Nutritional value, phytochemical composition, and antioxidant potential of Iranian fenugreeks for food applications

Fenugreeks (Trigonella L. spp.), are well-known herbs belonging to the family Fabaceae, whose fresh and dried leaves have nutritional and medicinal value. In the present study, the content of phytochemical traits (essential oil, diosgenin, trigonelline, total phenol, total flavonoid, total saponins, and total tannins), bitterness value, pigments (chlorophyll, carotenoid, β-carotene, and anthocyanin), vitamins (group B vitamins and ascorbic acid), minerals, and antioxidant activity of thirty cultivated populations belonging to ten Trigonella species were evaluated. The species and populations were significantly different in all studied parameters. A significant positive and negative correlation (p < 0.05) was also observed between the studied parameters. In total, T. teheranica, T. elliptica, and T. foenum-graecum were distinguished as superior species. The results showed that fenugreeks leaves can be considered as a valuable source of food and phytochemical compounds. The obtained data can be help to expand the inventory of wild and cultivated Trigonella species for further exploitation of rich chemotypes in the new foods and specific applications.


Extraction and HPLC-PDA determination of diosgenin and trigonelline
Diosgenin extraction was carried out as described by Aminkar et al. 36 with slight modification.For instance, the leaves of each sample (1 g) were added into a tube and 20 ml of 96% ethanol and then sonicated (Elma, S120H, Germany) for 30 min at room temperature.Then, 20 ml sulfuric acid (2N) was added and hydrolyzed under reflux conditions at 100 ℃ for 2 h.The suspension was centrifuged (centrifuge Rotanta 460r, Hettich, Germany) at 4400 rpm for 5 min.The mixture was partitioned with n-hexane.The n-hexane phase was dried under reduced pressure in a rotary evaporator (Heidolph Instruments GmbH, Schwabach Germany) at 35 ℃.Dried extract was solved in 3 ml acetonitrile and passed through the filter (0.22 μm).
Trigonelline extraction was performed as described previously 40 with some modifications.Briefly, the leaves of the studied samples (1 g) were added into a tube with 5 ml of acetonitrile and then placed in a bath ultrasonic for 15 min at room temperature.Then, 20 ml of phosphoric acid (5N) and 20 ml of methanol were added and sonicated for 20 min.The mixture was concentrated in a rotary at 35 ℃ for reduce.Finally, the dry extract was dissolved at 3 ml acetonitrile and passed through the filter (0.22 μm).
Employing HPLC equipped with a diode-array detector with a C 8 column (50 × 2 mm, 3 μm) and a UV detector (Waters 2487), the analysis was carried out.The following gradient system was used with acetonitrile/ water (90:10 v/v).The flow was maintained at 0.5 ml/min and column temperature at 25 ℃; sample injection was 20 μl.Absorbance was recorded at 210 and 263 nm for diosgenin and trigonelline, respectively.Total saponin content (TSC) of the studied populations was determined as per the reported procedure 41 .One gram of powdered leaves of each sample was extracted using a microwave-assisted extraction method under 3 min irradiation time, 572 Watt microwave power, 64% ethanol concentration, and 1:10 g/ml solid-to-solvent ratio (1g leaf sample/10 ml ethanol).The samples were extracted in a microwave system (Milestone ETHOS UP, Italy).Accurately 100 μl extract was mixed with 400 ml methanol and 200 μl vanillin/ethanol (10:90 w/v).Then, 600 μl sulfuric acid (70%) was mixed and heated at 100 ℃ for 10 min.Absorbance was taken against the reagent blank (methanol) at 544 nm using a spectrophotometer (Bio-Tek Instruments, Inc., USA).A standard curve was calculated using diosgenin solution in the range 100-500 mg/ml).Total saponin content was calculated as follows: [The volume of extraction solvent (ml) × The concentration measured from diosgenin standard curve (mg/ml)]/The dry weight of the sample (g).Total tannin content (TTC) was measured according to Abdouli et al. 24,42 with slight modification.Concisely, powdered leaves of each sample (100 mg) were mixed with 5 ml of diethyl ether containing 1% acetic acid and the mixture was placed on a magnetic stirrer for 15 min.The mixture was then centrifuged at 2000 rpm for 10 min.The supernatant was discarded and re-extraction was performed with 5 ml of acetone (70%) and stirring for 1 h.Finally, the extract was centrifuged at 2000 rpm for 20 min.
The total phenol content (TPC) in the extract was determined based on the Folin-Ciocalteu method and then 2 ml of diluted acetone extract was mixed with 100 mg of polyethylene glycol 4000 (for deposition Tannins).Total tannins content was calculated as the difference in total phenols content before and after the treatment.

