Trichosides A and B, new withanolide glucosides from Tricholepis eburnea

Trichosides 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.


Introduction
The genus Tricholepis (Asteraceae) comprises 18 annual or perennial herbs distributed in East and Central Asia (Bermer 1994), and are represented by 12 species in Pakistan. The various Tricholepis species have been used as traditional medicine for the treatment of several ailments including nerve tonic (Chittam & Deore 2012), seminal debility, urinary infections and cough (Khare 2007), antipyretic and skin diseases (Chopra et al. 1956;Nadkarni 1976), regularising the malfunction of pancreas, malaria, fever, skin grains, stomach pain, blood purification and dysentery (Khan et al. 2011). Sifting of the literature shows that diterpenes (Singhal et al. 1982), flavonoids (Yadava & Belwanshi 2009) and steroid (Chawla et al. 1976(Chawla et al. , 1978 have been reported from this genus. The chemotaxonomic and ethno-pharmacological importance of the genus Tricholepis prompted us to carry out phytochemical investigations of one of its species Tricholepis eburnea. No phytochemical work has so far been carried out on this species. We herein report the isolation and characterisation of two new withanolide glucosides named trichosides A (1) and B (2) from T. eburnea. The withanolides are ergostanolides which are chemically characterised by gor d-lactone-containing side chain at C-17. They have previously been reported from Solanaceae (Glotter et al. 1978), Taccaceae (Chen et al. 1987(Chen et al. , 1988 and Leguminosae (Srivastava et al. 1992) families and marine organisms (Ksebati & Schmitz 1988).

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 (CC) 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. Trichoside A (1) was isolated as a white amorphous solid, ½a 18 D ¼ þ54.2 (c ¼ 0.1, MeOH). The UV spectrum showed l max at 223 nm, which is characteristic of conjugated d-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, a,b-unsaturated d-lactone and cyclic sixmembered ketone (Pavia et al. 1979), respectively. The HR-ESI mass spectrum showed the [M þ H] þ ion peak at m/z 649.3216, corresponding to the molecular formula C 34 H 49 O 12 . The 13 C NMR [broad band decoupled (BB) and distortionless enhancement by polarisation transfer (DEPT)] (Table S1)  The 1 H NMR spectrum (Table S1) showed an olefinic proton doublet at d 5.68 (d, J ¼ 5.2 Hz, H-6). The oxymethine protons showed signals at d 4.84 (br s, H-22), 4.00 (m, H-3) and 3.82 (dd, J ¼ 11.7, 6.7 Hz, H-16). The methyl protons showed singlets at d 1.95, 1.84, 1.36, 1.33 and 1.31; however, the methyl signals at d 1.95 and 1.84 reveal their attachments to the unsaturated system. The anomeric proton showed signal at d 4.36 (d, J ¼ 7.8 Hz) and its large coupling constant (7.8 Hz) confirms b-configuration. The oxymethine and oxymethylene protons of hexose unit were resonated in the range of d 3.13 -3.83. The hexose unit was confirmed as b-D-glucose by its sign of optical rotation ([a ] D þ 52.4) (Mughal et al. 2012) through acid hydrolysis. The 1 H NMR spectral data were similar to the reported data for coagulin L (Atta-ur-Rehman et al. 1998) except for the downfield signal of an oxymethine proton at d 3.82 and methylene protons at d 2.91 and 1.59.
In the COSY experiment, the proton at d 3.82 showed 1 H-1 H COSY correlation with d 2.91 and 1.59, confirming protons were vicinal with each other. In the HMBC experiment, the proton at d 3.82 showed 3 J correlations with d 84.4 (C-14), 79.7 (C-20) and 55.0 (C-13), and 2 J correlations with d 89.4 (C-17) and 47.9 (C-15) ( Figure 2). Both COSY and HMBC correlations confirm the proton position of d 3.82 at H-16. The chemical shift values of C-14 (d 84.4) and C-16 (d 76.5) were close to the reported withanolides having hydroxyl groups at C-14 and C-16; however, in those reported compounds the shift value of C-15 was around d 37-38 (Damu et al. 2007). But in 1, the downfield chemical shift of C-15 was at d 47.9, which was only characterised by the etherification of hydroxyl groups at C-14 and C-16 which was further confirmed by the molecular ion peak. The stereochemistry of C-16 was confirmed as aconfiguration through NOESY correlation, as H-22 (d 4.84) did not show NOESY correlation with H-16 (d 3.82). The 3 J HMBC correlation of anomeric proton (d 4.36, H-1 0 ) with d 76.9 (C-3) confirms its attachment at C-3. Compound 1 is withanolide and the whole spectral data were in complete agreement to the assigned structure for trichoside A (1) (Figure 1).
was only characterised when it is directly attached with the other upfield quaternary carbon at 32.4 (C-8). In the HMBC experiment, the anomeric proton (d 4.94, H-1 0 ) showed 3 J HMBC correlation with d 63.3 (C-27) (Figure 2), 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 to the assigned structure of trichoside 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. TE-RBT-50).

Extraction and isolation
The freshly collected plants 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 into n-hexane (23 g), dichloromethane (92 g), ethyl acetate (34 g) and nbutanol (48 g) soluble fractions. The n-butanol fraction (35 g 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.

Supplementary material
Supplementary material relating to this paper is available online.

Disclosure statement
No potential conflict of interest was reported by the authors.