Seeds as Potential Sources of Phenolic Compounds and Minerals for the Indian Population

Seeds are major sources of nutrients and bioactive compounds for human beings. In this work, the chemical composition and physicochemical properties of 155 Indian seeds (belonging to 49 families) are reported. Moisture and ash were measured with reference protocols from AOAC; total polyphenols and flavonoids were measured with spectrophotometric methods after extraction with organic solvents, and mineral elements were determined by X-ray fluorescence spectrophotometry. Total phenolic compounds, flavonoids and mineral contents (Al, Ba, Ca, Cl, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, P, Rb, S, Sr, Ti, V and Zn) were found to vary in the ranges 182–5000, 110–4465 and 687–7904 mg/100 g (DW), respectively. Noticeably, polyphenol contents higher than 2750 mg/100 g were observed in 18 seeds. In addition, mineral contents >5000 mg/100 g were detected in the seeds from Cuminum cyminum, Foeniculum vulgare, Commiphora wightii, Parkia javanica, Putranjiva roxburghii, Santalum album and Strychnos potatorum. Botanical and taxonomical variations in the proximate characteristics of the examined seeds are also discussed.


Introduction
In the last decade, a growing interest in seeds as significant ingredients of the daily diet has been observed, since seeds are placed next to legumes as a source of plant proteins [1]. In addition, seeds contribute to meeting the increasing food demand, and in many cases, are also used as traditional medicines [2,3]. Even more, their seed cakes are used for animal feed and as green manures in organic agriculture [4,5].
Seeds are dried products with low water content [1]. Owing to their evolutionary adaptation to the embryonic nutrition of the plants they originate from, seeds are rich in different nutrients, such as proteins, carbohydrates and lipids [6]. In addition, seeds are also good sources of different bioactive compounds, such as carotenoids (vitamin A), tocopherols (vitamin E), xanthophylls and polyphenols [7,8]. Indeed, phenolic compounds such as phenolic acids, flavonoids, stilbenes and lignans are strong antioxidant compounds [9,10]. Polyphenols compounds are the subject of increasing scientific interest due to their potential applicability in the treatment of some chronic diseases, such as cardiovascular diseases, diabetes, osteoporosis or neurodegenerative disorders [11]. Moreover, The seeds from the species under study are shown in Figure S1, and their physicochemical characteristics are summarized in Table 1. Their mass ranged from 0.21 to 23,623 mg/seed, with the maximum value having been found for the seeds from Anthocephalus indicus (syn. Breonia chinensis). These values are in line with those reported by Cervera-Mata et al. [29], although in that paper, the highest masses were obtained for Cicer arietinum, Phaseolus vulgaris, Arachis hypogaea and Caesalpina crista. The mean mass values of the seeds from herbs (n = 57), shrubs (n = 14), vines (n = 20) and trees (n = 64) were 125, 556, 1264 and 1629 mg/seed, respectively. Among them, the seeds from Anthocephalus indicus, Anacardium occidentale, Areca catechu, Artocarpus heterophyllus, Bauhinia vahlii, Butea frondosa, Diospyros melanoxylon, Ficus racemosa, Gardenia thunbergia, Juglans regia, Litchi chinesis, Lagerstroemia parviflora, Mangifera indica, Nelumbo nucifera, Pistacia vera, Phoenix dactylifera, Pongamia pinnata, Prunus dulcis, Sapindus emarginatus, Semecarpus anacardium, Sterculia foetida, Syzygium cumini and Trapa natans (mostly trees) featured the largest seed sizes (>1000 mg/seed). Out of the 155 species studied, 81 had measurable seed coats, whose fractions varied from 4.0% to 95%, with the highest value having been found for Phyllanthus emblica. Such values are larger than those previously reported [29], since the coat fractions analyzed in that paper never exceeded 40%. The moisture and ash contents of the studied seeds were in the 1.0-35.5% and 1.0-7.8% ranges, respectively (Table 1). These results are in line with those of other Indian seeds [29]. In fact, moisture and ash content of Sesamum indicum seeds were similar to those reported for sesame seeds from Turkey, Sudan and Nigeria [30].
For comparison purposes, some of the highest values of total phenols (5460-15,188 mg/ 100 g) have been reported for clove (Syzygium aromaticum), peppermint (Mentha balsamea) and star anise (Illicium verum), according to Pérez-Jiménez et al. [9]. In turn, total phenolic compounds in the 21.2-417 mg/100 g range have been reported for the germinated peanut, Coriandrum sativum, cereals, pulses and other seeds [14,16]. These are important results, since Syed et al. [31] reported that peanuts have a high content of polyphenols, with a positive effect on non-communicable diseases such as cancer [32] or diabetes [33]. In fact, more than 20 bioactive compounds with phenol structure have been reported for this botanical species [34]. The total polyphenols content was also similar for other species, such as Allium cepa seeds, reported in the range of 200-400 mg/100 g by other authors [35,36]. In this respect, Žilic et al. [37] demonstrated that polyphenols play an important role in sunflower seed oil, by protecting it from oxidation during storage. Again, our total phenols value for Sesamum indicum are in line with those provided in other papers [30,38]. It is known that the total phenolic content can differ among the samples from different countries due to variations in the genotypes, ecological factors and cultivation practices [39].
Total phenols and flavonoids were also grouped by botanical families (Figure 1A,B, respectively). Taxonomically, total phenolic compounds ( Figure 1A

