Article

1.Introduction

The liver is an essential organ that normalizes metabolic functions and detoxifies many harmful substances. It is susceptible to external toxic substances and organic compounds¹. Exposure to these toxicants, including drugs, alcohol, and environmental pollutants, can lead to liver damage through metabolic activation, this leads to the creation of highly reactive substances. D-galactosamine is a proven hepatotoxin; it produces diffuse-type liver injury that closely mimics human viral hepatitis (viral hepatitis type) and acute self-limited hepatitis (injury with necrosis, inflammation, and regeneration), similar to human drug-induced liver diseases². The toxic effects of D-galactosamine are primarily due to uridine pool depletion, which results in inhibited RNA and protein synthesis in the liver, ultimately affecting hepatocellular activity³. Despite advances in modern medicine, pharmacotherapeutic treatment with synthetic drugs for liver protection has not yet been achieved. Therefore, alternative therapeutic strategies, such as Boerhavia diffusa leaf extract, are of great interest for liver protection⁴.

Diabetes is a common long-term metabolic disorder marked by higher levels of sugar in the blood. It is due to insufficient or ineffective insulin production and is associated with abnormalities in the metabolism of carbohydrate, protein, and fat^5-6. Recent findings have shown that high blood sugar can cause non-enzymic glycosylation of several macromolecules. The build up of harmful oxygen-containing molecules and the weakening of the build’s natural antioxidant scheme can performance a part in the long term health problems linked to diabetes⁷. The concept that oxidative stress significantly contributes to diabetes onset, alongside postprandial hyperglycemia, creates valuable opportunities for developing treatment and management strategies to decrease the risk of long-term vascular complications. One practical way to lower post-meal blood sugar spikes is to prevent carbohydrate absorption after eating. Complex polysaccharides are initially broken down by alpha-amylase into oligosaccharides, which are then further cleaved into glucose by intestinal α-glucosidase. The glucose molecules are absorbed through the lining of the intestine and then enter the bloodstream⁸.Constraints of α-amylase and α-glucosidase can help lower post-meal hyperglycemia by blocking the breakdown of carbohydrates, which slows glucose absorption⁹.Various synthetic drugs, including acarbose, voglibose, and miglitol, are commonly prescribed as enzyme inhibitors for managing type 2 diabetes. However, these medicines can often cause side effects like gas, stomach pain, bloating, and sometimes diarrhea¹⁰.Research is focused on exploring natural and safer alternatives for inhibiting alpha-amylase and alpha-glucosidase. Phytochemicals, especially phenolics with strong antioxidant potential, have been reported as effective enzyme inhibitors. This constitutes a complete strategy in the supervision of hyperglycaemia and the anticipation of oxidative stress-related sequelae in diabetes¹¹.

Medicinal plants (MPs) have proven to be effective in regulating metabolic functions, hyperglycemia, and metabolic syndrome. This effectiveness is primarily due to their high content of therapeutic phytochemicals, which include saponins, tannins, flavonoids, phenolics and glycosides. These compounds enhance various bioactivities¹², such as antioxidant and anti-inflammatory properties. Additionally, medicinal plants are often readily available, generally more affordable than commercial synthetic drugs, and tend to have fewer side effects. A classic example of such a medicinal plant is Boerhavia diffusa.¹⁰

Boerhavia diffusa, generally known as Punarnava in the Indian system of medicine, associated to the family Nyctaginaceae. It is a perennial creeping herb widely initiate in wastelands throughout India.¹³ The genus Boerhavia comprises 40 species, which are circulated across tropical and subtropical regions with warm climates. In India, six species of Boerhavia are present: B. diffusa, B. erecta, B. rependa, B. chinensis, B. hirsuta  and B. rubicunda. Boerhavia diffusa is typically initiate in the furnace parts of the country and can grow at altitudes equipped 2,000 meters in the Himalayan region.¹⁴ This plant is a perennial, creeping weed characterized by its spreading branches and tough, tapering roots. The stem is arboreal, sometimes purplish, and covered with hairs, becoming thicker at the nodes. The plant is fleshy and hairy, with leaves that grow in unequal pairs and produce small pinkish-red flowers. Boerhavia diffusa is commonly found in abundance in unused areas, waterways, and muddy spaces, particularly throughout the rainy season. Additionally, it is sophisticated to some range in West Bengal.

In India, diverse parts of B. diffusa, such as the leaves, roots, and aerial parts, are commonly used to treat kidney issues, rheumatism, and liver disorders, among other health problems¹⁵. Leaf extracts from the plant have been shown to have a diversity of beneficial properties, comprising hepatoprotective, anti-diabetic, anti-inflammatory, and antioxidant effects.^16-17 The leaves of Boerhavia diffusa are rich in phytochemicals, such as essential oils, rotenoids, alkaloids, flavonoids, alkamides, terpenoids, xanthones, and phenolic compounds.¹⁸The chemical profile of plant-derived extracts can differ significantly built on aspects such as species origin, extraction method (e.g., methanolic, aqueous), temperature, and solvent concentration.

