A new lupane-type triterpenoid fatty acid ester and other isolates from Ophiorrhiza shendurunii

Abstract A new pentacyclic triterpenoid fatty acid ester, lupan-20-ol-3(β)-yl hexadecanoate (1), together with lupan-20-ol-3(β)-yl acetate (2), olean-18-en-3(β)-yl hexadecanoate (3), dotriacontanoic acid (4), stigmasterol (5), rubiadin (6), nonadecanoic acid (7), palmitic acid (8) and camptothecin (9) were isolated from the hexane and chloroform extracts of Ophiorrhiza shendurunii from South India. Structures of the isolates were determined by 1H, 13C, 13C DEPT, 1H–1H COSY, HMBC, HSQC, NOESY NMR, FT-IR, DART-MS, ESI-MS, alkaline hydrolysis, derivatisation, GC–MS and HPTLC analyses. O. shendurunii hexane and chloroform extracts showed significant activities against Candida albicans and Fusarium oxysporum. Compounds 1 to 3 showed only moderate antiyeast/antifungal activities.

drug camptothecin along with other phytochemicals (Rajan et al. 2013). Ophiorrhiza shendurunii, A.E.S. Khan & al, is one of the species screened for camptothecin by us recently (Rajan et al. 2013). O. shendurunii is an undershrub with subfruticose stem endemic to the southern Western Ghats (Shanavas Khan et al. 1998). High degree of endemicity of O. shendurunii to southern Western Ghats prompted us to choose it for our studies. Here we report phytochemical investigation of hexane and chloroform extracts of O. shendurunii which led to the isolation of one new pentacyclic triterpenoid fatty acid ester, lupan-20-ol-3(β)-yl hexadecanoate (1), along with eight known compounds (2 to 9). Antifungal/antiyeast activities of O. shendurunii hexane, chloroform extracts and the major isolates (1), (2), (3) and (6) were also tested.

Plant material
O. shendurunii (whole plant) was collected from Shenduruny Wildlife Sanctuary of the Kerala region of southern Western Ghats in India in january 2011. Plant specimen was authenticated by Dr E.S. Santhosh Kumar of jNTBGRI and a voucher specimen was kept at the Institute Herbarium (TBGT 69911).

Extraction, isolation
O. shendurunii (whole plant) was shade dried and powdered. Powdered plant material (1.2 kg) was Soxhlet extracted using 4 L of hexane for 18 h. The plant powder was subsequently extracted with 4 L of chloroform for 18 h. Solvents were fully removed using a rotary evaporator to yield 64 g of hexane and 15 g of chloroform extracts.
Chloroform extract (14 g) was fractionated by silica gel column chromatography (5.5 × 90 cm) using 100% hexane, followed by increasing polarities of hexane-CHCl 3 , CHCl 3 -MeOH and 100% MeOH. These fractions were pooled to six final fractions (Cfr 001 to 006) based on their TLC profiles. Cfr 001 appeared as a waxy material and GC-MS confirmed it as a mixture of hydrocarbons with major components as octadecane, hexadecane and eicosane. Cfr 002 on repeated column chromatography and purification by preparative TLC resulted in a white waxy compound (10, 22 mg). Cfr 003 on repeated column chromatography and recrystallisation yielded a white solid (7, 31 mg). Compound (8, 18 mg) was isolated as a white solid from Cfr 004 by repeated column chromatography and recrystallisation. Cfr 005 on column chromatography and recrystallisation yielded a yellow-coloured solid (6, 15 mg). Cfr 006 on repeated column chromatography and preparative TLC yielded a fluorescent compound (9, 10 mg). The purity of all isolates was confirmed by HPTLC and melting point measurements. Compounds 1 to 10 were identified by 1 H, 13 C NMR, 2D NMR and mass spectral analyses (Figure 1, Figure S1).

Alkaline hydrolysis of (1)
Compound 1 (15 mg) was dissolved in 3 mL of benzene, 12 mL 3% of KOH/MeOH was added, reaction mixture was kept at room temperature for 15 min, then neutralized with 1 N HCl/ MeOH and extracted with chloroform. Triterpenoid alcohol (lupan-3,20-diol) and methyl ester of the fatty acid (hexadecanoic acid methyl ester) were separated from the residue by column chromatography (Al-Musayeib et al. 2013). Methyl ester of hexadecanoic acid was identified by GC-MS ( Figure S1).

Conclusion
A new pentacyclic triterpenoid fatty acid ester, lupan-20-ol-3(β)-yl hexadecanoate (1), together with eight known molecules were isolated from the hexane and chloroform extracts of O. shendurunii from South India. Several lupane-type terpenoids were isolated from plants and they are known for various biological activities, including antibacterial/antifungal/antiyeast activities (Abad et al. 2007;Nguyen & Nguyen 2013;Hill & Connolly 2015). O. shendurunii hexane and chloroform extracts showed significant activities against C. albicans and F. oxysporum on agar well diffusion assay. Isolated compounds 1 to 3 showed only moderate antiyeast and antifungal activities.