Metabolomic profiling of germinated and non-germinated Lablab purpureus seeds: antioxidant properties and α-amylase inhibitory activities for diabetes management

Abstract

Diabetes is characterized by prolonged hyperglycemia and disruptions in carbohydrate, lipid, and protein metabolism, stemming from inadequate insulin production, impaired insulin receptor functioning, or a combination of both. Conventional diabetes medications like biguanides and sulphonylureas, are widely used and raise concerns about potential side effects with prolonged usage. In this context, legumes emerge as promising candidates due to their significance in traditional diets globally and associated health benefits. Despite being challenging to digest due to anti-nutritive factors, germination, a simple bioprocessing technique, significantly enhances nutritional aspect of the seeds. This study focuses on Lablab purpureus, an underutilized legume, employing a metabolomic approach to explore compounds in germinated and non-germinated seeds. Metabolomic profiling identified 125 compounds in non-germinated and 80 compounds in germinated seeds, revealing unique compounds in each type with potential health benefits. The study identified therapeutically important metabolites such as alkaloids, flavonoids, terpenoids, and saponin in both the germinated and non-germinated seeds. A notable change in the phytochemical composition (total phenol, flavonoid, and total ascorbic acid content) of germinated seeds was observed compared to the non-germinated seeds flour. An increased fold change (1.15, 1.5 and 1.65) was observed in the total phenol, flavonoid, and total ascorbic acid content in germinated seeds compared to non-germinated seeds, alongside higher antioxidant levels in terms of DPPH, ABTS, and FRAP. The IC50 value for α-amylase inhibitory activity was noted to be 2.05 ± 0.05 mg/ml in germinated samples while 0.79 ± 0.00 mg/ml was observed in the non-germinated Lablab purpureus seeds. Therefore, displaying greater α-amylase inhibitory activity in the non-germinated seeds, possibly due to their unique biochemical composition. Nevertheless, even germinated seeds demonstrated appreciable α-amylase inhibitory activity. Therefore, these findings suggest that germination process significantly influences seed biochemistry and helps to raise the phytochemical composition, while the unique composition of the metabolites in the non-germinated seeds could have impact on the α-amylase inhibitory activity. Thus, study suggests Lablab purpureus as a promising functional food source with diverse health-promoting attributes, particularly in diabetes management.

Graphical Abstract

Introduction

Legumes are a staple in global diets owing to their rich nutritional content and they are highly recommended by organizations like the World Health Organization (Reyneke et al., 2022) and the Food and Agriculture Organization of the United Nations (FAO) (Hughes et al., 2022). They are renowned for their protein, dietary fiber, vitamins, minerals, complex carbohydrates, and caloric value (De Almeida Costa et al., 2006). Additionally, legumes are abundant in phenolic and polyphenolic compounds, such as phenolic acids, flavonoids, and lignin (Lin & Lai, 2006). A recent study on 15 different genotypes of Lablab purpureus L. revealed the presence of various phenolic and flavonoid compounds. Using HPLC analysis, approximately 10 phenolic compounds were quantified, including 8 phenolic acids and 2 flavonoids. Most of the genotypes contained trans-cinnamic acid, p-coumaric acid, and ferulic acid. Other phenolic acids identified included syringic acid, salicylic acid, and sinapic acid. Among the flavonoid compounds, quercetin and catechin were found in notable amounts (Das et al., 2023). Often, legumes are consumed after processing, which enhances both taste and nutrient availability (Tharanathan & Mahadevamma, 2003). Germination, a common processing method, can alter nutrient content and remove anti-nutrients, making sprouts suitable for consumption ( Lin & Lai, 2006).

Legume consumption is linked to various health benefits, including protection against cardiovascular diseases, cancer prevention, and diabetes management (Wang et al., 2009). Furthermore, it is associated with a reduced risk of obesity and metabolic syndrome (Singh & Pratap, 2016). These advantages are likely due to the synergy between the nutritional and non-nutritional components of legumes (Mitchell et al., 2009). Legumes have a low glycemic response, making them potentially beneficial in diabetes prevention or management due to their high fiber and resistant starch content (Ludwig, 2002). Some legumes, like Phaseolus vulgaris L. (Pinto beans), exhibit α-amylase inhibitory activity and anti-diabetic properties (Patil et al., 2020). In vivo, studies on sprouts of various legumes, including faba bean (Vicia faba L.), lentil (Lens esculenta L.), chickpea (Cicer arietinum L.), and fenugreek (Trigonella foenum-graecum L.), have shown anti-diabetic effects with significant reductions in blood glucose levels (Farag et al., 2022).

