Leaves of Tamarindus indica, Ricinus communis, and Calotropis gigantea were collected from the Govt. Medical College campus, Thiruvananthapuram, Kerala, and the leaves of Vitex negundo were obtained from Ayurveda Research Institute, Poojappura, Thiruvananthapuram. The collected specimens were examined and authenticated by Christil Lila. R, Senior Research Officer (Botany), Pharmacognosy unit, Ayurveda Research Institute, Poojappura, Thiruvananthapuram with voucher specimen numbers SKCPRC/COG/HS 2024/001 (Ricinus communis), SKCPRC/COG/HS 2024/002 (Tamarindus indica), SKCPRC/COG/HS 2024/003 (Vitex negundo) and SKCPRC/COG/HS 2024/004 (Calotropis gigantea). The herbarium was deposited at College of Pharmaceutical Sciences, Govt. Medical College, Thiruvananthapuram for later reference.

The leaves were chopped into small pieces after a thorough cleaning process, dried in the shade at room temperature and then ground to a fine powder in a mechanical blender. Dried leaf powder (30 g) was packed into a soxhlet apparatus and extracted with 500 ml of ethanol (95%) at 60-65⁰C for 4-6 hours. The extract was filtered, and the filtrate was concentrated by distillation. Each extract was then dried, weighed, and stored at 4⁰C.

All the chemicals and solvents of standard analytical grade were used for the study. Quercetin, PBS (phosphate buffered saline), Diclofenac sodium and Lupeol were purchased from Sigma–Aldrich (Mumbai-India). Bovine serum albumin and Folin-Ciocalteu (F.C) reagent were collected from HI media laboratories Pvt. Ltd Mumbai. Aluminium chloride, sodium nitrite and sodium hydroxide were obtained from Nice chemicals PVT LTD, Kochi, Gallic acid and DPPH from Sisco research laboratories PVT LTD Mumbai.

The phytoconstituents in the extracts were determined qualitatively according to standard procedures described previously 20. Phytochemicals tested included alkaloids, flavonoids, saponins, tannins, glycosides and terpenoids.

The total phenol content was determined using the Folin and Ciocalteu method, as described by Singleton and Rossi with slight modifications 21. Gallic acid was used as a standard and was prepared in methanol. From the stock solution (1 mg/ml), working solutions of gallic acid in concentrations of 20, 40, 60, 80, and 100 μg/ml were prepared and transferred to tubing (each in triplicate) and made up to 1ml with distilled water. 1 ml of alcoholic extract of TI, CG, RC, and VN at a concentration of 1000 μg/ml was used for the estimation. The standard and samples were mixed well with 5ml Folin-Ciocalteu Reagent (1:10 dilution), kept for 5 minutes, then treated with 4 ml 20% Na₂CO₃ solution, and after 30 minutes, absorbance was measured against the blank at 760 nm. The total phenol content for each extract was derived from the gallic acid calibration curve and expressed as mg of gallic acid equivalent/gm of the ethanolic extract (mg GAE/gm).

The total flavonoid content was determined according to the aluminium chloride method 22. From the quercetin standard (stock) solution in methanol containing 1000 µg/ml, working solutions of quercetin containing 10, 25, 50, 100, and 250 μg/ml quercetin were prepared. The alcoholic extracts of TI, CG, RC, and VN were tested at 1000 μg/ml concentration. Add 4 ml of water and 0.3 ml of 5% sodium nitrite solution to the test tubes and mix thoroughly. After 5 minutes, 0.3 ml of 10% aluminium chloride solution was added, and at the 6th minute, 2 ml of 1M sodium hydroxide was added. The total volume of the contents was then made up to 10 ml with distilled water and once again mixed well, and the absorbance was measured against the blank at 510 nm. The blank was prepared without the addition of aluminium chloride solution. The total phenol content for each extract was derived from the calibration curve of standard quercetin and expressed as milligram quercetin equivalent per gram of ethanolic extract (mg QE/gm).