Mineral Contents
Several vegetable species bear seeds during dry season in tropical countries such as India. This time of seed production is very important during periods of food scarcity [46] or for fast-growing populations such as the Indian one [27]. As seeds are excellent sources of micronutrients, their consumption may contribute to meeting the nutritional requirement and to overcoming the micronutrient deficiency at minimum cost [47,48]. Since the largest concentration of people with mineral elements deficiencies lives in low-income South Asian countries, including India [49], and due to the predominantly vegetarian diet of Indian inhabitants [50], a possible solution for this problem could be the increase in seed consumption [51].
The contents of 20 mineral elements (Al, Ba, Ca, Cl, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, P, Rb, S, Sr, Ti, V and Zn) in the seeds grown in India from the 155 species under study are shown in Table S1. The mineral content varied from 687 to 7904 mg/100 g (Figure 2). Although these values are generally in line with those previously reported [25,29,52], there are seeds with the largest mineral content exceeding by up to 3000 mg the limit of 4913 mg/100 g found in that paper; this could be related with the lower number of seeds analyzed in that study (60 species vs. 155 species in the current paper) and also related with the botanical nature of seeds. In ref. [29] the studied seeds were mainly beans, weeds and pulses, while in the current paper, a broad variety is found. In this sense, the seed mineral potentiality was categorized in two groups using cluster analysis (data not shown); cluster-I included seeds from 148 plants with mineral contents in the 687-5089 mg/100 g interval, while cluster-II consisted of seven species (Cuminum cyminum, Foeniculum vulgare, Commiphora wightii, Parkia javanica, Putranjiva roxburghii, Santalum album and Strychnos potatorum), which had mineral contents in the 5728-7904 mg/100 g range.
In relation to macronutrients, P and S were found to accumulate in all seeds, at concentrations ranging from 53 to 958 mg/100 g and from 50 to 5144 mg/100 g, respectively. These levels are similar to those of other seeds collected in India [29,40]. The highest P and S contents were found in Corchorus olitorius and Parkia javanica seeds, respectively. In another study [29] Crotalaria albida, Rorippa palustris and Cleome viscosa showed the highest S contents. High P contents (602-661 mg/100 g) and S contents (638-2067 mg/100 g) occurred in seeds belonging to the Moringaceae and Malvaceae families and to the Brassicaceae, Caricaceae, Fabaceae, Moringaceae and Putranjivaceae families, respectively ( Figure 2). Phosphorous is an important mineral required for the normal growth and maturity of plants, while S is another essential element for the synthesis of chlorophyll and proteins [53].
Chlorine is required in small amounts for plant metabolism and photosynthesis [53]. However, Cl accumulated in the seeds from 26 species (Table S1) at concentrations ranging from 14 to 1079 mg/100 g. The highest contents were found in Apiaceae seeds, followed by Lythraceae and Piperacee seeds (373-376 mg/100 g). The maximum value was observed in Trapa natans seeds, followed by Cuminum cyminum. In general, these values are in line with those of Heliotropium indicum (300 mg/100 g) [27].
Sodium was detected in the seeds from 17 species (78-6419 mg/100 g), with the highest contents in the seeds from Santalum album and Strychnos potatorum. In a previous publication [29], high Na accumulation was detected in the seeds from Eleusine corocana, Oryza sativa, Setaria italica and Zea mays. In general, some plants tend to accumulate Na when they grow in saline soils, secreting salts to regulate the ion balance, contributing to salinity tolerance [54]. In the case of potassium, it was identified in seeds from all species, at concentrations between 21 and 2625 mg/100 g, which are in line with the values found in weeds, pulses and beans from India [29]. The highest K accumulation (>2.5× mean value, 1944 mg/100 g) was identified in Coriandrum sativum, Cuminum cyminum, Foeniculum vulgare, Butea frondosa, Delonix regia, Leucaena leucocephala, Lagerstroemia parviflora, Artocarpus heterophyllus, Ficus racemosa and Withania coagulans. Remarkable K contents (1729-2349 mg/100 g) were detected in the seeds from the Apiaceae, Caricaceae, Moraceae, Piperacee and Zingiberaceae families.
Calcium is also an important mineral for human nutrition that was accumulated at concentrations in the 3.0-1786 mg/100 g range in the seeds from 155 species (Table S1)  Magnesium is the main component of chlorophyll [53] and was identified in the seeds from 147 species, at concentrations between 34 and 910 mg/100 g, in line with other seeds [29,40]. High Mg contents (>395 mg/100 g) were detected in the seeds from Abelmoschus esculentus, Acacia nilotica, Cassia fistula, Commiphora wightii, Corchorus olitorius, Parkia javanica, Pterocarpus marsupium and Urena lobata, which are similar to those reported for barley [55] and millet seeds [56]. The highest Mg accumulation was noticed in seeds belonging to the Amaryllidaceae, Burseraceae, Caricaceae and Moringaceae families.
Calcium is also an important mineral for human nutrition that was accumulated at concentrations in the 3.0-1786 mg/100 g range in the seeds from 155 species (Table S1). These are values quite similar to those for weeds such as Panicum sumatrense, Setaria italica, Cassia tora, Heliotropium indicum, Rorippa palustris and Ludwigia parviflora [14N]. Abundant Ca accumulation (919-1253 mg/100 g) was observed in the seeds from four families: Apiaceae, Caricaceae, Pedaliaceae and Putranjivaceae (Figure 2).
Strontium content in 125 cultivars varied from 0.1 to 27.4 mg/100 g. These are higher levels than those described for weeds, pulses and beans from India [29,40]. High Sr content (from 21 to 27 mg/100 g) was accumulated in two Apiaceae family species: Daucus carota spp. Sativus and Foeniculum vulgare. Rubidium concentration in the seeds under study ranged from 0.2 to 13.4 mg/100 g; such high Rb accumulation was registered in Lepidium sativum seeds. These levels are similar to those found in other seeds [40] and within the normal range in foods [57].
Barium accumulated in the seeds from 18 species at milligram levels (1.0-7.9 mg/100 g). The maximum Ba content was detected in Daucus carota spp. Sativus. Relatively high contents (2.3-2.4 mg/100 g) were observed in Apiaceae and Rubiaceae seeds (Figure 2). Aluminum accumulated in the seeds from eight species over a wide concentration range: from 41 to 269 m/100 g. Aluminum at high levels (240-269 mg/100 g) was found in the seeds from Trachyspermum ammi and Trapa natans.