The current study focuses on the metabolomics profiling of Lablab purpureus seeds and sprouts to explore their potential as anti-diabetic agents. Lablab purpureus, also known as field bean, country bean, Indian bean, and Egyptian bean, is widely grown globally and is particularly abundant in tropical regions like India (Pandey, 2023). Lablab beans possess a nutraceutical and pharmaceutical profile, offering protein, essential amino acids like lysine and leucine, essential fatty acids, dietary fibers, micronutrients, and minerals (Hossain et al., 2016). Given the increasing incidence of metabolic diseases and the side effects associated with synthetic drugs, plant-based remedies may offer a sustainable approach to manage these conditions.

Globally, the prevalence of diabetes is escalating, with an estimated 693 million people projected to have the condition by 2045 if effective interventions are not implemented (Cho et al., 2018). India is one of the epicenters of this epidemic, with the second-highest number of people living with diabetes mellitus (69 million as of 2015) (Unnikrishnan et al., 2016). Diabetes can lead to long-term damage to various organs, including the eyes, kidneys, nerves, heart, and blood vessels (American Diabetes Association. 2014). One approach to treat Type-2 diabetes mellitus is inhibiting carbohydrate-hydrolyzing enzymes like α-amylase and α-glycosidase to lower postprandial plasma glucose levels and prevent postprandial hyperglycemia (Taslimi et al., 2020).

Given the potential of Lablab purpureus beans in managing diabetes, this study aims to comprehensively analyze germinated and non-germinated Lablab purpureus beans, including their metabolomics profiling, quantitative phytochemical analysis, antioxidant activities, and α-amylase inhibitory potential. Metabolomics, an omics discipline, enables the analysis of low-molecular-weight compounds (metabolites) involved in metabolic pathways and cellular processes, offering insights into phenotypes and mechanistic aspects of conditions like diabetes mellitus (Segers et al., 2019; Shi et al., 2016). Numerous metabolomics studies have explored the relationship between various metabolites, insulin resistance, and Type 2 diabetes mellitus through in vivo and in vitro experiments (Hasanpour et al., 2020). Considering the benefits offered by Lablab purpureus beans, this study seeks to unveil their potential as an anti-diabetic resource.

Materials and methods

Chemicals

For the current investigation, the following chemicals such as α-Amylase enzyme from porcine pancreas (5 U/mg), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS), bovine serum albumin (BSA), Folin & Ciocalteu’s phenol reagent, gallic acid, quercetin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and tetra-2-pyridinyl pyrazine (TPTZ) were procured from SRL Pvt. Ltd. India. All other chemicals used in the study were of analytical grade.

Sample collection

Indian bean (Lablab purpureus L. GW-2) seeds were collected from the Pulses and Castor Research Station at Navsari Agricultural University, Navsari, India (Geographic coordinates: 20.9467° N, 72.9520° E) in March 2022. After collection, the seeds were cleaned, and any debris was removed. The cleaned seeds were then stored in an airtight container.

Sample processing and preparation

The germination process was carried out by (Shabbir et al., 2022) method. 500 g of seeds was soaked in 0.05% sodium hypochlorite solution (1:6 w/v) for 30 min at room temperature to disinfect the seeds. The seeds were further drained and washed with tap water. Afterward, seeds were soaked in distilled water (1:6 w/v) for 12 h at room temperature (30 ± 2 °C). Finally, hydrated seeds were placed in trays where a wet filter paper was extended and allowed to germinate. For germination, the seeds were kept at 37 °C in an incubator for 24 h. The moisture was maintained in the germinating seeds by watering the trays with distilled water. Every germination experiment was performed in triplicate. The germination percentage was determined by counting the number of germinated seeds relative to the total number of seedlings. The germination percentage was calculated using following formula: $\:Germination\:Percentage\:\%=\left[\frac{Germinated\:Seeds}{Total\:number\:of\:seeds}\right]\times\:100$

The sprouts were completely dried which typically took around 48 h in the hot air oven at 50 °C. The drying temperature was carefully selected to minimize any impact on the characteristics of the germinated seeds. Non-germinated seeds and germinated seeds were powdered in a commercial coffee grinder to obtain fine flour and further sieved. The seed flour was stored at -20 °C until further processing.

Aqueous sample preparation for quantitative analysis

1 gram of seed sample flour was dissolved in 10 ml of distilled water. The mixture was vortexed for 10 min and then centrifuged at 4000 g for 10 min. The supernatant was collected and stored at -20 °C until used.

Metabolomics analysis

Seeds extraction procedure

Around 100 g of legume seed flour obtained from germinated and non-germinated seeds was mixed with 80% methanol and further vortexed. The sample was sonicated using sonicator instrument (Ultra Sonic Bath-Loba Life) (using UAE) for 30 min in a volumetric flask and then centrifuged at 4000 g for 15 min. The supernatant liquid was then collected in a round-bottom flask and evaporated using rotary evaporation at 39 °C under vacuum. Finally, 80% methanol was used to dissolve the dry residue before it was filtered through a 0.22 μm syringe filter, this extract was used for further LCMS analysis (Abu-Reidah et al., 2012).

