Variability in Bulb Organosulfur Compounds, Sugars, Phenolics, and Pyruvate among Greek Garlic Genotypes: Association with Antioxidant Properties

In order to assess the diversity of Greek garlic (Allium sativum L.) landraces, 34 genotypes including commercial ones were grown in the same field and their content in organosulfur compounds, pyruvate, total sugars, and total phenolics, alongside antioxidant capacity, was determined. The organosulfur compounds were studied by Gas Chromatography–Mass Spectrometry (GC–MS) after ultrasound-assisted extraction in ethyl acetate, identifying 2-vinyl-4H-1,3-dithiin and 3-vinyl-4H-1,2-dithiin as the predominant compounds, albeit in different ratios among genotypes. The bioactivity and the polar metabolites were determined in hydromethanolic extracts. A great variability was revealed, and nearly one-third of landraces had higher concentration of compounds determining bioactivity and organoleptic traits than the imported ones. We recorded strong correlations between pyruvate and total organosulfur compounds, and between antioxidant capacity and phenolics. In conclusion, chemical characterization revealed great genotype-dependent variation in the antioxidant properties and the chemical characters, identifying specific landraces with superior traits and nutritional and pharmaceutical value.


Introduction
Common garlic (Allium sativum L., family Alliaceae) is the second most widely consumed bulb crop and one of the most cultivated bulb vegetables in Greece and worldwide, with an annual production of 28,494,130 tons and a total harvested area of 1,546,741 hectares [1,2]. It is consumed raw, cooked, or as an ingredient of herbal medicinal products and food supplements [3,4]. Garlic is considered effective and safe for the prevention and treatment of cardiovascular and other metabolic diseases, such as atherosclerosis, hyperlipidemia, thrombosis, hypertension, and diabetes; it also possesses antifungal, antibacterial, and antiviral properties and regulates blood sugar levels [5]. Among other biological mechanisms mediated by its components, garlic extracts also present significant in vitro and in vivo antioxidant properties [3].
Raw garlic bulbs contain mostly water, carbohydrates, and proteins but also trace elements and vitamins [1]. The main bioactive compounds are saponins, flavonoids, organic acids, and various organosulfur compounds [3]. The latter are present in intact bulbs as peptides, like γ-glutamyl-S-alk(en)yl-l-cysteine, and sulfoxides of S-alk(en)ylo-l-cysteine, like alliin, which is the predominant cysteine derivative. This compound is metabolized to allicin by the enzyme alliinase, when the bulb is crushed, also producing ammonia and pyruvic acid. Allicin and other sulfoxides may undergo many transformations both in vitro and in vivo, resulting in a wide variety of organosulfur volatiles, which are responsible for the flavor and aroma, as well as for most of the beneficial health effects of garlic [6,7]. Although many differences in the bioaccessibility and bioactivity of those compounds have been recorded so far, phenolics and saponins may also contribute to the antioxidant and anti-inflammatory properties of garlic, whereas polysaccharides (>85% fructose) have exhibited immunomodulatory effects [3,7]. Furthermore, the chemical composition and organoleptic characteristics of garlic are influenced by the genotype, the cultivation/environmental conditions, and the processing methods (temperature, pH, solvent) [8].
Garlic is a completely sterile diploid species, which has been clonally propagated for centuries [9]. Over time, cultivated garlic clones or clonal lineages have been established through domestication in several cultivation centers. These distinct genotypes have gained adaptation to different agroclimatic conditions and various ecotypes, exhibiting large-scale phenotypic diversity and variation in several traits [9]. Variation among garlic genotypes is the basis for breeding new varieties with superior traits. In this context, there is considerable interest for local genotypes (landraces and/or farmers' varieties) with respect to their content of bioactive compounds and the antioxidant properties of its cloves [10][11][12].
Crop landraces comprise an important part of agricultural biodiversity. Landraces are variable populations, genetically diverse, lacking "formal" crop improvement. They constitute an invaluable genetic pool due to their characteristics including local adaptation, resilience to biotic and abiotic conditions, and considerable organoleptic traits and nutritional value [13]. During the last years, they have been displaced by more productive and uniform improved varieties and hybrids, a trend which has led to a reduction of the crops' genetic base, and subsequently to genetic erosion, and to an increased threat of genetic vulnerability. Recently, due to the increased demand for natural, local, and high-quality products produced by traditional and environmentally friendly practices, landraces have been rediscovered as a source of value-added foods [14].
The phenotypic diversity and nutritional value of certain Greek garlic genotypes have recently been reported by our groups [11,12,15]. The aim of the present study was to determine the main bioactive compounds and to evaluate in vitro the antioxidant properties of Greek garlic germplasm; organosulfur compounds were determined for the first time and ultrasound-assisted extraction was adopted for the study of both volatiles and polar ingredients. For that purpose, we cultivated 34 garlic genotypes, including Greek landraces and commercial cultivars, under the same conditions (same location and cultivation practices). It was expected that the study of many local and imported garlic genotypes would reveal genotype-dependent diversity in chemical characters and antioxidant properties and contribute to the exploitation and valorization of this valuable genetic material.