Methanol has excellent solubility properties, allowing it to dissolve a varied range of polar and semi-polar compounds found in plants. Many bioactive substances in plants, such as phenolic acids, fall into these categories. Methanol’s ability to effectively dissolve these compounds enhances the extraction yield. For instance, when extracting flavonoids from plant leaves, methanol can penetrate the cell walls and dissolve the flavonoid molecules, making it easier to recover them from the extraction solution.¹⁹Similarly, water effectively extracts highly glacial compounds such as sugars, amino acids and some phenolics.²⁰

The current study aims to explore the biological potential of the leaf extract of Boerhavia diffusa. The study includes quantitative analysis to determine the phenolic and flavonoid content, as well as the evaluation of its hepatoprotective activity²¹on the HepG-2 cell line, antidiabetic potential (through α-amylase and α-glucosidase inhibition assays), anti-inflammatory potential²² (via protein denaturation(BSA) and proteinase inhibition(Trypsin) assays), and antioxidant activity²³ (using the DPPH assay) of the above ground parts of B. diffusa. This study search for to explore the likely of this plant as a capable source of innovative therapeutic candidates for managing contemporary health problems, especially those arising from oxidative stress, metabolic imbalance, and infectious conditions.

2. Materials and Methods

• Collection of Boerhavia diffusa leaves

In the months of July and October, new Boerhavia diffusa samples were unruffled from the Hudkeshwar region of Nagpur in Maharashtra. A taxonomist of Rashtrasant Tukadoji Maharaj Nagpur University’s Department of Botany identified the plant, and a herbarium specimen bearing voucher number 10923 was sent to the department. The leaves were splashed with distilled water to take away any surface impurities and then desiccated in the shadow at a temperature of 20-25°C for two weeks. The desiccated leaves were then powdered using a crusher and kept in hermetically sealed pliable containers at room temperature²⁴.

• Preparation of Plant Extracts

The leaf powder (10 g) was extracted using methanol and distilled water in a Soxhlet apparatus for a period of 12 to 14 hours. After being filtered, the methanol and aqueous extracts were kept for additional examination at 4°C. Whatman filter paper No. 1 was used to filter the extracts.

• Phytochemical analysis

To identify the livelyelements in the methanolic and aqueous extracts of Boerhavia diffusa, phytochemical screening was done. The extracts, namely the aqueous and the methanolic of Boerhavia diffusa leaves were screened for Alkaloids (Dragendorff’s reagent, Mayer’s reagent), Terpenoids (Salkowski test), Flavonoids (Shinoda’s test, Lead Acetate test),Carbohydrates (Molisch’s test, Fehling’s test and Benedict’s test), Anthraquinones (Borntrager’s test), Glycosides (Keller-Kiliani test).²⁵

Determination of Total Phenolic Content (TPC)

A partial of 12.5 μl of plant extract was cooperative with 12.5 μl of Folin-Ciocalteu reagent in a 300 μl reaction container. The combination was allowed to stand at room temperature for ten minutes. Subsequently, 125 μl of 7% sodium carbonate was added to the solution. The reaction mixture was nurtured in the dark at 37°C for 90 minutes. Following incubation, the volume was adjusted to 300 μl using distilled water. Finally, the optical density was sedate at 760 nm using a spectrophotometer. The phenolic concentration was determined using a standard calibration curve for gallic acid ranging from 20 to 100 μg/ml.²⁶

Determination of Total Flavonoid Content (TFC)

One hundred microliters of plant extract were mixed with one hundred microliters of 2% aluminum chloride and protected at room temperature for ten minutes. After gestation, a spectrophotometer was used to quantity the optical density at 367 nm. To determine the flavonoid content, a standard calibration curve for quercetin, stretching from 20 to 100 μg/ml, was utilized.²⁷

2.4 UPLC-QTOF-MS metabolite profiling of the extracts

 The occurrence of numerous bioactive combinations in the aqueous and methanolic leaf extracts of Boerhavia diffusa leaves was recognized using Liquid Chromatography Mass Spectrometry (LC-MS) of these extracts. The extract’s metabolites were profiled using quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) in both positive (ESI+) and negative (ESI-) modes using ultra-performance liquid chromatography.²⁸The aqueous and methanolic leaf extracts were clarified using 0.25 mm polyvinylidene fluoridemembrane nozzle sieves and then relocated straight into 2mL vials for analysis. Analytes were chromatographically separated using a 5 µL sample injection volume on an RPC-18 column that had proportions of 50 mm (length), 2.1 mm (internal diameter), and 2.7 µm (particle size). A 45 minute analytical run was planned, during which the movementcontour of the movable phase was determined. Covering gas temperature was 350°C, covering gas flow was 11, vaporizer pressure was 35 psig, and the scan rate was 2 spectra per minute in Acquisition approach with a range of 60-1700 (m/z).The movable phase followed a binary gradient system, using solvent A as 0.1% formic acid in deionized water and solvent B as acetonitrile. The elution was carried out at room temperature with a flow rate of 0.4 mL/min. From 0 to 18 minutes, the movable phase consisted of 95% solvent A and 5% solvent B. Between 18 and 25 minutes, the composition gradually shifted to 5% solvent A and 95% solvent B. At 25 minutes, the ratio briefly returned to 95% solvent A and 5% solvent B until 25.10 minutes. Finally, from 25.10 to 30 minutes, the gradient was changed back to 5% solvent A and 95% solvent B.