Determination of bitterness values
According to the WHO method 43 and Abdouli et al. 42 , the bitterness value was assessed by comparing the threshold concentration of aqueous leaf extract (minimum concentration that still tastes bitter) with a dilute solution of quinine hydrochloride.In summary, 50 mg quinine hydrochloride and 50 ml drinking-water were mixed.Afterward, 5 ml of the solution was diluted to 500 ml with drinking-water.The stock solution of quinine hydrochloride contains 0.01 mg/ml.For preparing of stoke solution, the leaves of each sample (1 g) were extracted with 1000 ml drinking-water.A test panel consisting of ten adult members (male and female) was assembled.Bitterness value of the test solutions resulted from calculating the average of the individual values.Bitterness value was determined by using the below formula 43 .
Bitterness value (unit/g) = [2000 × Quantity of quinine hydrochloride with the lowest bitter concentration (mg)]/[Concentration of the stock solution (mg/ml) × volume of stock solution with the lowest bitter concentration (ml)].The bitterness value of the solution was expressed as units/g.

Determination of pigments
Photosynthetic pigments (chlorophyll a, chlorophyll b, carotenoid, and anthocyanin) were determined according to Missaoui et al. 44 with minor modifications.For instance, fresh leaf tissues (0.1 g) were ground in acetone (10 ml, 80%).The mixtures were then centrifuged at 3,500 rpm for 10 min and supernatants were collected to determine Chla, Chlb, and carotenoid by reading the absorbance at 663, 645, and 470 nm, respectively, using a spectrophotometer (Shimadzu double beam UV-Visible spectrophotometer-1800, Japan).Leaf pigment concentrations were quantified according to the formulae of Lichtenthaler and Wellburn 45  β-Carotene content was determined by Negi and Roy 46 method.The absorbance was read at 440 nm.β-Carotene concentration (1-20 μg/ml) was determined from the standard curve.Anthocyanin content was calculated with grinding leaf tissues in 20 ml methanol/water/hydrochloric acid (16:3:1 v/v).The mixture was kept at room temperature for 2 days in the dark, then and centrifuged for 10 min at 13,000 rpm.The absorbance was read at 530 and 653 nm using a spectrophotometer.Anthocyanin content was calculated as follows: Anthocyanin = A 530 -(0.24 × A 653 ) with an extinction coefficient of 26,900 l/mol/cm 47 .

Measurement of vitamin content
Type of vitamin B (Thiamin, riboflavin, niacin, and pyridoxine) was also determined following AOAC method 48 .
Leaf powder (1 g) was used for the determination of vitamin B groups.For instance, thiamin was calculated with adding 65 ml hydrochloric acid (0.1 N) to the plant sample.The sample was heated at 100 ℃ for 30 min in a water bath followed by centrifugation for 5 min at 4400 rpm.For riboflavin quantification 65 ml acetic acid-water mixture (50:50 v/v) was added to the plant sample.The mixture was heated at 100 ℃ for 30 min and stirred for 10 min in the dark.The solution for niacin was 3 ml hydrochloric acid (5 N), 3 ml dichloromethane, and 64 ml water.For extraction of pyridoxine, the plant sample was mixed to 12 ml sodium acetate solution (0.05 M) and pH was adjusted to 5.5.Absorbance was taken at 531, 460, 410, and 291 nm using a spectrophotometer for thiamin, riboflavin, niacin, and pyridoxine, respectively.The amounts of the vitamins were calculated as follows.
Ascorbic acid was determined using AOAC method 49 .Briefly, powdered leaf samples (500 mg) were extracted with 5 ml of 3% metaphosphoric acid and centrifuged at 4400 rpm for 5 min.The supernatant was titrated against 2,6-dichlorophenolindophenol dye solution (0.25%) to faint pink color.The ascorbic acid content was determined as follows: Ascorbic acid (μg/100 g DW) = [(Concentration of the standard ascorbic acid (μg/ml) × Titre value

Elemental analysis
The leaves of the studied samples were prepared for digestion according to the method of Başgel and Erdemoğlu 50 and Kan et al. 51 with modifications.The leaf samples (500 mg) were digested using a microwave system (CEM, Mars X-Press, USA) with 6 ml pure nitric acid (69%) and 1 ml hydrogen peroxide (35%) in closed vessels with constant temperature heating at 140 °C for 3 h, and heated for digestion (