Analysis by UHPLC-ESI-QTOF-MS/MS

The extracted seeds samples of germinated and non-germinated were further analyzed for HR-LCMS at Sophisticated Analytical Instrument Facility (SAIF), IIT Bombay, Powai, and Mumbai. Identification of metabolite in legume seed flour extracts was conducted using Agilent high-resolution liquid chromatography and mass spectrometry model- G6550A. Both ESI (positive and negative) ion modes were used to conduct the mass spectrometric analysis. The acquisition method was set to be MS- minimum range 120 (M/Z) and maximum 1200 Dalton (M/Z). The scanning was done with a rate of each spectrum per second. The ejection speed was 100 µl /min and 3 µl injection volume was used for HR-LCMS. Acquisition time was 30 min.

Identification of components

Interpretation on mass spectrum HR-LCMS was conducted using the database of SAIF-IIT Bombay having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectra of the known components stored in the SAIF library. The name, molecular weight, and structure of the components of the test materials were ascertained.

Phytochemical analysis

Determination of total phenolic content

The total phenolic content of the germinated and non-germinated Lablab purpureus seeds flour aqueous extracts was determined by the Folin–Ciocalteu colorimetric method as described by Thimmaiah (1999) and Kamble and Jadhao (2020) with slight modifications. Briefly, gallic acid (10–100 µg/ml) was used as a standard. In a known quantity of aqueous sample extract, Folin–Ciocalteu reagent was added and incubated for 3 min at room temperature. Further, 20% of sodium carbonate was added to the reaction mixture and kept in a boiling water bath for 1 min. The tubes were cooled, and absorbance was measured at 650 nm against blank using a UV-Visible spectrophotometer (BR Biochem Lifesciences Pvt. Ltd, India). The total phenolic content was calculated from the calibration curve, and the results were expressed as milligram of gallic acid equivalents per gram dry weight.

Determination of total flavonoids

The total flavonoids were determined following the procedure modified by Zhishen et al. (1999) and Phuyal et al. (2020). Briefly, the flavonoid content in the extracts was determined by an aluminium chloride colorimetric assay. Quercetin (100–1000 µg/ml) was used as standard. A known quantity of aqueous seed extract was incubated with 5% NaNO₂ for 5 min at room temperature and a further 10% AlCl₃ and 1 M NaOH was added to the mixture after 6 min. Absorbance was measured at 510 nm using a UV-Visible spectrophotometer (BR Biochem Lifesciences Pvt. Ltd, India). The total flavonoid content was expressed as milligram of quercetin equivalents per gram dry weight using the linear equation based on the standard calibration curve.

Total ascorbic acid content

The aqueous seed flour samples’ ascorbic acid content was determined as described by Schaffert and Kingsley (1955) and Agarwal & Verma (2014). Ascorbic acid (10–100 µg/ml) prepared in 4% Trichloroacetic acid (TCA) was used as standard. 0.5 gram of seed flour were prepared in norit charcoal reagent containing TCA and activated charcoal and were oxidized. To the samples, standard and blank, 6% TCA, 10% thiourea, and 2% dinitrophenylhydrazine (DNPH) were added and placed in a boiling water bath for 15 min. After cooling, 85% H₂SO₄ was added to each tube and the mixture was allowed to stand for 15 min in ice cold water bath. Total ascorbic acid was estimated photometrically as dehydroascorbic acid at 515 nm wavelength UV-Visible spectrophotometer (BR Biochem Lifesciences Pvt. Ltd, India). The results were expressed as milligram of ascorbic acid equivalents per gram dry weight.

Determination of protein

Using BSA (50–400 µg/ml) as a standard, a suitable quantity of extract was assayed by Folin-Lowry method, and the respective protein value was expressed in milligram of BSA equivalents per gram dry weight (Lowry et al., 1951; Ramani et al., 2021). For the assay 0.1 ml of aqueous seed flour sample was allowed to interact with 5 ml of alkaline copper solution at room temperature and incubated for 10 min. Further 0.5 ml of folin’s reagent was added to each tube, mixed well, and incubated at room temperature in the dark for 30 min. The absorbance of the sample was read at 660 nm UV-Visible spectrophotometer (BR Biochem Lifesciences Pvt. Ltd, India).

Antioxidant analysis

Ferric-reducing antioxidant power (FRAP)

The ferric-reducing abilities of samples were determined using the method described by Benzie and Strain (1996). Briefly, ascorbic acid (10–100 µg/ml) was used as a standard. Different aliquots of standard, samples and FRAP reagent were incubated for 30 min at 37 °C. The blue colour so obtained by the samples and standard absorbance was measured at 593 nm. FRAP values were expressed as milligram of ascorbic acid equivalents per gram dry weight using the standard curve equation.

