Medicinal plants are widely used in both developed and developing countries for their healing properties (Bajpayeeet al., 2024). Before the 18^th century, the therapeutic qualities of many plants were recognized, though their active ingredients were not yet understood (Salmerón-Manzanoet al., 2020). Modern techniques now allow for the extraction and identification of these compounds. (Balkrishnaet al., 2024). Phytochemicals can help combat oxidative stress caused by reactive oxygen species, which poses a significant global health concern (Symanet al., 2024; Jomovaet al., 2024). Plant antioxidants can neutralize free radicals, reducing the risk of serious diseases and serving as potential therapeutic agents (Tiwanaet al., 2024; Chaachouayet al., 2024). India’s diverse climate makes it a prominent source of medicinal plants (Atanasovet al., 2021; Varahet al., 2023). D. palmatus (Shivlingi), from the Cucurbitaceae family, is traditionally used by tribal communities for various chronic ailments (Patelet al., 2020; Kauravet al., 2021). This study aimed to investigate the antioxidant potential of D. palmatus extract through in vitro and in vivo assays and to evaluate its bioactive compounds using RP-HPLC.

Plants were collected in October and November 2022 from Badheri, Uttarakhand, India (29°53’54.3″N 77°59’08.0″E) and matched with a specimen (Accession No. 14405, Patanjali Research Institute, Panchayanpur) and identified as D. palmatus.

Analytical grades of standards and other chemicals were procured from Sigma Aldrich Chemical Pvt. Ltd., India.

Air-dried seeds from healthy fruits were used to prepare SME by cold percolation method.

Three experimental replicates were used to calculate the mean value for the given below in vitro antioxidant assays.

Different concentrations (20-100 μg/mL) of extract and Ascorbic Acid (AA) were prepared from a stock solution (1 mg/mL) by dilution with methanol. A mixture of 1 mL of extract or AA and 3 mL of 0.2 mM DPPH was incubated for 30 min in the dark. Absorbance was measured at 517 nm against a methanol blank, with DPPH alone serving as the control. The percentage radical scavenging activity (% RSA) was calculated using the formula: % RSA=(abs. of control – abs. of sample)/abs. of control×100. The IC₅₀ value was determined for both standard and extract using their calibration curve equation “y=mx+c.” (Delarosaet al., 2023).

The assay involved mixing 7 mM ABTS solution with 2.45 mM potassium per sulfate and letting it sit for 12-16 hr at room temperature. After dilution with ethanol (1:89 v/v) and equilibrating at 30ºC, the absorbance was set to 0.700±0.02 at 734 nm. Then, 1 mL of the extract and antioxidant (from 1 mg/mL stock) were combined with 3.5 mL of ABTS, and absorbance was read at 734 nm (Hussenet al., 2023). % RSA and IC₅₀ values were calculated as given in DPPH assay.

To perform the assay, a 5 mM sodium nitroprusside solution was prepared, which generates nitric oxide in the presence of oxygen, forming nitrite ions that react with Griess Reagent to produce a colored compound. Antioxidants in the extract compete with oxygen, decreasing nitrite ion formation and colored compound levels. A reaction mixture was created using 5 mM nitroprusside in phosphate-buffered saline (0.2 mM, pH 7.4) along with varying concentrations of the extract and Gallic Acid (GA) as a standard, and incubated at 25ºC for 150 min. After adding 0.5 mL of Griess reagent, absorbance was measured at 546 nm (Daset al., 2023) and % RSA and IC₅₀ values were calculated as given in the DPPH assay.

The TPC and TFC of the extract were measured at 100 μg/mL. For TPC, 5 mL of 10% Folin-Ciocalteu’s reagent was added to the extract and Gallic Acid (GA) standard, incubated in the dark for 5-10 min, followed by 4 mL of 7% Na2CO3 and a 90-min incubation. Absorbance was recorded at 750 nm, with a blank sample for comparison. The GA calibration curve “y=mx+c” determined mg GA Equivalent (GAE) per gram dry weight of extract.

For TFC, 0.3 mL of 5% NaNO2 was mixed with the extract and quercetin standard, allowed to sit for 5-6 min, then 0.3 mL of 10% AlCl3, 4 mL of 1M NaOH, and 10 mL of water was added and shaken. Absorbance was measured at 510 nm, again using a blank for comparison. The quercetin calibration curve “y=mx +c” calculated mg Quercetin Equivalent (QE) per gram dry weight of the extract (Islamet al., 2023).

