Leaves of B. variegata were collected from Green Chem Private Limited, Bangalore and identified. The voucher specimen was preserved for future use. The plant material gathered was removed gently from the soil and any attached material and dried at room temperature for 5-6 days to complete dehydration. The dried plant material was powdered with a 60 # sieve and utilized for further research, such as determination of the extractive value.

Powdered leaves of B. variegata were subjected to successive solvent extraction to separate the active constituents. Petroleum ether, chloroform, ethyl acetate, and methanol were used as solvents. Extraction was performed stepwise in an extractor, with each solvent chosen according to increasing polarity.

Laboratory-scale extraction involved maceration of 200 g of powdered B. variegata leaves with various solvents, followed by filtration and concentration. Methanol extraction yielded a 2% extract after three-hour maceration with 200 ml of methanol, which was performed twice. Chloroform extraction using the same procedure yielded 1.2% yield. Ethyl acetate extraction, which gave the highest yield of 4.5%, used 200 ml of ethyl acetate. Petroleum ether extraction was performed using the same procedure to yield a 2.5% extract.

Ethyl acetate, petroleum ether, and methanol were used for pilot-scale extraction because of their higher yields. Petroleum ether extraction involved 3 kg of dried leaves and 6 L of solvent at a temperature of 55-60°C for two hours for three iterations, resulting in 100 g of powder. Methanol extraction under the same conditions at 65-75°C produced 70 g of powder. Ethyl acetate extraction at 80-85°C for two hours twice yielded 200 g of powder, which is the most efficient method. For this reason, the ethyl acetate extract was given higher priority for subsequent analysis because of its better yield.

Phytochemical screening was also carried out to detect secondary metabolites, such as carbohydrates, alkaloids, steroids, glycosides, saponins, flavonoids, triterpenoids, proteins, and amino acids. Various qualitative tests were conducted to ascertain their existence. Carbohydrates were identified through Molisch's, Fehling's, Benedict's, and Barfoed's tests, each of which showed characteristic color changes representing the presence of reducing sugars or monosaccharides. Alkaloids were ascertained by Dragendroff's, Wagner's, Mayer's, and Hager's tests, all of which gave characteristic precipitates. Steroids and sterols were detected by the Libermann-Burchard and Salkowski tests, which indicated color changes as proof of their presence.

Glycosides were detected using Legal's, Baljet, Borntrager's, and Killer-Killani tests, which yielded characteristic color reactions. The foam test established the presence of saponins, whereas flavonoids were detected by Shinoda, lead acetate, and alkaline reagent tests, which yielded red, yellow, and fading yellow colors, respectively. The tin chloride test revealed the presence of triterpenoids via a pink reaction. Proteins and amino acids were verified by the Biuret, Ninhydrin, and Xanthoprotein tests, in which color changes showed the presence of proteins and aromatic amino acids.

To isolate and identify further bioactive compounds, the ethyl acetate extract was subjected to column chromatography. Silica gel (60-200 mesh) was packed in a glass column and prepped with solvents prior to adsorbing the ethyl acetate extract onto the silica gel. Gradient elution was performed by stepwise increase in polarity using a solvent system containing hexane, benzene, toluene, ethyl acetate, and methanol. The obtained fractions were examined by Thin Layer Chromatography (TLC) to identify the constituents.

The separation and identification of the compounds in the extract were performed using TLC. Various solvents were used in this study. For the ethyl acetate extract, a solvent system of ethyl acetate: acetic acid: water (4:1:5) was used. TLC plates were developed, scanned at 366 nm, and detected using a vanillin-sulfuric acid reagent. High-Performance Thin Layer Chromatography (HPTLC) was performed on a CAMAG instrument using silica gel plates. The mobile phase employed was Benzene: Ethyl acetate: Formic acid (3.0:6.5:0.5). The sample was spotted on plates, which were developed in a saturated tank and scanned under UV light.

The isolated compounds were then subjected to High-Performance Liquid Chromatography (HPLC). The mobile phases used in the gradient system were 0.4% phosphoric acid (A) and acetonitrile (B). A Phenomenex C18 Luna, 5µm, 250 x 4.6 mm column was used, and detection was performed at 325 nm with a flow rate of 1.0 ml/min. The sample and standard solutions were prepared by dissolving the extracts in methanol and injecting them into the HPLC system for analysis.

