**Contents**





### **About the Editor**

#### **Stefania Lamponi**

Stefania Lamponi is a Researcher and Assistant Professor of General and Inorganic Chemistry at the Department of Biotechnology, Chemistry and Pharmacy (Department of Excellence 2018-2022) of University of Siena (Italy). Moreover, she is a co-founder of the academic spin-off company SienabioACTIVE of the University of Siena. She graduated in Biological Sciences and has a PhD in Biomaterials.

Her main research interests concern the extraction, chemical characterization and evaluation of *in vitro* bioactivity, both in cellular and acellular systems, of bioactive substances from plants; the study of nanocarriers for the intracellular release of natural antioxidants and the *in vitro* analysis of cytotoxicity and genotoxicity of new drugs. Furthermore, a significant part of her research activity focuses on the *in vitro* evaluation of the biocompatibility of medical devices, new materials, modified surfaces, nanomaterials and hydrogels for biomedical applications.

### **Preface to "Structural and Functional Analysis of Extracts in Plants"**

Today there is a growing interest in the study of classes of compounds obtained from plant species due to their specific activity, allowing their application for many selected purposes in humans. The role of their natural active principles is closely related to their chemical structure as the type and number of different classes of molecules in plant extracts influence their bioactivity and consequently their use. Moreover, agronomical and processing conditions, extraction techniques and solvents may influence the chemical profile of the herbal preparations and their pharmacological activities. For these reasons, chemical analysis should always be performed in order to correlate the type and amount of phytochemicals present with extract bioactivity so as to better select its specific applications.

> **Stefania Lamponi** *Editor*

### *Editorial* **The importance of Structural and Functional Analysis of Extracts in Plants**

**Stefania Lamponi**

Department of Biotechnologies, Chemistry and Pharmacy and SienabioACTIVE s.r.l., University of Siena, Via Aldo Moro 2, 53100 Siena, Italy; stefania.lamponi@unisi.it; Tel.: +39-0577-234386; Fax: +39-0577-234254

#### **1. Introduction**

Plants and their extracts have traditionally been used against various pathologies and in some regions are the only therapeutic source for the treatment and prevention of many chronic diseases [1]. Their numerous beneficial effects on human health are due to their content of natural bioactive compounds, molecules capable of modulating metabolic processes with positive properties such as antioxidant effects, inhibition of receptor activities and enzymes and induction of gene expression [2].

Many factors, such as agronomical and processing conditions used in plant cultivation, extraction methods and solvents, may influence the chemical profile of the herbal preparations and, consequently, their pharmacological activities [3–5]. For these reasons, chemical analysis should be always performed on each plant extract in order to correlate the type and the amount of phytochemicals present with its bioactivity so as to better select its specific applications [6].

Although numerous advances have been made in recent years regarding the structural and functional analysis of extracts in plants, this topic deserves more attention from the academic community; therefore, we have edited two Special Issues that include numerous research articles and reviews reflecting the advancements made thus far in this field.

#### **2. Plant Extracts' Phytochemicals and Their Bioactivity**

The health benefits of plant extracts mainly depend on their secondary metabolites, i.e., substances produced by plants that make them competitive in their own environment [7]. Secondary metabolites vary widely in chemical structure (types of functional groups, number and position with respect to the carbon skeleton, substitution in the aromatic ring, stereochemistry, side chain length, saturation, etc.) [8] and the most extensively studied are those with antioxidant properties that protect cellular systems from oxidative damage through a variety of mechanisms able to reduce the risk of chronic diseases such as cancer and cardiovascular disease [9].

The most important classes of secondary metabolites in plants and in their extracts are alkaloids, phytoestrogens, carotenoids, tocopherols, terpenes and phenolics.

Alkaloids are plant-derived compounds containing one or more nitrogen atoms, usually in a heterocyclic ring (amine functional group). They derive from amino acids as well as proteins, from which they differ in being alkaline [7]. Alkaloids demonstrate a large spectrum of activities and among them, there are compounds showing antibacterial, antiviral, anti-inflammatory and anticancer properties. For example, *Dicentra spectabilis*, *Corydalis lutea*, *Mahonia aquifolia*, *Fumaria officinalis*, *Meconopsis cambrica* and *Macleaya cordata* plant extracts are cytotoxic against human squamous carcinoma and adenocarcinoma cells and the extracts obtained from the stem bark of *Rutidea parviflora* against ovarian cancer [10]. The bisbenzylisoquinoline alkaloid, named curine, is able to modulate inflammatory effects in mice, by inhibiting macrophage activation, production of cytokines and neutrophil recruitment, and decreasing nitric oxide levels [11]. The antibacterial activity of alkaloids has been described for nigritanine, an alkaloid obtained from *Strychnos nigritana* belonging

**Citation:** Lamponi, S. The importance of Structural and Functional Analysis of Extracts in Plants. *Plants* **2021**, *10*, 1225. https:// doi.org/10.3390/plants10061225

Received: 11 June 2021 Accepted: 15 June 2021 Published: 16 June 2021

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

**Copyright:** © 2021 by the author. 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/).

to the family of Loganiaceae, against *Staphylococcus aureus*, one of the most important pathogenic bacteria diffused worldwide [12]. Moreover, extracts from *Lepidium meyenii*, a plant in the Brassicaceae family rich in alkaloids, shows a strong antioxidant effect, higher than that of phenols [13]. Many alkaloids act on the nervous system. For example, poppy is narcotic, caffeine and nicotine are stimulants, while cocaine is an anesthetic, and scopolamine induces "twilight sleep". Codeine is frequently used in medical practice to suppress severe coughing [7].

Phytoestrogens are polyphenolic and non-steroid compounds that have a similar structure and biological activity as human estrogens. They are divided into two main subgroups, isoflavonoids and lignans. Isoflavones are divided into isoflavones and cumestanes, and the most representative compound of this second subgroup is coumestrol. Lignans include matairesinol, secoisolariciresinol, lariciresinol, pinoresinol and their metabolites, enterodiol, enterolactone and equol. Numerous studies reported in the literature have shown that phytoestrogens can have protective effects in estrogen-dependent diseases. This bioactivity is due to their structural and/or functional similarity to estradiol and to their capacity to bind the human estrogen receptors. Furthermore, the use of phytoestrogens can have a positive effect on insomnia and cognitive function in neuronal pathologies such as Alzheimer's disease. Phytoestrogens also exhibit antioxidant activity by acting as scavengers of free radicals or forming chelating complexes with metal ions [14,15].

Carotenoids are natural pigments and one of the main classes of phytochemicals in plants. They are derived from acyclic C<sup>40</sup> isoprenoid lycopene, which can be classified as a tetraterpene [16]. Carotenoids are divided into carotenes (i.e., α-carotene, β-carotene, lycopene) and xanthophylls, which represent the oxygenated carotenoids fraction (lutein, zeaxanthin and β-cryptoxanthin). The importance of carotenoids is correlated, over their role as precursors of vitamin A (α-carotene, β-carotene and β-cryptoxanthin), with numerous bioactivities. In fact, it has been shown that carotenoids have antioxidant and antitumor activity, regulate gene function and gap-junction communication and modulate the immune response [17–19]. Carotenoids may protect light-exposed tissues from photooxidative damage which could be involved in the pathobiochemistry of several diseases affecting the skin and the eye. Lutein and zeaxanthin are the predominant carotenoids of the retina and are considered to act as photoprotectants, preventing retinal degeneration. Moreover, β-carotene is also used as an oral sun protectant for the prevention of sunburn and has been shown to be effective either alone or in combination with other carotenoids or antioxidant vitamins [20].

Tocopherols, together with carotenoids, are the most abundant group of lipid-soluble antioxidants in chloroplasts [21]. α-, β-, δ- and γ-tocopherols and α-, β-, δ- and γtocotrienols are different forms of vitamin E and, among them, α-tocopherol is the most predominant and active form in human tissues. Tocopherols are antioxidants, free-radical scavengers and membrane stabilizers, protect thylakoid components from oxidative damage, are involved in electron transport reactions, in the prevention of light-induced pathologies of skin and eyes and in photophosphorylation and also have hypocholestemic health benefits [22–24]. Other bioactivities of tocopherols, not correlated with their antioxidant effects, are inhibition of platelet aggregation and monocyte adhesion and anti-proliferative and neuroprotective effects [25].

Terpenes are hydrocarbons based on combinations of dimethylallyl pyrophosphate and isoprenyl diphosphate/pyrophosphate, while terpenoids (also known as isoprenoids) are terpenes with an oxygen moiety and additional structural rearrangements. Terpenoids are classified on the basis of the number of carbon atoms present in their structure in hemiterpenoids (C5), monoterpenoids (C10), homoterpenoids (C11,16), sesquiterpenoids (C15), diterpenoids (C20), sesterpenoids (C25), triterpenoids (C30), tetraterpenoids (C40) and polyterpenoids (C>40, higher-order terpenoids) [26]. Recent studies have shown that many triterpenoids are effective and have pharmacological activities against cancer and other pathologies, such as cardiovascular diseases, diabetes and neurological disorders [27]. The pharmacological properties of triterpenoids in cancer prevention are attributed to multiple

mechanisms, including antioxidant, anti-inflammatory and cell cycle regulatory properties, as well as epigenetic/epigenomic regulation.

Phenolic compounds are a class of molecules whose basic structural feature is an aromatic ring of hydroxyl groups. They include flavonoids (i.e., flavonols, flavones, flavan-3-ols, anthocyanidins, flavanones, isoflavones, condensed tannins) and nonflavonoids (i.e., phenolic acids, hydroxycinnamates, stilbenes, hydrolyzable tannins) depending on the number and arrangement of their carbon atoms. These compounds have high antioxidant activity, a protective effect against chronic pathologies such as cancer, inflammatory diseases and bacterial disorders and favorable effects in reducing the risks of coronary heart disease [28]. Much epidemiological evidence also reports their ability to reduce diabetes and human neurodegenerative pathologies, such as Parkinson's and Alzheimer's diseases. Moreover, anti-analgesic, anti-allergic, cardioprotective and anti-diabetic activities have also been documented for food phenolics [28].

#### **3. Future Perspectives**

All the studies reported in the literature demonstrated that natural plant products are an abundant source of biologically active compounds, many of which can be considered as the basis for the development of new lead chemicals for pharmaceuticals. Although numerous advances have been made in recent years in this field, further research is needed in order to translate experimental in vitro results into clinical applications. In particular, studying the in vivo mechanisms, identifying epigenetic regulatory switches, finding new analogs and increasing the bioavailability of plants metabolites, could help to identify more effective compounds to prevent and/or to treat chronic diseases. For the selection of natural compounds intended to represent the next generation of therapies based on natural formulations, and to enable them to compete with modern drugs, it is necessary to investigate different aspects of the processes necessary to obtain them, such as extraction techniques, and evaluation of the quality and of the bioactivity of crude extracts and their combinations. Moreover, new and advanced techniques for their purification and efficient animal studies along with appropriate clinical trials are required for justified use of these plant extracts with safety and efficacy.

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

**Acknowledgments:** I would like to thank Abigail Yuan for the guidance and support throughout the entire process of these Special Issues. I also would like to thank the numerous reviewers and authors who contributed to this challenge with their science and expertise.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**


### *Article* **Chemical Profile, Antioxidant, Anti-Proliferative, Anticoagulant and Mutagenic Effects of a Hydroalcoholic Extract of Tuscan** *Rosmarinus officinalis*

**Stefania Lamponi 1,\* , Maria Camilla Baratto <sup>1</sup> , Elisabetta Miraldi <sup>2</sup> , Giulia Baini <sup>2</sup> and Marco Biagi <sup>2</sup>**


**Abstract:** This study aimed to characterize the chemical profile of an ethanolic extract of Tuscan *Rosmarinus officinalis* (*Ro*ex) and to determine its in vitro bioactivity. The content of phenolic and flavonoid compounds, hydroxycinnamic acids and triterpenoids was determined, and highperformance liquid chromatography-diode array detection (HPLC-DAD) analysis revealed that rosmarinic acid and other hydroxycinnamic derivatives were the main constituents of the extract. *Ro*ex demonstrated to have both antioxidant activity and the capability to scavenge hydrogen peroxide in a concentration dependent manner. Moreover, NIH3T3 mouse fibroblasts and human breast adenocarcinoma cells MDA-MB-231 viability was influenced by the extract with an IC<sup>50</sup> of 2.4 × 10−<sup>1</sup> mg/mL and 4.8 × 10−<sup>1</sup> mg/mL, respectively. The addition of *Ro*ex to the culture medium of both the above cell lines, resulted also in the reduction of cell death after H2O<sup>2</sup> pretreatment. The Ames test demonstrated that *Ro*ex was not genotoxic towards both TA98 and TA100 strains, with and without S9 metabolic activation. The extract, by inactivating thrombin, showed to also have an anti-coagulating effect at low concentration values. All these biological activities exerted by *Ro*ex are tightly correlated to its phytochemical profile, rich in bioactive compounds.

**Keywords:** antioxidant activity; antiproliferative activity; mutagenicity; anticoagulant activity; *Rosmarinus officinalis*; hydroalcoholic extract

#### **1. Introduction**

Plants have been widely used all over the world for their numerous properties throughout the millennia, and today there is a growing interest in the study of classes of compounds obtained from plant species due to their specific activity, allowing their application for many selected purposes in humans [1]. The role of their natural active principles in the human organism is closely related to their chemical structure (functional group types, number and position related to carbon skeleton, substitution in aromatic ring, stereochemistry, side chain length, saturation, etc.) as the type and number of different classes of molecules in plant extracts influence their bioactivity and consequently their use [2]. For example, terpenoids show antimicrobial, antiviral, antibacterial, anticancer, antimalarial, anti-inflammatory effects; phenolics acids have anticarcinogenic and antimutagenic, anti-inflammation and anti-allergic activities; alkaloids have antispasmodic, antimalarial, analgesic and diuretic activities; flavonoids possess antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal, activities, are cardiovascular and hepato-protective; saponins are antitumor, anti-inflammatory, immunostimulant, anti-hypoglycemic, antihepatotoxic and hepatoprotective, anticoagulant, neuroprotective, antioxidant; tannins are antioxidant, anti-carcinogenic, diuretics, hemostatic, anti-mutagenic, metal ionchelators, antiseptic [2]

**Citation:** Lamponi, S.; Baratto, M.C.; Miraldi, E.; Baini, G.; Biagi, M. Chemical Profile, Antioxidant, Anti-Proliferative, Anticoagulant and Mutagenic Effects of a Hydroalcoholic Extract of Tuscan *Rosmarinus officinalis*. *Plants* **2021**, *10*, 97. https://doi.org/10.3390/ plants10010097

Received: 14 December 2020 Accepted: 31 December 2020 Published: 6 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/).

*Rosmarinus officinalis* L. is an aromatic, evergreen plant belonging to the family Lamiaceae, native of Mediterranean regions, where it grows wild, but now widely distributed all over the world. The dark green, needle-like leaves of the plant are usually used as spice for flavoring in food cooking, but rosemary is cultivated not only for its aromatic properties but mainly for its antioxidant activity [3–6]. Rosemary, in the form of extract derived from the leaves, contains several compounds which have been demonstrated to have antioxidant effects [7]. These compounds belong mainly to the classes of phenolic acids, flavonoids, diterpenes and triterpenes [8].