Variations in Mineral Levels as a Function of Plant Type and Family
The mineral concentration variation as a function of plant type is presented in Table 2.
Remarkably, high contents of Na and S, Mg-K-Fe-Mo, Al-Cl-Ca-Mn-Co-Ba, P-Cu-Zn were detected in the tree, shrub, herb and vine species, respectively. The taxonomical seed mineral concentration variations are shown in Figure 2 and Table S2. The highest total mineral contents were found in seeds from the Santalaceae, Burseraceae, Lythraceae, Moringaceae, Apiaceae, Moraceae, Papeveraceae, Solanaceae and Arecaceae families.  The nutrients in the soil solution can interact with each other, affecting their absorption and bioavailability [22]. The P/S (n = 155), K/P (n = 155) and Ca/Mg (n = 143) ratios in the seeds were in the 0.06-4.96, 0.24-21.07 and 0.04-12.61 ranges, respectively. Their minimum and maximum values were observed in Parkia javanica and Persicaria punctate; in Trapa natans and Gardenia thunbergia; and in Luffa aegyptiaca and Papaver somniferum seeds, respectively. These are ratios that have been previously described for other seeds from India [29,40].

Statistical Relationship among Mineral and Phytochemical Contents
Due to the large number of minerals and polyphenols studied (n = 22), a principal component analysis (PCA) was performed to obtain a low number of linear combinations of such parameters that explained data variability. We included those families that were the most representative in the study (with at least five different botanical species). Figure 3 depicts the results obtained, showing the main drivers on the separation of botanical families (total phenols, flavonoids, Ca, Cl, K, P and S). The combination of two components explained 88.77% of the variance. The PCA grouped the seeds from the Apiaceae family due to their high Cl and P contents. On the opposite, samples from the Brassicaceae family grouped in a different sector due to their high S levels. Fabaceae seeds also had an important content of S. Flavonoids and total phenols also played a role, especially for the Anacardiaceae family (Figure 3). the seeds were in the 0.06-4.96, 0.24-21.07 and 0.04-12.61 ranges, respectively. Their minimum and maximum values were observed in Parkia javanica and Persicaria punctate; in Trapa natans and Gardenia thunbergia; and in Luffa aegyptiaca and Papaver somniferum seeds, respectively. These are ratios that have been previously described for other seeds from India [29,40].

