Phytochemical Content and Antioxidant Activity of Ancient Majorca and Carosella (Triticum aestivum L.) Wheat Flours

Abstract

Two locally grown ancient wheat varieties named Carosella and Majorca (Triticum aestivum L.) were studied for their phytochemical contents and biological activities. Four different types of flour obtained from each variety were investigated. Carosella and Majorca samples showed high concentration of polyphenol content and high antioxidant activity. Among the different types of flour obtained by different grindings (whole wheat, 2, 1, and 0), whole wheat showed the highest antioxidant activity expressed as inhibition of DPPH radical, with an IC₅₀ value of 0.008 mg/mL for Carosella and 0.011 mg/mL for Majorca. The total polyphenol content ranged from 11.72 to 14.34 g/kg in Carosella samples and from 11.96 to 13.67 g/kg in Majorca samples. The results of this study reveal that the two wheat varieties, Carosella and Majorca, could be considered potential sources of antioxidant agent and could play a major role in human health.

1. Introduction

Consumed by billions of people, wheat (Triticum spp.) is the main basic food in many diets and accounts for a large percentage of the daily carbohydrate energy intake. It is a cereal currently cultivated all over the world [1]. In 2016, world wheat production exceeded 749 million tons, becoming the second most cultivated corn after maize. About 95% of the wheat produced is Triticum aestivum L., a hexaploid species [2]. The remaining portion mainly consists of Triticum durum L., or “hard” wheat, a tetraploid species mainly used in the production of pasta [3]. In the context of a balanced diet, wheat is a healthy source of multiple nutrients such as fibers and bioactive compounds, especially when consumed as whole grains. Cereals are plants belonging to the Poaceae family and are essential for sustenance, both for their nutritional value and for their chemical properties [4]. Wheat cultivation declined dramatically during the 1960s due to food and economic changes, in particular the introduction of bread and durum wheat [5]. Cereal products can be considered among the first foods included in the category of dietary foods. They are grown for their fruit, which is a caryopsis formed from the seed wrapped in a series of protective layers. The great interest in their cultivation is due to, on the one hand, the fact that they gather in a small volume important quantities of nutrients (proteins, carbohydrates, mineral salts, vitamins) and that they provide a large amount of calories (about 50% of the caloric value of the daily ration) and, on the other hand, the fact that they have economic advantages related to their short vegetation period and ease of transport and storage [1].

However, in recent years, the growing demand for organic and natural products has led to the rediscovery of ancient species of wheat [6]. This renewed interest in ancient grains is associated with the idea of a healthy and balanced diet, such as the Mediterranean diet. In fact, ancient wheat has been recognized as a healthy and dietary cereal and is recommended for the treatment of blood diseases, such as high cholesterol, but also colitis and allergies [7,8], in addition to its beneficial effects on insulin resistance [9]. The use of a lower quantity of fertilizers often makes ancient grains higher in height compared with conventional grains; moreover, they have a nutraceutical value superior to that of varieties patented after the green revolution [10]. The mechanisms by which the grain confers protective effects on human health are attributed to the physical properties and structure of the grains (granular size of semolina, quantity and type of fiber, quantity and quality of phytochemicals, amylose and amylopectin content) [11]. In recent years, there has been great interest in improving wheat yield because of the increase in wheat demand due to both population growth and the increasing consumption per capita. On the other hand, increase in yield should not compromise food safety, as wheat products contribute to dietary acrylamide intake. Selection and optimized crop management could reduce the formation of this carcinogen contaminant produced during the high-temperature cooking and processing of foods made from potatoes, cereals, and other crops [12]. In particular, ancient wheat species have acquired growing attention since numerous studies have suggested that they could present a healthier and better nutritional profile than modern grains, providing more vitamins, minerals, and nutraceutical compounds [13]. The most common species of antique wheat available on the market are Triticum monococcum and T. dicoccum Schrank or T. spelta L. and khorasan (T. turgidum L. ssp. turanicum). In addition, there are several historical cultivars of T. aestivum and T. durum Desf. that have remained unchanged over the years: Russello, Senatore Cappelli, Timilia or Tumminia, and Urria (T. durum), as well as Majorca, Sieve, Solina, Verna and Carosella (T. aestivum) [14,15,16,17].

