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INTRODUCTION
The global burden of chronic metabolic diseases, particularly Type 2 Diabetes Mellitus (T2DM) and inflammatory disorders, has reached epidemic proportions, affecting millions worldwide (Huang et al., 2025) and imposing substantial economic and healthcare challenges. Concurrently, chronic low-grade inflammation has been recognized as a pivotal pathophysiological mechanism underlying numerous diseases, including diabetes, cardiovascular disorders, and metabolic syndrome. The intricate interplay between hyperglycemia and inflammatory pathways has prompted researchers to explore multifunctional therapeutic approaches that can simultaneously target both conditions (Huang et al., 2025; Van Dijk et al., 2021).
Conventional pharmacological interventions, while effective, are often associated with adverse effects, high costs, and limited accessibility, particularly in developing nations. This has catalyzed growing interest in plant-based functional foods and nutraceuticals as complementary or alternative strategies for disease prevention and management (Gruda et al., 2024). Among the emerging class of functional foods, microgreens have garnered substantial scientific attention due to their exceptional nutritional density, bioactive phytochemical content, and potential health-promoting properties.
Microgreens are young, tender greens harvested at the cotyledon or first true-leaf stage, typically 7-21 days after germination. Despite their diminutive size, microgreens are nutritional powerhouses, containing significantly higher concentrations of vitamins, minerals, antioxidants, and bioactive compounds compared to their mature counterparts (Bhaswant et al., 2023).
Vigna mungo (L.) Hepper, commonly known as black gram or urad bean, is a vital leguminous crop extensively cultivated in South Asia, particularly in India, where it serves as a major source of dietary protein and essential nutrients. While mature urad beans have been well-characterized for their nutritional composition and health benefits, the microgreen stage of this legume remains relatively unexplored. Preliminary investigations suggest that urad microgreens possess substantial amounts of proteins, phenolic compounds, and antioxidant capacity, warranting comprehensive evaluation of their bioactive potential and therapeutic applications such as anti -inflammatory (Seth et al., 2025), anti-oxidant (Hadi et al., 2025), anti-diabetic (Partap et al., 2023), cardioprotective (Rizvi et al., 2023).
The biosynthesis and accumulation of phytochemicals in plants are significantly influenced by environmental conditions and stress factors. salinity, and light variations, trigger complex physiological and biochemical responses in plants as adaptive mechanisms. These stress-induced responses often result in the enhanced production of secondary metabolites, particularly phenolic compounds, flavonoids, and other antioxidants, as part of the plant's defense system against oxidative damage.
MATERIALS AND METHODS
Chemicals required
All chemicals used were of analytical grade and were procured from authorized suppliers. The chemicals were procured from Emplura and Emparta (Chennai). The standard chemicals were procured from Sigma-Aldrich, including quercetin, gallic acid, tannic acid, aspirin.
Instruments used
The instruments used included a UV-visible Spectrophotometer (JASCO Corporation, V-630, Tokyo, Japan); Digital Weighing Balance (Wensar Weighing Scales Ltd., PGB-200, Maharashtra, India); pH meter (SYSTRONICS, 335, Ahmedabad, India); Incubator (Remi Elektrotechnik Ltd., Mumbai, India) and Hot Water Bath (SSFW, LWB-12H/D, Kolkata, India).
Seed sample collection
About 150 seeds of Uradwhole procured from the local market of Sodepur, West Bengal, India in September 2025. The physical purity of the seeds was checked from the details mentioned on the company labels. Seeds were stored in air-tight containers under dry, cool conditions (~15±2ºC) before sowing in October.