Thanks to their chemical composition, rosemary extracts are used in food and in cosmetic industries for preventing food deterioration and protecting skin from free radical damage, respectively [9,10].

Butylated hydroxy anisole (BHA) and butylated hydroxy toluene (BHT) are the commonly used synthetic antioxidants added to foods to preserve the lipid components from quality deterioration, and to cosmetics to inhibit oxidation or reactions promoted by oxygen, peroxides or free radicals. Their synthetic quality, however, will not produce the same health benefits as natural antioxidants [11].

*R. officinalis*is also known to be employed in traditional and complementary alternative medicine in many countries, thanks to its broad range of beneficial health properties such as anti-inflammatory [4], anti-proliferative [12], antibacterial [13], antithrombotic [14], anticancer [15,16], hepatoprotective [17], antidiabetic [18], hypocolesterolemic [19] and antihypertensive [20].

It is well known that the antioxidant and biological properties of rosemary extracts are mainly due to their phytochemical composition which includes phenolcarboxylic acids (rosmarinic acid, caffeic acid, vanillic acid, quinic acid, syringic acid) as the major chemical constituents [21]. Other compounds present are: flavonoids [21,22], diterpenes, [21,23], and triterpenes [21]. Considering the co-presence of all the above classes of chemical compounds in rosemary extracts [6,24], their metabolization [25–27] and the bioavailability of their metabolites [28], the bioactivity of rosemary cannot be attributed to a single class of compounds but rather to the synergistic contribution of the various bioactive components and their metabolites (that is to its phytocomplex).

Moreover, agronomical and processing conditions, used in plant cultivation and extraction respectively, may influence the chemical profile of the herbal preparations and, consequently, their pharmacological activities [29–31]. For these reasons, chemical analysis should be always performed in order to correlate the type and amount of phytochemicals present with extract bioactivity so as to better select its specific applications.

This study aimed to analyze the chemical profile of an ethanolic extract obtained from an Italian rosemary collected in Tuscany and to determine its in vitro antioxidant activity, both in non-cellular and cellular systems, anti-proliferative effect towards NIH3T3 mouse fibroblasts and human breast adenocarcinoma cell line MDA-MB231, mutagenicity and interference with human blood coagulation factor, in order to correlate chemical profile of the extract with its wide range of in vitro bioactivity and to prevent unwanted effects when used in humans.

#### **2. Results**

#### *2.1. Chemical Analysis of Rosmarinus Officinalis Extract (Roex)*

Extraction Yield and Chemical Characterization of Tuscan *R. officinalis* Extract

The solvent utilized for the production of *Ro*ex was a hydro-alcoholic mixture with 60% *v*/*v* ethanol. As reported in Table 1, the amount of dry extract obtained from each gram of dried rosemary subjected to extraction was 112 mg, and 1 mL of suspension contained 24.3 mg of dry material corresponding to a percentage yield of 11.2%.

In order to quantify total polyphenols and total flavonoids, the extract was analyzed by means of Folin–Ciocalteu colorimetric assay and direct spectrophotometry, respectively. Total sulfuric-vanillin assay-reactive triterpenoids were also quantified. Rosmarinic acid content was evaluated by means of high-performance liquid chromatography-diode array

detection (HPLC-DAD) analysis, that was also used in order to identify other phenolic compounds in *Ro*ex.

**Table 1.** Extraction yield, total phenolic, flavonoids and triterpenoids content. Results are expressed as mean value ± standard deviation (SD) from three different preparations. β


The quantifications of total phenolic content, expressed as gallic acid equivalents, of total flavonoids expressed as hyperoside, and of total triterpenoids, expressed as βsitosterol, were reported in Table 1. The amount of each class of compounds was expressed as mg per gram of dry extract (d.e.). **β**

In order to identify its major components, the extract was analyzed by HPLC-DAD which recorded three main constituents at the following retention time (RT): 6.40 min, 6.95 min and 16.20 min. (Figure 1). By comparing RT and UV spectra of reference standards, it was possible to assign the peak at 16.20 min to rosmarinic acid (Figure 1), while the peak at 6.40 and 6.95 min were assigned to two other hydroxycinnamic derivatives, identified by characteristic UV spectra with λmax at 198–200, 288–298 and 322–332 nm (see Supplementary Materials). Other hydroxycinnamic derivatives identified in *Ro*ex were chlorogenic and caffeic acid, at RT = 7.80 and 10.00 min, respectively. The content of rosmarinic acid, chlorogenic acid, caffeic acid and total hydroxycinnamic derivatives (as the sum of rosmarinic, chlorogenic and caffeic acid and undefined hydroxycinnamic derivatives), expressed as rosmarinic acid, are reported in Table 2. Beside hydroxycinnamic acids, the chromatogram showed two main flavonoids at RT = 18.58 min and 20.55 min, identified by the characteristic UV profile of this class of metabolites with λmax at 194–200, the highest absorbance at 265–290 and a wide shoulder at 330–350 nm (see Supplementary Materials). These flavonoids could not be unambiguously identified, since RT and UV spectrum did not match any used reference standard. Nevertheless, by comparison with published literature on rosemary flavonoids [22,32] and polarity order of identified compounds, the main flavonoids in *Ro*ex could be likely related to luteolin glycosides. λ λ

**Figure 1.** HPLC-DAD analysis of *Ro*ex recorded at 280 nm. Rosmarinic acid is the phenolic compound of the extract with the highest content; other hydroxycinnamic derivatives are present in high concentration.


**Table 2.** Content of rosmarinic, chlorogenic and caffeic acid, and total hydroxycinnamic derivatives, expressed as mg of rosmarinic acid/g of d.e.

#### *2.2. Antiradical Activity: DPPH Assay and EPR Analysis*

The antioxidant behavior of the *Ro*ex was evaluated by its ability to scavenge the free DPPH radical by electron donation, by both spectrophotometric and EPR analysis. The results obtained by spectrophotometric measurements, demonstrated that the extract showed a great antiradical activity with the IC<sup>50</sup> value around 50 µg/mL.

By EPR analysis, as shown in Figure 2, the addition of the antioxidant to the DPPH radical solution determined a reduction in intensity of the signal with a scavenger percentage equal to 99%. *Ro*ex showed a gallic acid-equivalent antioxidant activity towards DPPH radical equal to 0.777 ± 0.013 (standard deviation).

ν **Figure 2.** Room temperature X-band spectra of (**a**) DPPH radical alone, (**b**) DPPH radical after the addition of *Rosmarinus officinalis* extract. *Experimental conditions:* The spectra were recorded at microwave frequency ν = 9.86 GHz, microwave power 2 mW and 0.1 mT modulation amplitude.

#### *2.3. Hydrogen Peroxide Scavenging Activity*

− − − − As shown in Table 3, the extract had the capability to scavenge hydrogen peroxide in a concentration dependent manner (0.024–0.96 mg/mL) with an EC<sup>50</sup> (half maximum effective concentration) of 0.48 mg/mL corresponding at a phenolic content of about 4.6 × 10−<sup>2</sup> mg/mL, hydroxycinnamic acids content of 2.2 × 10−<sup>2</sup> mg/mL, flavonoids 3.1 × 10−<sup>3</sup> mg/mL and triterpenoids 3.4 × 10−<sup>2</sup> mg/mL.


**Table 3.** Total phenolic content and percentage of scavenged hydrogen peroxide as a function of increasing concentrations of *Ro*ex.

#### *2.4. In Vitro Anti-Proliferative Activity*

Non-confluent adherent mouse fibroblasts NIH3T3 and human breast adenocarcinoma cells MDA-MB-231 were incubated with different concentrations of *Ro*ex diluted 1:5 with 60% ethanol. Cells were analyzed after 24 h of contact with the test samples and the results are reported in Figure 3. As shown, *Ro*ex exerted an anti-proliferative effect against both NIH3T3 and MDA-MB-231 cell lines in a concentration dependent manner, with lesser efficiency on breast adenocarcinoma cells compared to fibroblasts.

**Figure 3.** Percentage of viable NIH3T3 and MDA-MB-231 after 24 h of contact with increasing concentration of *Ro*ex dry extracts as determined by the Neutral Red Uptake. Date are mean ± SD of six replicates. \* Values are statistically different versus negative control (complete medium), *p* < 0.05. # Values are statistically differently versus percentage of viable MDA-MB-231, *p* < 0.05.

#### *2.5. Protective Effect against Hydrogen Peroxide Induced Oxidative Stress*

− Concentration-effect relationship for the cytotoxic action of H2O<sup>2</sup> towards NIH3T3 and MDA-MB-231 cells were obtained by testing concentration values of the hydrogen peroxide ranging from 0.082 to 1.60 mM. Cytotoxicity was determined as decreasing of cell viability after H2O<sup>2</sup> pre-treatment followed and not by contact with 7.20 × 10−<sup>3</sup> mg/mL *Ro*ex, the concentration value at which the extract showed the lowest degree of cytotoxicity for both cell lines.

As shown in Figure 4a, *Ro*ex was able to increase significantly NIH3T3 cell viability after pre-treatment with H2O<sup>2</sup> concentrations ranging from 0.33 and 1.3 mM but not at highest values, i.e., 1.5 and 1.6 mM.

**Figure 4.** Influence of hydrogen peroxide pre-treatment on viability of (**a**) NIH3T3 and (**b**) MDA-MB-231. \* Values are statistically different versus H2O<sup>2</sup> treated cells, *p* < 0.05.

The addition of *Ro*ex increased the percentage of viable MDA-MB-231 after pretreatment with hydrogen peroxide in a range of concentrations between 0.82 and 1.6 mM (Figure 4b). Human adenocarcinoma breast cells demonstrated to be more resistant to H2O<sup>2</sup> pre-treatment in comparison to NIH3T3 fibroblasts and the rosmarinic extract improved cell viability neutralizing the toxic effect of H2O<sup>2</sup> also at the highest concentrations, demonstrating to be able to protect cells and to reduce cell death.

#### *2.6. Mutagenicity Assay: Ames Test*

In *Salmonella* mutagenicity assay, six different concentrations of *Ro*ex were tested by Ames test on TA98, and TA100 strains with and without S9 metabolic activation. The results for the mutagenic effect of *R*oex reported in Table 4 demonstrated that all the concentrations tested were not genotoxic towards both TA98 and TA100 with and without S9 fraction. In fact, also at the highest concentration (24 mg/mL), the number of revertants was lower and statistically different in comparison to positive control (*p <* 0.01). The background level, as well as positive control values, were in all cases within the normal limit found in our laboratory and in accordance with literature data [33].

**Table 4.** Mean number of revertants in *S. typhimurium* strains 98 and 100, exposed to different concentrations of *Ro*ex with and without S9 fraction. The results are reported as the mean of revertants ± SD.


#### *2.7. Antithrombotic Activity: Thrombin Time (TT)*

By looking at the TT values reported in Table 5, it was possible to note that all the five *Ro*ex tested concentrations significantly increased clotting time in comparison to the control, demonstrating the extract to have anti-coagulating effect also at the lowest concentration value.

**Table 5.** Thrombin Time of different concentration of *Ro*ex. Data are mean ± SD of six replicates. (\*) Value statistical different versus control (human plasma diluted 1:1 with 60% *v*/*v* Et-OH solution).


#### **3. Discussion**

Herbal extracts bioactivity depends on their chemical profile and the choice of the solvent is crucial in extraction processes as it directly affects the chemical composition of the final extracts and the mass extraction yield. Among the alternative solvents available for plants extraction, hydro-alcoholic mixtures are good candidates since they are rather few selective and let to extract a wide range of compounds [34–37]. Considering global extraction yields of *Rosmarinus officinalis*, a ratio ranging from 50% to 80% ethanol gives highest mass extraction yields and total content in target compounds in comparison to lower Et-OH values. For this reason, the solvent that we utilized for the production of *Ro*ex was hydro-alcoholic mixture with 60% ethanol which yielded 112 mg of dry extract from

each gram of rosemary subjected to extraction, corresponding to a percentage yield of 11.2%. This lower percentage yield obtained in comparison to data reported by Jacotet-Navarro et al. [38] can be probably due to the environmental conditions as *Rosmarinus officinalis* is a sensitive plant to pedoclimatic variations that affect its chemical composition [30].

The *Ro*ex, showed a high total phenolic content, of about 95.8 mg/g dry extract, equivalent to 8.55 mg/g dry herbal material ca., in accordance with other extracts obtained from Mediterranean rosemary [21]. As previously reported for other rosemary [22,39], also in *Ro*ex the main phenolic subclass was identified in hydroxycinnamic acid derivatives, and rosmarinic acid was the main single compound, counting almost the half of total phenolic content. Still in accordance with other published papers, also in *Ro*ex total flavonoid content was low, about 6.5 mg/g. This analysis of the phenolic composition of the Italian rosemary extract, performed by means of HPLC-DAD technique, represents a good characterization of its phenolic fingerprint and demonstrated the range of molecules contributing to the definition of this matrix, and may assist in the study of its in vitro bioactive properties [8]. Surprisingly, we found also a high content in triterpenoids in *Ro*ex, 71.7 mg/g extract. Factors such as plant age, climate and stress conditions that inhibit or enhance the production of certain compounds might affect the chemical composition of the extracts [40]. These factors influence the metabolites qualitatively and quantitatively and may explain why certain compounds such as quercetin, rutin and other quercetin glycosides were not detectable in this study [40].

The good in vitro antioxidant activity in cell free assays showed by*Ro*ex, demonstrated both by its ability to scavenge the free DPPH radical via electron donation and by the capability to scavenge hydrogen peroxide in a concentration dependent manner (Figure 2), is very similar to others typical vegetal extracts with a good antiradical power [41], and is a consequence of its phytocompounds composition. Hydroxycinnamic acids, presents in high concentration in *Ro*ex, are potent antioxidants and, among them, rosmarinic acid and caffeic acid have a good dose-dependent DPPH scavenging activity and may contribute to the antiradical activity of the extract [42], together with rosmarinic acid. Moreover, also flavonoids and their metabolites are able to scavenge radicals and participate in antioxidant reactions as well as triterpenoids and total phenolic compounds [42].

A number of studies have demonstrated a good correlation of intrinsic antioxidant activity in a non-cellular assay with cytoprotection against an oxidant challenge in a cellular assay, demonstrating the ability of antioxidants to act intracellularly [43,44]. Cellular protection of *Ro*ex towards H2O<sup>2</sup> pre-treatment, was evaluated against both cancer and non-cancer cell line, adenocarcinoma breast cancer cells MDA-MB-231 and mouse fibroblasts NIH3T3, respectively (Figure 4). According to data reported in literature [45], *Ro*ex was able to increase significantly NIH3T3 cell viability after pre-treatment with H2O<sup>2</sup> concentrations ranging from 0.33 and 1.3 mM but not at highest values, i.e., 1.5 and 1.6 mM (Figure 4a). Human adenocarcinoma breast cells demonstrated to be more resistant to H2O<sup>2</sup> pre-treatment in comparison to NIH3T3 fibroblasts and the rosmarinic extract improved cell viability neutralizing the toxic effect of H2O<sup>2</sup> also at the highest concentrations (from 0.82 to 1.6 mM), demonstrating to be able to protect cells and to reduce cell death (Figure 4b). The importance of rosmarinic and hydrocynnamic derivative to protect cells in H2O2-induced cytotoxicity is well known. Rosmarinic acid exhibited substantial H2O<sup>2</sup> scavenging activity and inhibited H2O2-induced intracellular ROS production [44]. Caffeic acid was found to scavenge intracellular reactive oxygen species, and 1,1-diphenyl-2-picrylhydrazyl radical, and thus prevented lipid peroxidation. Chlorogenic acid protect cells against H2O2-induced oxidative stress and apoptosis [45]. Intracellular dose-dependent uptake of flavonoids has been demonstrated in various cell types in vitro and is believed to be even higher in vivo than under normal culture conditions [16].