This last wheat is a typical variety of Cilento that is also grown in southern Italy between the Calabria and Basilicata regions [18]. It is a soft wheat with a small, elongated, light, shiny, and golden grain [19]. The Carosella plant reaches at least 1 meter in height, while the roots branch off for tens of meters, making it possible to classify it as a very ancient type of wheat dating back to Roman times [18]. Carosella wheat is a semiwild seed, and the flour is consumed in its integral form. Over the centuries, this seed has been progressively set aside to make room for other types of wheat, which have presented characteristics more suited to mechanical threshing and other agricultural machinery, almost extinguished in favor of technological progress [19]. The Carosella flour is obtained by grinding an ancient variety of soft wheat (Triticum aestivum). Its name, which in local dialect is “Carusedda,” is derived from “wheat tosello” (without head sheared or “caruso”). The caryopsis has a very particular groove that differs from other grains; the outer shell can be white or red, giving rise to two different ecotypes: “red carousel” and “white” [18]. Carosella flour can be used for the preparation of pasta, bread, desserts, and pizzas [20].

Majorca wheat, on the other hand, is an ancient variety of soft wheat scientifically known as Triticum vulgare (this name is a synonym of T. aestivum, as shown in http://www.theplantlist.org/tpl1.1/record/kew-449183, accessed on 6 June 2021), characterized by its remarkable height of 180 cm. It has a quadrangular spike with reddish beards. It is suitable for dry soils and characterized by fast maturation. It was cultivated already in the Bourbon era not only in Puglia but also in Basilicata and Calabria and Sicily, where it was used regularly in the production of bread, pasta, and cakes until the 1930s. In 1927, there were only 52 indigenous varieties of wheat, and Majorca wheat accounted for 2.37% of the entire Italian production. However, with the advent of the improvement of consumption, it has not been taken into account because a more abundant production at lower prices is required [21].

Since 2002, whole grains have been included in the recommendations of the American Diabetes Association for the prevention of diabetes. Some studies on wheat cultivars demonstrated reduction in free radicals and inflammation in the blood plasma in rats and in liver tissues [22,23,24]. In several studies, antioxidant activities were analyzed according to different types of wheat grains, as well as their phenolic profiles. Adom and Liu [22] showed that ferulic acid is the main phenolic compound in grains. Choi and coworkers [25] separated 10 phenolic compounds from T. aestivum hydrophobic fractions, among which benzoic acid, quercetin, and luteolin were abundant. The consumption of whole grains or their components has beneficial effects in reducing the risk of a number of cardiovascular diseases and some forms of cancer [26]. Polyphenolic compounds and their associated antioxidant activity are one of the most important constituents of wheat widely studied in recent years. Phenolics exhibit strong antioxidant activity, scavenging or neutralizing free radicals, and this reduces or minimizes oxidative damage to DNA, proteins, and lipids. This reduction in oxidative damage to cells and cell components may explain their preventive effect against diseases related to oxidative stress. Free radicals, especially reactive oxygen species (ROS), are produced during aerobic metabolism in response to external stimuli or by environmental pollution. Excessive ROS generation or impaired endogenous antioxidants or a combination of these events produces oxidative stress, leading to protein oxidation, lipid peroxidation, DNA base modifications, and strand breaks [27].

Current interest in the health benefits provided by grain consumption has led to an increased focus on the phytochemical content of different grains and grain varieties, even to the extent of considering the use of some progenitor species of wheat, such as Aegilops ventricosa Tausch, which has high concentrations of microelements such as zinc, a very useful element for human health [28], very rare in many plant species, including the known wheat varieties. The aim of the present study was to evaluate the phytochemical content and the antioxidant activity of the two locally grown ancient wheat varieties, Carosella and Majorca (Triticum aestivum). Phytochemical contents and biological activities were evaluated in four different types of flour obtained from each variety.