Cultivation of microgreens
The microgreen is grown in soil of Sodepur, North 24 Parganas, West Bengal, under different stress condition (25%, 50%, 100% salinity, normal condition, 24 hr dark) in the month of October for 1-6 days in an open environment at a temperature of 20-25ºC and a relative humidity of 65-70% as measured by a digital thermohygrometer. Plants, when germinated to the two-leaf condition, were harvested and dried. The number of plants that germinated was also calculated. Harvesting was done during the early hours of the day (8:00-9:00 AM) to minimise moisture loss due to transpiration by using aseptic gloves to prevent external microbial contamination. The heights of leaves, shoots, and roots, as well as the leaf surface area, of 25 mature microgreens were measured. After harvesting, the microgreens were shade-dried under ambient room conditions (25±2ºC, protected from direct sunlight) in a well-ventilated, dust-free environment for 6 days. No artificial heat source was provided for the drying process. Drying was continued until a constant weight was achieved. The moisture content was measured by measuring the weight of the fresh and dried sample using the given formula (Balik et al., 2025).
Plant extract preparation
The dried plant material was crushed into a coarse powder using a pestle and mortar. About 2 g of the powdered sample of microgreen was taken and macerated in 10 mL of methanol for 7 days at a controlled temperature of 30±1ºC in the laboratory incubator with periodic shaking. The sample was sonicated daily for 30 min at 30ºC for 7 days. The macerated product was filtered and dried to a solid residue at room temperature and stored.
Preliminary phytoconstituent testing
The presence or absence of phytoconstituents was determined using different in vitro chemical testing procedures. Alkaloid was estimated by Dragendroff’s test and Mayer test, flavonoids by Lead acetate Test. Carbohydrate was estimated by Molisch test. The ferric chloride test was used to determine phenolic compounds and tannins. Glycoside was estimated by the Modified Brontrager Test. A foam test was used to determine saponins. Terpenoid was tested using chloroform and conc. image2. Identification of proteins was done by performing Ninhydrin test (Charoensiddhi et al., 2022).
Total phenolic content determination
The Total Phenolic Content (TPC) was determined using the established procedure. Different concentrations of gallic acid were used to form a calibration curve (20, 40, 60, 80 and 100 mg/mL) using 5 mL of 10% Folin-Ciocalteu and 4 mL of 7% Na2CO3 (Sodium carbonate) and measuring absorbance at 760 nm. Similarly, plant extract of 0.01, 0.05, and 0.1 mg/mL was used to determine TPC (Chakraborty et al., 2025; Dimita et al., 2022) (Figure 1).
Total flavonoid content calculation
The established procedure using quercetin as a standard was used to determine the calibration curve. Plant extract of 0.01, 0.05, and 0.1 mg/mL concentrations was used to determine TFC at 510 nm (Martins et al., 2021).
Total tannin content calculation
The established procedure of determination of Total Tannin Content (TTC) was performed. Tannic acid was used to form a calibration curve (20, 40, 60, 80, 100 mg/mL), and absorbance was measured at 725 nm. The TTC of plant extract 0.01, 0.05, and 0.1 mg/mL were determined (Galvão et al., 2018).
Total pigment content
Chlorophyll a, chlorophyll b, total chlorophyll content, and total carotenoid content were determined using 80% acetone as the solvent system. Anthocyanin content was estimated using a methanol-HCl-water mixture (90:1:9, v/v/v) according to the reported method and expressed as μg/g Fresh Weight (FW). All measurements were performed in triplicate (Gunjal et al., 2024).
Anti diabetic potential
The α-amylase inhibitory activity was evaluated using an established method. The sample solution was prepared at a concentration of 0.1 mg/mL and further diluted to obtain 0.05 and 0.01 mg/mL. Reaction mixtures containing different concentrations of the plant extract (test), acarbose (positive control), or ethanol (control) were incubated with α-amylase solution (0.5 mg/mL), 1% starch solution, and 0.2 M phosphate buffer (pH 6.9) for 5 min. The reaction was terminated by the addition of 1% 3,5-dinitrosalicylic acid, and the absorbance was measured at 540 nm (Kifle et al., 2021). The antidiabetic percentage was calculated by,
Where Ac=Absorbance of control solution and At=Absorbance of test Solution.