Determination of total phenol and total flavonoid content and antioxidant activity
TPC of the studied samples was determined by the method of Singleton et al. 52 with Folin-Ciocalteu's reagent using gallic acid as the standard.Absorbance was measured at 765 nm against methanol as a blank.The total flavonoid content (TFC) was determined using the method of Chang et al. 53 with aluminum chloride using rutin as the standard.Absorbance was determined at 510 nm versus prepared water blank.
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity and ferric reducing-antioxidant assay (FRAP) methods were tested in the present study due to the fact that antioxidants are classified into two categories as water-soluble and fat-soluble antioxidants.These methods are high sensitivity and accuracy, fast, cheap, well established and popular for measuring the antioxidant properties of crude extracts or purified compounds from plants.
The DPPH was determined based on the method of Akhlaghi and Najafpour-Darzi 29 .The absorbance values were recorded at 515 nm.The inhibition percentage of anti-oxidative activity was determined using the equation: DPPH clearance = A control -A sample )/A control × 100%.The DPPH radical scavenging activity of butylated hydroxytoluene (BHT) was also assayed for comparison.The concentration providing 50% inhibition (IC 50 ) was calculated using a calibration curve in the linear range by plotting the extract concentration vs the corresponding scavenging effect.
The FRAP solutions were prepared as described previously 54 .Ascorbic acid was used as the standard curve.The standard curve was constructed using iron (II) sulfate (FeSO 4 ) solution.The absorbance of the mixture was then read at 593 nm using a spectrophotometer.

Statistical analysis
All the analyses were run in three replicates.A one-way analysis of variance (ANOVA) was computed using the SPSS 25 software (SPSS Inc.Chicago, USA).The comparison between the data was evaluated using Duncan's test, considering p < 0.05 and expressed as mean ± standard deviations (SD).The heat map and the plots were created by R statistical software (4.4.1), using the pheatmap, RColorBrewer, Viridis, ggplot2, tidyr, and corrplot packages.Canonical correspondence analysis (CCA) was evaluated using PAST software (4.03).

Essential oil content and composition
The results showed that there is a significant difference (p < 0.05) between the species and populations in terms of content (% w/w) and identified components (%) of the oils.The content of the essential oils was in the range of 0.11 to 0.30% (Table 2).TEP2, TCL3, and TST1 had the highest essential oil content (0.30%), while the lowest (0.11%) was obtained in TAS1, TFG1, and TST3.According to previous reports, Trigonella species have low essential oil content.Ahmadiani et al. 55 have reported the content of the essential oil in T. foenum-graecum as 0.3%.
As can be seen in Table 2, oxygenated monoterpenes (0.00-7.95%) and oxygenated sesquiterpenes (4.49-10.11%)were the highest in oil.It has been reported that the plants of the legume family produce less mono-, sesqui-, and diterpenes than other plant families (Asteraceae and Lamiaceae) 56 .Heat map analysis based on the essential oil components is presented in Fig. 2. The studied fenugreeks populations were separated into two main groups.TEP1, TEP2, TEP3, and TCO3 were placed in one group and the other populations formed another group.
Riasat et al. 35 have identified thirty-six compounds in the essential oil of T. foenum graecum leaves grown in Iran with the majority of 2E-hexenal (26.61%) and n-hexadecanoic (10.14%).Pentacosane (27.3%) has also been reported as the major composition in the essential oil of T. disperma Vassilcz.leaves from Iran 57 .Esmaeili et al. 58 reported that dibutyl phthalate (10.3%), hexanal (9.5%), and nonanal (6.6%) were found to be the most common Table 2. Volatile compounds of the studied populations of Trigonella species identified by GC-MS analysis.For a detailed description of the plant populations code, cf.Table 1     In another investigation, n-hexadecanoic acid (20.84%), camphane (11.45%) and neo-menthol (5.05%) were found as the major essential oil compounds of T. teheranica (Bornm.)Grossh. 60.Furthermore, ten components were totally characterized in the volatile oil of T. foenum-graecum from Iran with the majority of δ-cadinene (27.6%), α-cadinol (12.1%), γ-eudesmol (11.2%), and α-bisabolol (10.5%) 55 .Variation in the composition of essential oil content can be attributed to the genetic and different environmental conditions of their origins 61 .2-Acetylfuran, is the most abundant flavoring compound in tamarind (Tamarindus indica L.), and its aroma in combination with α-terpineol, citral, and some rare pyrazines contributes to tamarind taste 62 .Octadecanol acetate, is also a chemical fraction of the sex pheromone of Heliothis virescens F., which prevents ovulation and is used in pest management 63 .Antibacterial 64 and antifungal 65 activity of T. foenum-graecum essential oil has also been reported.So, fenugreeks can be interestingly used to develop natural pesticides and fungicides to control pests and plant diseases.In the present study, TEP2, TEP3., and TCO3 are introduced as adequate populations rich in essential oil and 2-acetylfuran content.TST1 and TCL1 can also be considered for their high essential oil and octadecanol acetate content.