• Antioxidant Activity

The rummaging activity of DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals was evaluated with slight modifications to the protocols described by²⁹. In this protocol, 10 µL of the plant extract was mixed with 290 µL of the DPPH solution in a 96-well plate. The plate was protected in the dark for 20 minutes. After the evolution period, the absorbance was measured at 517 nm. A blank solution was used as the control, and the absorbance of the samples was compared to that of the blank solution to measure their free radical scavenging activity. Ascorbic acid was used as the standard. The IC50 value (µg/mL) was the concentration of the extract required to scavenge 50% of the DPPH free radicals.

Where, OD1 and OD2 are the Optical density of control and test sample respectively.

• Antidiabetic Activity

Alpha-amylase inhibition assay

Methanolic and water extracts were diluted to appropriate concentrations (0–200 µL). Each dilution was then supplemented with 0.4 mg/mL (800 µL) of porcine pancreatic alpha-amylase and incubated in a 0.02 M sodium phosphate buffer for ten minutes at 25°C (pH 6.9; 0.006 M NaCl). Following this, 600 µL of a 0.5% starch solution in the same sodium phosphate buffer was added to the tubes. After nursing the reaction mixtures for ten more minutes at 25°C, 0.5 mL of dinitrosalicylic acid color reagent was added to stop the reaction. After 10 minutes of boiling in a water bath, the combinations were allowed to cool to room temperature. After cooling, distilled water was added to dilute the reaction solutions to a total volume of 20 mL. The alpha-amylase inhibitory activity was measured by calculating the percentage of inhibition based on the absorbance readings obtained at 540 nm.³⁰

Alpha-glucosidase inhibition assay

Both the methanolic and aqueous extracts underwent repeated dilution. The mixture was then nurtured for 10 minutes at 25°C after 100 µL of a 0.1 M phosphate buffer (pH 6.9) containing an alpha-glucosidase solution (1.0 U/mL) was added. The solutions were then mixed with 50 µL of a 5 mM solution of p-nitrophenyl-alpha-d-glucopyranoside in 0.2 M phosphate buffer (pH 6.7). After that, the samples were once more nurtured at 25°C for ten minutes. A spectrophotometer was used to detect the absorbance at 405 nm following the incubation period. Based on these findings, the alpha-glucosidase inhibition percentage was computed³⁰.

Where As and Ac is the absorbance in the presence of the sample and the control respectively.

• Anti-inflammatory activity

Protein denaturation (BSA) inhibition assay

Bovine serum albumin was utilized as the test protein to evaluate the inhibition of protein denaturation. A 0.2% BSA solution was prepared in phosphate-buffered saline at pH 6.4, consisting of 10 mM Na₂HPO₄, NH₄PO₄, and 150 mM NaCl. Indomethacin was used as the standard drug at various concentrations. The mixed reaction volume contained 900 µL of BSA and 100 µL of plant extract. The solution was excited to 70°C for 15 minutes and then allowed to cool. All measurements were taken at 280 nm, except for rare instances where turbidity was used as a control. In these cases, only BSA was used as the control. The percentage inhibition of protein denaturation was calculated using a conventional formula.³¹

Where, OD1 and OD2 are the Optical density of the control and test sample, respectively.

Proteinase Inhibition(Trypsin) Assay

This work investigated the potential of plant extracts as trypsin inhibitors. The reaction mixture contained 60 mL of Tris-HCl buffer (20 mM, pH 6.4), 40 mL of 0.2% w/v casein (substrate) and 50 µL of each plant sample or a standard Indomethacin as the inhibitor. The Room temperature initial reaction was for 10 min. Subsequently, 50 µL of trypsin solution (concentration: 60 mg/mL) was introduced into the reaction mixture. This preparation was then kept at 38°C for 20 minutes to allow the enzymatic activity to proceed. After the incubation period, the solution’s absorbance at 280 nm was measured. For reference purposes, a blank sample containing only trypsin and casein was also prepared and analyzed under identical conditions.³¹

Where, OD₁ and OD2 are the optical density of Control and test sample, respectively.

• Cell Cytotoxicity

The National Cell Centre (NCCS) in Pune, India, provided the HepG-2 cell line. The tumorous cells were placed in a flask full of 1% antibiotic solution (Penicillin-Streptomycin-Sigma-Aldrich P0781), 2-10% (fetal bovine serum) FBS, and DMEM (Dulbecco’s Modified EagleMedium-AT149-1L) media. The flask was then incubated at 35°C with 5% CO2. These adherent cells were trypsinized for 3–5 minutes, nurtured at 37°C for 24 hours and then centrifuged (1,400 rpm for 5 min). Ten thousand cells were counted per 96-well ELISA plate. The plate was protected at 37°C with 5% CO₂ for 24 h to permit cells to be attached³².

