Eburneolins A and B, new withanolide glucosides from Tricholepis eburnea

Abstract Eburneolins A (1) and B (2), new withanolide glucosides, have been isolated from the n-butanolic fraction of the 75% methanolic extract of aerial parts of Tricholepis eburnea. Their structures were elucidated through spectroscopic analysis including ESI-MS, 2D NMR and acid hydrolysis.


Results and discussion
The 75% methanolic extract of aerial parts of T. eburnea was suspended into water and successively partitioned into n-hexane, dichloromethane, ethyl acetate and n-butanol fractions. A series of column chromatographic technique was applied on the n-butanol fraction on the Diaion hP-20, Sephadex lh-20, polyamide resin and silica gel to obtain compounds 1 and 2, respectively.
Eburneolin A (1) was isolated as a white amorphous solid, [ ] 18 D = +54.2 (c 0.1, MeOh). The UV spectrum showed λ max at 223 nm, which is characteristic of conjugated δ-lactone commonly present in withanolides (Scott 1964). The IR spectrum showed the absorption bands at 3513, 1713 and 1697 cm −1 for the presence of hydroxyl, α,β-unsaturated δ-lactone and cyclic six-membered ketone (Pavia et al. 1979), respectively. The hR-ESI mass spectrum showed the [M + h] + ion peak at m/z 667.3286, corresponding to the molecular formula C 34 h 51 O 13 . The 13 C NMR (broad band decoupled (BB) and distortionless enhancement by polarisation transfer (DEPT)) (Table S1) spectra showed 34 signals comprising 5 methyl, 8 methylene, 11 methine and 10 quaternary carbons. The carbon signals at δ 214.5 and 169.1 ppm suggest the presence of keto carbonyl and unsaturated ester moieties, respectively, while the olefinic carbons showed signals at δ 153.4, 136.4, 127.0 and 122.0. The oxymethine carbons appeared at δ 82.9, 76.9 and 76.5, whereas the oxygen-bearing quaternary carbons were resonated at δ 89.4, 84.4 and 79.7. The seven methylene carbons appeared in the range of δ 23.1-47.9 and five methyl carbons showed signals in the range of δ 12.3-20.6. The hexose-unit carbons resonated in the range of δ 62.7-103.1. The carbon signals at δ 169.1, 153.4, 122.0, 82.9 and 35.7 were typical of α,β-unsaturated lactone functionality which are mostly present in withanolides. The 13 C NMR data were close to the reported data for coagulin l (Atta-ur-Rahman et al. 1998) except for the downfield chemical shifts of C-15 and C-16, which appeared at δ 76.5 and 47.9 as oxymethine and methylene, respectively.
In the COSY experiment, the proton at δ 3.82 showed 1 h-1 h COSY correlation with δ 2.91 and 1.59, confirming protons were vicinal with each other. In the hMBC experiment, the proton at δ 3.82 showed 2 J correlations with δ 84.4 (C-14) and 47.9 (C-16), and 3 J correlations with δ 89.4 (C-17) and 55.0 (C-13) ( Figure S1) The 1 h NMR spectrum (Table S1)  The chemical shift values of C-1 to C-6 were very similar to the reported withanolide cilistol U (Zhu et al. 2001), which confirmed the presence of similar five-membered and three-membered rings at ring A and β-configuration at C-6. In the hMBC experiment ( Figure  S1), the anomeric proton (δ 4.94, h-1′) showed 3 J hMBC correlation with δ 63.3 (C-27) ( Figure  S1), confirming the attachment of glucose unit at C-27. On the basis of all spectral evidences, compound 2 is also withanolide and its spectral data were in complete agreement with the assigned structure of eburneolin B (2) (Figure 1).

Plant material
The aerial parts of T. eburnea were collected from Ziarat valley, Balochistan province of Pakistan, in March 2010 and identified by Prof Dr Rasool Bakhsh Tareen, Plant Taxonomist, Department of Botany, University of Quetta, where a voucher specimen has been deposited (voucher No. is TE-RBT-50).

Extraction and isolation
The freshly collected plant material of T. eburnea was shade dried (14 kg), ground and extracted with 75% MeOh (40 l × 3, 10 days each). The combined methanolic extract was evaporated under reduced pressure at room temperature to yield the residue (500 g), which was suspended in water and partitioned in n-hexane (23 g), dichloromethane (92 g), ethyl acetate (34 g) and n-butanol (48 g) soluble fractions. The n-butanol fraction (35 g) was subjected to CC on Diaion hP-20 and eluted with h 2 O, h 2 O-MeOh, MeOh in decreasing order of polarity to give five fractions. The fraction obtained with h 2 O-MeOh (1:1, 8.5 g) was rechromatographed on Sephadex lh-20 and eluted with various mixtures of h 2 O-MeOh in increasing order of polarity. The sub-fraction obtained with h 2 O-MeOh (6:4, 1.2 g) was rechromatographed over polyamide resin eluting with Ch 2 Cl 2 -MeOh to afford the semi-pure fractions, which were further purified by rechromatography over silica gel and eluted with ChCl 3 -MeOh (8.5:1.5) and ChCl 3 -MeOh (8.2:1.8) to afford compounds 2 (20 mg) and 1 (17 mg), respectively.

Acid hydrolysis of 1 and 2
Compound 1 or 2 (4 mg, each) in 10% hCl was refluxed for 45 min and the cooled reaction mixture was extracted with ethyl acetate. The aqueous phase was concentrated and the sugar was identified as D-glucose by the sign of its optical rotation ([ ] D + 52.4 (1); [ ] D + 52.7 (2)) [Mughal et al. 2012] and Co-TlC with an authentic sample of D-glucose using the solvent system BuOh-EtOAc-AcOh-h 2 O (12:2:2:2). Spot was visualised with aniline phthalate reagent. It was further confirmed by comparing the retention time of trimethylsilane ether with standard sample in GC [hara et al. 1987;Zhou et al. 2006]. The ethyl acetate residue could not be identified further due to paucity of material.