Diosgenin and trigonelline content
A significant difference (p < 0.05) was observed among the studied species and populations in terms of diosgenin and trigonelline content.Variation of the diosgenin and trigonelline among thirty populations of the ten studied Trigonella species are presented in Table 3.The content of diosgenin in leaf extract was varied from 5.05 ± 0.01 to 24.52 ± 0.18 mg/g DW, while the content of trigonelline was in the range of 1.00 ± 0.00 to 4.21 ± 0.05 mg/g DW.The highest content of diosgenin was found in TFG2, TEP1, TFG1, and TFG3, while the maximum level of Table 3. Variation of the diosgenin, trigonelline, total saponins, total tannins, and bitterness value among the populations of Trigonella species.Data expressed as mean ± standard deviation (SD) of three replicates.Different letters in column indicating statistically differences mean at p < 0.05 by Duncan's multiple range test.For a detailed description of the plant populations code, cf.www.nature.com/scientificreports/trigonelline was observed in TEP3, TFG3, and TFG2.Both TSG3 and TST3 had the lowest content of diosgenin and trigonelline.In a study on the leaves of four Trigonella species (T.foenum-graecum, T. maritima Delile ex Poir, T. hamosa L. and T. stellata Forssk.)from Egypt, the highest trigonelline content was found in the cultivated species.The compound was detected in all the leaf extract samples with the exception of T. maritima 37 .
Aminkar et al. 36 were measured the content of diosgenin in the leaves of twenty-two populations of T. foenumgraecum from Iran.They reported the highest diosgenin level of 23.8 mg/g DW, which was similar to the results measured in the present study for the same species, but the content of diosgenin in other studied species was lower than that of T. foenum-graecum species.Dangi et al. 31 reported the aerial parts of T. foenum-graecum contain 0.08 mg/g, while T. caerulea (L.) Ser. and T. anguina Delile had relatively higher diosgenin content (2.46 and 3.72 mg/g, respectively) than T. foenum-graecum.Trigonella can be a suitable and alternative source for the production of diosgenin.Therefore, finding the population of the plant with the highest potential can be of great importance for the production of diosgenin.
In a comparative study on diosgenin content in the aerial parts of the ten Trigonella species from Turkey, the highest diosgenin content (0.16 ± 0.00 mg/g) was reported in T. cilicica Hub.-Mor.Some of the studied fenugreeks including T. kotschyi Benth., T. filipes Boiss., and T. strangulata Boiss.lacked diosgenin in their aerial parts.They have also reported that T. spruneriana Boiss.contained 0.03 ± 0.00 mg/g diosgenin 26 while, in the present study, the content of diosgenin in the populations of T. filipes, T. spruneriana, and T. strangulata was ranged from 5.05 ± 0.01 to 5.74 ± 0.02 mg/g DW (Table 3).In another study, diosgenin content was studied in the aerial parts of eleven varieties of T. foenum-graecum and the lowest and highest value were reported as 187.3 ± 0.5 and 466.9 ± 0.3 mg/100 g DW, respectively 34 .The results showed that the studied species and populations of Trigonella are different in terms of the content of specialized metabolites.The diversity of phytochemicals in plant populations and also species can indeed be influenced by a variety of intrinsic and extrinsic factors 12 .Intrinsic factors may include genetic diversity within the plant population, as different genotypes can produce different phytochemical profiles.Understanding the interplay between the factors can provide valuable insights into the ecological and evolutionary dynamics of plant populations, as well as their potential applications in agriculture, medicine, and other fields.
In the present study, we identified that T. foenum-graecum, T. coerulescens (M.Bieb.), and T. elliptica Boiss.have more content of diosgenin and trigonelline.By identifying high-productive species and potent populations within these wild fenugreek species, researchers can contribute to the development of sustainable and diverse sources of diosgenin and trigonelline.This not only broadens the availability of these valuable compounds but also offers opportunities for pharmaceutical industries to diversify their sources of raw materials for steroid drug synthesis.Furthermore, the exploration of other wild fenugreek species for their diosgenin and trigonelline content can contribute to the conservation and sustainable utilization of plant genetic resources.Understanding the potential of these wild species can also lead to the development of new cultivation strategies and breeding programs aimed at enhancing the production of diosgenin and trigonelline.

