Introduction

Non-enzymatic glycation of proteins has important clinical implications in pathogenesis of diabetes, Alzheimer’s disease, atherosclerosis, renal failure, osteoporosis and cancer (Fournet et al. 2018). In diabetes, chronic hyperglycemia induced increased glycation of proteins which contributes significantly in the development of major diabetic complications such as retinopathy, nephropathy, neuropathy and coronary artery disease (Kim and Lee 2012). Chemically synthesized drugs with antiglycation activity cause adverse reactions and cannot be used in the long term (Song et al. 2021). Hence, natural compounds targeting specific steps of glycation cascade as safe therapeutic agents are gaining importance over conventional therapies for managing diabetic complications (Elosta et al. 2012; Sadowska-Bartosz and Bartosz 2015; Younus and Anwar 2016). T. foenum-graceum L. (commonly called fenugreek), known to targets several root causes of diabetic complications is widely used in botanical herbal preparations because of its reputed cardio tonic, hypoglycemic, diuretic and hypotensive activity. Therapeutic potential of fenugreek to manage the multifactorial nature of diabetes and its lifelong complications including glycation, oxidative stress, hyperlipidemia and insulin resistance are well documented (Abeysekera et al. 2018; Tabassum and Ahmad 2021; Salman and Qadeer 2012). Diosgenin, trigonelline, 4-hydroxyisoleucine and galactomannan (complex carbohydrate) are the most intensively studied bioactive constituents both clinically and in basic research settings for their anti-hyperglycemic and antiglycation activities to reduce diabetic complications (Naeem et al. 2021; Petropoulos 2002). Diosgenin, the major steroidal sapogenin in fenugreek, shows antidiabetic activity by inhibiting α-glucosidase and α-amylase, stimulating insulin secretion, enhancing insulin action in skeletal muscle and adipose tissue, upregulating the synthesis of antioxidant enzymes, reversing hyperlipidemia and downregulating the synthesis of enzymes involved in hepatic gluconeogenesis and glucose export (Fuller and Stephens 2015). Trigonelline, the principal alkaloid in fenugreek, shows antiglycation activity by inhibiting the formation of pentosidine compounds, Amadori product and advanced glycation end products (AGEs) in a dose-dependent manner (Chowdhury et al. 2018). Trigonelline also increases the levels of AGEs detoxification components such as glyoxalase1 (GLO-1-enzyme responsible for methylglyoxal detoxification) and AGE–receptor (AGE-R1- which promote degradation of AGE modified proteins) in the liver and kidneys (Costa et al. 2020). 4-hydroxyisoleucine, which comprises approximately 80% of the total free amino acid in fenugreek, exerts antidiabetic effect by stimulating insulin secretion, reducing hyperglycemia, restoring insulin sensitivity in skeletal muscle, decreasing plasma triglycerides, total cholesterol and free fatty acid levels and improving liver function (Fuller and Stephens 2015). The fiber component of fenugreek (galactomannans) enhances glycemic control by inhibiting lipid and carbohydrate hydrolyzing enzymes in the digestive system (Fuller and Stephens 2015).