In vitro antiproliferative activity of *Ro*ex towards NIH3T3 and MDA-MB-231, noncancer and cancer cells, respectively, was evaluated by NRU test. The NRU results showed that *Ro*ex exerted a weak anti-proliferative effect against both NIH3T3 and MDA-MB-231 cell lines in a concentration dependent manner, with lesser efficiency on breast

adenocarcinoma cells compared to fibroblasts (Figure 3). In particular, the concentration value of 2.4 × 10−<sup>1</sup> mg/mL reduced NIH3T3 viability by 50%, while the same effect with MDA-MB-231 was obtained at a *Ro*ex concentration of 4.8 × 10−<sup>1</sup> mg/mL, a higher value in comparison to data reported in literature where MDA-MB-231 cells proliferation is inhibited in a dose-dependent manner with an IC<sup>50</sup> of about 2.04 × 10−<sup>2</sup> mg/mL [46]. The antiproliferative effect of rosemary extracts towards cancer cells is influenced by their constituents and seems to be correlated with the presence of polyphenols, mainly caffeic and rosmarinic acid [47]. On the contrary, rosmarinic and caffeic acid demonstrated low cytotoxicity against non-cancer cells [48] and this could explain the weak antiproliferative activity of rosemary extract against non-cancer NIH3T3 cells.

A genotoxicity study is a key step for risk assessment during development of natural plant extracts for human applications because various genotoxic compounds can cause a DNA damage compromising human health [49].

In the bacterial reverse mutation assay (Ames test) performed on *Ro*ex, as expected, the positive control agents significantly induced genotoxicity but no genotoxic positive result was observed in *Ro*ex treated groups compared to control at any of the tested concentrations (Table 4). These results indicate that *Ro*ex and their phytochemical components, did not exhibit any genotoxic risk under the experimental conditions of this study. The preclinical evaluation of the antithrombotic potential of novel molecules requires the use of reliable reproducible experimental models. TT is one of the most commonly used tests to determine the efficacy of novel antithrombotic drugs. In our experiment, TT using pooled plasma from human healthy patients, was utilized to evaluate the anticoagulant effect of *Ro*ex. The results of TT assay (Table 5) showed that *Ro*ex prolonged coagulation times compared with the control sample, suggesting that the extract inhibited the activity of thrombin.

There is an evidence that coagulation and inflammation are related processes that may considerably affect each other [50]. On the basis of our current experimental studies, it can be hypothesized that inhibitory modulation of coagulation by extracts could give promising anti-inflammatory mediators. These anti-inflammatory and anticoagulant effects have been mostly attributed to the polyphenol and flavonoid compounds found in large quantities in these plants [51] as well as in our *Ro*ex. In the future, well-designed prospective studies are needed to prove this hypothesis.

#### **4. Materials and Methods**

#### *4.1. Materials*

Thrombin, Dulbecco's Modified Eagle's Medium (DMEM), trypsin solution, and all the solvents used for cell culture as well as Folin–Ciocalteu reactive, DPPH (2,2′ -diphenyl-1 picrylhydrazyl), gallic acid and all the other reagents were of analytical grade and purchased from Sigma-Aldrich (Milan, Italy). Mouse immortalized fibroblasts NIH3T3 and human breast adenocarcinoma cells MDA-MB-231 were from American Type Culture Collection (Manassaa, VA, USA). Ames test kit was supplied from Xenometrix (Allschwil, Switzerland).

#### *4.2. Rosmarinus officinalis Extract (Roex) Preparation*

*Rosmarinus officinalis* L. (Tuscan blue cultivar, Linnean Herbarium number LINN 41.1, authenticated at the Botanical Museum and Garden of University of Siena) fresh leaves were collected in spring (April) in Val d'Orcia (Tuscany, Italy, 42◦56′02′′ N 11◦38′17′′ E). The fresh leaves were washed three times in distilled water and left to dry at room temperature for 24 h. Then, the leaves were chopped with a scalpel. The extract was obtained by putting 20 g of chopped leaves in 80 g of 60% (*v*/*v*) ethanol (EtOH) for 48 h at room temperature in a shaker incubator. At the end of the incubation, the suspension was filtered by a 0.45 µm Whatman membrane filter and dried using a rotary evaporator. The dry extract obtained was weighted and the percentage yield was expressed as air-dried weight of plant material. Samples then were stored at 2–4 ◦C until it was time to conduct further analysis.

#### *4.3. Chemical Analysis*

#### 4.3.1. Determination of Total Phenolic and Flavonoid Content

Total polyphenols and flavonoids content of *Rosmarinus officinalis* ethanolic extract (*Ro*ex) was examined using spectrophotometric methods reported by Biagi et al., 2014 [52]. In detail, total polyphenols were determined by the colorimetric method of Folin-Ciocalteu. 0.01 mL of each extract were added to 2.990 mL of distilled water and 0.500 mL of Folin-Ciocalteu reagent 1:10 *v*/*v* in distilled water. After 30 s of shaking, 1.000 mL of Na2CO<sup>3</sup> 15% *m*/*m* in distilled water was added. After incubation at room temperature for 120 min, absorbance at 700 nm was read using a SAFAS UV-MC2 instrument (SAFAS, Monaco, Principality of Monaco). The polyphenols quantification was calculated by means of interpolation of calibration curve constructed using gallic acid.

Total flavonoids content of extracts was determined reading absorbance at 353 nm of 100 folds diluted extract according with Sosa et al. [53] and constructing calibration curve using hyperoside as standard.

#### 4.3.2. Determination of Total Triterpenes

A total of 10 µL of the sample solution was added to 190 µL of glacial acetic acid, after which 300 µL of a solution of glacial acetic acid with 5% *m*/*v* vanillin and, after mixing for 30 s, 1 mL of perchloric acid.

The mixture was heated to 60 ◦C for 45 min and, after cooling, the volume was brought to 5 mL with glacial acetic acid [54].

The absorbance was read at 548 nm and the quantification of the total triterpenes in the extract calculated according to the calibration curve, constructed using β-sitosterol.

#### 4.3.3. High-Performance Liquid Chromatography Analysis of Phenolics Compounds

With the aim of further investigating the polyphenolic fraction of the extract, a highperformance liquid chromatography-diode array detection (HPLC-DAD) analysis was carried out.

A Shimadzu Prominence LC 2030 3D instrument equipped with a Bondapak® C18 column, 10 µm, 125 Å, 3.9 mm × 300 mm column (Waters Corporation, Milford, MA, USA) was used.

Water + 0.1% *v*/*v* formic acid (A) and acetonitrile + 0.1% *v*/*v* formic acid (B) were used as mobile phase. The following program was applied: B from 10% at 0 min to 25% at 20 min, then B 50% at 26 min.; flow was set at 0.8 mL/min. Chromatograms were recorded at 280 nm.

Analyses were performed using 10 µL of extract; rosmarinic acid, caffeic acid, chlorogenic acid, quercetin, apigenin, luteolin, rutin and hyperoside were used as external standard. Calibration curves were established using reference standards ranging from 0.008 mg/mL to 0.500 mg/mL. The correlation coefficient (R 2 ) of each curve was > 0.99.

#### *4.4. Antiradical Capacity: DPPH Assay*

Antiradical capacity of *Ro*ex was evaluated both by spectrophotometric method using ascorbic acid as reference, and by EPR analysis using gallic acid as reference.

#### 4.4.1. Spectrophotometric Assay

The antiradical capacity of *Ro*ex was tested by means of the validated DPPH (2,2-diphenyl-1-picrylhydrazyl) test. The DPPH solution was prepared in methanol at a concentration of 1 × 10−<sup>4</sup> M. *Ro*ex was tested in seven 1:2 serial dilutions (ethanol 60% *v*/*v*) and ascorbic acid, used as reference.

All the samples were mixed with the DPPH solution (1:19), transferred into 1 cm path length cuvettes and incubated for 30 min at room temperature in the dark. Ethanol 60% *v*/*v* and DPPH (1:19) was used as positive control. The inhibition of DPPH was calculated according to the following Formula (1) where Absc is the absorbance of the positive control

and Absx the absorbance of tested samples IC<sup>50</sup> was calculated by constructing the curve of inhibition values for each tested concentration (in the linear range 10–75%) [55].

$$\% \text{ inhibition} = (\text{Absc} - \text{Absc}) / \text{Absc} \times 100 \tag{1}$$

4.4.2. Scavenger Activity by Electron Paramagnetic Resonance (EPR) Analysis

Continuous-wave X-band (CW, 9 GHz) EPR spectra were recorded using a Bruker E580 ELEXSYS Series spectrometer (Bruker, Rheinstetten, Germany), with the ER4122SHQE cavity. EPR measurements were performed filling 1.0 mm ID × 1.2 mm OD quartz capillaries placing them into a 3.0 mm ID × 4.0 mm OD suprasil tube.

A stock solution of DPPH was prepared (0.2 mM in ethanol) and the final concentration of the radical in each sample was 0.16 mM. The extract was diluted a hundred times in respect to the stock solution and it was added with one fourth volume in respect to that of the radical. The addition was incubated for 15 min.

The area of the EPR spectra was calculated by the double integral of the DPPH signal and the scavenger percentage was calculated using the Formula (2) where A<sup>0</sup> is the area of the DPPH signal without the addition of the extract, Aextract is the area the DPPH signal after the addition of the extract.

The addition of the antioxidant to the DPPH radical solution determines a reduction in intensity of the EPR signal with a scavenger percentage equal to 99%.

$$\text{scacvenger }\%= (\text{A}\_0 - \text{A}\_{\text{extract}}) / \text{A}\_0 \times 100\tag{2}$$

4.4.3. Gallic Acid-Equivalent Antioxidant Activity through DPPH Assay

A stock solution of DPPH radical 1 mM in EtOH was freshly prepared and used within 5 h. A stock solution of gallic acid 0.2 mM in EtOH was prepared. The calibration curves were built using a standard solution of gallic acid. The gallic acid standard solution with different linear increasing volumes (10–100 µL) and a known volume (100 µL) of the extract was added to a fixed volume of the DPPH solution (100 µL). After 15 min of incubation in the dark at room temperature, the EPR spectra were recorded. The antioxidant activity was plotted through the decay percentage of the area of the DPPH signal versus increasing concentrations of gallic acid standard solution. The area of the EPR spectra was calculated through the double integral of the DPPH signal.

The decay percentage for the plotting refers to the Formula (3) where A<sup>0</sup> is the area of the DPPH signal without the addition of the antioxidant or extract, A<sup>S</sup> is the area the DPPH signal after the addition of scavenger agent such as antioxidant gallic acid or the extract.

The decay area percentage expressed in gallic acid equivalent was obtained through the calibration curve built with the standard solution (R<sup>2</sup> = 0.928) and reporting the area of the DPPH EPR signal after the addition of the extract. The extract was diluted a hundred times in respect to the stock solution. The measurements were repeated in triplicate and the antioxidant activity of the extract was expressed as mole/g of acid gallic equivalent.

$$\mathbf{decay}\% = (\mathbf{A}\_0 - \mathbf{A}\_\mathbf{S}) / \mathbf{A}\_0 \times 100 \tag{3}$$

#### *4.5. Hydrogen Peroxide Scavenging Assay*

The ability of *Ro*ex to scavenge hydrogen peroxide was estimated according the method of Ruch et al. [56]. A solution of H2O<sup>2</sup> (2 mM) was prepared in phosphate buffer (50 mM, pH = 7.4). Aliquots (0.05, 0.1, 0.2, 0.3 and 0.4 mL) of *Ro*ex at a concentration of 24 mg/mL, were transferred into test tubes and their volumes were made up to 0.4 mL with 50 mM phosphate buffer at pH = 7.4. After adding 0.6 mL of hydrogen peroxide solution, tubes were vortexed and the absorbance of H2O<sup>2</sup> at 230 nm was determined after 10 min of incubation, against a blank containing phosphate buffer and Et-OH 60% without H2O2. The percentage of hydrogen peroxide scavenging was calculated as follows:

$$\text{\textbullet\text{\textbullet\textbullet\textbullet}}\,\text{scavenged\text{\textbullet H}}\,\text{2}\,\text{O}\_2\text{\textbullet}=\left[\text{\textbullet\textbullet}-\text{A}\_{\text{l}}\right)\,\text{\textbullet\textbullet}\,\text{1}\,\text{0}\,\tag{4}$$

where A<sup>i</sup> is the absorbance of control and A<sup>t</sup> is the absorbance of test samples.

#### *4.6. Anti-Proliferative Assay and Protective Effect against Hydrogen Peroxide Induced Oxidative Stress*

#### 4.6.1. Cell Cultures and Anti-Proliferative Test

In order to evaluate the in vitro anti-proliferative activity of new products, the direct contact test was used [57]. This test is suitable for sample with various shapes, sizes or physical status (i.e., liquid or solid). The evaluation of in vitro inhibition of cell growth does not depend on the final use for which the product is intended, and the document ISO 10995-5:2009 recommends many cell lines from American Type Collection. Among them, to test *Ro*ex cytotoxicity, NIH3T3 mouse fibroblasts were chosen [58]. Moreover, in order to evaluate the anti-proliferative activity of *Ro*ex towards tumoral cells, the same test was repeated by using human breast adenocarcinoma cells MDA-MB 231.

Both NIH3T3 and MDA-MB231 cells were propagated in DMEM supplemented with 10% fetal calf serum, 1% L-glutamine-penicillin-streptomycin solution, and 1% MEM non-essential amino acid solution, and incubated at 37 ◦C in a humidified atmosphere containing 5% CO2. Once at confluence, the cells were washed with PBS 0.1M, separated with trypsin-EDTA solution and centrifuged at 1000 r.p.m. for 5 min. The pellet was re-suspended in complete medium (dilution 1:15). Cells (1.5 × 10<sup>4</sup> ) suspended in 1 mL of complete medium were seeded in each well of a 24 well round multidish and incubated at 37 ◦C in an atmosphere of 5% CO2. Once reached the 50% of confluence (i.e., after 24 h of culture), the culture medium was discharged and the test compounds, properly diluted in completed medium, were added to each well. All samples were set up in six replicates. Complete medium was used as negative control. After 24 h of incubation, cell viability was evaluated by neutral red uptake (NRU) assay [59].

#### 4.6.2. Protective Effect against Hydrogen Peroxide Induced Oxidative Stress

To determine the protective effect of alcoholic extract of *Ro*ex against oxidative stress, NIH3T3 and MDA-MB-231 cells were pre-incubated with different concentrations of hydrogen peroxide (0.1, 0.2, 0.3, 0.9, 1.0, 1.1, 1.3, 1.5, 1.6 µM) for 15 min, then washed and incubated for 24 h with 0.3% *v*/*v Ro*ex at a concentration of 7.2 × 10−<sup>3</sup> mg/mL in complete culture medium. Cell viability was evaluated after 24 h of incubation at 37 ◦C in 5% CO<sup>2</sup> by NRU assay.