The study was approved by the Institutional Animal Ethical Committee (IAEC) of M.D. University, Rohtak (ref: IAEC/2023/46-60, dated 02/03/23). 15 rats were divided into Negative control, Positive control, Metformin and SME groups as shown in Figure 1. 12 rats received Letrozole (1 mg/kg/day) for 21 days to induce PCOS (Kakadia et al., 2021), which causes oxidative stress (Senguptaet al., 2024). After sacrificing one rat and confirming induction, each group contained 3 experimental replicates. Induced groups were treated with metformin (500 mg/kg) (Desaiet al., 2012) and SME (100 mg/kg) (Reddyet al., 2010; Chauhanet al., 2010; Sivakumaret al., 2004) for 21 days. Post-dissection, liver and kidney homogenates were prepared to evaluate the impact of treatments on oxidative stress, compared with the untreated PCOS group. Following assays were conducted to assess in vivo antioxidant activity. In all the assays three experimental replicates were used to calculate the mean.

Figure 1:
Experimental setup for in vivo oxidative stress evaluation.

The main reagent in the assay was Pyrogallol, which auto-oxidizes to form free radicals and a brown Pyrogallol-quinone complex that absorbs at 420 nm. This auto-oxidation is inhibited by SOD, with one unit defined as the amount of enzyme that prevents 50% of the reaction. A reaction mixture (3 mL) was prepared with 1 mM EDTA, 50 mM Tris-HCl buffer (pH 8.2), 100 µL tissue homogenate, and 0.4 mM pyrogallol, topped with distilled water. Blanks excluded pyrogallol, while controls included it. Absorbance was measured at 420 nm for 2 min at 30±2ºC. Following steps were used to calculate SOD activity (Siswoyoet al., 2021):

The rate of absorbance/min was calculated for the control and extract by using the equation:

SOD activity was calculated by using the equation.

SOD activity (U/min/mg protein)=% inhibition/0.5.

Hydrogen peroxide is converted into oxygen and water by the enzyme catalase, found in the peroxisomes of eukaryotic cells. The reduction in absorbance at 240 nm correlates with the decomposition rate of hydrogen peroxide. For the assay, a mixture was prepared with 50 µL of tissue homogenate, 1 mL of 30 mM H₂O₂, and 2 mL of phosphate buffer (pH 7.0). Phosphate buffer served as Blank, while the controls excluded the sample. Absorbance was measured at 240 nm for 1 min. Catalase activity (U/mg) was calculated by dividing the absorbance by the molar extinction coefficient (43.6 M^–¹cm^–¹), indicating the degradation of 1 mM H₂O₂ per min (Tekinet al., 2022).

GSH is an intracellular reductant that alleviates oxidative stress. When oxidized by DTNB (5,5′-dithio-bis-2-nitrobenzoic acid), it forms the yellow compound TNB, which has an absorbance peak at 412 nm. For the assay, 50 µL of tissue homogenate in 0.1 M phosphate buffer (pH 7.4) was mixed with an equal volume of 20% TCA and 1 mM EDTA to precipitate proteins. After standing for 5 min, the mixture was centrifuged at 2000 rpm for 10 min, and the supernatant was collected. Then, 1.8 mL of 0.1 mM Ellman’s reagent was added, and the volume was adjusted to 2 mL before measuring absorbance at 412 nm against a blank. GSH enzyme activity (U/mg protein) was calculated using the standard curve for GSH as shown in Figure 2 (Alahmadiet al., 2023).

Figure 2:
Standard curve for GSH.

HPLC (Agilent 1260 Infinity model, Agilent, USA), which was outfitted with a DAD G4212B detector, a G1311B quaternary pump, and an automatic injector (G1329B). The HPLC was operated by ChemStation software (Agilent, USA).

Preparation of solutions A standard stock solution of Quercetin and vanillic acid was prepared in methanol at 1 mg/mL. A calibration curve was created using a concentration range of 0.10-25 μg/mL. For each experiment, three experimental replicates were used. Extract solutions were made at 10 mg/mL in methanol. Both standard and sample solutions were homogenized for thirty seconds, and then filtered through a 0.22 μM membrane. An aliquot was placed in an amber vial and sealed before being injected into the HPLC unit.

Preparation of mobile phase Quercetin was separated from SME using an isocratic mobile phase of HPLC-grade acetonitrile and 0.3% Trichloroacetic Acid (TCA) in water (50:50, v/v) at a flow rate of 0.9 mL/min for 13 min, detected at 254 nm. Vanillic acid was separated using a mixture of distilled water, methanol, and acetic acid (700:300:10 mL) at a flow rate of 1.0 mL/min for 14 min, detected at 260 nm.

Method validation Following ICH requirements, the method’s accuracy, linearity, specificity, Limit of Detection (LOD), precision and Limit of Quantification (LOQ) were all validated.

Results were recorded as Mean±SD. Graph Pad (Version 3.0) was used to analyse the statistical data. Student’s t-test determined the significant differences between the outcomes. p<0.05 was considered as significant.