Besides fenugreek, in vitro pharmacological activities of some Trigonella species used in traditional medicine is scarcely documented (Petropoulos 2002). Experimental studies using animal models have evaluated the protective role of T. corniculata (L.) L. (synonym of T. balansae Boiss & Reut. commonly called wild trefoil, kasuri methi, kasturi methi or sickle shaped fenugreek) and T. glabra Thunb. (T. hamosa L. is a rejected name, T. hamosa Forssk. is a later homonym, Mittal et al. 2020) against diabetic perturbations and complications in rats (Salah-Eldin et al. 2007; Khan et al. 2014). However, there are no systematic studies evaluating the effect of these plants extracts on glycation induced modifications of albumin. Although included in Indian herbals along with T. foenum-grecum (Hardman and Fazil 1972), antiglycation potential of T. uncata Boiss. & Noë. and T. occulta Delile ex Ser has not been studied. Many naturally occurring polyphenols, polysaccharides, terpenoids and alkaloids from plants as new and potent inhibitors of glycation have attracted increasing attention in recent years because of their minimal toxic effects (Song et al. 2021). Therefore, the current study comparatively investigates antiglycation potential of seeds of T. foenum-graecum, T. corniculata, T. glabra, T. uncata and T. occulta to find out if they contain unique phytochemistry to manage diabetic complications. The multistage glycation markers fructosamines (early stage) and protein carbonyls (intermediate stage) are investigated along with measurement of thiols and β aggregation of albumin. Since inhibition of α-glucosidase slows carbohydrate breakdown in the small intestine and reduces the postprandial blood glucose rise in diabetes, the present work also compares α-glucosidase enzyme inhibition potential of these Trigonella species as new alternative in terms of natural bioactive compounds. Moreover, correlation of the protective effects of the extracts with total phenolics, total flavonoids and antioxidant potential is discussed.

Material and methods

Chemicals and plant material

Bovine serum albumin (BSA) [fraction V, initial fractionation by heat shock, purity- 98% (electrophoresis) and 2,2-Diphenyl-L-Picrylhydrazyl (DPPH) extra pure were obtained from Sigma Chemical Company (St. Louis, MO, USA). All other chemicals used were of analytical grade. T. foenum-graecum, T. corniculata, T. uncata, T. glabra and T. occulta were collected from Indian floristic regions. Authenticated voucher specimens of all collected species were submitted to Botanical Survey of India, Western Regional Centre, Pune, Maharashtra, India. The area of collection and herbarium numbers of all sampled species are outlined in Table 1.

Table 1 Summary of Trigonella species used in present study, area of collection and herbarium voucher numbers
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Preparation of seed extracts

10 to 15 g of seeds were crushed and ground into a fine powder using a dry grinder. For defatting, seed powder was extracted using hexane for 5 h followed by extraction with methanol for 6 h using Soxhlet (Zhang et al. 2018). The methanol solvent was evaporated by using rotary evaporator under reduced pressure to obtain crude methanolic extract. Aqueous and methanol extracts were prepared by dissolving the dried crude methanolic extract in water and methanol, respectively to make a stock solution of 500 mg/ml.

α-glucosidase inhibitory activity

One unit of α-glucosidase enzyme solution was prepared by adding 1 mg of α-glucosidase enzyme in 4 ml of phosphate buffer (0.1 M pH = 6. 9) and stored at 4 °C till further use. α-glucosidase inhibitory potential of aqueous seed extracts was carried out according to the standard method with minor modification (Telagari and Hullatti 2015). The assay mixture containing 240 µl phosphate buffer (100 mM, pH = 6. 8), 40 µl α-glucosidase (1 U/ml) and extracts at concentration from 1.25 to 7.5 mg/ml was pre incubated at room temperature for 5 min followed by the addition of 120 µl of 4-Nitrophenyl-β-D- glucopyranoside (5 mM) as substrate and incubated further at 37 °C for 15 min. The reaction was stopped by adding 800 µl of Na2 CO3 (0.1 M) and absorbance of released p-nitrophenol was measured at 400 nm. The known α-glucosidase inhibitory acarbose was used as a positive control. Results were expressed as percentage inhibition calculated using the formula:

$$\% {\text{ inhibition }} = \, \left( {1 \, - {\text{ As}}/{\text{Ac}}} \right) \, \times 100$$

where, As is the absorbance in the presence of extract and Ac is the absorbance of control. Concentration of extract required to achieve 50% enzyme inhibition (IC50) was determined graphically using Microsoft Excel.