2,2-Diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH)

The DPPH scavenging activity of the seed flour samples was measured using Brand-Williams et al. (1995). 0.1 mM DPPH solution was added to the samples and the mixtures were shaken vigorously and left to stand for 30 min in the dark at 37 °C. The absorbance of the mixture was read at 517 nm against methanol. As per the principle of the assay DPPH in oxidized form gives a deep violet colour in methanol. When DPPH receives an electron donation from an antioxidant chemical, it is reduced and turns from deep violet to yellow. The percentage of DPPH scavenging activity was calculated by the following equation.

$$\:\%DPPH\:radical\:scavenging\:activity=\left[\frac{A\:of\:control-A\:of\:sample}{A\:of\:control}\right]\times\:100$$

ABTS activity

ABTS assay was performed following the method described by Arnao et al. (2001) and González-Montelongo et al. (2010). Ascorbic acid was used as standard. To the 0.1 ml aqueous sample and standard add 2.9 ml of ABTS radical solution and were allowed to incubate for 6 min in the dark at room temperature and the absorbance was read at 734 nm. The percentage of ABTS was calculated by the following equation.

$$\:\varvec{\%}\:ABTS\:radical\:scavenging\:activity=\left[\frac{\left(A\:of\:control-A\:of\:sample\right)}{A\:of\:control}\:\right]\times\:100$$

α-amylase inhibitory activity

The assay was conducted as described by Vilcacundo et al. (2017) with slight modifications. The assay mixture contained α-amylase enzyme from porcine pancreas (2 U /ml) prepared in 20 mM sodium phosphate buffer (pH 6.8) and a known quantity of sample extract was incubated for 10 min at 37 °C. Thereafter 100 µl of 1% starch was added in all test tubes followed by incubation at 37 °C for 10 min. The reaction was terminated with the addition of 100 µl of 96 mM 3,5-dinitrosalicylic acid (DNS) reagent. The reaction was stopped by putting the tubes in a boiling water bath for 5 min. Finally, the reaction mixture was diluted with the addition of 800 µl of distilled water, and the absorbance was measured at 540 nm in a spectrophotometer. The α-amylase inhibitory activity was expressed as percent inhibition as calculated using the following equation and further IC50 values were calculated:

$$\%\;\mathrm{inhibition}\;\mathrm{of}\;\mathrm\alpha-\mathrm{amylase}=\left[\mathrm A\;\mathrm{of}\;\mathrm{control}-\frac{\left(\mathrm A\;\mathrm{of}\;\mathrm{sample}-\mathrm A\;\mathrm{of}\;\mathrm{sample}\;\mathrm{blank}\right)}{\mathrm A\;\mathrm{of}\;\mathrm{control}}\right]\times100$$

Statistical analysis

Phytochemical content, antioxidant activity, and α-amylase inhibitory activity analyses were performed in triplicate and the results are presented as Mean ± SEM. Data analysis was performed by independent samples t- test by using SPSS for Windows (version 16.0) and p value of < 0.05 was considered to be significant. The metabolomic studies were carried out for a single representative sample of seeds and explored for their metabolite compounds.

Results and discussion

Germination percentage

An easy way to improve the biochemical makeup of legumes is through germination. Additionally, the process of germination aids in enhancing the legumes’ nutritional qualities, palatability, and digestibility. In the current study, Lablab purpureus seeds were soaked for 12 h and then allowed to sprout for a further 24 h, as this is the most typical method for domestic use. The radicle size was measured using ruler, the radicle length from its tips to the point it emerges from the seed was calculated. A germination percentage of approximately 97.66 ± 1.52% and a maximum radicle length of 4.2 cm were observed in the seeds (Fig. 1). However, the radicle length in more than 80% of germinated seeds represented in the range of 3.5–4.2 cm. The appropriate achievement in the radical length stated initiation of germination in the Lablab purpureus seeds and further the seeds were studied for their metabolite, phytochemical, antioxidant profiles, and α-amylase inhibitory activity.