2. Materials and Methods

2.1. Samples and Preparation of the Extracts

Two locally grown ancient wheat varieties named Carosella and Majorca (Triticum aestivum) were studied. Four different types of flour obtained from each variety were taken into account: whole wheat, 2, 1, and 0. The samples were provided by Fattoria Biò Soc. Semp. Agricola, Camigliatello Silano, Cosenza, Italy (geographic coordinates 39°23′37.5″ N–16°27′53.7″ E), and the identification was done by F. Conforti, University of Calabria.

Extraction was performed using the maceration technique. A fixed quantity of flour was put in contact with a specific volume of the most appropriate solvent for a variable time; in this case, it was 48 h for extraction, for a total of two consecutive extractions. In this work, the selected solvent was methanol. According to the extraction procedure, 100 g of flour was left in contact with 200 mL of methanol for about 48 hr. After being filtered, the methanol extracts were thus obtained and dried under reduced pressure using a rotary evaporator with a bath of water at 40 °C to preserve the chemical composition of the extract.

2.2. Determination of Total Phenolic Content

The total phenolic compound content was determined using the Folin–Ciocalteu reagent. An amount of 1 mL of this reagent was added to 1 mL of Na₂CO₃ 7.5% w/v and to 200 mL of sample previously prepared at a concentration of 2 mg/mL in a mixture of acetone/methanol/water/acetic acid (40:40:20:0.1). Two hours later, the absorbance was measured at 726 nm [29]. The results were expressed as mg of chlorogenic acid equivalents per g of dry matter (g CAE/kg DM).

2.3. Determination of Total Flavonoid Contents

Flavonoid content was achieved using an aluminum chloride colorimetric method. An amount of 1 mL of AlCl₃ 2% was added to 1 mL of sample previously prepared at a concentration of 2 mg/mL in EtOH 80%. After 15 min, the absorbance was measured at 430 nm [30]. The results were expressed as mg of quercetin equivalents per g of dry matter (g QE/kg DM).

2.4. Free Radical Scavenging Activity (FRSA) Assay

The antioxidant activity of all the extracts was assessed by DPPH free radical scavenging ability according to the previous protocol [31]. The test was based on the reduction of a purple methanolic solution of the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). An amount of 0.8 mL of methanolic solution of DPPH (0.1 mM) was mixed with 0.2 mL of various concentrations (5–200 μg/mL) of sample solutions. After 30 min in the dark at room temperature, the absorbance of the samples was measured at 517 nm by using a Perkin Elmer Lambda 40 UV–VIS spectrophotometer. The solution containing methanol and DPPH without sample was used as a blank. The percentage inhibition of DPPH free radical was calculated by using the following formula:

Percentage of inhibition = {1 − [(DPPH absorbance with extract)/(DPPH absorbance without extract)] × 100}.

Ascorbic acid was used as a positive control.

2.5. Statistical Analyses

Each in vitro test was performed in triplicate. Data were expressed as mean ± SEM. Normality of data and homogeneity of variances were assessed with D’Agostino–Pearson’s K2 test and Levene’s test, respectively.

Raw biological data were fitted through nonlinear regression in order to deduce the IC₅₀ values (GraphPad Prism Software version 5.00, San Diego, CA, USA). Statistical differences between treated groups and the control and among treated group means were estimated by one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test and the Bonferroni post hoc test, respectively (p < 0.05, SigmaStat Software version 3.5, Jantel Scientific Software, San Rafael, CA, USA).