#### 4.6.3. Evaluation of Cell Viability: NRU Assay

In order to determine the percentage of viable cells as follows, the following solutions were prepared:


At the end of incubation, the routine culture medium was removed from each plate and the cells were carefully rinsed with 1 mL pre-warmed D-PBS 0.1M. Plates were then gently blotted with paper towels. 1.0 mL NR medium was added to each dish and further incubated at 37 ◦C, 95% humidity, 5.0% CO<sup>2</sup> for 3 h. The cells were checked during incubation for NR crystal formation. After incubation, the NR medium was removed and the cells were carefully rinsed with 1 mL pre-warmed D-PBS 0.1M. PBS was decanted and blotted from the dishes and exactly 1 mL NR desorb solution was added to each sample. Plates were placed on a shaker for 20–45 min to extract NR from the cells and form a homogeneous solution. During this step the samples were covered to protect them from light. Five minutes after removal from the shaker, absorbance was read at 540 nm with a UV/visible spectrophotometer (Varian Cary 1E).

#### *4.7. Mutagenicity Assay: Ames Test*

The TA100 and TA98 strains of *Salmonella typhimurium* were utilized for mutagenicity assay in absence and presence of metabolic activation, i.e., with and without S9 liver fraction. The tester strains used were selected because they are sensitive and detect a large proportion of known bacterial mutagens and are most commonly used routinely within the pharmaceutical industry [60]. The following specific positive controls were used, respectively, with and without S9 fraction: 2-nitrofluorene (2-NF) 2 µg/mL + 4-nitroquinoline N-oxide (4-NQO) 0.1 µg/mL, and 2-aminoanthracene (2-AA) 5 µg/mL. The final concentration of S9 in the culture was 4.5%.

Approximately 10<sup>7</sup> bacteria were exposed to 6 concentrations of *Ro*ex, as well as to positive and negative controls, for 90 min in medium containing sufficient histidine to support approximately two cell divisions. After 90 min, the exposure cultures were diluted in pH indicator medium lacking histidine, and aliquoted into 48 wells of a 384 well plate. Within two days, cells which had undergone the reversion to His grew into colonies. Metabolism by the bacterial colonies reduced the pH of the medium, changing the color of that well. This color change can be detected visually. The number of wells containing revertant colonies were counted for each dose and compared to a zero-dose control. Each dose was tested in six replicates.

The material was regarded mutagenic if the number of histidine revertant colonies was twice or more than the spontaneous revertant colonies.

#### *4.8. Antithrombotic Activity: Thrombin Time (TT)*

The selected blood donors were normal, healthy men who had fasted for more than 8 h and had not received medication for at least 14 days. Blood samples were collected into 3.8% (m/V) tri-sodium citrate as anticoagulant at a volume ratio of 9 parts blood to 1-part citrate. The blood samples were then centrifuged at 3500 r.p.m. for 15 min to obtain PPP which was utilized to perform TT. In particular, 0.2 mL of each samples were added to 0.2 mL of a solution obtained by diluting 1:1 human plasma with a solution of 60% Et-OH (% *v*/*v*). TT was determined by incubating the aliquot (0.2 mL) of human plasma containing the sample at 37 ◦C for 2 min, after which 0.2 mL of thrombin (0.6 NIH) was added. The clotting time was revealed by an Automatic ElviDigiclot 2 Coagulimeter (from Logos SpA, Milan, Italy).

#### *4.9. Statistical Analysis*

All assays were carried out in six replicates and their results were expressed as mean ± standard deviation (SD). Multiple comparisons were performed by one-way ANOVA and individual differences tested by Fisher's test after the demonstration of significant intergroup differences by ANOVA. Differences with *p <* 0.05 were considered significant.

#### **5. Conclusions**

Taken together, the data obtained demonstrated that hydro-alcoholic extraction of Tuscan rosemary can be a good method for obtaining active compounds with high potential for application in many different fields, such as cosmetics, food or pharmaceutical research. The results of this study have demonstrated, indeed, that the *Ro*ex analyzed possessed good antioxidant and radical scavenging activities when tested in cellular and non-cellular assays, as well as a weak, anti-proliferative effects towards both cancer and noncancer cells, absence of genotoxic and ability to prolong thrombin time. All these bioactivities are tightly correlated to the *Ro*ex chemical profile in a dose dependent manner. In order to prevent unwanted effects when used in humans, it is essential that multiple aspects of the bioactivity of each *Rosmarinus officinalis* extract are tested.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2223-774 7/10/1/97/s1.

**Author Contributions:** S.L. obtained the extract, performed biological experiments, and data/evidence collection; wrote the manuscript; M.C.B. performed the chemical characterization and data/evidence collection, revised the manuscript; E.M. performed the chemical characterization and data/evidence collection, revised the manuscript; G.B. performed antiradical capacity DPPH assay and spectrophotometric assay and data/evidence collection; M.B. performed biological experiments, and data/evidence collection, revised the manuscript. All authors have read and agreed to the published version of the manuscript.

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

**Institutional Review Board Statement:** Ethical review and approval were waived for this study, due to the research involves no risk to subjects.

**Informed Consent Statement:** Patient consent was waived due to the research involves no risk to subjects.

**Data Availability Statement:** The data presented in this study are available in supplementary material.

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

#### **References**


### *Article* **Application of Deep Eutectic Solvents for the Extraction of Carnosic Acid and Carnosol from Sage (***Salvia officinalis* **L.) with Response Surface Methodology Optimization**

**Martina Jakovljevi´c <sup>1</sup> , Stela Joki´c <sup>1</sup> , Maja Molnar <sup>1</sup> and Igor Jerkovi´c 2,\***


**\*** Correspondence: igor@ktf-split.hr; Tel.: +385-21-329-434

**Abstract:** *Salvia officinalis* L. is a good source of antioxidant compounds such as phenolic diterpenes carnosic acid and carnosol. From 17 deep eutectic solvents (DESs) used, choline chloride: lactic acid (1:2 molar ratio) was found to be the most suitable for the extraction of targeted compounds. The influence of H2O content, extraction time, and temperature (for stirring and heating and for ultrasoundassisted extraction (UAE)), H2O content, extraction time, and vibration speed for mechanochemical extraction on the content of targeted compounds were investigated. Carnosic acid content obtained by the extraction assisted by stirring and heating was from 2.55 ± 0.04 to 14.43 ± 0.28 µg mg−<sup>1</sup> , for UAE it was from 1.62 ± 0.29 to 14.00 ± 0.02 µg mg−<sup>1</sup> , and for mechanochemical extraction the yield was from 1.80 ± 0.02 to 8.26 ± 0.45 µg mg−<sup>1</sup> . Determined carnosol content was in the range 0.81 ± 0.01 to 4.83 ± 0.09 µg mg−<sup>1</sup> for the extraction with stirring and for UAE it was from 0.56 ± 0.02 to 4.18 ± 0.05 µg mg−<sup>1</sup> , and for mechanochemical extraction the yield was from 0.57 ± 0.11 to 2.01 ± 0.16 µg mg−<sup>1</sup> . Optimal extraction conditions determined by response surface methodology (RSM) were in accordance with experimentally demonstrated values. In comparison with previously published or own results using conventional solvents or supercritical CO<sup>2</sup> , used DES provided more efficient extraction of both targeted compounds.

**Keywords:** sage; optimization; stirring and heating extraction; ultrasound-assisted extraction; mechanochemical extraction

#### **1. Introduction**

The growth of the pharmaceutical industry and increased need for bioactive components has led to the increased development of new extraction and isolation methods [1]. The most important differences between these methods are better efficiency and shorter extraction time for modern techniques compared to the conventional ones. Furthermore, conventional solvents are very often flammable and toxic with their manufacture depending on fossil resources [2]. However, there are also certain issues associated with the modern techniques, such as poor selectivity and solubility of targeted components in the solvents used, such as H2O, ethanol, or CO2, as well as recovery of bioactive components and their chemical changes during the extraction period due to the reactions such as ionization, hydrolysis, and oxidation [3,4]. Over the past few years, deep eutectic solvents (DESs), first proposed by Abbott et al. [5,6], have been developed as analogues of ionic liquids (ILs), although they differ from them in the starting material and the method of preparation. DESs are mixtures of hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD) with a lower melting point relative to the starting components. Their green character is also attributed to their low price, easy preparation, biodegradability, and low toxicity [7]. In addition to these properties, different studies reported that DES could dissolve several components better than organic solvents, due to dissolving lignocellulose which causes

**Citation:** Jakovljevi´c, M.; Joki´c, S.; Molnar, M.; Jerkovi´c, I. Application of Deep Eutectic Solvents for the Extraction of Carnosic Acid and Carnosol from Sage (*Salvia officinalis* L.) with Response Surface Methodology Optimization. *Plants* **2021**, *10*, 80. https://doi.org/ 10.3390/plants10010080

Received: 4 November 2020 Accepted: 24 December 2020 Published: 2 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/).

damage of plant cell wall and strengthens the mass transfer process [8]. DESs can be prepared from different combinations of the starting compounds, thus being tunable solvents with different functionality and solubility for various compounds. Therefore, a suitable combination of the starting solvents and their molar ratio can increase the solubility and the extraction efficiency of DESs for desired compounds [9]. However, their shortcomings should also be taken into account with an emphasis on viscosity and low vapor pressure which makes it difficult to isolate and purify desired components. In addition, high viscosity complicates industrial application due to the high energy consumption needed to ensure their liquid state [10].

One of the most commonly used HBA is choline chloride (ChCl), since it is an inexpensive, biodegradable, and non-toxic quaternary ammonium salt. ChCl can form DES with different nontoxic components as carboxylic acids, sugars, sugar alcohols, or amines which act as HBDs [5,6]. In the last few years, deep eutectic solvents are increasingly used for the extraction of phenolic compounds including phenolic acids, flavonoids, stilbenes, anthocyanins, and furanocoumarins [1,2,11–13]. In the research performed by Bi et al. [11] DESs were used to extract flavonoids such as myricetin and amentoflavone from *Chamaecyparis obtusa* (Siebold and Zucc.) Endl. leaves by alcohol-based DESs. Wang et al. [13] extracted polyphenols and furanocoumarins from fig (*Ficus carica* L.) with tailor-made DESs and showed that DESs were effective for the extraction of these components. Anthocyanins in the flower petals of *Catharanthus roseus* L. were extracted with natural deep eutectic solvents (NADESs) such as lactic acid—glucose and propane-1,2-diol—choline chloride, which provided higher stability for anthocyanins [12]. The extraction with DES can be improved by combination with ultrasound (UAE), microwaves and heating as well as by mechanochemical extraction (MCE). Therefore, Bosiljkov et al. [14] successfully extracted anthocyanins with UAE combined with DESs. Wang et al. [4] developed fast, efficient, and ecofriendly MCE for tanshinones as well as bioactive compounds from tea leaves [15]. Due to the desirability of DESs in the extraction of phenolic components, we decided to extract phenolic diterpenes, carnosic acid, and carnosol from sage (*Salvia officinalis* L.) applying DESs. These compounds have been suggested to account for over 90% of the sage antioxidant properties [16]. In addition to antioxidant activity, carnosic acid, and carnosol showed proapoptotic [17], antiproliferative [18], anti-angiogenic [19] and antitumor activity. So far, there are no available data on DESs extraction and optimization of the parameters for carnosic acid and carnosol from sage. There are few reports dealing with DESs extraction of sage by Bakirtzi et al. [2] and Georgantzi et al. [20] who investigated the influence of different DESs on the extraction of polyphenols from medicinal plants including sage. In paper by Bakirtzi et al. [2] lactic acid-based natural deep eutectic solvents in combination with ultrasound were used for the extraction of total polyphenols and flavonoids from sage, while Georgantzi et al. [20] investigated combination of lactic acid-based DES with cyclodextrin for UAE of total polyphenols and flavonoids.

Taking into account all the above mentioned, the objectives of this study were focused on (1) investigation on finding appropriate choline chloride based deep eutectic solvent for the extraction of carnosic acid and carnosol as well as (2) suitable extraction techniques (stirring with heating, UAE or MCE). Afterwards, the influence of various DES extraction parameters (H2O content, time, and temperature for stirring and heating and UAE; H2O content, extraction time and vibration speed for mechanochemical extraction) on the (3) content of carnosic acid and carnosol in sage extract analyzed by HPLC was investigated. In addition, (4) the optimal extraction conditions by RSM for desired antioxidant components (carnosic acid and carnosol) were determined.

#### **2. Results and Discussion**

#### *2.1. Influence of DESs on the Obtained Amount of Carnosic Acid and Carnosol in the Extracts*

Due to the different effects of viscosity, surface tension, polarity, and HBD interaction it is hard to estimate the suitability of a DES for the extraction of targeted compounds. Therefore, in order to select the best DES for the extraction of carnosic acid and carnosol

from sage, the extraction was performed with different solvents and different H2O addition at 30 ◦C (Figure A1). According to Dai et al. [12] and Bosiljkov et al. [14], H2O addition in organic acid-based DESs causes decrease of the solvent polarity since these solvents are more polar than H2O. Therefore, for targeted components it would be more suitable to lower H2O addition (which is consistent with the results obtained). As can be seen from Figure A1, the solvents substantially differ in their ability to extract carnosic acid and carnosol. In addition to the influence of HBD, the amount of H2O added also plays an important role for the extraction efficiency. For certain solvents, like choline chloride:malonic acid (1:1 molar ratio), the amount of carnosic acid was increased with increased H2O content that may be due to the viscosity lowering effect. For DES choline chloride:citric acid (1:1 molar ratio), the highest amount of carnosic acid and carnosol was obtained with 30% H2O addition that may be the consequence of a high viscosity of the solvent with 10% H2O addition. H2O amount was changed to reduce the viscosity which causes a slow mass transfer, thus affecting the extraction process. The viscosity of DESs can be reduced by the addition of a certain amount of H2O as well as by increasing the temperature [12]. Several solvents such as choline chloride:glucose (1:1 molar ratio) were too viscous even with the addition of H2O at 50% (*v*/*v*). Although the addition of H2O can decrease the viscosity, an excessive concentration of H2O can decrease the interactions between the components of DES as well the interactions between DES and desired components [11]. This is the reason why the focus was on H2O addition in the range of 10–50% (*v*/*v*).

Carnosol can be extracted with all DESs applied, but choline chloride based DESs with butane-1,4-diol (1:2 molar ratio) (ChClB) and ethane-1,4-diol (1:2 molar ratio) (ChClE) were the most effective. The result of extraction with choline chloride:glucose (1:1 molar ratio) with 10% H2O were not shown because of the high viscosity preventing further analysis. The same limitation was observed with choline chloride:fructose (1:1 molar ratio) (ChClF) and choline chloride:citric acid (1:1 molar ratio) (ChClC) at 10% of H2O and at 30 ◦C. The higher amount of carnosol was observed at lower H2O content (Figure A1). With choline chloride:urea (1:2) (ChClU) as the solvent, the highest amount of carnosol was extracted at 30 ◦C. According to the literature, higher content of carnosol is usually present in the extracts obtained at higher temperatures since it is one of the degradation products of carnosic acid [20].