Total phenolic content (TPC)

Total Phenolic content of methanol extracts was determined in triplicate using Folin–Ciocalteau reagent. In brief, Folin–Ciocalteau reagent was diluted to tenfold with deionised water. 500 µl of plant extract (2.5–25 mg/ml) was mixed with 500 µl of the 1 M Na2CO3 and 500 µl diluted Folin–Ciocalteau reagent. The mixture was incubated at room temperature for 15 min in dark condition. The absorbance of all the samples was measured at 765 nm. TPC was determined as mg of gallic acid equivalents (GAE) per g of sample by computing it with standard calibration curve. Concentrations of 10, 20, 40, 60, 80, 100 µg/ml were taken for gallic acid (Singleton et al. 1999). The standard graph was obtained for Y = 0.009x + 0.0384; R2 = 0.9994.

Total flavonoids content (TFC)

Quantitative analysis of total flavonoids in methanolic seed extracts was done using AlCl3 assay. 100 µl of plant extract (2.5–25 mg/ml) was mixed with 400 µl of methanol, 100 µl of 10% AlCl3 and 100 µl of 1 M CH3CO2K. The tubes were incubated at room temperature for 30 min in dark condition. The absorbance of the reaction mixture was measured at 415 nm. TFC was determined from extrapolation of calibration curve which was made by preparing quercetin solution in methanol (10, 20, 40, 60, 80 and 100 µg/ml). The standard graph was obtained for Y = 0.0093x + 0.148; R2 = 0.9993. TFC was expressed as mg of quercetin equivalents (QE) per g of seed extract (Kalita et al. 2013).

DPPH radical scavenging activity

The capacity of seed extracts to react with and quench free radicals was determined by comparing it with ascorbic acid as a standard (Standard graph was obtained for Y = 1.0481x + 21.705, R2 = 0.9441; Brand-Williams et al. 1995). A stock solution of DPPH (100 µM) was prepared in methanol. The reaction mixture containing 100 µl of DPPH, 100 µl of seed methanolic extracts at different concentrations (2.5–25 mg/ml) and 200 µl of methanol was incubated at 37 °C for 30 min. The absorbance was measured at 515 nm. DPPH scavenging activity was determined according to the formula:

$$\% {\text{ inhibition }} = {\text{ A}}_{{{\text{control}}}} {-}{\text{ A}}_{{{\text{sample}}}} /{\text{ A}}_{{{\text{control}}}} \times 100$$

where the Acontrol is absorbance of the control (DPPH solution without sample) and Asample is absorbance of the test sample (DPPH solution plus test sample).

Total antioxidant activity (TAA) by phosphomolybdenum method

The total antioxidant capacity of methanolic seed extracts was evaluated using phosphomolybdenum method (Jamuna et al. 2011). Seed extracts at various concentrations (15–25 mg/ml) was combined with 1000 µl of reagent solution [0.6 M H2SO4, 28 mM Na3PO4 and 4 mM (NH4)2MoO4] . The tubes were capped and incubated in a water bath at 95 °C for 90 min. After cooling absorbance was measured at 695 nm against blank. Results were expressed as GAE (mg/g of seed extract).

Determination of reducing power assay (RP)

Reagents were prepared as per the original method (Oyaizu 1986). In 96-well plates, 10 µl of sample solution (15–25 mg/ml), 25 µl of buffer and 25 µl of Potassium Hexacyanoferrete II K4[Fe(CN)6] (1%) were added sequentially. The mixture was incubated for 20 min at room temperature, and reaction was stopped by adding 25 µl of trichloroacetic acid (TCA) solution. Further, 85 µl of water and 8.5 µl of FeCl3 was added to each well. The contents were mixed, incubated for another 15 min at room temperature and absorbance was measured at 750 nm. Ascorbic acid was used as a standard at concentration range from 10 to 100 µg/ml. Higher absorbance indicated higher reducing potential.