Plant Material
Thirty-four garlic genotypes, including 29 local and commercialized landraces and 4 commercial cultivars, were examined in the present study. The geographical coordinates of the genotypes' collection sites are presented in Table 1. The garlic genotypes were planted and cultivated in the experimental field of Kavasila, Ileia Regional Unit (37 • 52 N, 21 • 17 E) during the growing period 2016-2017 (all the accessions were planted on 5 December 2016 and harvested on 15 June 2017), as previously described [15].

Preparation of Extracts
Cloves of fresh garlic bulbs were separated and skinned; 10 g of each accession were weighed and ground to a paste with a mortar and a pestle. The obtained garlic paste was subjected twice to ultrasound-assisted extraction (UAE) in an ultrasound bath (40 kHz, ISOLAB Laborgeräte GmbH, Wertheim, Germany) for 30 min with 60 mL ethyl acetate each time. The ethyl acetate extracts were collected and extracted further with water, while the remaining garlic paste was extracted with 100 mL methanol:water (50/50, v/v) under stirring for 24 h. The aqueous phase and the hydromethanolic extract were pooled and lyophilized (polar extract), while the ethyl acetate extract (nonpolar extract) was concentrated with nitrogen. The extracts were stored at −20 • C until further use.

Determination of Dry Weight
Dry weight (D.W.) was calculated by heating approximately 10 g of fresh sample (5-10 cloves) in preweighed porcelains at 105 ± 2 • C for 22-24 h, until constant weight. Samples were cooled down for 30 min in laboratory desiccators containing silica gel and then weighed.

GC-MS Analysis of Volatiles in Nonpolar Extracts
Analysis was performed by GC-MS on Agilent 6890N GC apparatus coupled to an Agilent 5975 B mass spectrometer (Agilent Technologies, CA, USA), with a nonpolar column HP-5MS (30.0 m × 250.00 µm, film thickness 0.25 µm), with electron impact ionization energy at 70 eV. Helium was used as a carrier gas at 1.0 mL/min flow rate. Injection volume was 1 µL in splitless mode; scan range was 50-1050 m/z. Injector temperature was set at 300 • C, and source temperature at 230 • C. Solvent delay was set at 3 min, initial oven temperature was 50 • C and then was ramped at 1 • C min −1 to 61 • C, remained at 61 • C for 4 min, ramped at 1 • C min −1 to 115 • C, and then at 2 • C min −1 to 191 • C and at 15 • C min −1 to 281 • C, remained at 281 • C for 3 min, and finally ramped at 25 • C min −1 to 300 • C.
Tentative identification was performed by examination and comparison to the literature of their MS spectra and retention indices (AI), using the Van den Dool and Kratz equation based on a series of linear alkanes, C8-C20 and C21-C40 [16]. Octane was used as both an internal and external standard. Concentration (from duplicate analyses) was determined as n-octane equivalents through the equation where y = µg n-octane/mL and x = response factor of the analytes (i.e., the ratio of peak area of each analyte to that of the internal standard at the concentration of 1.20 g L −1 ); the calibration curve was established with seven different n-octane concentrations (0.15, 0.30, 0.60, 1.20, 1.60, 2.00, and 2.50 g L −1 ). The coefficient of variation of the analyses never exceeded 14.8%. Detection level was set at 0.1% of total peak area. Peaks were quantified only if their response factor was higher than 0.025.