Spectral identification of the isolated compounds was performed using FT-IR, NMR spectroscopy, and mass spectrometry (MS). FT-IR spectroscopy was performed using the KBr pellet technique in the range of 4000-400 cm⁻¹ to identify functional groups. NMR spectroscopy was employed to establish the chemical structures of the isolated compounds based on their chemical shifts, integration, and multiplicity of the peaks. Furthermore, mass spectrometry was used to identify the mass-to-charge ratio of the isolated compounds, which could contribute to their structural identification. This approach enabled the extraction, isolation, and thorough analysis of bioactive compounds from B. variegata leaves, providing useful information regarding their chemical makeup and potential pharmacological activities.

Thin-layer chromatography (TLC) showed three violet spots that were visually detected, with an Rf value of 0.72. Sterols were detected by phytochemical tests. The compound was found to have good solubility in ethanol and a melting point range of 144-146º C. No spots or residues were found on TLC in fractions 1-3 to 43-46. All these fractions gave fractions 53-74 showed a brown residue with three violet spots (Figure 1), with Rf values of 0.72, 0.50, and 0.28. Similarly, fractions 54-57 too possessed a brown residue with a solitary spot with an Rf value of 0.72.

Figure 2 shows the HP-TLC profile of the B. variegata leaf extract, which was detected under UV light at 366 nm and visualized with vanillin sulfuric acid reagent. The chromatographic plate showed three lanes, A, B, and C, representing Bauhinia leaf extract, an isolated compound (BLE/RD/01), and a reference standard (BLE/REF/01), respectively. Under UV 366 nm detection, clear bands were observed, indicating the presence of different phytochemical constituents.

Figure 3 depicts the HP-TLC pattern of B. variegata leaf extract after treatment with the vanillin sulfuric acid spraying reagent. The chromatography plate showed three lanes, A, B, and C, showing Bauhinia leaf extract, an isolated compound (BLE/RD/01), and a reference standard (BLE/REF/01), respectively. Upon the application of reagent spraying, well-defined bands with different shades and intensities were observed, indicating the presence of various phytochemical constituents. The bands in lanes B and C seemed comparable in position and color, indicative of the existence of the same or structurally homologous compounds. The Bauhinia leaf extract sample (lane A) showed multiple bands, consistent with its multi-component phytochemical nature. The reagent-induced color distinction of the compounds facilitated easy comparison among the samples.

The IR absorption spectrum revealed absorption peaks at 3439.6cm^‐1 (O‐H stretching.); 2924.7 cm^‐1 and 2867.9 cm^‐1 (aliphatic C‐H stretching); 1631.6 cm^‐1 (C=C absorption peak); other absorption peaks include 1410.3 cm^‐1 (CH2); 1043.7 cm^‐1 (cycloalkane) and 881.6 cm^‐1 (Figure 4).

1 HNMR (CDCl3) of isolated compound: 1HNMR has provided signals at δ3.2 (1H, m, H‐3), 5.26 (1H, m, H‐6), 5.19(1H, m, H‐23), 4.68 (1H, m, H‐22), 3.638 (1H, m, H‐3), 2.38 (1H, m, H‐20), 1.8‐2.0 (5H, m) ppm. Other peaks are found at δ 0.76‐0.89 (m, 9H), 0.91‐1.05 (m, 5H), 1.35‐1.42 (m, 4H), 0.69‐0.73 (m, 3H), 1.8‐2.00 (m, 5H), 1.07‐1.13 (m, 3H), 1.35‐1.6 (m, 9H) ppm. The mass spectrum of the compound exhibited a molecular ion peak at m/z 414.38, which is equivalent to the molecular formula of C29H52O. Mol. Weight: 414.707. MS indicated molecular ion peaks at 414 equivalents to the molecular formula C29H50O. There were also ion peaks at m/z 367, 271, 255, 229,189, 175, 161, 133, 121, 105, 107, 95, 81, 69, 55, 41. The structure of the isolated compound is shown in Figure 2.