Antiglycation potential

Albumin glycation was performed with some modifications (McPherson et al. 1988). Glycated BSA samples were prepared using BSA (20 mg/ml), Glucose (166.5 mM) in potassium phosphate buffer (100 mM, pH 7.4 containing 0.02% sodium azide) along with aqueous seed extracts (final concentration 200 µg/ml). Positive control (BSA + Glucose) and standard (BSA + Glucose + Quercetin final concentration 10 µg/ml) was maintained under similar conditions. Before incubation, all the solutions were filtered through 0.22 µm membrane filters in sterile plastic-capped vials to maintain sterility and strict asepsis was maintained during incubation period. Glycation sealed tubes were incubated in the dark at 37 °C for 28 days. All the incubations were performed in triplicates. The unbound glucose was removed by dialysis against phosphate buffer (100 mM, pH 7.4) and stored at 4 °C. The dialysates were used to determine the antiglycation activity of aqueous extract by estimation of (i) Fructosamines adducts (ii) Protein carbonyls (iii) Protein thiols and (iv) Congo red absorbance.

Nitroblue tetrazolium assay

Nitroblue tetrazolium assay was used to determine the fructosamine (Baker et al. 1994). Aliquots of glycated samples and positive control (40 µl) was added to the 800 µl of nitroblue tetrazolium (0.75 mM) in sodium carbonate buffer (100 mM, pH 10.4) and incubated at 37 °C for 45 min. The absorbance was measured at 530 nm and the percent inhibition of fructosamines formation by aqueous seed extracts was calculated using the following equation:

$${\text{Inhibitory activity }}\left( \% \right) \, = \, \left[ {1 - \left( {{\text{A}}_{{0}} - {\text{A}}_{{1}} } \right)/{\text{A}}_{{0}} } \right] \, \times \, 100$$

where A0 is the absorbance value of the positive control and A1 is absorbance of the glycated albumin samples co-incubated with seed extracts.

Protein carbonyl content assay

Protein carbonyl content assay was conducted using standard protocol with minor modifications (Uchida et al. 1998). A total of 500 µl of 2,4-dinitrophenylhydrazine (0.1% w/v in 2.5 M HCl) was added with 500 µl of glycated samples and incubated for 60 min in the dark. After incubation, 500 µl of 20% (w/v) chilled TCA was added to the solution for protein precipitation and left at 4 °C for 5 min. The solution was centrifuged at 3000 rpm for 10 min at 4 °C and the protein pellet was rinsed with a mixture of ethanol/ethyl acetate (1:1) for three times and finally re-suspended in 500 µl of 6 M guanidine hydrochloride. Absorbance was captured at 365 nm. Protein carbonyl concentration was calculated by using the molar extinction coefficient (á ʹ 365 nm = 21 mM per cm). The results are expressed as percent inhibition and calculated by the formula stated above.

Protein thiol estimation

Thiol groups of glycated albumin samples (positive control), glycated albumin samples co-incubated with aqueous seed extracts and glycated albumin samples co-incubated with quercetin was measured using 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB). For this assay, 250 µl samples was incubated with three volumes of 0·5 mM DTNB (750 µl) for 15 min. The absorbance was measured at 410 nm. The free thiol concentration was calculated using standard curve performed with various BSA concentrations (0·8–4 mg/ml) as nM thiol/mg protein (Ellman 1995). The results are expressed as percent protection and calculated by the formula

$${\text{Protection }}\% \, = \, \left[ {\left( {{\text{A}}_{{0}} {-}{\text{A}}_{{1}} } \right)/ \, \left( {{\text{A}}_{{0}} } \right)} \right] \, \times \, 100$$

Were A0 is the absorbance of the positive control and A1 is the absorbance of the glycated albumin samples co-incubated with seed extracts/quercetin.

Estimation of amyloid β aggregation

Aggregation in glycated sample was measured using amyloid specific Congo red dye according to the method described previously (Klunk et al. 1999). In this assay, glycated samples (500 µl) were incubated with 100 µl of Congo red (100 µM) in PBS with 10% (v/v) ethanol for 20 min. at room temperature. Absorbance was recorded for the Congo red-incubated samples as well as for Congo red background at 530 nm. The results were expressed as percent inhibition calculated by the formula used in Nitroblue tetrazolium assay.