Total saponin and tannin content and bitterness value
TSC, TTC, and bitterness value of the studied fenugreeks are presented in Table 3.A significant difference (p < 0.01 and p < 0.05) was observed between species and populations.The TSC of the leaf samples (17.52 ± 0.11-66.37± 0.59 mg DE/g DW) varied greatly among the studied species and populations.The highest TSC content was in TFG1, TFG2, and TFG3.The lowest content was observed in TSG3 (Table 3).
In a study on optimizing the extraction conditions of TSC in T. foenum-graecum seeds from Malaysia, the highest reported content was 195.89 ± 1.07 mg DE/g DW 41 .This finding underscores the potential of fenugreek seeds as a rich source of saponins.Shawky et al. 37 reported that saponin and saponin glycosides are abundantly found in the leaves of Trigonella genus.In another study, TSC and TTC in the leaves of T. foenum-graecum from Tunisia at the maturity stage have been reported as 0.33 and 7.02 g/100 g DW, respectively 24 .The lack of studies investigating the TTC and TSC, as well as the bitterness value of fenugreek leaves, highlights a significant gap in our understanding of this plant's nutritional and sensory properties.
Previous studies have indicated that a range of factors, including the origin, plant species, environmental factors, and growing practices, influence the type and quantity of saponins present in food 72,73 .Saponin levels in crops can differ based on geographical location, plant species, and various stages of plant growth 67 .Considering the cultivation of fenugreek species and populations under the same agricultural conditions, the cause of variation obtained in the present study can be attributed to genetic factors.
The bitter taste of fenugreek leaves, attributed to compounds like saponins, can indeed pose a challenge for consumer acceptance and usage in daily diets 22,74 .Given the importance of sensory attributes in food acceptance, especially bitterness, it is crucial to explore debittering processes to enhance the palatability of fenugreek leaves 75 .By removing or reducing the compounds responsible for bitterness, such as saponins, the culinary appeal of fenugreek can be enhanced, potentially expanding its utilization in various food recipes.
Introducing less bitter populations of fenugreek species like T. stellata, T. strangulata, and T. filipes could be a promising approach to attract consumers who may have been deterred by the strong bitterness of traditional fenugreek varieties.By incorporating these less bitter alternatives into the food basket, consumers may discover new ways to enjoy the nutritional benefits of fenugreek without being put off by its bitter taste.