Fig. 1

Germination characteristics of Lablab purpureus seeds

Metabolomic profiling

Plant-derived metabolites have biological activity and are known to alleviate acute and chronic disease. The present study was undertaken to elucidate the wide spectrum of bioactive metabolites present in both non-germinated and germinated seeds of Lablab purpureus. Metabolomic profiling using UHPLC-ESI-QTOF-MS/MS in both positive and negative ion modes identified a total of 125 compounds in non-germinated seeds and 80 compounds in germinated seeds. A comprehensive list of these identified compounds is provided in Tables 1 and 2. The tables include molecular formula, retention time (RT), m/z, base peak, score, ion mode, and their suggested identities. Notably, 39 compounds were found to be common in both germinated and non-germinated seeds in the metabolomic profiling. All the identified compounds were further categorized into ten major classes, which included amino acids, peptides, and analogues, carbohydrates and carbohydrate conjugates, lipids and lipid-like molecules, phytochemicals, nucleic acids, benzenoids, carboxylic acids, amines, organic compounds, and other compounds. As illustrated in Fig. 2A, the composition of major classes in non-germinated seeds is depicted, with approximately 34 metabolites contributing to the largest segment, characterized by their phytochemical properties. There were 26 compounds classified as lipids and lipid-like molecules, and 15 compounds fall under amino acids, peptides, and analogs. The carbohydrates class encompassed about 12 metabolites, while the remaining segments encompass various compound types, including nucleic acids (6), benzenoids (6), carboxylic acids (4), amines (2), organic compounds (10), and other compounds (10). The metabolite profiling of germinated seeds, as shown in Fig. 2B, unveils a distinct composition of compounds. In the case of germinated seeds, approximately 24 compounds were categorized as phytochemicals. Followed by the second-largest group of lipids and lipid-like molecules constituting around 20 compounds. There were 11 compounds categorized under amino acids, peptides, and analogs. Furthermore, 6 compounds were present in the carbohydrates and organic compounds classes, while a reduced number of compounds, specifically 5 and 1, belong to amines and benzenoids, respectively. Among the identified compound classes, most of the compounds belong to phytochemicals in both the seed samples. The phytochemical compounds such as phenylethylamine, vernolepin, brompheniramine, cynaroside A, cinncassiol A, pisumsaponin II, leucodelphinidin 3- [galactosyl-(1->4)-glucoside] belonging to alkaloid, terpenoid, saponin, and flavonoid respectively were noted in non-germinated seeds Table 1; while the following compounds veracevine, salannin cynarasaponin C, azukisaponin IV, flavonol 3-O-[α-L- rhamnosyl-(1->6)-beta-D- glucoside] belonging to alkaloid, terpenoid, saponin and flavonoid category were noted in germinated seeds Table 2. Moreover, as per Fig. 2A and B phytochemicals class dominated the composition in both the seed samples, followed by lipids and amino acids, while other compound types make up smaller but still noteworthy portions. Suggesting bioactive compound phytochemicals present in both the sets could help in the prevention of chronic diseases, as well as add health-promoting benefits such as providing antioxidant and anti-inflammatory properties. The metabolites identified were searched using the KEGG (Kyoto Encyclopaedia of Genes and Genomes) pathway to understand the metabolites and their association with the biosynthetic pathways and activity (Kanehisa & Goto, 2000). Wherein several compounds in non-germinated seeds and germinated seeds were noted to exhibit a role in the biosynthesis of secondary metabolites. This group comprises 4-hydroxybutanoic acid, methyl N-methyl anthranilate, dihydrozeatin, acetyl tyrosine ethyl ester, isocitrate, caffeic acid 3-glucoside, aryl beta-D-glucoside, citronellyl hexanoate, auxin a, and avermectin B1b aglycone. Furthermore, several other compounds referring to their role in other associated pathways were miraxanthin-I (implicated in betalain biosynthesis), 4-hydroxybutanoic acid (linked to carbon metabolism), L-ascorbate 6-phosphate (related to the biosynthesis of cofactor in hydroxylation and in antioxidant pathways), and 9,10-dihydroxy-12,13-epoxyoctadecanoate (associated with linoleic acid metabolism) were noted. In contrast, the germinated compounds identified in this study, namely miraxanthin-I, methyl N-methyl anthranilate, and isocitrate, were noted to be integral component in biosynthesis of secondary metabolites. Additionally, 4-hydroxybutanoic acid is noted for its implication in carbon metabolism, C16 sphinganine is known to contribute to sphingolipid signalling pathway, and methyl 2-furoate is known to exhibit a role in furfural degradation. Notably, oleanolic acid 3-[rhamnosyl-(1->4)-glucosyl-(1->6)-glucoside] is noted to be linked to photosynthesis. Therefore, the observed metabolites in both the seed samples are noted to play a key role in different pathways associated with plant metabolism.