MTT Assay

The MTT colorimetric test was used to assess cell viability with a few modest adjustments³². In short, 200 µl of MTT [3-(4, 5-dimethylthiazol-2)-2, 5-diphenyl tetrazolium bromide] without phenol red (yellowish-coloured solution; 5 mg/ml in PBS) was added per well at a volume of 20 µl and the plates were nurtured for 3 hours under a standard atmosphere containing 5% CO₂ to enable metabolically active cells reduce MTT by dehydrogenase enzymes which produces reducing equivalents (NADH and NADPH). The corresponding purple insoluble formazan crystals were then solubilized and supernatants removed prior to spectrophometric measurement.

100 µL of DMSO was added to each well to dissolve the MTT crystals. After shuddering for 15 minutes, the optical densities were measured at 580 nm using a Thermo Scientific Multiskan Sky Plate Reader spectrophotometer. The absorbance levels correlate with the number of viable cells. Each experiment was performed in duplicate. Percentage cell viability was calculated by dividing the test sample absorbance by the control absorbance (medium without sample) and multiplying by 100.

Where, A(test) and A(Control)  is Absorbance of the test sample and the Absorbance of the control.

• Statistical Analysis

The mean ± standard deviation (SD) of two separate experiments, each carried out in triplicate, are used to display the data. Using GraphPad Prism 8 Statistical Software (San Diego, California, USA), one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison tests were used to evaluate statistical differences between the treatments and the control group. A statistically significant P-value was defined as < 0.05. The primary results were also calculated using Microsoft Excel formulae.

3.  Results

3.1. Phytochemical Screening

Several phytochemicals listed below were found in the aqueous and methanolic sources of Boerhavia diffusa leaves after qualitative screening. Both of the mines contained the same phytochemicals³³. But the methanolic extract only failed the anthraquinones test (Table 1).

Determination of Total Phenolic and Flavonoid Content

The B. diffusa methanolic and aqueous extracts had total phenolic values of 93.34 ± 0.024 and 73.34 ± 0.035 mg of gallic acid equivalents per gram of extract (mg GAE/g), respectively. The B. diffusa aqueous and methanolic extracts were found to have total flavonoid concentrations of 43.03 ± 0.023 and 52.30 ± 0.009 mg quercetin equivalents per gram of extract (mg QE/g), respectively. These values were determined using calibration curves constructed using gallic acid and quercetin as standard references. The detailed results are presented in Table 2.

3.2Metabolite profiling of the extracts by UPLC-QTOF-MS

LCMS analysis was conducted on both plant extracts, revealing the occurrence of the compounds listed in Table 3 and Table 4.

3.3 Antioxidant Assay

In this study, plant extracts were tested at deliberations of 20, 40, 60, 80, and 100 μg/ml for their free radical scavenging activity. Results (Table 5) showed that the highest concentration (100 µg/ml) exhibited strong antioxidant properties, effectively neutralizing free radicals. Both methanolic and aqueous extracts showed enhanced scavenging abilities compared to lower concentrations, as illustrated in Figure 1.

3.4 Antidiabetic Activity

Figure 2 illustrates the interaction of Boerhavia diffusa leaf extracts with α-amylase, specifically focusing on both aqueous and methanolic extracts. Both extracts exhibit a dose-dependent inhibition of enzyme activity across the deliberation range of 20 to 100 μg/ml. The IC50 values, which indicate the potency of the inhibitory effect, are as follows: the aqueous extract has an IC50 of 71.85 ± 0.003 μg/ml, while the methanolic extract shows a lower IC50 of 56.29 ± 0.003 μg/ml. For comparison, the standard reference compound acarbose has an IC50 of 59.27 ± 0.001 μg/ml. Additionally, Figure 2 presents the effects of both aqueous and methanolic Boerhavia diffusa leaf extracts on α-glucosidase. As with α-amylase, both extracts inhibit the activity of α-glucosidase in a dose-dependent manner (20–100 μg/ml). The IC50 values for α-glucosidase inhibition are 79.34 ± 0.001 μg/ml for the aqueous extract and 58.59 ± 0.004 μg/ml for the methanolic extract.

The extract exhibited a strong, dose-dependent reticence of both α-amylase and α-glucosidase activity. In fact, it performed similarly to, and in some cases even exceeded, the efficacy of acarbose. These results clearly indicate that the B. diffusa extract contains potent constituents that inhibit both α-amylase and α-glucosidase. Complete data can be found in Tables 6 and Table 7.

3.5 Anti-Inflammatory Activity

Protein denaturation inhibition assay(Bovine Serum Albumin)

Protein denaturation is a major source of inflammation. Ability of extracts to deter protein denaturation was determined as a further part of the investigation for the anti-inflammatory mechanism. Protein denaturation or its breakdown at higher temperature is enhanced and will become formation of peptides aggregates. At the various quantities shown in Figure 3, the plant demonstrated effectiveness against heat-induced albumin denaturation. With an IC50 value of 75.25 ± 0.003 μg/ml, the aqueous extract shows the maximum level of protein denaturation, whereas the methanolic extract has an IC50 value of 58.89 ± 0.011μg/ml.

Proteinase inhibition assay(Trypsin)

There is information linking proteinases to the arthritic response. Figure 3 shows the substantial antiprotease activity of the B. difusa plant parts in both aqueous and methanolic extracts at different doses.Aqueous and methanolic extracts exhibited their highest inhibitory effect at  IC50 – 80.93 ± 0.003 μg/ml and IC50 -57.60 ± 0.003 μg/ml, respectively.