Pigments data
The results showed that there is a significant difference (p < 0.05) among the studied fenugreeks in terms of leaf pigments content (chlorophyll a and b, carotenoid, β-carotene, and anthocyanin).The pigment content in the studied fenugreeks is shown in Fig. 3.The content of chlorophyll a in the studied fenugreeks was found in the range of 0.94 ± 0.03 to 2.43 ± 0.07 mg/g FW.TFG1 and TCO2 had the highest chlorophyll a content.The content of chlorophyll b in ranging 0.31 ± 0.04 to 1.34 ± 0.12 mg/g FW was lower than chlorophyll a.The highest and lowest chlorophyll b content was obtained in TCO1 and TAS1, respectively.
The carotenoid content in the leaves of the studied Trigonella species and populations was ranged from 0.12 ± 0.05 to 0.70 ± 0.14 mg/g FW, which showed the highest level in TCO1.β-Carotene content was in the range of 0.05 ± 0.01 to 0.27 ± 0.03 mg/g FW, and the highest value was obtained in TCO1, TCO2, and TCO3.
Hussain et al 76 have reported that the leaf of T. foenum-graecum from India contained 128.1 ± 5.2 and 71.2 ± 2.5 mg/100 g of total chlorophyll and carotenoid content respectively.Joshi and Kulshrestha 77 reported the β-carotene content of fenugreek leaves as 625 mg/100 g DW.In a study, β-carotene content in fenugreek leaves was obtained 28.1 mg/100 g DW 46 .In another study, β-carotene content of fresh T. foenum-graecum leaves was reported as 19 mg/100 g 78 .The anthocyanin content was found in the range of 0.16 ± 0.02 to 0.95 ± 0.01 mg/g FW and the highest value belonged to the TFP1.In many studies, chlorophyll, carotenoid, and anthocyanin contents of T. foenum-graecum were investigated and similar values were reported [79][80][81][82] .In a study, leaf chlorophyll a and b, and carotenoid content of T. corniculata Sibth.& Sm. were reported 1.36, 0.64, and 0.6 mg/g, respectively 33 .www.nature.com/scientificreports/ The effect of genetic and climatic conditions such as light, temperature, and precipitation on the content of pigments has been extensively reported 83,84 .Since the pigments are natural antioxidants, as a result, the protective and therapeutic role of Trigonella seems to be different in the studied fenugreeks.This issue is very important in the food industry, especially the production of edible pigments, as well as the pharmaceutical industry.The findings regarding the high carotenoids, β-carotene and anthocyanin content in the populations of T. coerulescens, T. foenum-graecum, and T. teheranica species suggest that these species have potential for further exploitation in cultivation and breeding programs to meet the demands for food and pharmaceutical applications.
It has been reported that the leaves of T. foenum-graecum contain 40, 310, and 800 µg/100 g of thiamine, riboflavin, and niacin, respectively.The leaves of the species also contained 52 mg/100 g ascorbic acid 22,27 .In a study, the ascorbic acid content of T. foenum-graecum leaves from India was found to be 1047.4mg/100 g DW 46 .
Yadav and Sehgal 78 stated that fresh T. foenum-graecum leaves contain about 220.97 mg of ascorbic acid per 100 g.According to Hussain et al 76 total ascorbic acid of T. foenum-graecum leaves from India was determined as 51.4 ± 1.2 mg/100 g.
It is estimated that more than two billion people in the world, most of them in developing countries, are vitamin deficient.Vitamin deficiency leads to low quality of life and reduced economic productivity 85 .The present study showed that the leaf of the Trigonella species especially T. foenum-graecum has high vitamin content that can be used in different diets and the formulation of various food supplements.
Uras Güngör et al. 26 have measured the content of macro-and micro-elements in the aerial parts of the ten Trigonella species grown in Turkey.In their report, K content varied from 5225 to 13327 μg/g DW with the majority in T. cilicica.Ca content was in the range of 7466 to 13754 μg/g DW.The Fe level was varied from 35 to 285 μg/g DW in their plant materials studied.They have been reported the highest content of Ca and Fe in T. spruneriana and T. smyrnaea Boiss., respectively 26 .
Macro-and microelement content in the leaf of the studied fenugreeks populations is largely an inherent feature, although they are also determined by climate and cultivation practices, which explains the significant differences in the results noted by various authors 26,32,86,87 .
In the present study, it was found that the leaves of the studied fenugreeks have a high content of macro-and micro-elements, which increases the nutritional value.The leaves of the studied species and populations were also a good source of Fe, so it can be recommended in the diet of individuals with iron deficiency.According to the maximum allowed level reported by the WHO 88 , the concentration of heavy metals was not higher in any of the studied samples.Soil factors such as pH, organic matter content, and mineral composition can indeed play a significant role in determining the trace element composition of plants 89 .Understanding the interactions between the properties and plant uptake of trace elements is important for managing the levels of the elements   The Iranian Trigonella species and populations evaluated in this study showed significant variability in terms of TPC, TFC, and antioxidant activity.These results are similar to the findings of other researchers.For example, antioxidant activity in the leaves extract of T. foenum-graecum has been already reported as 44.89 μg/ml 29 .In another study, antioxidant activity of T. arabica Delile and T. berythea Boiss.has been reported as 12.50 ± 0.54 and 6.02 ± 0.44 µg/ml.TPC were also obtained as 885.34 ± 1.14 and 64.44 ± 1.44 mg GAE/g, and TFC were 93.97 ± 0.4 and 76.67 ± 1.1 mg RE/g, respectively 90 .Gupta and Prakash 91 obtained the antioxidant activity of T. foenum-graecum as 27 mg/ml.A wide range of TPC, TFC, and antioxidant activity has been reported in Trigonella species so far 22,92 .Hussain et al 76 reported the TPC and TFC of T. foenum-graecum leaves from India as 425.4 ± 10.6 and 205.1 ± 8.9 mg/100 g, respectively.
Our results revealed that the studied samples have a high TPC and TFC, which led to an increase in their antioxidant activity.Correlation analysis showed a significant relationship between TPC, TFC, and antioxidant properties of the studied Trigonella species and populations (Fig. 7a-f).A significant relationship was found www.nature.com/scientificreports/ between TPC and antioxidant properties by DPPH (R2 = −0.83)and FRAP method (R2 = 0.86).TFC also had a significant relationship with the antioxidant properties by the DPPH (R2 = −0.61)and FRAP (R2 = 0.65).Nowadays, most of the researches are focused on the use of new and safe antioxidants from plant sources 93 .Due to cancer incidence increasing globally, the importance of studying antioxidant compounds, especially in plant sources, has been clarified 94 .Alternatively, through targeted breeding efforts and selection processes, breeders can create new fenugreek cultivars that not only meet specific nutritional needs but also useful for special pharmaceutical purposes.