Table 1 Comprehensive Metabolite Profiling of Non-germinated Lablab purpureus seeds using LCMS in positive and negative Ion Modes

Table 2 Comprehensive Metabolite profiling of Germinated Lablab purpureus seeds using LCMS in positive and negative Ion Modes

Fig. 2

A Composition and distribution of compounds in non-germinated Lablab purpureus seeds. B Composition and distribution of compounds in germinated Lablab purpureus seeds

Furthermore, this study delved into a list of common compounds that exhibited a score of over 90% in both non-germinated and germinated seeds. The abundance of these compounds was analyzed and represented in Fig. 3. Among the 10 shared compounds, 4-amino 2-methylene butanoic acid and galactinol dihydrate were found to be more abundant in germinated seeds than in non-germinated seeds. Conversely, non-germinated seeds displayed higher levels of isocitrate, arvensoside A, calenduloside H, cynarasaponin C, oleanolic acid 3-[rhamnosyl-(1->4)-glucosyl-(1->6)-glucoside], 28-glucosyl-3b-hydroxy-12-oleanene-30-methoxy-28-oic acid 3-[arabinosyl-(1->3)-glucuronide], and azukisaponin IV compared to germinated seeds. Many studies reported consumption of saponins leads to lower blood glucose, and blood cholesterol levels and additionally enhances protection against cancer. Furthermore, saponins have been shown to have anti-inflammatory and immune-stimulating properties (Singh et al., 2017; Liu et al., 2017). Figure 3 represents the double bar graph indicating the common compounds identified in both the samples and further depicts the abundance of the respective compounds such as 4-amino 2-methylenebutanoic acid and galactinol dihydrate in the germinated Lablab purpureus seeds. As per the literature, 4-amino 2-methylenebutanoic acid is reported to be analog and possibly serves as an agonist for the GABA receptor (Duke et al., 2004; Chebib et al., 1997). On the other hand, galactinol dihydrate is known to exhibit raise seed vigor, carry hydroxyl radical scavenging activity, and protect plant cells (Salvi et al., 2020). A few compounds such as fagopyritol B3, gamma-glutamyl-S-methylcysteine, 9 S,12 S,13 S-trihydroxy-10E-octadecenoic acid, and oleanolic acid 3-O-beta-D-glucosiduronic acid were identified only in the sprouted seeds. Among the reported compounds, for instance, fagopyritol B3 is reported to be present in the seeds of buckwheat and certain genotypes of soybean (Steadman et al., 2000, 2001; Obendorf et al., 2009). Also higher temperature favours, fagopyritol B3 accumulation in seeds during maturation (Horbowicz & Obendorf, 2005). In turn increases the supply of D-chiro-inositol to seeds, which is noted to act as an insulin-sensitizing agent (Cheang et al., 2008; Galazis et al., 2011). While gamma-glutamyl-S-methylcysteine is known to be a component of the seed storage protein, modification of the compounds yields to improve the nutritional quality of the legumes (Taylor et al., 2008). Breakdown of the storage protein can generate bioactive peptides that in turn can help to alleviate the blood glucose by displaying α-amylase inhibitory activity. Lastly, oleanolic acid 3-O-beta-D-glucosiduronic acid based on an in-silico study was reported to exhibit DPP-IV inhibitory and in turn, could be a potential anti-diabetic compound (Kalhotra et al., 2020). Therefore, the presence of these unique compounds displayed in both non-germinated and germinated seeds suggest Lablab purpureus is a potential source in managing diabetes and its complications.

Fig. 3

Comparative analysis of major common compounds identified in metabolomic profiling with a score exceeding 90 and its abundance in non-germinated and germinated Lablab purpureus seeds