Statistical Relationship among Mineral and Phytochemical Contents
Due to the large number of minerals and polyphenols studied (n = 22), a principal component analysis (PCA) was performed to obtain a low number of linear combinations of such parameters that explained data variability. We included those families that were the most representative in the study (with at least five different botanical species). Figure  3 depicts the results obtained, showing the main drivers on the separation of botanical families (total phenols, flavonoids, Ca, Cl, K, P and S). The combination of two components explained 88.77% of the variance. The PCA grouped the seeds from the Apiaceae family due to their high Cl and P contents. On the opposite, samples from the Brassicaceae family grouped in a different sector due to their high S levels. Fabaceae seeds also had an important content of S. Flavonoids and total phenols also played a role, especially for the Anacardiaceae family (Figure 3).

Sample Collection and Preparation
Seeds from 155 species (listed in Table 1), belonging to 49 families, were collected in 2018 in the Raipur area (21.25 • N 81.63 • E), India, and were identified using standard monographs [52]. For each species, samples from 4 different plants and growing in 4 different locations were collected and were mixed to form composite samples.
The samples were sundried for one week in a glass room and stored in glass bottles. They were further dried for 12 h in a hot-air oven at 50 • C (then, the seeds were stored at −20 • C until analysis). These drying steps could be easily performed by local farmers as well as from small to large companies. The mass was measured using a Mettler-Toledo (Columbus, OH, USA) electronic balance. The testa of 74 examined seeds was carefully removed (manually), and both kernel and seed coat were weighted. Dried seeds or kernels were crushed into powder form using an agate mortar, and particles ≤0.1 mm were sieved out.
Moisture content was determined by drying the samples at 105 ± 2 • C in an air oven for 3 h, and the reported mean values were calculated using the following equation [61]: where w1 and w2 denote the initial and the dry weight (DW) of the sample, respectively. Ash residue was evaluated by heating to 550 ± 25 • C in a muffle furnace till constant weight was reached [61] and was reported according to the expression: where Wash and Wseed denote the weight of the ash residue and the dry weight of the sample, respectively.

Total Phenolic Compound and Flavonoid Determination
The seed samples in powder form (100 mg) were dispersed in 5 mL of an acetone/water (7:3, v:v) solution and were sonicated in an ultra-sonic bath for 20 min at 20 • C [26]. Then, 5 mL of fresh acetone:water (7:3, v:v) solution was added to the mixture, and the extraction was repeated for 20 min at 20 • C. After centrifugation, the supernatant was collected [62]. Sigma-Aldrich AR-grade Folin-Ciocalteu reagent, aluminum chloride, tannic acid, gallic acid and quercetin were used for the spectrophotometric determination of phenols. The total phenolic content of each extract was determined as tannic acid equivalents using the Folin-Ciocalteu method [19]. The reaction was carried out in an alkaline medium at room temperature (27 ± 2 • C). After a standing time of 40 min, the absorbance was measured at λ = 725 nm against the reagent blank. A calibration curve was prepared for the absorbance of 2.0, 4.0, 6.0 and 8.0 mg tannin/L. The detection limit (>3 std. dev.) of the method was 0.80 µg/mL as tannic acid. The slope (13.4) and intercept (0) obtained were used for computing the concentration of total phenolic compounds in the sample solution.
The flavonoid content was determined by the aluminum chloride method as quercetin equivalents [63]. Aluminum ions were allowed to react with flavonoids in the presence of tartrate buffer, and the absorbance of the complex formed after a 30 min incubation period was measured at λ = 415 nm against the reagent blank. A calibration curve for 4.0, 6.0, 8.0 and 10.0 mg quercetin/L was prepared. The detection limit of the method (>3 std. dev.) was 0.54 µg/mL as quercetin. The derived slope (20.5) and intercept (0.85) were employed for estimating the concentration of flavonoids in the sample solution.

Statistical Analysis
Each analysis was carried out in triplicate. A principal component analysis was used to classify the seeds from the 155 species as a function of their mineral and phytochemical contents using Statistica 10.0 (StatSoft, Tulsa, OK, USA) software.

Conclusions
A wide variety of seeds belonging to herb (57), tree (64), shrub (14) and vine (20) plant types were investigated, focusing on their phenolic and mineral contents. Remarkably, high contents of total phenolic compounds (>4000 mg/100 g) were detected in the seeds from Argemone mexicana (Mexican poppy), Nigella sativa (black cumin), Papaver somniferum (opium poppy), Sesamum radiatum (benniseed) and Solena amplexicaulis (creeping cucumber). A. mexicana and S. radiatum also featured the highest flavonoid contents. Regarding mineral levels, Parkia javanica and Santalum album (Indian sandalwood) featured the highest total concentrations (around 7900 mg/100 g). Taking all this information into account, seeds from different species could be a valuable source of mineral elements and phenolic compounds for the Indian population if such seeds were properly cooked or used as condiments for regular foods.