A dose-dependent increase in inhibition was observed for both the extract and the standard compound, as presented in Tables 8 and 9. These findings indicate that the B. difusa extract exhibits moderate inhibitory activity against BSA denaturation and trypsin inhibition assays compared to indomethacin. Complete data are presented in Table 8 and 9.

Table 8 : In vitro Protein denaturation inhibition assay (Bovine Serum Albumin) effect of B. diffusa aqueous and methanolic extract and IC50 value in Comparison to the standard. Data are presented as the Mean Value ± SEM (n = 3).

Table 9 : In vitro Proteinase inhibition assay (Trypsin) effect of B. diffusa aqueous and methanolic extract, and IC50 value in Comparison to the standard. Data are presented as the Mean Value ± SEM (n = 3).

Figure 3: Assessment of the anti-inflammatory potential of Boerhavia diffusa leaf extracts was carried out through protein denaturation inhibition and proteinase inhibition assays, utilizing both methanolic and aqueous solvents for extraction.

3.6 Cytotoxic Assay

To exclude that inhibitory effects of selected parts of B. diffusa extract are connected to cytotoxicity, we have assessed cell viability by the MTT test. Cell viability was measured using the MTT assay, based on conversion of yellow tetrazolium salt to a purple formazan product (Fig. 4). As seen in Table 10, the viability of HepG-2 cells was more than 60% for treatments relative to control (HepG-2 cells not treated), which demonstrated that the different parts of B. diffusa plant were not toxic to the cells at the concentrations used. IC50 of aqueous extract of B. diffusa leaves was 351.1 ± 0.1258 µg/ml.

Table 10: Cell cytotoxicity of B. diffusa leaf aqueous extract on HepG-2 cell line using MTT Assay. Data are presented as the Mean Value ± SEM (n = 3).

4. Discussion

The current study explored the in vitro antidiabetic, anti-inflammatory, and antioxidant potential of aqueous and methanolic extracts, as well as assessed cell cytotoxicity on the HepG-2 cell line using the aqueous leaf extract.Phytochemical studies depicted the presence of phytoconstituents. Quantitative phytochemical evaluation of B. diffusa reveals a higher concentration of phenolic compounds compared to flavonoids. The total phenolic content was measured at  93.34 ± 0.024mg/g in the methanolic extract and  73.34 ± 0.035mg/g in the aqueous extract. In contrast, the flavonoid content was found to be  43.03 ± 0.023mg/g in the methanolic extract and  52.30 ± 0.009mg/g in the aqueous extract. These findings suggest that phenolics are the predominant class of secondary metabolites in B. diffusa^34-37, with potential implications for its antioxidant and therapeutic properties.²³Our study used a methanolic and aqueous extract on traditional spectroscopic methods,³⁸ or advanced techniques like LC-MS, or UPLC-QTOF-MS/MS.³⁹

Among the various plant parts of B. diffusa, the leaves exhibit the maximum concentration of phenolic compounds, correlating with superior free radical scavenging activity⁴⁰. The antioxidant efficacy of these compounds is attributed to their intrinsic redox properties, which assist them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers. This activity is further enhanced by the presence of hydroxyl functional groups, which play a critical role in neutralizing reactive oxygen species^41-42.The IC50 value for the antioxidant potential of aqueous extract showing 75.233 ± 0.001 μg/ml and that of methanolic extract showing 49.374 ± 0.003 μg/ml. Therefore, the methanolic extractis more effective in scavenging free radicals and exhibits higher antioxidant activity than the aqueous extract.

The extract also displayed moderate anti-inflammatory activity, as evidenced by its inhibition of BSA denaturation and trypsin inhibition assay. This effect may be attributed to compounds Tyrosol 4-sulfate, Dihydroferulic acid 4-sulfate, 5-Sulfosalicylic acid, 2,4-Dihydroxyacetophenone 5 sulfate, Hexahydro-6,7-dihydroxy-5-(hydroxymethyl)-3-(2-hydroxyphenyl)-2H-pyrano[2,3-d]oxazol-2-one, Kaempferol 3-O-β-D glucosyl-(1->2)-β-D glucosyl-(1->2)-β-D glucoside, Quercetin-3-sophoroside, Rhamnetin-3-laminaribioside, Isoorientin-2”-[feruloyl-(>6)-glucoside] known for their anti-inflammatoryproperties.⁴³ However, while this assay is preliminarily acceptable for assessing anti-inflammatory activity.