To create a uniform mixture, the four extracts were taken in different amounts and constantly agitated. Table 1 shows the amounts of extracts needed to make 1 g of the polyherbal mixture. The mixtures were developed on the basis of the results of phytochemical screening and quantitative analysis.

HPTLC finger printing studies were performed according to the method of Wagner and Baldt 23 and Harbone 24. For HPTLC profiling, a CAMAG HPTLC system was utilized, which included a Linomat 5 automatic applicator with a 100 μL syringe, a twin trough plate development chamber (20X10 cm), a Camag TLC scanner, and Win CATS planar chromatography manager software.

The HPTLC was performed on 9.0 × 10.0 cm precoated silica gel 60 F 254 HPTLC plate (E. MERCK KGaA). The sample solution was applied as bands to the plate by CAMAG Linomat applicator fitted with 100 μl syringe. Five microliters of the sample solution and 2 μl lupeol (1000 mg/l) were spotted as bands with bandwidth of 8mm. After the application of the spots, the chromatogram was developed in a twin trough glass chamber (20×10 cm) that had previously been saturated with the solvent system n-Hexane: Ethyl Acetate (80:20). The solvent system was chosen from the literature 25. The plate was produced vertically rising to a height of 70.0 mm. A TLC scanner with winCATS Planar chromatography manager software was used for densitometric scanning. Images were taken in visible light and UV wavelengths of 366 nm and 254 nm while the plate was in the photo-documentation chamber. The Peak numbers were detected with the height, peak area and peak densitogram. Following scanning, the plates were post-derivatized by subjecting them to an anisaldehyde sulfuric acid reagent and then drying for 20 minutes at 120 ⁰C in an oven. Following derivatization, the plates were examined at visible light and UV under 366 nm and 254 nm.

DPPH inhibition in the plant extracts and mixtures was determined by the method of Brand-Williams et al., 26, 27 with some modifications. The DPPH radical scavenging assay depends upon the colour change of 1-diphenyl-2-picryl hydrazyl (DPPH) radical, which is red in colour, to 2,2'-diphenyl-1-picryl hydrazine (yellow coloured) on scavenging. The decrease in absorbance thus occurs is directly proportional to the anti-oxidant capacity of the test substance. 3 ml of 0.1 mM solution of DPPH (4 mg DPPH in 100ml ethanol) was incubated with 1 ml each of 25, 50, 100, 200, and 400 µg/ml extract in ethanol. Quercetin similar concentrations was used as the standard. The control consisted of 3 ml DPPH and 1 ml ethanol only. The percentage inhibition was calculated by the formula,

Ethanolic extracts of TI, RC, CG, and VN, as well as the reference standard (Diclofenac sodium) and control (no standard/test sample), were prepared at various quantities (25, 50, 100, and 200 µg/mL) and added to 4 mL of phosphate buffer solution (0.2 M, pH 7.4). All the tubes were combined with 1 mL of a 1 mM albumin solution in phosphate buffer, and the tubes were then incubated for 15 minutes at 37⁰C. The reaction mixture was maintained at 60°C in a water bath for 15 minutes in order to produce denaturation. The turbidity was measured at 660 nm after cooling 28.

The following formula was utilized to determine the percentage inhibition of denaturation.

Values were expressed as Mean ± SD. The statistical analysis was performed by one -way analysis of variance (ANOVA) followed by Tukey’s test. The p-value < 0.05 was considered statistically significant when compared with the control. The statistical analysis was made with the Graph pad prism version 8.

The preliminary phytochemical analysis showed that the ethanolic extracts of leaves of TI, RC, CG, and VN contained carbohydrates, alkaloids, tannins, flavonoids and phenols. The results of the phytochemical screening are shown in Table 2.

The absorbance values obtained at different concentrations of gallic acid (20, 40, 60, 80 and 100 mg/ml) were used for constructing the calibration curve. The result is expressed as Gallic acid equivalents. The total phenolic content of the extracts was calculated from the regression equation of the calibration curve (y = 0.0104x + 0.0422, R² = 0.9985) and expressed as mg gallic acid equivalents (GAE) per gram of extract (mg/g). The results are given in Table 3.