On the other hand, not all applied solvents could extract carnosic acid. Basic solvents, such as ChCl:U, choline chloride:*N*-methlyurea (1:3 molar ratio) (ChClmU), and choline chloride:thiourea (1:2 molar ratio) (ChCltU), were not efficient for carnosic acid extraction. The highest amount of carnosic acid was extracted using acidic DESs, with emphasis on choline chloride:lactic acid (1:2 molar ratio) (ChClLa). Such solvents are significantly acidic (pH < 3) compared to the solvents where HBDs were sugars or alcohols (pH > 6) [7,12]. Additionally, the polarity of DESs should also be considered as an important criterion for the evaluation and selection of the solvents to achieve maximum extraction efficiency. Carnosic acid and carnosol are polar constituents, soluble in polar solvents (which is in agreement with the obtained results), such as DESs with organic acids as HBDs, which are more polar than the DESs with sugars as HBDs [12]. The highest amount of carnosic acid was obtained with the lowest amount of added H2O (10%) in most solvents. Okamura et al. [21] have investigated the effect of temperature on the degradation of carnosic acid in acetone solution and reported that the increase of temperature affected the degradation of carnosic acid. In case of the extraction with DESs such as ChClLa and choline chloride:levulinic acid (1:2 molar ratio) (ChClL) the maximum amount of carnosic acid was extracted at 30 ◦C.

Since carnosol can be extracted with all DESs applied and due to the highest amount of extracted carnosic acid in the case of choline chloride:lactic acid (1:2 molar ratio) and the lactic acid properties as natural component, this solvent was selected for further optimization of the extraction with three extraction methods (stirring and heating, UAE, and MCE).

#### *2.2. Comparison of the Used Extraction Methods*

After selection of the appropriate solvent, the extractions performed by stirring and heating and UAE, applying the same temperature and H2O content as well as MCE were compared. Both, stirring and heating and UAE increase mass transfer and speed up diffusion of the compounds. In the case of ultrasound, acoustic cavitation phenomenon leads to the disruption of cell walls and consequently improves the yield of extraction compared to maceration [22]. However, MCE can decrease the processing time and solvent consumption and reduce noise and radiation compared to UAE and to stirring and heating extraction.

Table 1 shows that slightly higher amounts of carnosol and carnosic acid were extracted by stirring and heating, compared to UAE. Such results can be explained by the positive influence of stirring on the mass transfer in such viscous solvent. For MCE, the utilization of the glass beads led to much better mixing of the plant material and solvent, thus extracting significant amounts of carnosic acid and carnosol in a shorter time compared to the other two extractions (Table 2).

**Table 1.** Experimental matrix and values (µg mg−<sup>1</sup> of the plant material) of observed response for the extraction with choline chloride:lactic acid (1:2 molar ratio) obtained by stirring with heating and by ultrasound-assisted extraction (UAE). The results are expressed as mean value ± standard deviation (*n* = 3).


For the constant H2O content in all extraction methods, the extracted amounts of carnosic acid and carnosol obtained by different extractions were compared. The extracted amounts of selected compounds (8.26 and 7.92 µg mg−<sup>1</sup> for carnosic acid and 1.87 and 2.02 µg mg−<sup>1</sup> for carnosol) obtained by MCE at Run 12 and 17 can be compared to Run 6 and 9 obtained with stirring and heating extraction and UAE. The difference between the parameters of these extractions was the extraction time, so at 10% H2O for MCE, 2 min were enough to obtain similar amounts of targeted compounds as for 30 min of stirring and heating extraction and UAE. In the case of 30% H2O by MCE, 3 min were sufficient to obtain the amount of extracted components similar to the amount extracted for 90 min by stirring and heating extraction and UAE. It is important to note that MCE was carried out at room temperature (24–28 ◦C). In fact, by using mill, we wanted to show how much time was needed for the extraction at room temperature, and prolonging the extraction time would result in warming of the samples without the possibility of heating control.


**Table 2.** Experimental matrix and values (µg mg−<sup>1</sup> of the plant material) of observed response for the extraction with choline chloride:lactic acid (1:2 molar ratio) obtained by mechanochemical extraction (MCE). The results are expressed as mean value ± standard deviation (*n* = 3).

#### *2.3. Influence of Various DES Extraction Parameters on the Content of Carnosol and Carnosic Acid*

The effect of H2O addition, temperature or vibration speed and extraction time on carnosol and carnosic acid was investigated for three extraction techniques using DES choline chloride:lactic acid (1:2 molar ratio). In these experiments, the content of carnosic acid in sage extract obtained by stirring and heating was 2.55–14.43 µg mg−<sup>1</sup> , depending on the applied extraction parameters. The lowest content of carnosic acid was obtained at 50% (*v*/*v*) H2O added at 30 ◦C and 60 min, while the highest content was obtained at 10% (*v*/*v*) H2O added at 50 ◦C and 90 min (Table 1). The content of carnosic acid obtained by UAE varied, depending on the parameters used, in the range 1.62–13.99 µg mg−<sup>1</sup> . The lowest content of carnosic acid was obtained at 30% (*v*/*v*) H2O added, 70 ◦C, and 30 min and the highest yield at 10% (*v*/*v*) H2O added, 70 ◦C and 60 min (Table 1). The content of carnosic acid obtained by MCE varied depending on the parameters used in the range 1.80–8.26 µg mg−<sup>1</sup> . The lowest content of carnosic acid was at 50% (*v*/*v*) H2O added, vibration speed of 1 m/s and 2 min and the highest yield at 10% (*v*/*v*) H2O added, 5 m/s and 2 min (Table 2). The content of carnosol obtained by mixing and heating was 0.81–4.83 µg mg−<sup>1</sup> depending on the applied extraction parameters. The lowest content of carnosol was obtained at 50% (*v*/*v*) H2O addition, 50 ◦C and 30 min, while the highest yield was obtained at 10% (*v*/*v*) of H2O, 50 ◦C and 90 min. The content of carnosol, depending on the parameters used in UAE, was 0.56–4.18 µg mg−<sup>1</sup> with the lowest content at 30% (*v*/*v*) H2O addition, 30 ◦C and 30 min and the highest yield at 10% H2O addition, 70 ◦C and 60 min (Table 1). The content of carnosol obtained by MCE was 0.57–2.02 µg mg−<sup>1</sup> depending on the applied extraction parameters (Table 2). The lowest content of carnosol was obtained at 50% of H2O (*v*/*v*), vibration speed of 1 m/s and time of 1 min, while the highest content was obtained at 30% of H2O (*v*/*v*), 5 m/s and 3 min.

The addition of H2O and extraction time (Figure 1 and Table A1) showed statistically significant influence on the content of carnosic acid (*p* < 0.0001; *p* = 0.0202) in the extracts obtained by stirring and mixing. The content of carnosic acid increased with prolonged extraction time and decreased with the increase of H2O amount.

−

−

−

**Figure 1.** Three-dimensional plots for obtained content of carnosic acid as a function of the extraction time, temperature, and H2O content for the extraction with mixing and heating (**a**,**b**), UAE (**c**,**d**) and for the extraction time, vibrational speed, and H2O content for MCE (**e**,**f**).

The interactions between amount of H2O added and extraction time (*p* = 0.0259) also showed a significant influence on the content of carnosic acid. In the extracts obtained by UAE, H2O addition and temperature showed statistically significant influence on the content of carnosic acid (*p* = 0.0025; *p* = 0.0144). For this extraction technique, interactions between the amount of added H2O and temperature (*p* = 0.0433) also showed a significant influence in terms of content of carnosic acid. The content of carnosic acid increased with increased extraction temperature and decreased with the increase of H2O amount. In the extracts obtained by MCE, H2O addition, time, and vibration speed (*p* = 0.0006; *p* = 0.0266; *p* = 0.0002) as well as the interactions between H2O addition and vibration speed (*p* = 0.0221) showed statistically significant influence on the content of carnosic acid. The content of carnosic acid increased with prolonged extraction time and vibration speed and decreased with the increase of H2O amount.

As can be seen from Figure 2 and Table A2, H2O addition, extraction time, and temperature showed statistically significant influence on the content of carnosol (*p* < 0.0001; *p* = 0.0008; *p* = 0.0003) in the extracts obtained by stirring and mixing. The content of carnosol is increased with increased time and temperature of the extraction and with decreased H2O amount.

**Figure 2.** Three-dimensional plots for obtained content of carnosol as a function of the extraction time, temperature, and H2O content in the extraction with mixing and heating (**a**,**b**) and MCE (**c**,**d**).

Interactions between amount of H2O added and the extraction time and between the amount of H2O added and temperature (*p* = 0.0184; *p* = 0.0234) also showed a significant influence for the content of carnosol. In the extracts obtained by MCE, H2O addition, time, and vibration speed (*p* = 0.0055; *p* = 0.0187; *p* = 0.0012) showed statistically significant influence on the content of carnosol. The content of carnosol increased with prolonged extraction time and vibration speed and decreased with the increase of H2O amount. Since model according to RSM is not significant for the extraction of carnosol with ultrasound (*p* = 0.0708), the results obtained for that extraction are not discussed. To optimize the extraction conditions of two different phenolic diterpenes 17 runs determined by BBD with three variables (percentage of H2O added, time and temperature or vibration speed) at three levels were used to fit a second-order response surface. The amount of carnosic acid and carnosol were observed as the response (Tables A1 and A2).

The data describing the optimal conditions for the extraction of carnosic acid and carnosol from sage using DESs are not available in the literature, but there are few papers investigating the optimal conditions with other solvents. In paper by Fatma Ebru et al. [23] it was shown that 70% of ethanol was the most efficient solvent since it extracted 3.45 mg carnosol + carnosic acid per g of the extract. According to the optimization carried out, they showed that the amount of these bioactive components was in the function of extraction time. In addition, they also demonstrated that carnosol and carnosic acid degraded easily at higher temperatures over a longer period of time. Therefore, they have shown that the optimum conditions were temperatures of 40–50 ◦C, the extraction time 3–6 h, solventto-sage ratio 6:1 (*v*/*w*) and 70–80 wt.% ethanol for maceration. Similar results were also showed in paper by Durling et al. [24]. According to the optimization carried out, the amount of targeted components depended on several parameters such as particle size, temperature, time, and a solvent-to-sage ratio. The highest concentration of targeted components was obtained with the particle size 1 mm, 40 ◦C, the extraction time of 3 h, the solvent-to-sage ratio of 6:1 (*v/w*) and 55–75 wt.% ethanol. Under these conditions, the extract containing 10.6% carnosic compounds was obtained.

The optimization process of extraction is important for determining the most favorable conditions for achieving maximum yields of desired components in the extracts. Based on BBD, estimated coefficients of second order response models for carnosol and carnosic acid in sage extracts are given in Tables A1 and A2. *R* 2 for carnosic acid was 0.9630 and for carnosol was 0.9607 in the extracts obtained by stirring and heating, and for UAE *R* 2 for carnosic acid was 0.8660. In the case of MCE, *R* 2 for carnosic acid was 0.9442 and for carnosol *R* <sup>2</sup> was 0.9032. According to ANOVA, statistically significant models for carnosic acid (Table A3) and carnosol in the extraction by stirring and heating (Table A4) (*p* ≤ 0.05) were obtained. Additionally, the obtained models showed non-significant lack of fit (*p* = 0.2042–0.4491), except in the case of MCE for carnosic acid (*p* = 0.0008).

According to RSM, optimum conditions are expressed as those at which it is possible to achieve the maximum amount of carnosic acid and carnosol. They are slightly different depending on the extraction technique used, so for the extraction with heating and mixing they were 10% H2O addition, 90 min and 70 ◦C, while for UAE they were 11.05% of H2O addition, 82.36 min and 69.84 ◦C. Under these optimal conditions, the content of carnosic acid and carnosol was calculated as 14.20 µg mg−<sup>1</sup> and 6.47 µg mg−<sup>1</sup> in case of stirring and heating and 14.72 µg mg−<sup>1</sup> of carnosic acid for ultrasound extraction. The desirability for these optimizations was 0.990 and 1.0, respectively. The experimental results for the amount of carnosic acid and carnosol obtained at optimum conditions were 13.73 ± 0.26 and 6.15 ± 0.33 µg mg−<sup>1</sup> for the extraction with stirring and heating, while for UAE this amount was 14.24 ± 0.21 µg mg−<sup>1</sup> . Optimum conditions for MCE were 11.13% H2O addition, time of extraction 2.90 min, and vibration speed 4.98 m s−<sup>1</sup> . Under these optimal conditions, the content of carnosic acid and carnosol is calculated as 8.95 µg mg−<sup>1</sup> and 2.02 µg mg−<sup>1</sup> with the desirability 1.0 which was confirmed experimentally (8.90 ± 0.10; 2.03 ± 0.04 µg mg−<sup>1</sup> ).

#### *2.4. Comparison with Other Extraction Methods*

According to the literature, the most common solid–liquid extraction of sage has been performed with methanol. Due to the toxic effect of methanol, it is preferable to use ethanol which can be classified as bio-solvent and is much safer for the use [25,26]. In the paper by Abreu et al. [27] the content of carnosic acid and carnosol in methanolic extract of sage was 14.6 mg g−<sup>1</sup> of dry weight and 0.4 mg g−<sup>1</sup> , respectively. This is similar to our results for Run 2 (mixing and heating) and Run 3 (UAE), but with a significantly higher amount of carnosol in our case. Sage extraction with 80% methanol over 24 h at room temperature led to the extraction of carnosic acid only with the content of 273.8 mg 100 g−<sup>1</sup> of the plant dry weight [28], much lower than our results. In other case, the extraction with 50% methanol during 60 min in ultrasound bath has brought carnosic acid content of 2.1 g kg−<sup>1</sup> extract and carnosol content of 4.1 to 15.1 mg g−<sup>1</sup> of plant dry weight [29].

According to Table 3, which shows our results obtained by the stirring with heating extraction of the same sage material with common solvents, it is observed that the most effective solvent is absolute ethanol, while H2O is the least effective solvent for the extraction of carnosic acid and carnosol. The preparation of aqueous solutions of ethanol in the range of 30–70% (*v*/*v*) shows that the increase in the volume of ethanol (*v*/*v*) increased the amount of extracted components. In this case, methanol as the extraction solvent shows lower extraction efficiency compared to ethanol. In addition, the influence of extraction parameters such as extraction time and temperature can be observed in Table 3. However, when ethanol is used as the extraction solvent and with the most efficient extraction conditions applied (50 ◦ C and 90 min), lower amount of carnosic acid and carnosol was obtained compared to the selected DES (choline chloride:lactic acid 1:2).