Statistical analysis

Data was expressed as mean and standard deviations of triplicate values. Statistical analysis was carried out using the Microsoft Excel software package. The Pearson correlation matrix was applied to find the relationships between the different analytical methods, which were expressed as the correlation coefficient ‘R’.

Results

α-glucosidase inhibitory potential

Aqueous seed extracts of five Trigonella species were evaluated for their inhibitory effect on α-glucosidase enzyme by in-vitro method. All the tested extracts were able to suppress the activity of α-glycosidase in a dose-dependent manner where a dose of 2.5 mg/ml caused percent inhibition of α-glycosidase by 45.72 ± 0.08, 21.76 ± 0.56, 25.73 ± 0.24, 62.81 ± 0.68 and 47.00 ± 0.13 for T. foenum-graecum, T. corniculata, T. glabra, T. uncata and T. occulta, respectively. Comparing the IC50 values of the extracts against α-glycosidase revealed that T. uncata possessed the lowest IC50 value (1.14 ± 0.04 mg/ml) with highest enzyme inhibitory potential among the tested Trigonella species while T. corniculata possessed the highest IC50 value (7.91 ± 0.01 mg/ml) indicating its lesser activity against the tested enzyme (Fig. 1).

Fig. 1
figure 1

Percent α-glucosidase inhibition of aqueous seed extracts (values are expressed as mean ± SD, n = 3)

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TPC of seed extracts

TPC varied from 5.09 to 12.91 mg GAE/g of seed extract in the following increasing order: T. balansae < T. foenum-graecum < T. occulta < T. glabra < T. uncata. The extracts of T. occulta, T. glabra and T. uncata contained almost two fold total phenolics as compared to T. foenum-graecum extract (Fig. 2).

Fig. 2
figure 2

Total Phenolic and Flavonoid contents of tested plants. Results expressed as mg of Gallic acid equivalence (GAE) and mg of Quercetin equivalence (QE) respectively. Values are expressed as mean ± SD, n = 3

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TFC of seed extracts

TFC varied from 7.08 to 13.87 mg QE/g of seed extract in the following increasing order: T. occulta < T. balansae < T. foenum-graecum < T. glabra < T. uncata. In addition to TPC, extracts of T. glabra and T. uncata also showed higher TFC content as compared to T. foenum-graecum extract (Fig. 2).

DPPH radical scavenging activity

The scavenging ability of methanolic extracts on DPPH free radical is shown in Fig. 3 and expressed as IC50. It can be seen that both extracts and standard exhibited effective concentration dependent DPPH radical scavenging ability. Result showed that methanolic extract of T. uncata (IC50 = 2.46 ± 0.160 mg/ml) and T. corniculata (IC50 = 2.34 ± 0.038 mg/ml) were most active. However, both the extracts showed a much lower radical scavenging activity as compared to ascorbic acid (IC50 = 0.029 ± 0.002 mg/ml) which was used as a standard.

Fig. 3
figure 3

2,2-Diphenyl-L-Picrylhydrazyl (DPPH) free radical scavenging activity of selected plants

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TAA

TAA of seed methanolic extracts increased with increasing concentration of seed extract from 7.5 to 12.5 mg/ml and was highest for T. glabra (14.91 mg GAE/g of extract) and lowest for T. foenum-graecum (9.91 mg GAE/g of extract, Fig. 4).

Fig. 4
figure 4

Total Antioxidant activity (TAA) and Reducing power (RP) of tested plants. Results expressed as mg of Gallic acid equivalence (GAE) and mg of Ascorbic acid equivalence (AAE), respectively. Values are expressed as mean ± SD, n = 3

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RP

Among the tested plants, RP of methanolic extract of T. glabra was highest (121.744 mg of Ascorbic acid equivalence (AAE)/g of extract), indicating that it has strong capacity of donating electrons. Moderately high RP value was shown by methanolic extract of T. occulta (91.54 mg AAE/g of plant extract) and T. uncata (77.08 mg AAE/g of plant extract) while lowest reducing capacity was demonstrated by T. corniculata and T. foenum-graecum which had least TPC and TFC (Fig. 4).