The total Flavonoid content was determined by aluminium chloride colourimetric method and standard used was quercetin with concentrations of 10, 25, 50,100, 250 µg/ml. The total flavonoid content of the extracts and mixtures were estimated, and the results are shown in Table 3. The absorbance values obtained at different concentrations of quercetin were used for constructing the calibration curve. The result is expressed as quercetin equivalents. The total flavonoid content of the extracts was calculated from the regression equation of the calibration curve (y = 0.0008x + 0.0031, R² = 0.9984) and expressed as quercetin equivalents (QE) per gram of extract (mg/g). The total flavonoid content in the case of plant extracts was found to be CG ˃ TI ˃ VN ˃RC. Among mixtures MX A ˃ MX B ˃ MX C.

For the HPTLC study, the ethanol extracts of TI, RC, CG, VN, MX C and standard lupeol was developed using the solvent system n-Hexane: Ethyl acetate (80:20) and visualized under white ray, UV 254 and 366 nm pre-derivatization and post derivatization with anisaldehyde sulfuric acid spray reagent (Figure 1). The Rf values and area percentage of different bands obtained post derivatization with anisaldehyde sulfuric acid and visualization under 366 nm are presented in Table 4. Figure 3, Figure 4 indicating the chromatogram and 3D overlay of densitogram. A number of 5 bands were observed in TI, CG, VN, MX C and 11 bands in RC. A spot for lupeol was obtained at Rf value of 0.48. Among extracts, the peak area for lupeol was maximum in CG. No spot of lupeol was seen in VN contrary to the literature reference. Seasonal variation in lupeol content has been reported in many plants 29, 30. The absence of spot may be due to seasonal variation. A common spot with Rf value 0.31 was observed in all the plant extracts and in the mixture when the spots are observed pre-derivatization under 366 nm, as shown in Figure 1 (A).

Figure 2

Figure 3

Figure 4

The antioxidant activity of TI, RC, CG, VN, MX A, MX B and MX C was determined by the DPPH radical scavenging assay. The percentage DPPH radical scavenging activity of the standard quercetin and the plant extracts at concentration of 25, 50,100, 200, 400 µg/ml was performed in triplicate and the results were expressed as mean ± standard deviation. The results are shown in Table 5, and Figure 3. IC₅₀ values of TI, RC, CG, VN, MX A, MX B, MX C and standard quercetin was found to be 213.43, 65.26, 355.55, 123.78, 190.90, 83.28, 36.45 and 34.70 µg/ml, respectively. The anti-oxidant activity in the case of plant extracts followed the order RC ˃VN ˃TI ˃CG and in the case of mixtures, MX C ˃ MX B ˃MX A.

Denaturation of tissue proteins may be the cause of the production of autoantigens in certain arthritic diseases. Therefore, tissue protein denaturation is a marker for inflammatory and arthritic diseases and agents that can prevent protein denaturation, therefore, would be possible candidates for anti-inflammatory drug development.

The anti-inflammatory activity of TI, RC, CG, VN and MX C was determined by the protein denaturation assay. The percentage denaturation of protein activity of standard diclofenac sodium and the plant extracts at concentrations of 25, 50,100 and 200µg/ml was performed in triplicate and the results were expressed as mean ± standard deviation. The results are shown in Table 6, and Figure 4. The IC₅₀ values of standard diclofenac sodium, TI, RC, CG, VN, MX C and sodium was found to be 10.7, 134.7, 127.6, 16.29, 35.80, and 28.30 µg/ml, respectively. The anti-inflammatory activity in the case of plant extracts followed the order VN˃RC ˃TI ˃CG.

CG- Calotropis gigantea, RC- Ricinus communis, TI- Tamarindus indica, VN- Vitex negundo, MX- Mixture, IC₅₀- Half maximal inhibitory concentration, DPPH- 2,2-Diphenyl-1-picrylhydrazyl, HPTLC- High performance thin layer chromatography

The authors would like to acknowledge Care Kerala Ltd, KINFRA Park, Koratty, Kerala, India for providing facilities to carry out the HPTLC study.