The extract demonstrated notable inhibitory activity against key carbohydrate-hydrolyzing enzymes, specifically α-amylase and α-glucosidase.⁴⁴ The methanolic extract exhibited superior α-amylase inhibition (IC50 = 56.29 ± 0.003 μg/mL) compared to the pharmaceutical reference standard acarbose (IC50 = 59.27 ± 0.001 μg/mL), while the aqueous extract also showed appreciable activity (IC50 = 71.85 ± 0.003 μg/mL). Similarly, α-glucosidase inhibition was more pronounced in the methanolic extract (IC50 = 58.59 ± 0.004 μg/mL) relative to acarbose (IC50 = 71.27 ± 0.002 μg/mL), with the aqueous extract yielding an IC50 of 79.34 ± 0.001 μg/mL. These findings suggest potential antidiabetic properties of the extract, likely attributable to its bioactive constituents. However, the current evidence is confined to in vitro assays. Therefore, further investigations are warranted to evaluate in vivo efficacy and safety, isolate and characterize the active phytochemicals, and assess the therapeutic potential through clinical studies.⁴⁵

The MTT assay was utilized to evaluate the cytotoxic effects of B. diffusa aqueous extract on HepG-2 cells⁴⁶. Figure 4 illustrates the dose-dependent cytotoxic effect of the aqueous extract of B. diffusa on HepG-2 cells. Acquaintance to 1 μg/mL of the extract for 3 hours resulted in a modest reduction in cell viability to 91.99%. Increasing the concentration to 500 μg/mL and 1000 μg/mL led to a more pronounced decline in viability, reaching 46.81% and 41.35%, respectively. The half-maximal inhibitory concentration (IC50) of the aqueous extract was calculated to be 351.1 ± 0.1258 μg/mL, indicating significant antiproliferative activity against HepG-2 cells.⁴⁷

The study demonstrates that B. diffusa leaves contain substantial levels of phenolics and flavonoids, which contribute to their notable antioxidant, antidiabetic and anti-inflammatory properties. The extracts also exhibited measurable cytotoxicity against the HepG-2 cell line, indicating additional therapeutic relevance. Collectively, the findings highlight B. diffusa as a promising candidate for developing plant-based therapeutics. Future research should focus on isolating the key active constituents, clarifying their mechanisms of action, and validating their safety and efficacy through comprehensive in vivo and clinical evaluations.

Conclusion

The current investigation aimed to assess the biological activities of Boerhavia diffusa leaf extract sourced from the arid region of Nagpur, Maharashtra. The results indicate that the extract possesses substantial antioxidant activity, demonstrated by its ability to scavenge free radicals, which may contribute to its overall pharmacological benefits. Additionally, a notable anti-inflammatory effect was observed, suggesting that the extract could influence inflammatory pathways associated with chronic metabolic and degenerative conditions. Furthermore, the study highlighted promising antidiabetic effects, indicating that the phytochemicals found in B. diffusa leaves may help regulate blood sugar levels and maintain metabolic balance. The extract also demonstrated hepatoprotective properties, supporting its traditional use in medicine for liver-related issues. It appears to mitigate oxidative stress and biochemical changes linked to liver dysfunction. This protective effect may be accredited to the existence of various bioactive phytochemicals, including flavonoids and phenolics, which were characterized using UPLC-QTOF-MS analysis.

Despite these promising findings, the study has certain limitations. All biological evaluations were conducted in vitro, and the use of plant material from a single geographical location may not fully represent the chemotypic diversity of B. diffusa. Therefore, the results should be interpreted as preliminary.

Collectively, the present findings substantiate the potential of B. diffusa as a promising source of bioactive phytochemicals. Future investigations should emphasize the isolation and characterization of individual compounds, elucidation of underlying molecular mechanisms through target-based studies, and validation of pharmacological efficacy and safety using in vivo and clinical models.

Acknowledgment

The authors sincerely thank Rashtrasant Tukadoji Maharaj Nagpur University for providing essential resources and valuable assistance. They also express their gratitude to Aakaar Biotechnologies Private Limited for their support in conducting anticancer activity studies. Additionally, the authors appreciate the efforts of Venture Center in Pune for performing the LCMS analysis.

Ethical Approval

This study does not apply to human or animal studies that require ethical approval.

Credit author statements

Shivani R. Sharma (Conceptualization, designing the experiments, phytochemical analysis, biological activity assays, data collection, analysis and interpretation, writing-review and editing,Nilima M. Dhote (provided critical guidance in the experimental design and methodology, optimizing the phytochemical extraction and analysis technique), Mamta S. Wagh (Conceptualization, formal analysis, Data curation, visualization, Supervision, writing-original draft).

Conflict of interest statement

The authors declare that there are no conflicts of interest amongst them.

Data availability statement

The relevant authors can provide the data used to support the study’s conclusions upon request.

Funding Statement

No agencies specifically provided support for this study.

References

[1] Bhatt B, Dey A, Kanjilal S, Srikanth H, Biswas R, Chakraborty P, et al. In vitro hepatoprotective activity of a polyherbal formulation on HepG2 cell line. Anc Sci Life. 2017;37(2):99.

[2] Praneetha P, Durgaiah G, Narsimha Reddy Y, Ravi Kumar B. In vitro hepatoprotective effect of Echinochloa Colona on ethanol-induced oxidative damage in HEPG2 cells. Asian Journal Pharm Clin Res. 2017;10(9):259–261.

[3] Álvarez DV, Dimas VM, Velázquez GV, Romero L del CA, Bátaz ER, Cruz YC. Hepatoprotective activity of Mimosa lacerata and Croton lechleri on the HepG2 cell line. Int Journal Biol Nat Sci. 2023;3(6):2–7.