Anti-inflammatory potential
The anti-inflammatory activity was assessed using an egg albumin denaturation assay following an established method. The reaction mixture consisted of 0.2 mL egg albumin, 2.8 mL phosphate buffer (pH 6.4), and 0.2 mL of the sample at concentrations of 0.1, 0.05, and 0.01 mg/mL (aspirin as the positive control, plant extract as the test sample, and appropriate control). The mixtures were incubated at 27ºC for 10 min, followed by heating in a water bath at 70ºC for 10 min. The absorbance was measured at 660 nm (Vučetić et al., 2025, Balik et al., 2024). Anti-inflammatory potential was estimated by using the formula:
Where, AC=Absorbance of control solution, AT=Absorbance of test solution.
Ethical Statement
Ethical approval was not required for this study as it involved only plant materials and did not include human participants or experimental animals.
Statistical Analysis
All experiments were performed in triplicate, and the results are expressed as Mean±Standard Deviation (SD). The mean and standard deviation values were calculated using Microsoft Excel 2021 version. No inferential statistical tests were applied.
RESULTS
The microgreens were grown in a controlled laboratory environment with periodic watering.
Germination details
The germination rate and details of germination for different stress condition microgreens are presented in Figures 2 and 3.
Preliminary Phytochemical Test
The phytochemical screening was performed to assess the presence or absence of various classes of phytoconstituents. The results are shown in Table 1.
| Sl. No. | Identification | Test Name | Normal condition | 25% salinity | 50% salinity | 100% salinity | 24 hr Dark |
|---|---|---|---|---|---|---|---|
| 1 | Alkaloid | Dragendroff’s Test | - | - | - | - | - |
| Mayer Test | - | - | + | + | + | ||
| 2 | Carbohydrate | Molisch Test | + | + | + | + | + |
| 3 | Glycoside | Modified Brontrager | - | - | - | - | + |
| 4 | Flavonoids | Lead acetate Test | + | + | + | + | - |
| 5 | Tanins and Phenolics | Ferric Chloride Test | - | - | + | + | + |
| 6 | Protein | Ninhydrin Test | - | - | - | - | - |
| 7 | Steroid | Salkowski Test | + | + | + | + | + |
| 8 | Saponin | Frothing’s Test | - | - | - | - | - |
Phytochemical quantification
Standard curve of quercetin, gallic acid and Tannic acid was prepared at different concentration. Calibration curves of standard quercetin, gallic acid and tannic acid and demonstrated high Regression Correlation (R2) of 0.9519, 0.9253, 0.99 with linear equations
y=0.0003x+0.1941, y=0.0058x-0.0007 and y=0.0014x+0.023
Respectively, as presented in Figures 4a-c.
Phytochemical Quantification and Pigment Content Estimation
For phytochemical quantification, total Flavonoid Content increased in 50% salinity (0.5969)>24 hr Dark (0.5656)>25% salinity (0.2946)>Normal (0.2502)>100% salinity (0.0714). Total Phenolic Content increases in 100% salinity (0.1422)>50% salinity (0.1345)>25% salinity (0.0537)>Normal (0.0414)>24 hr Dark (0.0329). Total Tannin Content 25% salinity (27.9)>Normal (14.02)>100% salinity (11.59)>24 hr Dark (9.04)>50% salinity (0.80) as presented in Table 2.