Considering the adverse properties of organic solvents and in order to overcome their disadvantages, such as low selectivity for antioxidant compounds [30], safe or green solvents and processes have been used. Supercritical fluid extraction (SFE) has been used in the plant material extraction due to its ability to provide clean extracts without residual solvent [31]. In addition, SFE can be performed at low temperatures in short time, which is suitable for carnosic acid oxidation prevention during the extraction, also supported by the absence of air and light during the extraction process thus reducing its degradation [32]. In our previous work [33] we used the same herbal material for carnosic acid and carnosol extraction using SFE with CO<sup>2</sup> (SC-CO2). Comparing the results, the highest amount of extracted carnosic acid using SC-CO<sup>2</sup> was 855.8 mg 100 g−<sup>1</sup> of the plant material (30 MPa, 50 ◦C, 1 kg h−<sup>1</sup> CO2), while the extraction yield using DESs was 1443.22 mg 100 g−<sup>1</sup> and 1399.22 mg 100 g−<sup>1</sup> , depending on the extraction technique employed. In the case of carnosol, the highest amount was extracted under the same conditions of SC-CO<sup>2</sup> (446.35 mg 100 g−<sup>1</sup> ), and similar results were achieved using DESs (483.34 and 418.39 mg 100 g−<sup>1</sup> of plant, depending on the extraction technique employed). However, certified reference material was not used and therefore minor changes in the composition of the plant material are possible with respect to the same sample used in our previously published data. In the paper published by Babovic et al. [34] the content of carnosic acid obtained by SC-CO<sup>2</sup> was 13.76 g per 100 g of the extract and carnosol content was 6.97 g per 100 g of the extract similar to our results (11.63 g carnosic acid per 100 g and 8.55 g carnosol per 100 g) [32]. Despite the fact that SC-CO<sup>2</sup> extraction conditions may reduce carnosic acid degradation, we still notice that more carnosic acid was extracted and preserved by DESs extraction even at higher extraction temperatures. On the other hand, preparing DESs is simple and inexpensive, i.e., the price is comparable to the cost of the conventional solvents. Moreover, this is sustainable process theoretically without generated waste [10] which makes this extraction process suitable for the extraction of bioactive components including carnosic acid and carnosol.


**Table 3.** The values (µg mg−<sup>1</sup> of the plant material) of carnosic acid and carnosol for the extraction obtained by stirring with heating. The results are expressed as mean value ± standard deviation (*n* = 3).

#### **3. Materials and Methods**

*3.1. Chemicals*

The standard compounds carnosic acid (≥95.0%) and carnosol (99.2%) (Sigma Chemical Co., St. Louis, MO, USA) were used for the chemical analyses. All solvents were of analytical grade and purchased from J.T. Baker (Avantor, Phillipsburg, NJ, USA).

#### *3.2. Plant Material*

Dried sage leaves (*Salvia officinalis* L.) were used for experiments. Prior to the extraction, the dried leaves were grounded and sieved using a vertical vibratory sieve shaker (LabortechnikGmbh, Ilmenau, Germany) as described in paper by Joki´c et al. [35].

#### *3.3. Preparation of DES*

The choline chloride based DESs were prepared as described in our paper [36]. In this study, seventeen different choline chloride based DESs were prepared using inexpensive components as shown in Table 4.


**Table 4.** Preparation of deep eutectic solvents (DESs).

#### *3.4. Extraction of Carnosic Acid and Carnosol with DESs*

Grounded *Salvia officinalis* L. dried leaves (50 mg) were mixed with 1 mL of the selected solvent, a pure DES or a mixture of DES and ultrapure H2O (Millipore Simplicity 185, Darmstadt, Germany). Prepared samples were stirred at 1500 rpm in aluminum block (Stuart SHB) on a magnetic stirrer or ultrasound treated in temperature-controlled ultrasonic bath at specified temperature for the certain time (Table 1). The temperaturecontrolled ultrasonic bath (Elma P70 H, Singen, Germany) was set with frequency at 37 Hz and power at 50 W at the same temperature over the same time as in case of mixing in aluminum block (Table 1). Prepared samples (50 mg of plant + 1 g of glass beads with 1 mL of solvent) were also extracted on the BeadRuptor 12 ball mill (Omni International, Kennesaw, GA, USA) according to parameters in Table 2 at room temperature (24–28 ◦C). After the extraction, the mixture was centrifuged for 15 min and then decanted. The supernatant liquid was then diluted with methanol, filtered through a PTFE 0.45 µm filter, and subjected to HPLC analysis.

#### *3.5. Extraction of Carnosic Acid and Carnosol with Conventional Solvents*

Grounded *Salvia officinalis* L. dried leaves (50 mg) were mixed with 1 mL of selected solvent (Millipore Simplicity 185, Darmstadt, Germany). Prepared samples were stirred at 1500 rpm in aluminum block (Stuart SHB) on a magnetic stirrer at specified temperature for the certain time (Table 3).

#### *3.6. Chemical Characterization of the Extracts*

HPLC analyses of carnosic acid and carnosol was performed on an Agilent 1260 Infinity II (Agilent, Santa Clara, California, USA) with chromatographic separation obtained on a ZORBAX Eclipse Plus C18 (Agilent, Santa Clara, CA, USA) column (100 × 4.6 mm, 5 µm).

The separation of analyzed compounds was made with method described in our previous paper [31], but since analysis was performed on different device, linearity of the calibration curve, LOQ and LOD was confirmed. Standard stock solutions for carnosic acid and carnosol were prepared in a methanol and calibration was obtained at eight concentrations (concentration range 10.0, 20.0, 30.0, 50.0, 75.0, 100.0, 150.0, and 200.0 mg L−<sup>1</sup> ). Due to *R* <sup>2</sup> = 0.99789 for carnosic acid and *R* <sup>2</sup> = 0.99968 for carnosol, calibration curve was confirmed. Limit of detection were 0.795 mg L−<sup>1</sup> and 0.971 mg L−<sup>1</sup> for carnosic acid and carnosol, respectively. Limit of quantification were 2.648 mg L−<sup>1</sup> and 7.416 mg L−<sup>1</sup> for carnosic acid and carnosol, respectively. Retention time for carnosic acid was 7.416 min, while for carnosol was 4.217 min. The chromatograms of the standard and real sample are shown in the Appendix A (Figure A2). For the validation of the HPLC method for the determination of carnosic acid and carnosol, in addition to linearity, retention time comparison and absorption spectrum comparison with standards, repeatability of measurements and solution preparation as well as accuracy were performed, which is also shown in the Appendix A (Table A5).

#### *3.7. Statistical Experimental Design*

BBD explained in detail by Bas and Boyaci [37] was used for determination of optimal DES (stirring and heating), UAE-DES and MCE-DES extraction conditions in terms of getting higher amount of carnosic acid and carnosol in the *S. officinalis* extracts. Independent variables in design were H2O content (X1), time (X2) and temperature (X3) and vibration speed (X3) and tested levels were reported in Table 5. Design-Expert® Commercial Software (ver. 9, Stat-Ease Inc., Minneapolis, MN, USA) was used for data analysis. The analysis of variance (ANOVA) was also used to evaluate the quality of the fitted model, and the test of statistical difference was based on the total error criteria with a confidence level of 95.0%.


**Table 5.** Coded and real levels of independent variables for the designed experiment.

#### **4. Conclusions**

In present study, determination of suitable deep eutectic solvent and optimization of the extraction of carnosol and carnosic acid from sage were performed. Among 17 different solvents, choline chloride:lactic acid (1:2 molar ratio) was selected for the extraction by heating and mixing, as well as for ultrasound and mechanochemical extraction. The content of carnosic acid and carnosol was slightly higher in the extracts obtained by stirring and heating and mechanochemical extraction. The influence of H2O content, extraction time and temperature (for stirring and heating and for ultrasound-assisted extraction (UAE)), H2O content, extraction time and vibration speed for mechanochemical extraction on the content of targeted compounds were investigated. Optimal extraction conditions determined by response surface methodology (RSM) were in accordance with experimentally demonstrated values.

Compared to SC-CO<sup>2</sup> extraction, we observed that more carnosic acid is extracted using DESs, with emphasis on ChClLa, while the amount of carnosol detected in the extract

obtained by ChClLa is similar to that obtained by SC-CO2. In addition, the comparison with the solvents such as ethanol, H2O, aqueous solutions of ethanol (30–70% (*v*/*v*)) and methanol under the same extraction conditions, showed that choline chloride:lactic acid (1:2 molar ratio) was more efficient for the extraction of carnosic acid and carnosol compared to used conventional solvents.

Given the amounts of carnosic acid achieved at high temperatures in DES in further research it would be useful to examine the stability of the component over the certain period of time.

**Author Contributions:** Conceptualization, M.M. and M.J.; methodology, M.J., S.J., M.M., and I.J.; formal analysis, M.J. and S.J.; investigation, M.J. and M.M.; resources, I.J.; data curation, M.M.; writing—original draft preparation, M.J.; writing—review and editing, S.J., M.M., and I.J.; visualization, S.J., M.M., and I.J.; supervision, M.M., S.J., and I.J.; funding acquisition, I.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Croatian Government and the European Union (European Regional Development Fund—the Competitiveness and Cohesion Operational Programme— KK.01.2.2.03) for funding this research through project CEKOM 3LJ (KK.01.2.2.03.0017), granted to Institution for research and knowledge development of nutrition and health CEKOM 3LJ.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available from the authors.

**Acknowledgments:** We would like to thank Croatian Government and the European Union (European Regional Development Fund—the Competitiveness and Cohesion Operational Programme— KK.01.2.2.03.0017) for funding this research through project CEKOM 3LJ (KK.01.2.2.03.0017), granted to Institution for research and knowledge development of nutrition and health CEKOM 3LJ.

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

#### **Appendix A**

**Table A1.** Regression coefficient of polynomial function of all response surfaces for carnosic acid.



**Table A1.** *Cont.*

**Table A2.** Regression coefficient of polynomial function of all response surfaces for carnosol.


**Table A3.** Analysis of variance (ANOVA) of the modelled responses for carnosic acid.



**Table A3.** *Cont.*

**\*** *p* < 0.01 highly significant; 0.01 ≤ *p* < 0.05 significant; *p* ≥ 0.05 not significant.

**Table A4.** Analysis of variance (ANOVA) of the modelled responses for carnosol.


**\*** *p* < 0.01 highly significant; 0.01 ≤ *p* < 0.05 significant; *p* ≥ 0.05 not significant.


**TableA5.**HPLCmethodvalidationparameters.

**re A1.** Carnosol and carnosic acid content obtained by stirring and heating with DESs depending on the temperatur **Figure A1.** Carnosol and carnosic acid content obtained by stirring and heating with DESs depending on the temperature and H2O content (*n* = 3). The columns represent determined amount of carnosol and carnosic acid in the samples, and the color of the column indicates DES used according to the abbreviations recorded in the legend to the right and Table 4.

**Figure A2.** Obtained chromatogram of carnosol and carnosic acid standards (picture **above**) and chromatogram of real sample (picture **below**).

#### **References**


*Article*

## **The Impact of the Method Extraction and Di**ff**erent Carrot Variety on the Carotenoid Profile, Total Phenolic Content and Antioxidant Properties of Juices**

**Aleksandra Purkiewicz <sup>1</sup> , Joanna Ciborska <sup>1</sup> , Małgorzata Ta ´nska <sup>2</sup> , Agnieszka Narwojsz <sup>1</sup> , Małgorzata Starowicz <sup>3</sup> , Katarzyna E. Przybyłowicz <sup>1</sup> and Tomasz Sawicki 1,\***


Received: 29 November 2020; Accepted: 9 December 2020; Published: 11 December 2020 -

**Abstract:** The study assesses the antioxidant activity (AA), carotenoid profile and total phenolic content (TPC) of carrot juices obtained from three different varieties (black, orange and yellow) and prepared using high- (HSJ) and low-speed juicer (LSJ). The AA assessment was carried out using four assays (DPPH, ABTS, PCL ACW and PCL ACL). The content of carotenoids was conducted by high performance liquid chromatography equipped with a diode array detector (HPLC-DAD) method, while the total phenolic content by the spectrophotometric method. It was shown that orange carrot juices contain more carotenoids than yellow and black carrot juices, approximately ten and three times more, respectively. The total carotenoid content in orange carrot juice made by the HSJ was higher (by over 11%) compared to juice prepared by the LSJ. The highest total phenolic content was noticed in black carrot juices, while the lowest in orange carrot juices. In black carrot juices, a higher range of TPC was found in juices made by HSJ, while in the case of the orange and yellow carrots, the highest content of TPC was detected in juices prepared by the LSJ. AA of the juices was highly dependent on the carrot variety, juice extraction method. The most assays confirmed the highest AA values in black carrot juices. Furthermore, it was shown that the HSJ method is more preferred to obtain orange and yellow carrot juices with higher antioxidant properties, while the LSJ method is more suitable for black carrot juice extraction.

**Keywords:** carrot juices; carotenoids; polyphenols; antioxidant activity; high-speed juicer; low-speed juicer; food processing

#### **1. Introduction**

Carrot is a root vegetable [1] that contains many bioactive compounds, for example, carotenoids (α-carotene, β-carotene lutein, zeaxanthin and lycopene) [2], phenolic acids (chlorogenic, ferulic, p-coumaric, caffeic) [3] and anthocyanins (cyanidin-3-O-xylosyl (sinapoylglucosyl) galactoside, cyanidin-3-O-xylosyl (feruolylglucosyl) galactoside, cyanidin-3-O-xylosyl (coumaroylglucosyl) galactoside) [1]. In addition, carrots are great sources of vitamins (ascorbic acid, thiamine, riboflavin,

niacin, pyridoxine, folic acid, vitamin K and A) as well as minerals (calcium, iron, magnesium, phosphorus, potassium, sodium and zinc) [2]. Thanks to the presence of the mentioned bioactive compounds, a carrot has significant health-promoting properties. It was demonstrated that carrots display powerful antioxidant and radical scavenging activities. Moreover, the consumption of carrots has been linked with a lower risk of diseases such as atherosclerosis, cataract, diabetes and cancer [1].

Carrots are commonly classified by the color of roots into white, black, orange, yellow, purple and red [4]. The most common carrot variety is the orange ones which is a genetic crossword between purple, white and yellow carrots [5]. The color of the root has a significant impact on the presence of bioactive compounds. The orange carrot root contains high amounts of α-carotene and is the richest source of β-carotene (precursor of vitamin A). The black carrot is an excellent source of anthocyanins, red carrot root is rich in lycopene, the yellow ones, in turn, was demonstrated to accumulate lutein [6,7]. In addition to the root's color, the growing and season conditions, the ripeness of carrots as well as part of the root also influence the presence of bioactive compounds [8].

It should be emphasized that the content of bioactive compounds and their biological activity is also influenced by technological processes. However, effect of technological parameters and carrot variety interactions on juice nutrition value is still not fully understood Determination of the composition and content of biologically active substances in both fresh and processed vegetables is crucial for the food industry. Such information is also essential for consumers with an increasingly developing nutritional awareness.

The available literature data show that to date, only a high-speed juicer and traditional blenders were used to examine the impact of the extraction method on the juices' antioxidant activity. No studies have evaluated the impact of using a high-speed juicer or a low-speed juicer on the antioxidant activity of carrot juices [9,10]. In addition, in the previously published papers, the juices' antioxidant activity was tested only by DPPH and ABTS assays [9,11,12]. Therefore we intended to examine the influence of the extraction methods on the concentration of main bioactive compounds in the juices obtained from roots of different carrot varieties. To meet this goal we applied low- and high-speed juicer and roots of yellow, orange and black carrots. In addition, to examine the effects of the extraction methods on the antioxidant activity of carrot juices four different methods (DPPH, ABTS, PCL ACW and PCL ACL) were used.

#### **2. Results**

#### *2.1. Content of Carotenoids in Carrot Juices*

The results of the total and individual compound content of carotenoids in the tested carrot juices are presented in Table 1. The carotenoid profile in tested carrot juices were clearly more dependent on the carrot variety and this differentiation is visible in Figure 1.