Phytochemical analysis

The aqueous extracts from the seed flour were compared for their phenolic, flavonoids, ascorbic acid, and protein content. The results are presented in Table 3. The total phenolic content values of the non-germinated and germinated seed were 5.89 ± 0.01 and 6.83 ± 0.21 mg/ g of gallic acid equivalents. Specifically, there was a substantial 1.15-fold surge in the phenolic content of the germinated seeds compared to the non-germinated seeds. Fouad & Rehab reported increased phenolic content in lentil seeds with germination time (3, 4, 5, and 6 days). The increase in phenolic content is attributed to the biosynthesis and bioaccumulation of phenolic compounds as a defensive mechanism to survive under environmental stresses (Fouad & Rehab, 2015). Additionally, another study by Aguilera also reported total phenol levels were increased with germination in Vigna unguiculata (Cowpea), Canavalia ensiformis (Jack bean), Lablab purpureus (Dolichos) and Stizolobium niveum (Mucuna). Endogenous enzymes are activated during the process of germination and thus lead to an increase in the phenolic content in the seeds (Aguilera et al., 2013). The total flavonoid content was 19.82 ± 0.16 and 29.68 ± 0.21 mg/g quercetin equivalents in the non-germinated seeds and germinated seeds. Therefore, a 1.5-fold elevation in flavonoid content was observed in the germinated seeds when contrasted with the non-germinated seeds extracts The raised flavonoid content in the germinated seeds was also noted in Vigna radiata samples with germination (1,3, and 5 days of germination) (Krishnappa et al., 2017). Moreover, Sharma et al. reported that pigeon peas with germination (12, 24, 36, and 48 h) and temperature (at 25, 30, and 35 °C) lead to an increase in the total phenol and flavonoid content (Sharma et al., 2019). In the present study, the total ascorbic acid content in the non-germinated and germinated seeds extract was noted to be 2.03 ± 0.01and 3.35 ± 0.00 mg/g ascorbic acid equivalents. Around 1.65-fold increment of ascorbic acid content was observed in the germinated seeds when contrasted with the non-germinated seeds extracts. Previous study also noted increase in the total ascorbic content in the chickpea with germination time (24, 48,72, 96, and 120) and it is also related to the impact of light on germination (Khattak et al., 2007). Another study conducted by Suryanti et al. (2016), determined the effect of germination time on the antioxidant activity of Leucaena leucocephala (lmk.) de Wit (lead tree) sprouts, reported higher ascorbic acid content with germination stage. Germination for 4 days affected the greatest enhancement in antioxidant activity by increasing total ascorbic acid activity (Suryanti et al., 2016). Additionally, Sibian et al. (2016) analyzed Bengal gram seeds germination effect and found that ascorbic acid content was increased with germination time, they suggested that germination is the only process reported that caused an enhancement in the ascorbic acid content. Increases in ascorbic acid levels were a consequence of the reactivation of ascorbic acid biosynthesis undergone in the seeds during germination. The differences in the effect of germination on ascorbic acid content could be due to the genetic variation, age of the grain, climatic conditions, lighting conditions, harvesting and storage methods (Sibian et al., 2016). Thus, from the present data substantial increase in total ascorbic acid content in germinated underscores the significant impact of the germination process on the accumulation of this vital antioxidant compound. The levels of protein in the non-germinated seeds and germinated seeds were 162.95 ± 1.79 and 147.87 ± 2.40 mg of BSA equivalents per gram dry weight. A decrease in total protein content after germination was observed in the present study, which could be due to the increased level of protease activity during germination (Torres et al., 2007). Also, a study by Shastry and John (1991) reported a decrease in the soluble protein levels with an increase in the germination days (2, 4, 6, and 8 days) (Shastry & John, 1991). Conversely, another study by Borijindakul and Phimolsiripol (2013) reported Dolichos lablab seeds protein contents were found to be increased with germination time (12, 24, 36, and 48 h) (Borijindakul & Phimolsiripol, 2013). The difference in protein level can be due to germination time, as in present case the seeds were sprouted for short span of 24 h. There is possibility of gradual increase in protein synthesis in seeds and thus possibly increasing the germination time could further help in breakdown of the storage protein in turn help production of amino acids and further increase the protein synthesis could raise the protein content on germination. In present investigation, a significant difference was noted in terms of total phenol, total flavonoid, total ascorbic acid, and protein content between the germinated and non-germinated samples at p < 0.05. Thus, it can be concluded that the germination bioprocessing positivity effects to raise the level of total phenol, total flavonoid, and total ascorbic acid in the present study.

Table 3 Comparative Analysis of Phenol, Flavonoid, total ascorbic acid content and protein (mg/g) in non-germinated and germinated Lablab purpureus seeds

Antioxidant content

The antioxidant activities were studied using FRAP, DPPH, and ABTS assays for both the germinated and non-germinated samples, as depicted in Fig. 4A and B, and C. Both sets of samples exhibited considerable antioxidant activity, which was statistically different at p < 0.05. Notably, the germinated seed samples showcased higher FRAP content, and their DPPH and ABTS inhibitory activities exceeded those of the non-germinated counterparts. Specifically, the DPPH inhibitory activity for aqueous samples at a concentration of 100 mg/ml was approximately 38.49% for germinated seeds and 22.48% for non-germinated seeds. Furthermore, A significant difference at p < 0.05 was noted for the ABTS activity between the germinated and non-germinated seed samples. The 20 mg/ml aqueous seed samples from both germinated and non-germinated sources demonstrated around 7.3% and 2.2% ABTS inhibitory activity, respectively. Studies by Aguilera et al. reported DPPH and FRAP activities were increased with germination in Vigna unguiculata (Cowpea), Canavalia ensiformis (Jack bean), Lablab purpureus (Dolichos) and Stizolobium niveum (Mucuna) (Aguilera et al., 2013). The raised phytochemical levels in the germinated seeds can be directly correlated with the increased antioxidant profiles in the germinated seeds. Guzmán-Ortiz et al. reported increased ABTS and FRAP activities in the soybeans on increasing the germination time (2, 6 days) (Guzmán-Ortiz et al., 2017). These findings highlight the positive influence of germination on the antioxidant properties of the Lablab purpureus and its potential as a valuable source of natural antioxidants.