[4] Gupta S, Ranade A, Gayakwad S, Acharya R, Pawar S. Hepatoprotective activity of ficus semicordata buch.-ham. ex sm. leaves aqueous extract on d-galactosamine induced toxicity in hepg2 cell line. Indian Journal Nat Prod Resour. 2020;11(4):239–243.

[5] Roglic G. WHO Global report on diabetes: A summary. Int Journal Noncommunicable Dis. 2016;1(1):3.

[6] Nair SS, Kavrekar V, Mishra A. In vitro studies on alpha amylase and alpha glucosidase inhibitory activities of selected plant extracts. Eur Journal Exp Biol. 2013;3(1):128–132.

[7] Jain PK, Jain S, Sharma S, Paliwal S, Singh G. Evaluation of anti-diabetic and antihypertensive activity of Phoenix sylvestris (L.)Roxb leaves extract and quantification of biomarker Quercetin by HPTLC. Phytomed Plus. 2021;1(4).

[8] Oyebode OA, Erukainure OL, Chukwuma CI, Ibeji CU, Koorbanally NA, Islam S. Boerhaavia diffusa inhibits key enzymes linked to type 2 diabetes in vitro and in silico; and modulates abdominal glucose absorption and muscle glucose uptake ex vivo. Biomed Pharmacother. 2018;106:1116–1125.

[9] Ademiluyi AO, Oboh G. Soybean phenolic-rich extracts inhibit key-enzymes linked to type 2 diabetes (α-amylase and α-glucosidase) and hypertension (angiotensin I converting enzyme) in vitro. Exp Toxicol Pathol. 2013;65(3):305–309.

[10] Karri SK, Sheela A. Comparative in vitro antidiabetic and immunomodulatory evaluation of standardized five select medicinal herbs and spectral analysis of boerhavia erecta L. (Nyctaginaceae). Pharmacogn J. 2017;9(3):336–344.

[11] Pari L, Amarnath Satheesh M. Antidiabetic activity of Boerhaavia diffusa L.: Effect on hepatic key enzymes in experimental diabetes. Journal Ethnopharmacol. 2004;91(1):109–113.

[12] Patil KS, Bhalsing SR. Ethnomedicinal uses, phytochemistry and pharmacological properties of the genus Boerhavia. Journal Ethnopharmacol. 2016;182:200–220

[13] Nayak P, Thirunavoukkarasu M. A review of the plant Boerhaavia diffusa: its chemistry, pharmacology and therapeutical potential. Journal Phytopharm. 2016;5(2):83–92.

[14] Aslam MS. An Update Review on Ethnomedicinal, Phytochemical and Pharmacological Profile of genus Boerhavia. Int Journal Complement Altern Med. 2017;6(3).

[15] Mahesh A, Kumar H, Mk R, Devkar RA. Detail Study on Boerhaavia Diffusa Plant for its Medicinal Importance-A Review. Res Journal Pharm Sci J Pharm Sci. 2012;1(1):28–36.

[16] Gaur PK, Rastogi S, Lata K. Correlation between phytocompounds and pharmacological activities of Boerhavia diffusa Linn with traditional-ethnopharmacological insights. Phytomed Plus.2022;2(2):100260

[17] Kale S, Kirdat P, Kale S, Dandge P. Phytochemical Screening With LC-HRMS Profiling And In Vitro Biological Activities Of Argyreia Cuneata (L.) And Argyreia Setosa (L.). Asian Journal Pharm Clin Res. 2022;72–80.

[18] Kaur H. Boerhaavia diffusa: Bioactive compounds and pharmacological activities. Biomed Pharmacol Journal 2019;12(4):1675–1682.

[19] Odion EE, Favour JC, Osigwe CC, Umeji TC, Uwaeme U. Boerhavia diffusa ( Linn .) Nyctaginaceae : Phytochemical profiling and sub-chronic toxicity study of the methanolic leaf extract in rodents. Cameroon Journal of Experimental Biology. 2025;19:9-16.

[20] Apu AS, Liza MS, Jamaluddin ATM, et al. Phytochemical screening and in vitro bioactivities of the extracts of aerial part of Boerhavia diffusa Linn. Asian Pac Journal TropBiomed. 2012;2(9):673-678.

[21] Monali P, Ramtej V. Hepatoprotective activity of boerhavia diffusa extract. Int Journal Pharm Clin Res. 2014;6(3):233–240.

[22] Anyasor GN, Okanlawon AA, Ogunbiyi B. Evaluation of anti-inflammatory activity of Justicia secunda Vahl leaf extract using in vitro and in vivo inflammation models. Clin Phytoscience. 2019;5(1).

[23] Malhotra D, Ishaq F, Khan A, Gupta AK. Analysis of bioactive phytochemicals and evaluation of antioxidant activity of a medicinal plant, Boerhaavia diffusa L. Journal Appl Nat Sci. 2013;5(2):357–360.

[24] Sinan KI, Aldahish AA, Celik Altunoglu Y, et al. LC-MS/HRMS analysis, anti-cancer, anti-enzymatic and anti-oxidant effects of Boerhavia diffusa extracts: a potential raw material for functional applications. Antioxidants. 2021;10(12):2003.