**Compounds Black Carrot Orange Carrot Yellow Carrot HSJ LSJ HSJ LSJ HSJ LSJ** lutein 1.15 <sup>±</sup> 0.49 <sup>a</sup> 1.98 <sup>±</sup> 0.08 <sup>A</sup> 0.61 <sup>±</sup> 0.03 <sup>c</sup> \* 0.14 <sup>±</sup> 0.05 <sup>C</sup> 0.81 <sup>±</sup> 0.18 <sup>b</sup> 0.77 <sup>±</sup> 0.02 <sup>B</sup> zeaxanthin 0.31 <sup>±</sup> 0.06 <sup>b</sup> 0.14 <sup>±</sup> 0.03 <sup>A</sup> 0.61 <sup>±</sup>0.00 <sup>a</sup> \* 0.12 <sup>±</sup> 0.03 <sup>A</sup> 0.06 <sup>±</sup> 0.08 <sup>c</sup> 0.14 <sup>±</sup> 0.02 <sup>A</sup> <sup>α</sup>-carotene 0.14 <sup>±</sup> 0.05 <sup>b</sup> 0.16 <sup>±</sup> 0.04 <sup>B</sup> 2.90 <sup>±</sup>0.34 <sup>a</sup> 2.70 <sup>±</sup> 0.66 <sup>A</sup> ND ND 13-*cis*-β-carotene 0.24 <sup>±</sup> 0.14 <sup>b</sup> \* 0.35 <sup>±</sup> 0.03 <sup>B</sup> 8.30 <sup>±</sup> 1.33 <sup>a</sup> \* 7.88 <sup>±</sup> 0.08 <sup>A</sup> ND ND <sup>β</sup>-carotene ND 0.68 <sup>±</sup> 0.06 <sup>B</sup> 15.08 <sup>±</sup> 2.84 <sup>a</sup> \* 13.85 <sup>±</sup> 0.01 <sup>A</sup> 0.08 <sup>±</sup> 0.01 <sup>b</sup> 0.12 <sup>±</sup> 0.02 <sup>C</sup> Total 1.84 <sup>±</sup> 0.47 <sup>b</sup> \* 3.31 <sup>±</sup> 0.79 <sup>B</sup> 27.50 <sup>±</sup> 0.71 <sup>a</sup> \* 24.69 <sup>±</sup> 0.51 <sup>A</sup> 0.95 <sup>±</sup> 0.06 <sup>c</sup> 1.03 <sup>±</sup> 0.06 <sup>C</sup>

**Table 1.** The content (mg/100 mL) of carotenoids in black, orange and yellow carrot juices.

Abbreviations: Rt—retention time; HSJ—high-speed juicer; LSJ—low-speed juicer; ND—non-detected. Data are expressed as means ± SD (*<sup>n</sup>* <sup>=</sup> 3), means in the line with different letters (abc/ABC) are significantly different (*p* < 0.05). Statistically significant differences (*p* < 0.05) between method of extraction each carrot variety are marked by \*.

**Figure 1.** The high performance liquid chromatography (HPLC) chromatograms of carotenoid compounds identified in the carrot juices. Identified peaks are as follows: 1—lutein, 2—zeaxantin, 3—β-apo-8′ carotenal (used as an internal standard), 4—13-*cis*-β-carotene, 5—α-carotene and 6—β-carotene. Abbreviations: BC—black carrot, OC—orange carrot, YC—yellow carrot, HSJ—high-speed juicer, LSJ—low-speed juicer.

The highest carotenoid content was found in orange carrot juices, while the lowest in yellow ones. The content of t in orange carrot juices was significantly different (*p* < 0.05) from the content in black and yellow carrot juices.

Orange carrot juices contained almost thirty and twenty four times more carotenoids in juices obtained by the use of high-speed juicer and a low-speed juicer than yellow ones and fifteen times (for high speed juicer (HSJ) method) and seven times (for low speed juicer (LSJ) method) more than black carrot juices. The significant difference (*p* < 0.05) was also detected in the carotenoid content of black and yellow carrot juices. Black carrot juices were characterized by two times and over three times more carotenoids content in juices prepared by HSJ and LSJ, respectively, than yellow ones.

In the examined carrot juices five different carotenoids were detected (Table 1). Three compounds belong to the carotenes group (α-carotene, β-carotene and 13-*cis*-β-carotene) and two compounds belong to the xanthophylls group (lutein and zeaxanthin). The richest profile of carotenoids was found in the orange carrot juices. Five compounds (α-carotene, β-carotene, 13-*cis*-β-carotene, lutein and zeaxanthin) were found in the carrot juice obtained by high-speed juicer (HSJ) and by low-speed juicer (LSJ).

The dominant carotenoid in orange carrot juices was β-carotene, constituting 55% and 56% of the total content of carotenoids in juices prepared by the HSJ and LSJ methods, respectively. The second dominant compound, in juices obtained from orange carrots, was 13-*cis*-β-carotene, which was 30% of the total content of carotenoids for juice obtained by the use of HSJ and 32% obtained by the use of LSJ. Juices obtained from yellow and black carrots contained three (lutein, zeaxanthin and β-carotene) and five compounds (lutein, zeaxanthin, β-carotene and 13-*cis*-β-carotene), respectively. In the yellow carrot juices, the dominant carotenoid was lutein which constituted 85% of the total carotenoids content in juices prepared by the use of high-speed juicer and 75% by low-speed juicer. The dominant compounds in the black carrot juices were lutein (63% for HSJ method and 60% for LSJ method) and zeaxanthin (17% for HSJ method and 4% for LSJ method) and 13-*cis*-β-carotene (13% for HSJ method and 11% for LSJ method). Significantly higher amounts (*p* < 0.05) of β-carotene and 13-*cis*-β-carotene were determined in the orange carrot juices compared to the black carrot ones. In turn, the orange carrot juices obtained by HSJ method contained thirty five times more 13-*cis*-β-carotene, whereas juices made by LSJ method contained twenty three times and twenty times more of 13-*cis*-β-carotene and β-carotene, respectively. The highest amounts of lutein were determined in the black carrot juices. The content of this compound was two and fourteen times higher in the black carrot juices than in orange carrot juices made by HSJ and LSJ, respectively. It should be emphasized that, in comparison to other carrot varieties, orange carrot juice contained the lowest percentage of lutein (*p* < 0.05).

It was showed that method of juice extraction significantly affected the content of lutein, zeaxanthin, 13-*cis*-β-carotene and β-carotene in orange carrot juices (*p* < 0.05). Juice prepared by the high-speed juicer contained 77%, 80%, 5% and 8% more these compounds compared to juice obtained using the low-speed juicer (Table 1). Furthermore, the total content of carotenoids in the juice obtained from orange carrots prepared by the HSJ method was significantly higher (by over 111%) than in juice made by the LSJ method (*p* < 0.05).

#### *2.2. Total Phenolic Content in Carrot Juices*

The total phenolic content (TPC) in the tested juices differed among carrot varieties (Figure 2). The significantly highest TPC was demonstrated in the juices obtained from black carrots (*p* < 0.05), while the lowest content of these compounds was detected in the orange carrot juices (*p* < 0.05). The black carrot juice prepared with the use of LSJ method contained 7% more total phenolic content than the juice prepared with the use of HSJ (*p* < 0.05). In comparison, the juice obtained from orange carrot using HSJ method was characterized by a higher TPC and contained 10% more these compounds than the juice obtained by LSJ. The TPC demonstrated in the juice obtained from yellow carrot did not differ between the extraction method. The TPC demonstrated in black carrot juices prepared with the HSJ and LSJ methods was eight and nine times higher than TPC demonstrated in orange carrot juices prepared with the same methods, respectively. and six times higher than in yellow carrot juices.

**Figure 2.** Results of total phenolic content (TPC) in black, orange and yellow carrot juices. Abbreviations: BC—black carrot, OC—orange carrot, YC—yellow carrot, HSJ—high-speed juicer, LSJ—low-speed juicer. Each bar corresponds to the mean of three independent replicates with error bars indicating the standard deviations. Different letters (abc/ABC) indicate significant differences among samples (*p* < 0.05). Statistically significant differences (*p* < 0.05) between method of extraction each carrot variety are marked by \*.

#### *2.3. Antioxidant Capacity of Carrot Juices*

Four methods (ABTS, DPPH, PCL ACW and PCL ACL) were used to determine the antioxidant activity of the carrot juices (Table 2). The results of the PCL methods were presented both separately and as a sum of ACL and ACE measurements.


**Table 2.** The antioxidant activity of black, orange and yellow carrot juices determined by different assays.

Abbreviations: HSJ—high-speed juicer; LSJ—low-speed juicer. Data are expressed as means ± SD (*n* = 3), means in the line with different letters (abc/ABC) are significantly different (*p* < 0.05). Statistically significant differences (*p* < 0.05) between method of extraction each carrot variety are marked by \*.

The obtained juices were characterized by different antioxidant activity depending on the carrot variety and extraction method. The results of DPPH assay showed that yellow carrot juices demonstrated the highest antioxidant activity among examined varieties (*p* < 0.05) and the juice made by the LSJ method was characterized by slightly higher values of antioxidant activity than those made by the HSJ. In comparison, black and orange carrot juices obtained by the HSJ method were characterized by higher values of antioxidant activity than the juices obtained by the LSJ. A significant relationship (*p* < 0.05) between the extraction method and the antioxidant activity was demonstrated only for black carrot juices (Table 2). The antioxidant activity demonstrated in juice obtained from orange and yellow carrots did not differ between the extraction method (*p* > 0.05).

The results of ABTS assay demonstrated that the highest antioxidant activity was shown for the black carrot juices and the lowest activity was demonstrated for the yellow ones. The antioxidant activity of black carrot juice obtained by HSJ method was 17% and 21% higher than the antioxidant activity of orange and yellow carrot juices, respectively. A similar tendency was observed for the black carrot juices obtained with the use of LSJ. Moreover, the significant relationship between the extraction method and the antioxidant activity estimated by the ABTS test of juices obtained from each carrot variety was noted. The antioxidant activity demonstrated in juices obtained by HSJ method was higher than those shown in juice obtained with the use of LSJ (1.5%-black, 5.5%-orange and 4%-yellow carrot juice) (*p* < 0.05).

The highest antioxidant activity were demonstrated for the black carrot juices, while the lowest values were demonstrated for the orange carrot juices (*p* < 0.05) in all applied assays (PLC ACW, PLC ACL, PLC). The results of PCL ACW assay demonstrated that differences in juices' antioxidant activity values depends on the carrot variety. The antioxidant activity of black carrot juices method was higher in comparison to the antioxidant activity of orange and yellow carrot juices made (regardless of the extraction method) (*p* < 0.05). It should be noticed that, in case of black and orange carrot, the extraction method had a significant effect on the antioxidant activity determined by all applied assays. The results of all assays showed that the black carrot juice obtained by LSJ had higher antioxidant activity than the juice obtained by HSJ (*p* < 0.05). The opposite situation was observed in case of orange and yellow carrot juices, where the higher antioxidant values were measured in juices obtained by HSJ (Table 2).

#### *2.4. Association Between Obtained Data*

#### 2.4.1. Linear Pearson's Correlation Coefficients

The values of antioxidant activity of carrot juices analyzed by the ABTS assay was strongly positively correlated with the values obtained by the PCL ACW (*r* = 0.89), PCL ACL (*r* = 0.96) assays and the sum of PCL assay (*r* = 0.96). While the results of the AC measured by the DPPH assay were only slightly correlated with other assays; negatively with all PCL assays and positively with ABTS assay (Table 3).


**Table 3.** Correlation coefficients (r) calculated for the relationships between carotenoids, total phenolic content and antioxidant activity assays (r marked by \* are statistically significant at *p* < 0.05).

Relationships between the content of carotenoids and antioxidant activity were also estimated (Table 3). Statistically significant (*p* < 0.05) and positive correlation coefficients were shown between the content of lutein and the results of ABTS (*r* = 0.91), PCL ACW (*r* = 0.89) and PCL ACL assays (*r* = 0.96) and sum of PCL (*r* = 0.96), while the negative correlation coefficient between the content of lutein and results of the DPPH assay (*r* = −0.91). In turn, average negative correlation coefficients (but not significant at *p* < 0.05) for relationships between content of total carotenoids, α-, β- and 13-*cis*-β-carotene and results of the ABTS, PCL ACW, PCL ACL and PCL assays were noted (r values ranging from −0.29 to −0.51). It was also shown that TPC was highly positively correlated with most applied antioxidant assays; r values in range of 0.79–0.98 for ABTS, PCL ACW, PCL ACL and total PCL assays. However, a strong negative correlation between TPC and DPPH (*r* = −0.83) was found.

#### 2.4.2. Principal Component Analysis (PCA)

Principal component analysis (PCA) was performed on all samples and variables (individual carotenoid concentration, total phenolic content, antioxidant capacity (ABTS, DPPH, PCL ACW, PCL ACL assays and the sum of PCL) to investigate the structure and regularity in the relationships between variables and cases. The first two principal components (PC) explained 90.61% of total data variance. The correlations between the original variables and the obtained principal components are shown in Figure 3a. Each of the variables is represented by a vector. The direction and lengths of the vectors indicate to what extent the given variables affect the principal components. In our study, most input variables are located near the circle, which means that the information in these variables is transferred by principal components. PCA analysis showed a strong positive correlation between α-carotene, β-carotene, 13-*cis*-β-carotene and between lutein and TPC, ABTS, PCL ACW, PCL ACL and the sum of PCL. However, the opposite variables are negatively correlated. The strong negative correlation between lutein and zeaxanthin and the DPPH assay and between ABTS, PCL ACW, PCL ACL, the sum of PCL and DPPH were noted. Moreover, the graph shows that TPC was also negatively correlated with the antioxidant capacity determined by the DPPH assay and positively correlated with the ABTS, PCL ACW, PCL ACL assays and sum of the PCL.

**Figure 3.** Principal components plot, variations in the parameters (ABTS, DPPH, PCL ACW, PCLACL assays and sum of the PCL) of the analyzed carrot juices (**A**) score plot of the obtained juices (**B**). Abbreviations: BC—black carrot, OC—orange carrot, YC—yellow carrot, HSJ—high-speed juicer, LSJ—low-speed juicer.

Figure 3b presents the score plot on the plane of principal components, which shows the similarity between the types of carrot juices tested. The analyzed cases' position concerning each other proves different antioxidant properties of juices obtained from black, orange and yellow carrots. Moreover, based on the analysis, it was observed that the juices from black carrots were the most diverse (LSJ and HSJ methods), while the juices from yellow carrots were the most similar. There was no similarity between the juices from different carrot cultivars.

#### **3. Discussion**

The carotenoid content in carrot juices varies and depends on many factors, for example, type of raw material and/or storage time [13]. In this study, the highest content of carotenoids was demonstrated in juices made of orange carrot 27.50 mg/100 mL in the juice prepared by the use of HSJ and 24.69 mg/100 mL in the juice obtained by the use of LSJ. In comparison, Amal et al. [13] shown that the total content of carotenoids in the juice from orange carrot was 6.6 mg/100 mL. The difference in the results may arise from the variety of carrot use in the research, growing conditioning and cultivation practice [14]. The lowest carotenoid content was identified in juices from the yellow carrot. Similarly, Sun et al. [15] presented that yellow (as well as white) carrot varieties contain the lowest amount of carotenoid pigments. Therefore, the carrot root color used to juice preparation has an essential contribution to the content of selected compounds from the carotenoid group.