3. Results and Discussion

3.1. Percentage Yields

The composition of the flour is influenced by the grinding process. The grinding process begins with wetting (conditioning), which increases the moisture of the grain (48 h). Subsequently, the cereals are conveyed to the mill, which begins to strip the grain of the external part through a system of pairs of metal cylinders that rotate in opposite directions (cylinder mill). Subsequently, the grinding proceeds with the refining, which is the removal of the bran from the flour: the operation is called sifting. In relation to the degree of sifting, which is the percentage of residual minerals and bran present in the ground grain, wheat flours are classified by law into five basic types. We analyzed four types of flour: wholemeal; flour type 0, which has a sifting of 72%; semi-wholemeal type 1, which has a sifting of 80%; and semi-wholemeal type 2, which has a sifting of 85%. Finally, the wholemeal flour only undergoes the first grinding phase and has a 100% sifting rate; therefore, it contains the ground kernels entirely. The last one is the most fiber-rich flour ever but also the most dangerous, as residues of pesticide treatments can be found on the outside of the kernels, which would pass entirely into the flour. For this reason, especially when choosing wholemeal flour, it is important that it is an organic product or grown with natural methods [32]. The phytochemical study of samples was initially provided for the extraction of the secondary metabolites (the active principles of the plant). Extraction is an operation that allows the separation of the active ingredients from vegetable or food matrices and can be carried out in different ways according to the nature of the plant. Previous studies evaluated different solvents of extraction, such as diethyl ether, acetone, ethanol, methanol, and a combination of these solvents with water at different proportions [33].

In this study, all samples were extracted with methanol through the maceration procedure. Methanol is a polar solvent able to solubilize, in a single solution, almost all the active ingredients present in the vegetable flour. The extractive yield of the selected samples obtained from the four different types of flour obtained from two wheat varieties is presented in

Table 1. Yield percentage of methanolic extracts obtained from Carosella and Majorca samples (% dry matter).

Sample	Flour Type	Yield %
Carosella	Whole wheat	6.3 ± 0.3 a
	2	5.4 ± 0.3 a,b
	1	5.2 ± 0.3 b
	0	4.2 ± 0.2 c
Majorca	Whole wheat	6.1 ± 0.3 a
	2	5.6 ± 0.3 a,b
	1	5.1 ± 0.3 b
	0	4.5 ± 0.2 c

Results are expressed as mean ± SD of three replicates. Values followed by different letters are significantly different according to the Bonferroni post hoc test (p < 0.05).

. It is worth noting that a higher yield percentage was observed in whole wheat with the highest content of hull with a yield of 6.3% in the Carosella sample and 6.1% in the Majorca sample (Figure 1).

3.2. Total Phenolic Compound Content

The content of total phenolics in flour extracts was determined using the Folin–Ciocalteu assay and expressed as chlorogenic acid equivalents (CAEs). The colorimetric dosage revealed the presence of a high quantity of phenolic compounds.

Whole wheat samples showed the highest total phenolic content, with values of 14.34 ± 0.28 and 13.90 ± 0.33 g per kg of dry matter (DM). The lowest content was observed in the other three flour types (Bonferroni post hoc test, p < 0.05,

Table 2. Total polyphenol content of Carosella and Majorca samples.

Sample	Flour Type	Total Polyphenols (g CAE/kg DM)
Carosella	Whole wheat	14.34 ± 0.28 a
	2	12.43 ± 0.33 b
	1	12.28 ± 0.35 b
	0	11.72 ± 0.52 b
Majorca	Whole wheat	13.90 ± 0.33 a
	2	12.67 ± 0.25 b
	1	12.27 ± 0.34 b
	0	11.96 ± 0.47 b

Total phenols are expressed as equivalents of chlorogenic acid (CAE) per kg of dry matter (DM). Data are expressed as means ± SD (n = 3). Values followed by different letters are significantly different (Bonferroni post hoc test, p < 0.05).

, Figure 2).

The flavonoid content followed the same trend. The two whole wheat samples showed the highest content, with values of 0.410 ± 0.018 and 0.404 ± 0.015 g per kg of dry matter (DM) (

Table 3. Total flavonoid content of Carosella and Majorca samples.

Sample	Flour Type	Total Flavonoids (g QE/kg DM)
Carosella	Whole wheat	0.410 ± 0.018 a
	2	0.312 ± 0.013 b
	1	0.270 ± 0.014 b,c
	0	0.242 ± 0.015 c
Majorca	Whole wheat	0.404 ± 0.015 a
	2	0.372 ± 0.017 a,b
	1	0.342 ± 0.010 b
	0	0.320 ± 0.023 b

Data are expressed as equivalents of quercetin (QE) per kg of dry matter (DM). Data are expressed as means ±SD (n = 3). Values followed by different letters are significantly different (Bonferroni post hoc test, p < 0.05).

