ISOLATION AND NMR-BASED STRUCTURAL ELUCIDATION OF A LUPEOL DERIVATIVE FROM Tapinanthus dodoneifolius

Abstract

The present study investigated the phytochemical composition, chromatographic profile, structural characterization and safety of Tapinanthus dodoneifolius leaf extracts. Extraction of 1700 g of powdered leaves with methanol yielded 287.82 g (16.93%) of crude extract, which upon solvent partitioning afforded n-hexane (5.08 g; 2.54%), chloroform (5.80 g; 2.90%), ethyl acetate (16.27 g; 8.14%), and n-butanol (4.67 g; 2.30%) fractions. Preliminary phytochemical screening of the crude extract revealed tannins, terpenoids, triterpenes, flavonoids, saponins, carbohydrates, cardiac glycosides and alkaloids, while anthraquinones were absent. Fractional analysis showed distinct metabolite distribution: terpenoids and triterpenes were prominent in non-polar (n-hexane and chloroform) fractions, whereas ethyl acetate and n-butanol fractions were enriched with tannins, flavonoids, carbohydrates and cardiac glycosides. Thin layer chromatography (TLC) demonstrated chemical diversity, with the crude extract producing ten bands (Rf = 0.06–0.89) compared to seven to nine bands across fractions, suggesting multiple phytoconstituents. Column chromatography of the ethyl acetate fraction yielded eighteen pooled subfractions, which were purified to a single compound designated as STVE. The compound appeared as a single homogeneous spot across multiple solvent systems with Rf values between 0.36 and 0.56, confirming purity. Physicochemical analysis indicated solubility in methanol and chloroform, insolubility in hexane, and a melting point of 196–198 °C. Spectroscopic characterization revealed key functional groups: UV absorption at 210–255 nm indicated a conjugated system, IR absorption at 3429 cm⁻¹ (O–H stretch), 2933–2866 cm⁻¹ (C–H stretch), 1461 cm⁻¹ (C=C), and 883–957 cm⁻¹ (olefinic/methylenic vibrations) suggested a triterpenoid skeleton. The ¹H-NMR spectrum displayed seven tertiary methyl singlets, carbinylic protons at δ 3.66 and 2.99 and olefinic protons at δ 4.57 and 4.69, characteristic of a lupane-type triterpene. ¹³C- and APT-NMR confirmed 30 carbons, including seven methyl, ten methylene, seven methane and six quaternary carbons. Two-dimensional NMR (COSY, HSQC, and HMBC) established connectivities consistent with a lupeol derivative. Comparative analysis with literature confirmed the structure as Lup-20(29)-ene-2,3-diol. Toxicological evaluation indicated an oral LD₅₀ > 5000 mg/kg, suggesting a wide margin of safety. Overall, this study provides the first report of isolating a lupeol derivative from Tapinanthus dodoneifolius, substantiating its ethnomedicinal relevance and highlighting its potential as a bioactive triterpenoid for future pharmacological applications.