In the conducted research the amount of β-carotene in juices made from orange carrot represented more than 50% of total carotenoids content. In juices made from black carrot the β-carotene content was 20% and in juices from yellow carrot the amount of β-carotene represented 8% (HSJ) and 12 % (LSJ). Juices made of black and yellow carrot contained a higher amount of xanthophylls than carotenes. The group of xanthophylls includes lutein. The available literature data confirms that lutein is the main compound of the yellow carrot and the yellow carrot juices can contain from 0.1 to 0.5 mg/100 mL of lutein [5]. In our experiment, the lutein content in yellow carrot juice was from 0.77 mg/100 mL (LSJ method) to 0.81 mg/100 mL (HSJ method). Black carrot juice possesses 1.5 (from HSJ method) and 2.5 (from LSJ method) times higher content of lutein in comparison to yellow carrot juice. Black carrot juices are considered poor source carotenes but a better source of xanthophylls [5]. More than 79% of carotenoids in juices made of black carrots are zeaxanthin and lutein, which are assigned to the xanthophylls group. Higher content of β-carotene and 13-*cis*-β-carotene was demonstrated in orange juices prepared by the HSJ method and in yellow and black carrot juices prepared by the LSJ method. Similarly to the result obtained in the present study, the result of other researchers demonstrated that the juice preparation method did not significantly influence carotenoid content in juices [11]. On the other hand, the study conducted by Ma et al. [16] showed that the peeling method, blanching and enzyme liquefaction treatment had an impact on β-carotene, α-carotene and lutein contents in the carrot juice. In the case of peeling and blanching, the authors showed a decrease in carotenoid compounds content. On the other hand, use of enzymes in the carrot juice production may significantly increase the content of carotenoids [16].In the present study, the total polyphenols' content fluctuates in the wide range and depends on carrot variety and juices extraction methods. The highest amount of TPC was observed in black carrot juices. The high content of total phenolic (TPC) in black carrot juices is result of their high concentration in raw material [17]. Total phenolic content in carrot juices determined in the present study was similar to those demonstrated previously by the other authors [18].

The study showed that the extraction method affected phenolics' content in black and orange carrot juices. In the black carrot juice more phenolics were found in juices prepared with the use of LSJ, while in the orange carrot juices more phenolics contained juices obtained by HSJ. The studies of the other authors also confirm the relationship between the method of juice extraction and the content of polyphenols. For example, the studies of Pyo et al. [9] and Mphahlele et al. [19] demonstrated that juices prepared with the use of blender contain a significantly higher total phenolic content in compared to squeeze juice. Juices made with a blender contain more flesh which is rich in polyphenols, whereas squeezed juices are devoid of pomace to which most polyphenolic compounds pass [9,20].

In presented study, the strong correlations between the content of phenolics and antioxidant capacity measured by ABTS, PCL ACW, PCL ACL and the sum of PCL assays were found. It showed that phenolics play a significant role as antioxidants in carrot juices. The literature data also provided some information on the antioxidant capacity of carrot juices. Kim et al. [12] indicated the higher antioxidant activity of juices from the low-speed juicer. MacDonald-Wicks et al. [21], noticed that antioxidant capacity is the sum of the antioxidant activity of all types of antioxidant compounds present in the product that trap free radicals. In carrots, these compounds include polyphenols, carotenoids and antioxidant vitamins—C and E. The number of values of antioxidant activity of carrot juices for the methods used was as follows: sum of PCL > PCL, ACL > DPPH > PCL, ACW > ABTS. The highest antioxidant activity was established in ABTS, PCL ACW, PCL ACL and the sum of PCL for black carrot juices. According to the literature, methanolic extracts from black carrots possess the highest antioxidant activity determined with the use of DPPH assay [22]. Similarly to the results of present study, the results demonstrated by the other authors showed that, in comparison to juice obtained from yellow and orange carrots, black carrot juices exhibited the highest antioxidants activity [15,22–24].

The ratio of antioxidant potential of the lipophilic fraction (ACL) and hydrophilic fraction (ACW) was established at 12.4 and 4.4 in black carrot juices, 21.3 and 20.0 in orange carrot juices and 13.6 and 13.3 in orange carrot juices, obtained by the HSJ and LSJ methods, respectively. These proportions indicated that in a carrot juice, in which hydrophobic antioxidants are predominated, a species-diverse antioxidant profile is presented. The juices' antiradical activity for fat-soluble compounds (ACL) was significantly higher than the antiradical activity of water-soluble compounds (ACW). The PCL ACL assay indicated antioxidant activity in lipids, in which the soluble carotenoids contained in carrots were found; therefore, a strong positive correlation with the ABTS test was demonstrated. Due to the presence of water-soluble antioxidants in carrot juices (polyphenols and vitamin C) the PCL ACL assay was negatively correlated with the DPPH assay. The strong positive correlation between TPC and the ABTS, PCL ACL, PCL ACW assays and the sum of PCL and the strong negative correlation between TPC and the DPPH assay indicate that polyphenolic compounds that contributed to the increase in the activity of scavenging free oxygen radicals were characterized by different hydrophilicity [22]. In study, the antioxidant activity of carrot juices, measured with the ABTS, PCL ACL, PCL ACW assays and sum of PCL, is positively correlated mostly with the content of lutein, which indicates an important role of this carotenoid in formation of the antioxidant potential of carrot juices.

The juice preparation method had a significant impact on the antioxidant activity of black carrot juices. In the DPPH and ABTS assays, juices obtained with the use of HSJ were characterized by higher antioxidant activity than those obtained with the use of LSJ. In the PCL ACW, PCL ACL and total PCL assays, significantly higher antioxidant activity was confirmed in juices obtained with the use of LSJ method. The highest differences in antioxidant activity in the black carrot juices were demonstrated in the PCL ACW assay and LSJ-obtained juices showed three times higher antioxidant activity than HSJ-obtained juices. In orange carrot juices, in all the performed assays (except DPPH assay), a significant correlation was found between the method of juice extraction and antioxidant activity. In contrast to black carrot juices, in orange carrot juices in each of the assays, higher antioxidant activity was found for juices prepared by the use of HSJ. In yellow carrot juices, only in the ABTS test, there was a significant correlation between the method of juice extraction and the level of antioxidant activity. Higher values of antioxidant activity were obtained in juices prepared with the use of HSJ. In the studies on the influence of the juice extraction method on juices' antioxidant activity, various relationships were noted—the higher activity of juices prepared with LSJ method [25] or no significant influence of the extraction method on the antioxidant activity of juices [11].

#### **4. Materials and Methods**

#### *4.1. Chemicals and Reagents*

2,2′ -azinobis(3-ethylbenzothiazoline-6-sulphonic-acid) diammonium salt (ABTS), 2,2-diphenyl-1 picrylhydrazyl (DPPH) and 6-hydrozy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). ACW (hydrophilic condition) and ACL (lipophilic condition) kits for the photochemiluminescence (PCL) assay were received from Analytik Jena AG (Jena, Germany). Hexane, acetone, ethanol, methanol, toluene, sodium thiosulfate, isopropanol, Folin-Ciocalteu reagent, gallic acid, methanol, methylene chloride were purchased from Sigma Chemical Co. (St. Louis, MO, USA). B-apo-8′ -carotenal was received from Sigma-Aldrich (St. Louis, MO, USA).

#### *4.2. Plant Material and Juices Extraction*

The carrot (*Daucus carota* L.) varieties (Bangor—orange, Gele peen—yellow, Peen zwart—black) were obtained from a local market in Warsaw (Poland). The obtained carrots (5 kg for each variety) were thoroughly cleaned and washed. Carrot extracts were obtained using a low-speed (Kenwood Pure Juice JMP600WH, Warsaw, Poland) and high-speed (Waring Commercial Juice Extractor WJX50, China) juicers. The obtained juices were collected for analysis in appropriately labelled tubes. Carrot extracts were stored at −20 ◦C until the analysis (up to 2 days).

#### *4.3. Determination of Carotenoids Content*

The method described by Marx et al. [26] was used to determine the content of carotenoids. Carrot juice sample (5 g) was extracted three times in an amber glass separatory funnel with a 30 mL mixture of acetone and hexane (1:1, *v*/*v*). The emulsion formed was removed by adding 50 mL sodium

chloride solution (10%, *w*/*v*). After separation, the hexane layer was washed three times with water (50 mL) to remove acetone. Butylated hydroxytoluene (BHT) was added as an antioxidant (0.1%) and the extract was dried with sodium sulfate (2 g). The separated hexane phase was evaporated to dryness under a nitrogen stream at 40 ◦C and dissolved in 10 mL of methanol-methylene chloride (45:55, *v*/*v*). The solution was filtered through a membrane filter (ReZist® syringe filter 30 mm, pore size 0.2 µm) and was analyzed by high performance liquid chromatography (HPLC) according to the procedure described by Czaplicki et al. [27]. For analysis it was used an Agilent Technologies 1200 series apparatus (Palo Alto, CA, USA) equipped with a diode array detector (DAD) from the same manufacturer. The separation was carried out on a YMC-C30 150 × 4.6 mm, 3 µm column (YMC-Europe GmbH, Dinslaken, Germany) at 30 ◦C. The mobile phases consisted of methanol (solvent A) and methyl tert-butyl ether (MTBE) (solvent B) were used. The solvent gradient was as follows: 0–5 min, 95% A, 1 mL/min; 25 min, 72% A, 1.25 mL/min; 33 min, 5% A, 1.25 mL/min; 40–60 min, 95% A, 1 mL/min. The absorbance was measured at the wavelength of 450 nm. Carotenoids were identified based on the retention times of available standards and by comparing the UV-Visible absorption spectra. The content of carotenoids (mg/100 mL) was calculated based on the concentration of the internal standard and expressed in mg/100 mL juice.

#### *4.4. Determination of Total Phenolic Content (TPC)*

The TPC assay was adapted from the method described by Singleton and Rossi [28]. 0.1 mL of juice sample was mixed with 0.5 mL of Folin-Ciocalteu reagent, diluted 1: 2 with water. Then 1.5 mL of a 14% sodium carbonate solution was added and mixed. The prepared solution was kept in the dark at room temperature for 2 h. Absorbance was measured at 765 nm using an Optizen Pop UV/VIS spectrophotometer (Metasys Co. Ltd., Daejeon, Korea) against a blank sample. The total phenolics content was calculated based on the gallic acid calibration curve (concentrations in the ranges of 10–500 mg/L) and expressed in mg/100 mL juice.

#### *4.5. Antioxidant Activity Assays*

#### 4.5.1. DPPH Assay

Determination of antioxidant properties by the DPPH method was conducted according to the procedure developed by Brand-Williams et al. [29] and modified by Thaipong et al. [30]. The DPPH radical solution was prepared by dissolving 10 mg of DPPH in 250 mL of 80% methanol. To perform the spectrophotometric test, 300 µL of DPPH solution and 20 µL of juice sample or Trolox solution were mixed. The resulting mixture was left for 30 min at room temperature in the dark. Decreasing absorbance of the resulting solution was monitored at 517 nm using an Infinite M1000 plate reader (Tecan Group AG, Switzerland). The standard curve was determined based on the lag phase's length compared to Trolox concentrations in the range of 0.01–0.75 mM. The antioxidant activity was expressed as µmol Trolox/mL juice.

#### 4.5.2. ABTS Assay

The ABTS test described by Re et al. [31] was used to determine carrot extract's antioxidant activity. The measurement required dilution of the ABTS solution using a methanol/water mixture (80:20, *v*/*v*) to achieve an absorbance level of 0.70 ± 0.02 at 734 nm. For the spectrophotometric test, 290 µL of ABTS solution and 10 µL of the Trolox or juice sample were mixed and absorbance was measured directly after 6 min The standard curve was determined based on the lag phase's length compared to Trolox concentrations in the range of 0.01–0.75 mM. Measurements were carried out using the Infinite M1000 PRO plate reader (Tecan Group AG, Männedorf, Switzerland). The antioxidant activity was expressed as µmol Trolox/mL juice.

#### 4.5.3. Photochemiluminescence (PCL) Assays

The PCL method with the Photochem apparatus (Analytik Jena, Leipzig, Germany) was used to measure antioxidants' effectiveness against superoxide anion radicals. Antioxidant activity was analyzed using the ACW (antioxidative capacities of water-soluble) and ACL (antioxidative capacities of lipid-soluble) kits. The assay was conducted as previously described by Sawicki et al. [32]. For ACW and ACL tests, the luminal reagent and Trolox working solution were prepared according to the protocol. Juice solution concentration added that the generated luminescence was in the range limits of the standard curve. Therefore, the lag time (180 s) for the ACW test was used as a free radical scavenging activity. The antioxidant activity was calculated by comparing it with the Trolox standard curve (0.5–3 nmol) and expressed as µmol Trolox/mL juice. In the ACL test, the kinetic light emission curve, showing no lag phase, was monitored for 180 s and expressed in µmol Trolox/mL. The antioxidant test was performed in triplicate for each sample.

#### *4.6. Statistical Analysis*

The values were expressed as mean ± standard deviation (SD). The results were subjected to a one-way analysis of variation (ANOVA) using Duncan's test. A linear Pearson's correlation coefficients were calculated to show relationship between bioactive compounds and antioxidant activity and *p* < 0.05 was considered significant. Principal Component Analysis (PCA) was also carried out to show differences between juice samples. The statistical analysis was performed using Statistica 13.1 (Statsoft Inc., Tulsa, OH, USA).

#### **5. Conclusions**

The carrot juices obtained in the current study were a rich source of carotenoids and phenolic compounds and the content of these bioactive depended on carrot variety and juice extraction method. It was shown that the orange carrot juices were most abundant in the carotenoids, while black carrot juice was characterized by the highest TPC. The orange carrot juices obtained with the use of HSJ contained a higher amount of carotenoids and phenolic compounds in comparison to the juice obtained using LSJ. Contrary, black carrot juices prepared with the use of HSJ contained significantly lower content of phenolic compounds compared to the juice obtained with the use of LSJ. Moreover, the extraction method had a significant impact on the antioxidant activity of the obtained juices. Among the carrot varieties, the highest antioxidant activity exhibited black carrot juice. The experiments performed with the use of PCL antioxidant activity assays demonstrated that black carrot juice obtained with the use of LSJ was characterized by a higher antioxidant activity in comparison to that obtained with HSJ. Contrary, for orange and yellow carrots juices, a higher antioxidant activity was demonstrated for juices obtained with the use of HSJ. It may be concluded that juices prepared with the use of a low-speed juicer were not always characterized by the higher content of bioactive compounds and antioxidant potential.

**Author Contributions:** Conceptualization, J.C., M.T. and T.S.; methodology, J.C., M.T., A.N., M.S. and T.S.; formal analysis, A.P., M.T., A.N., M.S. and T.S.; investigation, A.P., M.T., A.N., M.S. and T.S.; writing—original draft preparation, A.P., J.C., M.T., A.N. and T.S.; writing—review and editing, M.S. and T.S.; visualization, A.P. and T.S.; supervision, J.C., M.T. and K.E.P.; funding acquisition, K.E.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by Minister of Science and Higher Education in the range of the program entitled "Regional Initiative of Excellence" for the years 2019–2022, Project No. 010/RID/2018/19, amount of funding 12.000.000 PLN.

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

#### **References**


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