| Sl. No. | Stress Condition | TFC (mg QE/g) | TPC (mg GAE/g) | TTC (mg TAE/g) | Chlorophyll a content (µg/g) | Chlorophyll b content (µg/g) | Total Chlorophyll content (µg/g) | Total anthocyanin content (µg/g) | Total carotenoid content (µg/g) |
|---|---|---|---|---|---|---|---|---|---|
| 1 | Normal (Vigna mungo) | 0.25015±0.1203 | 0.0414±0.0046 | 14.02±0.96 | 0.955±0.0086 | 7.26±0.0653 | 8.291±0.0703 | 0.263±0.0010 | 1.8014±0.8014 |
| 2 | 25% salinity (Vigna mungo) | 0.2946±0.0011 | 0.0537±0.0017 | 27.9±1.00 | 1.600±0.0096 | 0.7045±0.0116 | 2.318±0.0091 | 0.2001±0.0005 | 0.5937±0.0069 |
| 3 | 50% salinity (Vigna mungo) | 0.5969±0.2238 | 0.1345±0.2295 | 0.80±0.53 | 1.6502±0.0039 | 3.5675±0.0054 | 5.2600±0.0044 | 0.3197±0.0011 | 1.5366±0.0062 |
| 4 | 100% salinity (Vigna mungo) | 0.0714±0.0033 | 0.1422±0.0012 | 11.59±0.73 | 1.8914±0.0106 | 0.4518±0.0033 | 1.4429±0.0138 | 0.0889±0.0687 | 1.1416±0.0117 |
| 5 | 24 hr Dark (Vigna mungo) | 0.5656±0.0030 | 0.0329±0.0005 | 9.04±0.64 | 1.89 ±0.0104 | 0.76±0.0059 | 1.44±0.0135 | 0.00017±0.00001 | 23.12±0.1900 |
Pigment content estimation identified Total Anthocyanin content increases in 50% salinity (0.3197)>Normal (0.263)>25% salinity (0.2001)>100% salinity (0.0889)>24 hr Dark (0.00017) and chlorophyll a increases 100% salinity (1.8914) ≈ 24 hr Dark (1.89)>50% salinity (1.6502)>25% salinity (1.600)>Normal (0.955) chlorophyll b increases in Normal (7.26)>50% salinity (3.5675)>24 hr Dark (0.76)>25% salinity (0.7045)>100% salinity (0.4518) and total chlorophyll content increases in Normal (8.291)>50% salinity (5.2600)>25% salinity (2.318)>100% salinity (1.4429) ≈ 24 hr Dark (1.44) and total carotenoid content increases in 24 hr Dark (23.12)>Normal (1.8014)>50% salinity (1.5366)>100% salinity (1.1416)>25% salinity (0.5937). Quantification of different pigment contents like anthocyanin, Chlorophyll, and Carotenoid is represented in Table 2.
In vitro Assay
Anti-diabetic activity
The anti-diabetic activity of different stress condition of Uradwhole microgreens was calculated and compared against the standard compound (Acarbose). The antidiabetic potential of the Uradwhole Microgreen is represented in Table 3. In Table anti-diabetic effect increases in Normal (55.98)>50% salinity (56.44)>100% salinity (62.46)>25% salinity (74.81)>24 hr Dark (92.23) (Figure 1).
| Antidiabetic activity | |||||
|---|---|---|---|---|---|
| Sl. No. | Stress Condition | 0.01 mg/mL | 0.05 mg/mL | 0.1 mg/mL | IC50 (µg/mL) |
| 1 | Normal | 2.07±1.0142 | 88.64±2.0556 | 77.25±0.0230 | 55.9866 |
| 2 | 25% salinity | 87.48±0.1905 | 41.94±0.4271 | 95.02±0.0152 | 74.8166 |
| 3 | 50% salinity | 6.78±0.1212 | 38.72±0.7825 | 23.84±0.5024 | 56.4477 |
| 4 | 100% salinity | 57.49±0.5164 | 34.77±0.2203 | 95.11±1.1983 | 62.4611 |
| 5 | 24 hr Dark (Vigna mungo) | 99.48±0.09 | 98.97±1.56 | 78.24±0.25 | 92.23 |
| Anti- Inflammatory activity | |||||
| Sl. No. | Stress Condition | 0.01 mg/mL | 0.05 mg/mL | 0.1 mg/mL | IC50 (µg/mL) |
| 1 | Normal | 20.3±0.6519 | 2.67±0.9351 | 4.77±0.4099 | 9.2488 |
| 2 | 25% salinity | 67.32±0.0642 | 67.13±56.66 | 6.56±0.0721 | 47.0077 |
| 3 | 50% salinity | 62.41±0.3435 | 55.59±0.0230 | 34.53±0.0360 | 50.8477 |
| 4 | 100% salinity) | 28.69±0.0351 | 3.23±0.1242 | 2.55±0.0230 | 11.4955 |
| 5 | 24 hr Dark (Vigna mungo) | 54.60±4.29 | 95.7±3.48 | 88.24±0.56 | 79.51 |
Anti-inflammatory activity