Determination of Pyruvic Acid, Total Sugars, Total Phenolics, and Antioxidant Activity of Hydromethanolic Extracts
Pyruvic acid, total phenolics, total sugars content, and antioxidant capacity were measured in the dry aqueous methanolic extracts (twice in triplicates). All methods except for that of hydrogen peroxide scavenging were adapted for 96-well plates and the absorbance was measured in a UV/vis microplate reader (Sunrise, Tecan, Männedorf, Switzerland).
Total sugars were determined by the anthrone method [18,19]. Forty µL of samples (50, 80, and 100 mg dry extract L −1 ) or standard sucrose (0.015, 0.030, 0.060, 0.120, 0.240, and 0.480 g L −1 ) or blank were cooled at 4 • C for 15 min and then were mixed with 100 µL of freshly prepared anthrone reagent (2 g L −1 in concentrated sulfuric acid). After 3 min in a water bath at 92 • C, the microplate was immersed in a water bath at 25 • C for 5 min and then was placed in an oven at 45 • C for 15 min. Absorbance was measured at 620 nm and concentration is expressed as mg sucrose equivalents per 100 g of F.W., according to the equation y = 0.409x − 0.002 (R 2 = 0.999).
The antioxidant activity of the dry methanolic extracts was evaluated with two different assays:  [21]. For this purpose, an H 2 O 2 (43 mM) solution was prepared in phosphate buffer (0.1 M, pH 7.4). Extracts (4 g dry extract L −1 ) as well as ascorbic acid (0.1 to 0.8 g L −1 ) in 3.4 mL phosphate buffer were added to 0.6 mL of H 2 O 2 solution. The percentage of H 2 O 2 scavenging of ascorbic acid and extracts was calculated by measuring the absorbance at 230 nm, subtracting that of their respective blanks (extracts only), and comparing to that of H 2 O 2 alone. H 2 O 2 scavenging effect is expressed as g ascorbic acid equivalents/100 g F.W.

Statistical Analysis
Spearman's correlation was performed for all variable pairs at a significance level of 95% (α = 0.05) and r > 0.90, r > 0.70, r > 0.50, r > 0.30 are interpreted as very high, high, moderate, and low coefficients, respectively. The SPSS software version 25.0 (IBM Corp., Armonk, NY, USA) was used for data analysis. Value standardization and heatmap were performed with PRISM 8 (Graph Pad, San Diego, CA, USA).

Extraction Protocol, Volatiles, and Pyruvic Acid
UAE was used for the extraction of garlic volatiles based on the methodology earlier described [22]; in that study, the authors demonstrated that UAE diminishes the danger of thermal decomposition of sensitive aroma compounds. In the present study, a slight modification in the extraction protocol was applied, that is, the garlic homogenate was firstly extracted with ethyl acetate and then with aqueous methanol. Moreover, the extractions of the organic solvent phase were performed only with water to collect all aqueous phases and then to determine the polar ingredients and the antioxidant properties. As a result, with the above described pretreatment modification, we managed to determine both polar and nonpolar ingredients with the same amount of plant material.
Among those organosulfur compounds, 2-VDT, 3-VDT, and DDS were detected and quantified in all genotypes examined, while 3-VDT and 2-VDT were the predominant compounds (45.7 ± 7.5% and 30.9 ± 10.2%, respectively) ( Table 2). An organosulfur compound that could not be fully identified (compound 13) was detected in all tested genotypes in relatively high amounts (6.3 ± 3.0%); it reached nearly 15.0% of organosulfur compounds in AS01 and AS10 ( Table 2). The ratio of 3-VDT to 2-VDT in most genotypes was about 1, except for AS04, AS05, AS08, AS10, AS25, AS31, AS35, and AS36 genotypes in which the ratio ranged from 3.4 to 3.9. Our results contribute to the quest for garlic genotypes and processing methods which can provide high 3-VDT content [30,31]. Since 3-VDT is more lipophilic and inhibits the differentiation of preadipocytes, it can be a beneficial agent against obesity, along with its other beneficial antioxidant and cholesterol-lowering properties [32]. Based on our results, genotypes AS36 and AS25 could be good candidates for that purpose.
The detection of vinyl-dithiins in most of the tested genotypes is in accordance with studies performed in raw garlic where the plant material is not subjected to high temperatures. The cyclic dithiins are presumed to be the first products of allicin transformation, while acyclic compounds are produced during the thermal degradation of cyclic dithiins [22]. Indeed, other researchers who used various distillation methods for the extraction of garlic volatiles also found that organosulfur compounds such as DDS, diallyl trisulfide, and methyl allyl trisulfide were among the four most abundant ones [4,23,28,29]. In our study, DDS was also an important volatile constituent detected in percentages ranging from 1.81 to 8.55% (4.34 ± 1.47%). This finding is in agreement with earlier observations that only DDS was present in extracts obtained under mild conditions and not with thermal treatment [28].   (9), 69 (7), 59 (7), 64 (6), 58 (6) Even if a part of allicin is converted during GC analysis to divinylthiins and other organosulfur compounds [22], the differences described above (e.g., the ratio of 3-VDT to 2-VDT) among the genotypes indicate that this process is highly complex and matrix-dependent. Recent studies reported that organosulfur compounds are also formed nonenzymatically in the aqueous environment of raw garlic at room temperature and thus are naturally occurring and are responsible for its distinct aroma [33]. In particular, 2-VDT has the highest flavor dilution factor among other volatiles and thus determines aroma of fresh garlic samples [6]. Therefore, GC profiling gives information not only on the different quantities of alliin and other γ-glutamylalk(en)ylcysteine precursors, but also on the aroma-responsible transformation products which are naturally occurring in the untreated (raw) plant material, while any observed differences are also genotype-dependent.
In a recent study, pyruvate constituted up to 61% of total organic acids in garlic [11]. Determination of pyruvate has been used for the indirect estimation of allicin in fresh raw garlic since it is the by-product of alliin transformation to allicin [34]. In the present study, the pyruvic acid content in the hydromethanolic extracts varied greatly among genotypes (Figure 1,   Table 4.
To investigate the relationships among the determined compounds and the antioxidant capacity of the tested garlic genotypes, a correlation analysis was performed, and the results are presented in Table 5. A strong positive correlation between total phenolics and FRAP assay was observed. Accordingly, scavenging activity towards H2O2 was moderately correlated with total phenolics and the FRAP assay. Similarly, pyruvic acid content was strongly correlated with the FRAP assay and moderately correlated with sugars, total phenolics, and the H2O2 scavenging activity.
The total phenolic concentration in the Allium genus is possibly correlated with its strong antioxidant, anti-inflammatory, and anticancer properties [38]. Furthermore, the total phenolics content in garlic was positively and strongly correlated with antioxidant capacity regardless of their  Table 4. compounds content could be attributed to the growing location as well as to genotypic differences and the cultivation practices. Herein, considering that all the tested genotypes were cultivated at the same location and under the same cultivation practices, any variation found could be attributed to differences in the genetic background of the genotypes.
To investigate the relationships among the determined compounds and the antioxidant capacity of the tested garlic genotypes, a correlation analysis was performed, and the results are presented in Table 5. A strong positive correlation between total phenolics and FRAP assay was observed. Accordingly, scavenging activity towards H 2 O 2 was moderately correlated with total phenolics and the FRAP assay. Similarly, pyruvic acid content was strongly correlated with the FRAP assay and moderately correlated with sugars, total phenolics, and the H 2 O 2 scavenging activity. The total phenolic concentration in the Allium genus is possibly correlated with its strong antioxidant, anti-inflammatory, and anticancer properties [38]. Furthermore, the total phenolics content in garlic was positively and strongly correlated with antioxidant capacity regardless of their individual phenolic compounds' composition [10,38]. In the present study, there is strong evidence of such correlation between the total phenolic compounds content and the antioxidant properties estimated by the FRAP and H 2 O 2 scavenging activity assays.