Antiglycation potential of aqueous seed extracts

The potential of aqueous seed extracts of five Trigonella species to inhibit the glycation reaction was analysed at three levels, early and late glycation reactions, albumin oxidation and amyloid aggregation. Early glycation products (fructosamines) were inhibited in the range of 17–26%. T. glabra showed least inhibition with the remaining species showing almost similar inhibition in the range of 21–26% (Fig. 5a). Protein carbonyl compounds were moderately inhibited in the range of 14–23% as compared to standard (Quercetin 10 µg/ml) with T. glabra extract showing maximum inhibition (Fig. 5b). As shown in Fig. 5c, glycation in presence of T. corniculata and T. glabra extract increased free thiol groups by 29.8% and 28.4%, respectively. T. uncata showed least increase (19.1%) in thiol groups as compared to other species. Extracts of T. occulta exhibited maximum reduction of β-amyloid aggregation compared with other plant extracts (Fig. 5d). A good correlation was observed between inhibition of fructosamine and amyloid formation (R = 0.950, p < 0.05). Correlation was also observed between reduction in carbonyl levels and increase in free thiol groups (R = 0.684, p < 0.05).

Fig. 5
figure 5

The effect of seed extracts and standard (Quercetin) on glycation induced albumin modifications in terms of a Fructosamine inhibition, b Protein carbonyls inhibition, c Protein thiol protection, d β- aggregation inhibition. Values are means (n = 3), with standard deviations represented by vertical bars. Results of the measured parameter expressed as percent inhibition with respect to positive control

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Discussion

Natural products as inhibitors of carbohydrate hydrolyzing enzymes (α-amylase and α-glucosidase) are useful in the control of hyperglycemia. Some Trigonella species are more potent inhibitors of α glycosidase activity as compared to α-amylase activity (Shawky et al. 2022). Hence, in the present work, α glycosidase inhibitory potential of T. corniculata, T. uncata, T. glabra and T. occulta was tested and compared to T. foenum-graecum, a complementary herbal drug used since ancient times in existing medications for the treatment of diabetes and its complications. Potent α-glucosidase inhibitory potential of seeds as well as leaves of T. foenum-graecum is well documented in literature (Ganeshpurkar et al. 2013; Shawky et al. 2022) with a single report on α-glucosidase inhibitory potential of leaves of T. glabra (Shawky et al. 2022). No data was found about inhibition of α-glycosidase enzyme activity by seeds of T. corniculata, T. uncata and T. occulta. Results obtained are in agreement with previous reports demonstrating inhibitory effect of T. foenum-graecum and T. glabra extracts on α-glycosidase activity with T. foemun-graecum being a better inhibitor than T. glabra (Shawky et al. 2022). However, in the present study, the IC50 values were higher. This may be due to less diversity and concentration of bioactive compounds in seeds as compared to leaves. In the present analysis, T. uncata showed a much higher α-glycosidase inhibition potential as compared to T. foenum-graecum. Many studies have reported flavones, isoflavones, and pterocarpans as potent α-glycosidase inhibitors (Proença et al. 2017; Dendup et al. 2014; Riyaphan et al. 2017) with quercetin, quercetin hexoside and isoschaftoside, β-chacotriosyllilagen, para hydroxybenzoic acid and dihydocaffeic acid having the highest positive correlation to α-glycosidase inhibitory effect in Trigonella (Shawky et al. 2022). The higher α-glycosidase inhibitory potential of T. uncata as compared to T. foenum-graecum, T. corniculata, T. glabra and T. occulta can be correlated to higher TPC and TFC of its seed extracts as compared to other species.