[25] Shaikh JR, Patil M. Qualitative tests for preliminary phytochemical screening: An overview. Int Journal Chem Stud. 2020;8(2):603-608.

[26] Blainski A, Lopes GC, De Mello JCP. Application and analysis of the folin ciocalteu method for the determination of the total phenolic content from limonium brasiliense L. Molecules. 2013;18(6):6852-6865.

[27] Chia-Chi C, Ming-Hua Y, Hwei-Mei W, Jiing-Chuan C. Estimation of total flavonoid content in propolis by two complementary colometric methods. Journal Food Drug Anal. 2002;10(3):178-182.

[28] Jiji KN, Muralidharan P. Identification and characterization of phytoconstituents of ethanolic root extract of Clitoria ternatea L. Utilizing HR-LCMS analysis. Plant Sci Today. 2021;8(3):535–540.

[29] Bhardwaj R, Yadav A, and Sharma RA. Phytochemicals and Antioxidant Activity In Boerhavia Diffusa. Sciences. 2014; 6(1): 4–8.

[30] Mechchate H, Es-Safi I, Louba A, Alqahtani AS, Nasr FA, Noman OM, et al. In vitro alpha-amylase and alpha-glucosidase inhibitory activity and in vivo antidiabetic activity of withania frutescens L. Foliar extract. Molecules. 2021;26(2):293

[31] Gunathilake K, Ranaweera K, Rupasinghe H. In vitro anti-inflammatory properties of selected green leafy vegetables. Biomedicines. 2018;6(4).

[32] Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal Immunol Methods. 1983;65(1-2):55-63.

[33] Banu KS, Cathrine L. General Techniques Involved in Phytochemical Analysis. Int Journal Adv Res Chem Sci. 2015;2(4):25-32.

[34]Mishra S, Aeri V, Gaur PK, Jachak SM. Phytochemical, therapeutic, andethnopharmacological overview for a traditionally important herb: Boerhavia diffusa linn. Biomed Res Int. 2014.

[35] Nair A, Chattopadhyay D, Saha B. Plant-Derived Immunomodulators. In: New Look to Phytomedicine: Advancements in Herbal Products as Novel Drug Leads. ; 2018:435-499.

[36] Milošević N, Milanović M, Pavlović N, et al. Indian ayurvedic herb, Boerhaavia diffusa as BCPR inhibitor: The story behind the curtains. Journal Mol Struct. 2022;1249.

[37] Pereira DM, Faria J, Gaspar L, Valentão P, Andrade PB. Boerhaavia diffusa: Metabolite profiling of a medicinal plant from Nyctaginaceae. Food Chem Toxicol. 2009;47(8):2142-2149.

[38] Anokwuru CP, Anyasor GN, Ajibaye O, Fakoya O, Okebugwo P. Effect of Extraction Solvents on Phenolic, Flavonoid and Antioxidant activities of Three Nigerian Medicinal Plants. Nat Sci. 2011;9(7):53-61.

[39] Mane MP, Patil RS, Magdum AB, Kakade SS, Patil DN, Nimbalkar MS. Chemo-profiling by UPLC-QTOF MS analysis and in vitro assessment of Anti-inflammatory activity of Field Milkwort (Polygala arvensis Willd.). South African J Bot. 2022;149:49-59.

[40] Sirivibulkovit K, Nouanthavong S, Sameenoi Y. Paper-based DPPH assay for antioxidant activity analysis. Anal Sci. 2018;34(7):795–800.

[41] Baba SA, Malik SA. Determination of total phenolic and flavonoid content, antimicrobial and antioxidant activity of a root extract of Arisaema jacquemontii Blume. Journal Taibah Univ Sci. 2015;9(4):449-454.

[42] Adebiyi OE, Olayemi FO, Ning-Hua T, Guang-Zhi Z. In vitro antioxidant activity, total phenolic and flavonoid contents of ethanol extract of stem and leaf of Grewia carpinifolia. Beni-Suef Univ Journal Basic Appl Sci. 2017;6(1):10-14.

[43] Anyasor GN, Okanlawon AA, Ogunbiyi B. Evaluation of anti-inflammatory activity of Justicia secunda Vahl leaf extract using in vitro and in vivo inflammation models. Clin Phytoscience. 2019;5(1).

[44] Sangeetha G, Vidhya R. In vitro anti-inflammatory activity of different parts of Pedalium murex (L.). Int Journal Herb Med. 2016;4(3):31–36.

[45] Manikandan R, Anand AV, Kumar S, Pushpa. Phytochemical and in vitro antidiabetic activity of psidium guajava leaves. Pharmacogn Journal. 2016;8(4):392-394.

[46] Shinde KV, Magdum AB, Mane MP, et al. Green synthesis of silver nanoparticles using Argyreia pilosa leaf extract : Characterization and bioactivity assessment. Next Mater. 2025;8(5):100832.

[47] Deepak HB, Chandrasekaran CV, Dethe S, Mundkinajeddu D, Pandre MK, Balachandran J, et al. Hepatoprotective and antioxidant activity of standardized herbal extracts. Pharmacogn Mag. 2012;8(30):116–123.