Conclusions
The chemical characterization of garlic genotypes performed in the present study revealed important correlations among the content of volatiles, polar constituents (total sugars, total phenolics, pyruvate), and antioxidant properties enabling us to identify local Greek landraces with superior characteristics which could be further exploited. It has been earlier demonstrated that the successive accumulation of somatic mutations in ancestral cultivars combined with clonal propagation leads to heterogeneity of cultivated clones. This could be the case for the dissimilarities observed in landraces obtained from the same or nearby regions in our study. Another explanation could be the exchange of germplasm between farmers and the deliberate introduction of genetic material from remote origin with different organoleptic traits.
Nearly one-third of the tested genotypes had higher pyruvate and total organosulfur concentration than the imported cultivated varieties. Among the tested genotypes, AS12 and AS36 had the highest pyruvate content and high concentrations of total sugars, AS15, AS36, and AS25 were the most abundant in organosulfur volatiles, while AS15 had the best overall performance in all the measurements. On the other hand, the high prevalence of superior characteristics in landraces originating from the Ionian islands, that is, Kefalonia and Lefkada and the neighboring Peloponnese areas of Arkadia and Messinia prompts us to further valorize these localities for the identification of promising garlic landraces. The selection of superior genotypes could be used in breeding efforts to produce distinct garlic varieties of specific origin with high content of bioactive ingredients and great nutritional, nutraceutical, and pharmaceutical value. I.A., K.Z., S.A.P., P.J.B., V.P., and F.N.L.; Writing-original draft, K.Z.; Writing-review & editing, I.A., K.Z., S.A.P., P.J.B., V.P., and F.N.L. All authors have read and agreed to the published version of the manuscript.
Funding: This work was funded by the Operational Program "Competitiveness, Entrepreneurship and Innovation" (NSRF, 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund), project "PlantUP" [MIS 5002803] which is implemented under the Action "Reinforcement of the Research and Innovation Infrastructure".