T. corniculata, used as a spice and flavoring agent, is cultivated in India and Pakistan and used medicinally in Ayurveda, Unani and Siddha systems (Quattrocchi 2016). In the present work, with comparable level of TPC and TFC, T. corniculata seed extract had a better DPPH radical scavenging and TAA as compared to T. foenum-graecum indicating presence of phytoconstituents with better antioxidant potential in T. corniculata. Results agree to Semalty et al. (2009) who demonstrated a stronger DPPH radical scavenging activity in methanolic seed extracts of T. corniculata as compared to T. foenum-graecum. T. uncata, T. glabra and T. occulta with their higher TPC and TFC showed better overall antioxidant potential (DPPH radical scavenging activity, TAA and RP) as compared to T. foenum-graecum. Higher antioxidant potential of T. glabra (TAA and RP) as compared to T. uncata (with higher TPC and TFC) could be contributed by other non-phenolic antioxidants.

Protein glycation is the major contributor of diabetic complications. Medicinal plants with antiglycation potential provide promising opportunity as complementary herbal interventions to reduce diabetic complications. There are number of reports about antidiabetic and antioxidant activities of T. foenum-graecum with a single report of inhibition of AGEs by seeds of fenugreek (Naeem et al. 2021; Abeysekera et al. 2018). However, there are no reports regarding its effect on oxidative modifications of BSA and inhibition of protein aggregation. Antiglycation potential of seed extract of T. corniculata. T. uncata, T. glabra and T. occulta is reported for the first time. All the tested plants exerted noticeable but lesser antiglycation potential as compared to standard (Fig. 5a–d).

T. foenum-graecum, T. corniculata and T. glabra possesses antidiabetic and antioxidant activities (Naeem et al. 2021; Khan et al. 2014; Kaur et al. 2018; Salah-Eldin et al. 2007; Khalil et al. 2022). Likewise, in our study T. foenum-graecum and T. corniculata moderately inhibited glycation of albumin at multiple stages being more effective in inhibiting fructosamine formation which may further lead to inhibition of amyloid formation (R = 0.950; p < 0.05). T. glabra showed poor inhibition of fructosamine and amyloid formation. However, it was more effective in preventing protein oxidation (R = 0.684; p < 0.05, reduction in carbonyl levels along with an increase in free thiol groups). This difference may be attributed to variation in chemical profile of tested species. Flavonoids, pterocrpans and aliphatic acids (Hamoside B, sarsaponin, trigocoumarin, piscigenin, quercetin diglycoside and trigoxazonane) are the main chemical classes detected in T.glabra while amino acids and isoflavones (hyroxyisoleucine and ketoleucine, trigonelline, kaempferol hexoside and kaempferol diglycoside, panasenoside, soyasaponin I, medicarpin, demethyl medicarpin and methyl vestitol) are the main discriminatory phyto constituents present in T. foenum-graecum (Shawky et al. 2022).

T. glabra, a recent addition to flora of India and known as Hasawi gardener is eaten fresh and uncooked as a refreshment in salad (Mittal et al. 2020; Mandaville 2013). It exerts its antidiabetic activities by improving insulin synthesis, secretion and action, enhancing lipid and protein metabolism and removing the oxidative stress concomitant with glucose homeostasis (Salah-Eldin et al. 2007; Hamed 2007). The plant contains volatile oils with antimicrobial and antioxidant activities (Qari and Fahmy 2017; Shahat et al. 2015). Recently, orientin, a bio-flavonoid isolated from T. glabra was shown to possess anticancer activity (Khalil et al. 2022). During glycation, toxicity of free radicals leads to oxidation induced carbonyl protein formation accompanied by the loss of free thiols in albumin (Ardestani and Yazdanparast 2007; Nunthanawanich et al. 2016). In the present study, a moderately better protection of albumin against oxidation by T. glabra can be partly correlated to its stronger antioxidant potential (TAA and RP). Among the tested species, T. glabra, with its α-glycosidase inhibitory potential, higher antioxidant activity coupled with stronger protection of albumin against oxidation can be combined with T. foenum-graecum in polyherbal formulations for treating diabetic complications. The additional protective effects of such polyherbal preparations to minimize albumin modifications needs to be investigated further by in vivo studies.