concentrations were considerably lower than those reported in weaning formulas which were not categorized as organic. Krejcova et al. [19] showed slightly higher mean Cd concentrations in conventional carrots (0.066 µg/g) versus organic ones (0.060 µg/g), and Hoefkens et al. [11] indicated significant higher or lower concentrations and even non-significant differences in Cd concentrations, depending on the food matrix.

A factor that again influenced the Cd concentrations in *Brassicaceae* was the soil in which they were grown. Thus, we found statistically significant differences in Cd levels (*p* < 0.05) for conventional *Eruca vesicaria* grown on Farm I (0.72 µg/g d.w.) and those conventionally grown on Farm II (0.48 µg/g d.w.)

According to Karavoltsos et al. [25], organic agriculture could eventually lead to the production of foodstuffs with a lower heavy metal content, although organic agriculture as such is not able to secure low metal contents in its products. Our results show that this final content is also influenced by other factors such as soil, vegetable variety or even the presence of these elements in the air and in irrigation waters.

#### *3.2. Probabilistic Assessment: Conventional Versus Organic*

As already mentioned in the Material and methods section, a probabilistic model approach was developed to estimate the intake level of trace elements, which were derived from the consumption of 15–20 g (around 150–200 g of fresh matter) of these *Brassicas*. It should be pointed out that in developing the model, only the concentrations of trace elements for the *Brassica rapa* species (turnip greens and turnip top) were considered as they are the one most consumed.

Dietary reference intakes (DRI) for the Spanish population were considered [12]. There is not DRI for Co; however, this element represents approximately a 4.3% of vitamin B12. Considering a DRI for vitamin B12 between 2–2.4 µg, this corresponds to around 0.10 µg/day [27]. In the case of Ni [14] and Cd [13], considering that they are heavy metals,

the tolerable intake (TI) of 2.8 µg/kg body weight·day and 2.5 µg/kg body weight·week (0.36 µg/kg body weight·day) was used. For Pb a benchmark dose (BMDL01) for cardiovascular effects (1.50 µg/kg body weight·day) was considered [15]. It should also be noted that the present statistical tool was completed using the variability of inorganic elements present in plants as well as the variability of the *Brassica* vegetable ingested. Both aspects determine the total intake of the inorganic elements.

Thus, the results obtained from the simulation of the probabilistic model with conventional *Brassicas,* indicated values for Co, Cr, Cu, Fe, Mn and Zn of 2.58 µg, 17.41 µg, 0.14 mg, 1.74 mg, 0.55 mg and 0.56 mg, respectively, for 50th percentile (Figure 2). This shows that the intake of trace elements of at least half of the population consuming these conventional *Brassicas* will not be lower than these values. That intake fulfills Co DRI and complies with the following percentages of DRI for studied elements: Cr 69.6%, Cu 12.7%, Fe: 9.7%, Mn 30.6% and Zn 8.0%. When the same simulation of the probabilistic model is developed with concentrations belonging to organic *Brassicas*, the values obtained for 50th percentile were 3.61 µg, 9.60 µg, 0.12 mg, 1.20 mg, 0.65 mg and 0.49 mg for Co, Cr, Cu, Fe, Mn and Zn, respectively (Figure 3), which satisfy similarly to the conventional *Brassicas* the DRI percentages for these trace elements.

Regarding heavy metals, we have considered intakes for 95th percentile as being the most unfavorable situation. Thus, Ni, Pb and Cd intakes for 95th percentile with conventional *Brassicas* were 39.00, 47.34 and 6.82 µg, respectively (Figure 4), whereas intakes with organic *Brassicas* were 25.70, 15.55 and 4.56 µg for Ni, Pb and Cd, respectively (Figure 5). According to these values, organic *Brassicas* led to a decrease in the intake of these three elements metals. However, when these results were expressed as percentages of TI for these elements, we only found slight differences (mainly in the case of Pb) between both agriculture systems. The TI percentages for a mean body weight of 70 kg per person were 19.9, 45.1 and 27.1% for Ni, Pb and Cd, respectively, with conventional *Brassicas,* and 13.1, 14.8 and 18.1 % for organic *Brassicas*. These results indicate that Cd, Ni and Pb contents in vegetable *Brassica rapa* harvested under both conventional and organic farming conditions are below the accepted safety limits and do not represent any toxicologic risk.

**Figure 2.** Simulated data and fitted probabilistic distribution for Co, Cr, Cu, Fe, Mn and Zn in conventional *Brassicas*.

**Figure 3.** Simulated data and fitted probabilistic distribution for Co, Cr, Cu, Fe, Mn and Zn in organic *Brassicas*.

**Figure 4.** Simulated data and fitted probabilistic distribution for Cd, Ni and Pb in conventional Brassicas.

**Figure 5.** Simulated data and fitted probabilistic distribution for Cd, Ni and Pb in organic Brassicas.

**Author Contributions:** F.C.-M., conceptualization, methodology, validation, formal analysis, investigation, resources, writing, visualization, supervision, project administration, funding acquisition; J.S.-M., formal analysis; L.R.-P., investigation, resources, supervision; J.B.-H., resources, supervision; A.d.H.-B., conceptualization, methodology, investigation, resources, writing, visualization, supervision, project administration. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Project "Desarrollo de Alimentos Funcionales y Nutracéuticos Con Efectos Antioxidantes, Antitumorales y Cardiosaludables a partir de una Selección de Crucíferas Mediterráneas" Ref. UCO–1261749-R of the University of Cordoba-Regional Council for the Economic and Knowledge of Junta de Andalucía, which was co-financed by the European Regional Development Fund (ERDF 2014–2020).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Acknowledgments:** The authors thank Diana Badder, English speaker, for linguistic review of the manuscript.

**Conflicts of Interest:** Declarations of interest from all the authors: none.

#### **References**


*Review*