, Figure 3).

The obtained results are very interesting when compared with those of other studies. Abozed and coworkers [34] evaluated the phenolic content of two varieties of T. aestivum and found a content of phenolic of less than 3 mg per g of dry matter, while in the Carosella and Majorca varieties, we found a content of phenolics greater than 11 mg per g of dry matter. Vaher et al. evaluated also the phenolic content of different wheat varieties and found the highest content in the bran layers with values ranging from 1.258 to 3.157 mg/g. The authors also identified the phenolics present in the flours, with sinapic acid, vanillic acid, and ferulic acid being the major compounds [35]. Brandolini et al. investigated different Triticum species, including T. aestivum, and assessed the presence of p-coumaric, vanillic, syringic, ferulic, p-hydroxybenzoic, and caffeic acids and syringaldehyde [36].

A different trend, on the other hand, was found in a study by Amessis-Ouchemoukh et al. [37], who studied the whole grains of T. aestivum and T. vulgare from Algeria. In this study, cereal powders were extracted by maceration with ethanol, and the obtained extracts contained about 44 mg per g of dry matter in T. vulgare and about 47 mg per g of dry matter in T. aestivum of total phenolics.

Interestingly, Liyana-Pathirana and colleagues reported the phenolic content of soft and hard wheat both as such and subjected to simulated gastric conditions, obtaining values of 22 and 23 µg of ferulic acid equivalents per gram of defatted material for untreated samples and 74 and 118 µg/g for treated samples [38].

3.3. Antioxidant Activity

The free radical scavenging activity can be measured using the DPPH assay. This assay is based on the use of a radical compound, 2,2-diphenyl-1-picrylhydrazyl (DPPH). The DPPH radical is of purple-red color, but the addition of an antioxidant agent reduces the radical, making it yellow. The percentage of discoloration is proportional to the antioxidant “scavenger” activity of the examined extracts. This spectrophotometric analysis allows for evaluating the antioxidant capacity based on the absorbance decreases that are observed following the binding of the antioxidants present in the sample to the radical for donation of hydrogen to the radical [29]. The capacity of flour extracts to scavenge the stable DPPH radical is shown in

Table 4. Free radical scavenging activity of Carosella and Majorca samples.

Sample	Flour Type	DPPH IC50 (mg/mL)
Carosella	Whole wheat	0.0080 ± 0.0001 b
	2	0.0202 ± 0.0008 c,d
	1	0.0220 ± 0.0010 d
	0	0.0240 ± 0.0013 d
Majorca	Whole wheat	0.0110 ± 0.0010 b
	2	0.0161 ± 0.0011 c
	1	0.0192 ± 0.0012 c,d
	0	0.0203 ± 0.0015 c,d
Ascorbic acid *		0.0020 ± 0.0001 a

Data are expressed as mean ± SEM (n = 3). Different letters indicate statistically significant differences at p < 0.05 (Bonferroni post hoc test). * Positive control.

, which summarizes the results for quenching of important reactive oxygen species (ROS), such as hydroxyl radical (HO ^·) and superoxide radical anion (O₂ ^·−), as well as hydrogen peroxide (H₂O₂).

All samples showed high antioxidant activity (Figure 4). In particular, whole wheat flours were the most active ones with IC₅₀ values of 0.0080 ± 0.0001 and 0.0110 ± 0.0010 mg/mL for Carosella and Majorca samples, respectively (Table 4). The other samples showed also good radical scavenging activity with IC₅₀ values of 0.0202 ± 0.0008, 0.0220 ± 0.0010, and 0.0240 ± 0.0013 for Carosella 2, 1, and 0 samples, and IC₅₀ values of 0.0161 ± 0.0011, 0.0192 ± 0.0012, and 0.0203 ± 0.0015 for Majorca 2, 1, and 0 samples, respectively.