The DPPH, ABTS, and NO assays showed an increase in % RSA with increasing concentrations of the standard and extract (Figures 3–5). The estimated IC₅₀ values were 28.69 µg/mL for AA and 53.82 µg/mL for SME in the DPPH assay, 21.2 µg/mL for AA and 37.43 µg/mL for SME in the ABTS assay, and 13.37 µg/mL for GA and 26.16 µg/mL for SME in the NO assay.

Figure 3:
Calibration curve for AA and SME by DPPH assay.

Figure 4:
Calibration curve for AA and SME by ABTS assay.

Figure 5:
Calibration curve for GA and SME by NO assay.

In TPC analysis, the calibration curve for Gallic Acid as in Figure 6(a) yielded the equation “y=0.001x+0.0492” (R²=0.9901) for calculating mg GAE/g of extract. For TFC analysis, the Quercetin calibration curve as in Figure 6(b) provided “y=0.0873x+0.0579” (R²=0.996) for calculating mg QE/g of extract. The results indicated that SME contains 28.13±0.2 mg GAE/g and 10.51±0.01 mg QE/g of dry extract

Figure 6:
Calibration curve for (a) Gallic Acid (TPC assay) and (b) Quercetin (TFC assay).

The PCOS induction in female Wistar rats has reduced the levels of oxidative stress markers SOD, GSH, and catalase in all groups except the Negative control. kidney and Liver tissue homogenate of different groups when subjected to SOD, GSH and Catalase assay the results indicated that the 21 days extract treatment had significantly raised the levels of these markers in all the treated groups compared to PC as shown in Tables 1 and 2 respectively.

Linearity A range of 0.10-25 μg/mL was taken as the calibration range. Vanillic acid and quercetin were determined to have 0.997 and 0.994 as coefficients of determination (R²), respectively.

Specificity According to the specificity test, the well-shaped peak showed that the main peak of bioactive chemicals is not impacted by other components found in the extract.

LOD and LOQ Quercetin’s LOD and LOQ were determined to be 0.184±0.6 and 0.723±0.9 µg/mL, respectively, while that for vanillic acid were 0.171±0.2 and 0.732±0.5 µg/mL, respectively.

Accuracy: The high recovery values demonstrated the method’s accuracy for vanillic acid and quercetin, which ranged from 99.40 to 99.91% and 99.53 to 99.90%, respectively.

Precision: The remarkable repeatability of the procedure is confirmed by the finding that the percentage RSD of both intra-day and inter-day precision was less than 0.20%.

Robustness: The robustness was assessed by taking the standard solution (n=3) of quercetin and vanillic acid with slight variations of ± 2 in flow rate, column temperature, pH, and wavelength. However, no appreciable variations in the recovery, peak area, or retention time were noted.

The RP-HPLC analysis of SME showed Quercetin peak at a retention time of 2.643 min with a peak area of 24424 and the calculated value of the bioactive compound was 3.342%. Figure 7 represents the peaks for (a) Quercetin standard and (b) SME. Figure 8 represents the peaks for (a) Vanillic acid standard and (b) SME in similar chromatographic conditions. SME showed a vanillic acid peak at a retention time of 6.258 with a peak area of 3283 and the calculated value of the bioactive compound was 2.312%.

Figure 7:
HPLC Peak for (a) Quercetin standard (b) SME.

Figure 8:
HPLC peak for (a) Vanillic acid standard (b) SME.

Phyto-antioxidants protect cells from oxidative stress by scavenging free radicals, with various phytochemicals playing a role in health promotion and disease prevention (Dibaet al., 2021; Mishraet al., 2020; Pandeyet al., 2009; Bhattacharjeeet al., 2022). Research indicates essential phytochemicals are present in the methanolic extract of D. palmatus (Attaret al., 2017). In vitro assays (DPPH, ABTS, Nitric oxide) evaluated the antioxidant potential of SME, revealing IC₅₀ values comparable to standards. TPC and TFC analysis showed SME has 28.13±0.2 mg GAE/g and 10.51±0.01 mg QE/g dry weight. The results were consistent with the earlier research demonstrating the presence of polyphenols, a class of antioxidants in the methanolic extract of D. palmatus (Upadhyayet al., 2023). In vivo assays demonstrated reduced oxidative stress markers in PCOS female Wistar rats treated with SME, along with increased levels in liver and kidney tissue homogenate. RP-HPLC analysis confirmed vanillic acid and quercetin as the primary antioxidants in SME, supporting their high antioxidant capacity.

The study concluded that the SME of D. palmatus exhibits significant antioxidant activity with vanillic acid and quercetin identified as the main antioxidants by RP-HPLC. Therefore, the research promises to create a safe, all-natural health supplement.

The authors are thankful to the Head, Department of Genetics and Animal House, Maharshi Dayanand University, Rohtak, Haryana, India, for providing the facilities to carry out the trial.