The egg albumin denaturation assay was employed to evaluate the inhibitory effect of the plant extract on protein denaturation, a key event in inflammatory responses. The anti-inflammatory activity of the plant samples was analyzed at three concentrations (0.1, 0.05, and 0.01 mg/mL) and compared with aspirin as a reference standard. The findings indicated significant differences in activity both among the concentrations tested and between the Microgreen extracts and aspirin. The anti-inflammatory potential of the Uradwhole Microgreen is represented in Table 3. In this Table anti-inflammatory effect increases in Normal (9.24)>100% salinity (11.49)>25% salinity (47.00)>50% salinity (50.84)>24 hr Dark (79.51).
DISCUSSION
The present study evaluated how different stress conditions influence the phytochemical composition and biological activities of Vigna mungo microgreens. The findings clearly demonstrate that environmental stress plays a crucial role in modulating secondary metabolite production and associated bioactivities.
Salinity stress significantly affected phytochemical accumulation. Moderate salinity (50%) markedly enhanced total flavonoid and anthocyanin content, suggesting that controlled stress can stimulate secondary metabolism as a defensive response. In contrast, severe salinity (100%) resulted in the highest total phenolic content, indicating activation of protective antioxidant mechanisms under higher stress intensity. Mild salinity (25%) favored tannin accumulation, showing that different stress levels selectively influence specific phytochemical pathways.
Pigment analysis further supported these observations. Normal growing conditions maintained the highest total chlorophyll content, reflecting optimal photosynthetic activity. However, chlorophyll a increased under severe salinity, while anthocyanin accumulation peaked at 50% salinity, indicating stress-induced pigment modulation. Interestingly, 24-hr dark treatment drastically reduced anthocyanin content but showed unusually high carotenoid levels, possibly due to altered plastid development under light deprivation.
Biological assays revealed meaningful differences among treatments. Although phytochemical enhancement was observed under stress, the strongest antidiabetic and anti-inflammatory activities were associated primarily with normal and high-salinity conditions. In contrast, microgreens grown under complete darkness showed comparatively weaker enzyme inhibition and anti-inflammatory activity despite elevated flavonoid levels. This suggests that overall bioactivity depends not merely on total flavonoid quantity but on the specific composition and interaction of phenolic compounds, which are influenced by light exposure and stress intensity.
Taken together, these results highlight the importance of optimizing environmental conditions to enhance the nutraceutical potential of microgreens.
CONCLUSION
This study demonstrates that environmental stress significantly influences the phytochemical profile and therapeutic potential of Vigna mungo microgreens. Moderate salinity stress effectively enhanced flavonoid and anthocyanin accumulation, while severe salinity maximized total phenolic content and showed strong biological activity. Normal conditions maintained optimal chlorophyll levels and also exhibited considerable bioactivity.
Although dark treatment stimulated flavonoid accumulation and morphological elongation, it reduced phenolic and anthocyanin synthesis and resulted in weaker antidiabetic and anti-inflammatory activities. Therefore, complete light deprivation does not enhance functional properties. Overall, controlled salinity stress-particularly moderate to high levels-appears to be a promising strategy for improving the phytochemical richness and medicinal value of urad microgreens. Further in vivo studies and bioavailability assessments are recommended to validate their potential as functional foods or nutraceuticals.