Fig. 4

A Comparative analysis of ferric reducing antioxidant power (frap) in Lablab purpureus seeds. B Evaluation of 2,2-Diphenyl-1-picrylhydrazyl radical scavenging activity (dpph) in Lablab purpureus seeds. C Assessment of 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic Acid (ABTS) percent inhibition

α-Amylase inhibitory activity

Considering the pivotal role of α-amylase in diabetes mellitus, the α-amylase inhibitory activity of aqueous samples derived from both germinated and non-germinated seeds was studied. The IC50 value was calculated to measure the inhibitory potency. A significant difference was noted between the test samples at p < 0.05. The findings indicated that the non-germinated seeds exhibited an IC50 value of approximately 0.79 ± 0.00 mg/ml, while the germinated seeds displayed an IC50 value of 2.05 ± 0.05 mg/ml, as depicted in Fig. 5. The standard drug acarbose was noted to have an 82.26 µg/ml IC50 value.

Fig. 5

Comparative alpha amylase percent inhibition

As per previous studies, black turtle bean crude extracts showed an IC50 value of approximately 2.69 mg/ml, and black soybean reported an IC50 value of around 2.25 mg/ml for α-amylase inhibition (Tan et al., 2017). Additionally, studies on chickpeas resulted in an IC50 value of approximately 0.167 mg/ml for α-amylase inhibition (Ercan & El, 2016). Previous study by Sharma & Giri, reported IC50 values for α-amylase enzyme inhibition in various legumes, finding values such as 0.401 mg/ml in peas, 0.376 mg/ml in chickpeas, 0.425 mg/ml in mung beans, 0.351 mg/ml in common beans, 0.349 mg/ml in lentils, 0.275 mg/ml in lima beans, 0.217 mg/ml in soybeans, and 0.264 mg/ml in broad beans (Sharma & Giri, 2022). When comparing these findings with our investigation, Lab Lab purpureus (non-germinated) aqueous seeds extract exhibited an IC50 value of 0.79 ± 0.00 mg/ml, aligning well with existing literature. Previous studies on sprouts of lentils, black medick, and mung beans reported IC50 values for α-amylase inhibition of around 88.4 mg/ml, 4.9 mg/ml, and 5.3 mg/ml, respectively (Wojdyło et al., 2020). In our study, Lab Lab purpureus sprouts revealed an IC50 value of 2.05 mg/ml, indicating their potential as an α-amylase inhibitor. These comparisons highlight that while the seeds studied are less potent than acarbose, they are comparable to other natural α-amylase inhibitors, supporting their potential role in diabetes management. The non-germinated seeds show α-amylase inhibitory activity comparable to other legume extracts like black beans and black soybean. Although the germinated seeds have higher IC50 values, they still demonstrate substantial inhibitory activity. These findings suggest that both Lab Lab purpureus seeds and sprouts can be potential candidates for functional foods and diabetes management by inhibiting α-amylase and thus reducing postprandial hyperglycemia.

The study also observed that no improvement in the inhibition activity of the starch digestion enzyme α-amylase was seen upon germination. In fact, a decrease in α-amylase inhibitory activity was noted in germinated samples. The germinated and non-germinated seeds were found to have different metabolites compositions, which likely explains the lower IC50 value in the non-germinated seeds. This could be attributed to a higher number of metabolites as noted in the metabolomic profiling, and a unique combination of bioactive molecules leading to higher α-amylase inhibitory activity. Supporting this, Mulimani & Rudrappas (1994) reported a decrease in α-amylase inhibition activity in chickpeas as the days of germination increased (1 day to 6 days of germination) (Mulimani & Rudrappa, 1994). Thus, germinated and non-germinated seeds of Lablab purpureus possess different compositions of bioactive molecules, leading to varying α-amylase inhibitory activities. While the present investigation underscores the utilization of both germinated and non-germinated seeds for their substantial α-amylase inhibitory activity, it highlights their potential contribution to managing diabetes.

Conclusion

In conclusion, the comparative analysis of germinated and non-germinated Lablab purpureus seeds unveiled distinctive metabolite profiles, emphasizing the influence of the germination process. The germinated seeds exhibited a heightened phytochemical and antioxidant profile, showcasing their potential health-promoting benefits. Notably, the α-amylase inhibitory activity was more pronounced in the non-germinated seed samples, as evidenced by a lower IC50 value, indicating a stronger inhibitory effect on α-amylase. This intriguing finding suggests that the unique biochemical composition of non-germinated seeds contributes to their superior α-amylase inhibitory activity. However, it is noteworthy that even the germinated seeds displayed appreciable α-amylase inhibitory activity, highlighting their potential as a functional food ingredient with benefits for glycemic control. Further investigation into the ideal germination period for Lablab purpureus seeds is warranted to boost their bioactive properties, thereby amplifying their therapeutic efficacy and suitability for integration into functional food products.