Principal component analysis and correlations among traits
Biplot analysis was performed using PC1 and PC2, which accounted a total of 64.75 and 46.44% of the variance for phytochemical compounds and elements, respectively (Fig. 8).According to the biplot of phytochemical compounds, the populations were divided into four groups (Fig. 8a).TFG1, TFG2, TTH1, TTH2, TTH3, TCO3, and TEP1 characterized with high values in riboflavin, β-carotene, carotenoid, TSC, chlorophyll a, bitterness value, and diosgenin were placed in the first group, while TCO1, TCO2, TEP2, TEP3, TFG3, TCL3, TSP1, and TSP2 formed the second group that characterized by high value in chlorophyll b, niacin, and trigonelline.The third group including some populations of T. strangulata (TSG1, TSG2, TSG3), T. astroides Fisch.& C.A.Mey.(TAS1, TAS3), and T. spruneriana (TSP3) were not characterized by any phytochemical compounds studied.The highest content of total tannins, anthocyanin, ascorbic acid, thiamine, and pyridoxine was found in TAS2, TCL1, TCL2, TST1, TST2, TST3, TFP1, TFP2, and TFP3, which were placed them in the fourth group.The studied samples were separated into four groups based on element content (Fig. 8b).TCL1, TCL2, TCL3, TSG1, TSG2, TSG3, TEP1, and TAS2 were formed the first group which were associated with high value in macro-elements.In addition, nine populations formed the second group, which were characterized by high content of Ni and Co. TFG1, TFG2, TFG3, TST2, TTH1, TTH3, TEP2, TEP3, and TCO3 were placed in the third group that characterized by Zn, Al, Fe, Mo, and Cd content.The fourth group including TSP1, TSP2, TSP3, and TST3 was related to high values in Cr, Se, Pb, Cu, and Mn.
Correlation analysis showed a significant positive and negative relationship (p < 0.05) among the studied phytochemical traits.The content of niacin, chlorophyll b, diosgenin, trigonelline, TTC, TSC, and bitterness value had the most significant relationship between the studied traits (Fig. 9).β-Carotene content had the highest positive and significant relationship with carotenoid (r = 0.95) followed by diosgenin content with bitterness value (r = 0.92).The strongest negative and significant relationship was observed between TTC and TSC (r = − 0.86) and bitterness value (r = − 0.83).As mentioned before, correlation analysis was also showed that the bitterness value of fenugreek leaves is due to saponin compounds including diosgenin 74 .A significant relationship between phytochemical traits in plants has been previously reported [95][96][97] .In addition to genetic, plant growth conditions play a key role in the value of compounds 11 .