Various previous studies have demonstrated the high radical scavenging activities of wheat grain, such as Amessis-Ouchemoukh et al. [37], who found a percentage of inhibition of 67% for T. aestivum and 40% of T. vulgare extracts using 0.08 mg/mL concentration. In our study, all the flours showed high activity. Lower inhibition rates were obtained also in the study of Abozed and coworkers [34], who evidenced a percentage of inhibition lower than 30% for all the tested samples.

Liyana-Pathirana and coworkers reported the DPPH radical scavenging activity of commercial soft and hard T. aestivum subjected to simulated gastric conditions. Defatted soft and hard wheat flours were demonstrated to be able to scavenge 1.8 and 1.9 µmol/g DPPH radical, while the corresponding values increased when wheat samples were subjected to simulated gastric pH conditions [38].

Brandolini and colleagues evaluated the antioxidant activity of T. aestivum subsp. aestivum and subsp. spelta soluble-conjugated and insoluble-bound extracts and found that the antioxidant activity was highly correlated to phenolic acid content [36].

The antioxidant properties of T. aestivum [39] and of several wheat samples have been also investigated by other authors [34,40,41,42,43]. However, a comparison of our results with those reported in the literature is difficult because of the different combinations of concentration parameters and diverse methods of evaluating the antioxidant activity.

4. Conclusions

The major sources of energy, protein, B vitamins, and minerals of the population of the world are cereals and cereal-based foods. The health benefit (lower risk of cardiovascular disease, ischemic stroke, type II diabetes, metabolic syndrome, and gastrointestinal cancers) of the consumption of whole grains may be due to their phytochemical composition. The consumption of whole grain cereals can have health effects because it reduces diabetes, obesity, constipation, cardiovascular disease, and other lifestyle disorders, as different studies have reported [44].

The structure of all grains includes the endosperm, germ, and bran. The endosperm comprises <80% of the whole grain, whereas the percentages accounted for by the germ and bran components vary among different grains [33]. Most cereals are converted to flour before usage. Milling is defined as an act or process of grinding, especially the grinding of grain into flour or meal. It is an important and intermediate step in the postproduction of grain. In the milling process, the husk and sometimes the bran layers are removed and produce an edible portion without impurities and in the form of a powder with varying particle sizes. The milling process changes the composition and matrix of the grain. The grade of milling and refining can produce very fine flour that has different amounts of nutrients in comparison with its original sources. In whole grain can be found different elements that are associated with health status, such as lignans, tocotrienols, phenolic compounds, and antinutrients, including phytic acid, tannins, and enzyme inhibitors. In the process of refining the grain, the bran is separated, resulting in the loss of the elements cited previously. The refined grains are more concentrated in starch. The phytochemicals are involved in health-improving activities, which are very important for health. Therefore, using whole grain or milled flour without sieving and separating different portions can be beneficial for human health [45]. The outer layer of grains is rich in antinutrients, which can be reduced by dehulling. Previous work evidenced that the major compositional difference between whole grains and their milled form is the reduction of all nutrients, which are contained in the external layer, fiber, phytic acid, tannin, polyphenols, minerals, and some vitamins [34]. The reduction of phytate, tannin, and phenolic elements could improve the availability of minerals and the digestibility of protein and carbohydrates, but these components also exhibit strong antioxidant properties, which may stop free radical activity and reduce oxidative stress in the human body [46].

The domestication of wheat, together with breeding efforts, allowed an increase in yields but resulted in grain quality deterioration. It has been demonstrated that old wheats have higher micronutrient content compared with modern ones [14]. Furthermore, many crops originate from segetal weeds, and both the coevolution of this kind of wild species and crops and the management practices of crop fields that affect the sustainability of agriculture should be deeply investigated, as underlined by Perrino and Calabrese [47].

In our study, we confirmed that the grade of milling and refining produces different phenolic compositions and different antioxidant activities. We also demonstrated that the studied ancient varieties showed better phenolic composition and antioxidant activity and so could be good sources of active compounds with beneficial effects in reducing the risk of a number of diseases.