Canonical correspondence analysis
The studied fenugreeks populations are distributed within the latitude of 27° 15′ N to 38° 40′ N and longitude of 44° 77′ E to 60° 18′ E encompassing different geographical regions.Precipitation levels fall between 98.2 and 1892.0 mm/year, while the annual temperature averages 9.3 to 27.2 ℃.Relative humidity ranges from 28.0 to 84.2%.CCA was conducted to assess the relationship between the populations' environmental factors and the forty-seven studied parameters of phytochemical and nutritional compounds (Fig. 10).The environmental factors that were considered included altitude, relative humidity (RH), mean annual precipitation (MAP), and mean annual temperature (MAT).The first CCA variable (CC1) concerning environmental parameters showed that MAT, MAP, and RT had a positive share, while altitude had a negative share on this CCA construction.The first canonical variable in connection to the studied characteristics showed that the value of ascorbic acid, anthocyanin, thiamin, riboflavin, pyridoxine, total tannins, and most macro-elements had a negative share in the formation of CCA1 variables.The most important factor of the second CCA (CCA2) was altitude.The majority of micro-elements, heavy metals, essential oil components, and other important traits such as trigonelline, diosgenin, and total saponins, niacin, carotenoid, TPC, TFC, and FRAP had a negative share with altitude.
The findings suggest that while environmental and climatic factors such as temperature, altitude, and precipitation do impact the variability of nutritional and phytochemical compounds in the studied fenugreeks, genotype plays the most crucial role.Interestingly, our study revealed no significant differences among the populations of the same species in terms of the most studied parameters, indicating that these factors are not solely determined by fenugreek genetic combinations and climate properties.For example, the similarity of the content of nutrients and phytochemical compounds in the populations of T. coerulescens, T. filipes, T. calliceras, and T. teheranica species can be due to the geographical and climatic origin of close distance populations.
According to the results, it can be concluded that in most cases, the differences in nutritional and phytochemical compositions among fenugreek species and populations are due to genetic factors and are less related to the geographical origin of the populations.Zhang et al. 98 analyzed the phytochemical constituents of 747 plant species and concluded that while environmental and climatic conditions do influence the phytochemical composition of plants, the genotype is the most essential determinant, aligning with our findings.In addition, Tripodi et al. 99 , and Neugart et al. 100 also achieved similar results.Metabolome and genome analysis can determine whether geographic patterns of variation observed in phytochemical levels are simply due to environmental flexibility or are actually due to genetic differentiation due to isolation by geographic distances.

Conclusions
Extracts of medicinal plants and vegetables are increasingly receiving attention due to their industrial applications as natural compounds.Therefore, it is very important to identify the different masses that are the richest in terms of specialized metabolites.In this study, phytochemical diversity as well as considerable variations in vitamins and essential elements were observed among Trigonella species.Therefore, it is possible to improve nutritional, biochemical and phytochemical compounds by designing breeding programs and introducing new species and varieties.In summary, populations of T. teheranica, T. elliptica, T. coerulescens, and T. foenum-graecum were identified with high values of the studied traits, depending on different purposes, including functional foods as well as a therapeutic agent can be exploited.
. a CRI: calculated retention indices determined in the present work relative to n-alkanes C

Fig. 1 .
Fig. 1.A typical GC-MS chromatogram of the studied essential oil sample.The numbers correspond to the compound ordered in Table2.

Fig. 2 .
Fig. 2. Heat map showing the essential oil profiles of thirty Trigonella populations.Mean values refer to colors from minimum displayed in dark blue to maximum represented with yellow.Created by R statistical software (version 4.4.1)using the pheatmap, RColorBrewer, and Viridis packages.
https://doi.org/10.1038/s41598-024-71949-4www.nature.com/scientificreports/ in crops and ensuring food safety.The present study showed that the leaves of the studied fenugreeks are a good source of nutrients that can play an important role in diets and prevention of various diseases.Total phenol content, total flavonoid content, and antioxidant propertiesTPC, TFC, and antioxidant properties of the studied samples are shown in Fig.6a-d.The TPC was ranged from 87.43 ± 1.23 to 179.10 ± 1.06 mg GAE/g DW.The highest content of TPC was measured in some populations of T. teheranica (TTH1, TTH2, TTH3) followed by TCL2.

Fig. 6 .
Fig. 6.Total phenol (a) and flavonoids (b) content, and antioxidant activates (c, d) in the leaf of the studied populations of Trigonella species.n = 3, error bars represent standard deviation.

Fig. 8 .
Fig. 8. Principal component analysis (PCA) graph of quantified studied parameters: (a) specialized metabolites and (b) elements.For a detailed description of the plant populations code, cf.Table 1.

Fig. 9 .
Fig. 9. Correlation plot based on the specialized metabolites for the studied populations of Trigonella species (Significant level: 0.05).

Fig. 10 .
Fig. 10.Canonical correspondence analysis (CCA) of the studied populations of Trigonella species collected from different environmental conditions.MAP mean annual precipitation, MAT mean annual temperature.

Table 1 .
Geographic location of thirty studied populations of the ten Trigonella species from Iran. a RH: relative humidity.b MAP: mean annual precipitation.c MAT: mean annual temperature.