New cytotoxic steroid from Stachyurus himalaicus var. himalaicus

A phytochemical study of the ethanolic extract of Stachyurus imalaicus var. himalaicus was undertaken and as a result a new polyoxygenated steroid, named stachsterol ((20S)-20, 25-dihydroxy-4-cholesten-3-one, 1) and three known ecdysteroids, 20-hydroxyecdysone (2), 20-hydroxyecdysone-20, 22-monoacetonide (3) and polypodine B-20,22-monoacetonide (4), were isolated. Their structures were elucidated by spectroscopic methods, including UV, NMR, MS and HR-MS. The purified product 1 was found to have in vitro cytotoxic activity against human Hela cell lines with an IC50 value of 2.5 microg/mL. This is the first time that phytoecdysteroids have been found in the genus Stachyurus.


Introduction
Since ancient times, medicinal plants have been used in Asian cultures as a source of remedies and for healthcare preparations. In fact, the word medicine was used for herbal remedies for centuries.
Only after the 1950s has the use of synthetic drugs became so dominant as to totally eclipse herbal cures. Recently, however, renewed interest in medicinal plants for restoring and maintaining health has emerged. Members of the genus Stachyurus have been used in Chinese folk medicines for a long time and earlier work reported the isolation of tannins from the genus [1,2]. Stachyurus himalaicus var. himalaicus, known as 'Tong-Cao" in Chinese folklore, is used for the treatment of dropsy and gonorrhea [3]. However, to our knowledge no work has been reported on the biologically active constituents of this species. A preliminary pharmacological study on this plant showed that the 95% EtOH extract had cytotoxic activity against human Hela cell lines at a concentration of 10 µg/mL. A bio-assay guided study revealed that an EtOAc fraction of the extract also displayed strong cytotoxic activity. In the previous phytochemical study, we isolated two new polyoxygenated triterpenoids, one of which was found to have mild cytotoxic activity against human Hela cell lines in vitro [4].
In this study, we report the bioactivity-guided isolation and structural determination of four steroids (1-4; Figure 1) from Stachyurus himalaicus var. himalaicus. One of these steroids, 1, is a new 3-oxo-4-ene steroid and the structure was elucidated in detail. The purified product 1 was found to have cytotoxic activity against human Hela cell lines in vitro with an IC 50 value, 2.5 µg/mL. Phytoecdysteroids 2, 3, and 4 were isolated for the first time from the genus Stachyurus.

Results and Discussion
In order to obtain more information about the bioactive compositions of Stachyurus himalaicus var. himalaicus, the EtOAc-soluble fraction of a crude ethanolic extract was analyzed by HPLC-UV. The early-running compounds possessed the UV spectra of flavonoids, whilst the later-running compounds had simple UV spectra and could not be correlated with a specific chemical class. The present study focused on the purification and identification of the latter minor components. The twigs and leaves of Stachyurus himalaicus var. himalaicus, collected from Wenshan County, Yunnan province, were extracted with 95% ethanol and then the concentrated extract suspended in water was partitioned successively with petroleum ether, EtOAc and n-BuOH. The EtOAc extract was subjected to chromatography on silica gel, Sephadex LH-20, and RP-18 to yield compounds 1, 2, 3 and 4 ( Figure 1).
Compound 1 was isolated as white powder. The color test indicated 1 to be a steroid. The HR-ESI/MS determined the molecular formula to be C 27 H 44 O 3 (m/z 417.3377 [M+H] + ), indicating six degrees of instauration, ascribed to α, β-unsaturated ketone and four steroid rings. The UV absorption at 243 nm suggested the presence of an α, β-unsaturated ketone, which was indicative of a conjugated 4-ene-3-one [5]. The IR spectrum (KBr) displayed absorption bands for hydroxyl (3443 cm -1 ) and α,βunsaturated ketone (1667, 1652 cm -1 ) groups. The 1 H-NMR and 13 C-NMR (DEPT) of 1 revealed signals due to five methyls, eleven methylenes, five methines and six quaternary carbons. Its 1 H-NMR indicated the presence of five methyl groups at δ H 0.86 (3H, s, CH 3 -18), 1.16 (3H, s, CH 3 -19), 1.20 (6H, s, CH 3 -26, 27), 1.27 (3H, s, CH 3 -21). Twenty-seven carbons including five methyls suggested that 1 was an analogue of cholesterol. A series of proton signals at δ 0.9-2.5 were attributed to resonances of overlapping of methylenes and methines of framwork of steroid. All of the protonated carbons were assigned by HMQC experimental. The proton NMR spectrum of 1 displayed a vinyl signal at δ 5.70 (1H, s), which is fully consistent with those reported for 3-keto-4-ene steroids [5,6]. In the HMBC spectrum, the correlations between δ H 5.70 (1H, s, H-4) and δ C 39.0 (C-10), 172.0 (C-5), 200.1(C-3), 33.3 (C-2) supported the presence of 3-keto-4-ene unit. The 1 H-NMR signal at δ H 1.20 (6H, s) indicated the side chain C-26/C-27 was intact. Its 13 C NMR spectrum showed two oxygenbearing quaternary carbon signals at δ C 75.5 and 71.4. The presence in the 1 H-NMR spectrum of a methyl singlet at δ 1.27 suggested the location of one hydroxyl function at C-20, and this was supported by a quaternary carbon signal at δ 75.5 ppm in the 13 C-NMR spectrum. Furthermore, in the HMBC experiments, the methyl proton signals at δ H 1.27 (CH 3 -21) correlated with the signals at δ C 58.1 (C-17), 75.5 (C-20) and 44.7 (C-22), and that at δ H 1.20 (6H, s, C-26, C-27-CH 3 ) correlated with the signals at δ C 44.7 (C-24) and 71.4 (C-25). Thus, two hydroxyl groups were assigned to C-20 and C-25, respectively. The ESI + MS of 1 gave a significant base peak at m/z 315 ([M + -C 6 H 14 O], derived from the cleavage at the C-20-C-22 bond from [M+H] + (as shown in Figure 2), which also confirmed that the hydroxyl groups were located on the side chain. The value of the chemical shift of CH 3 -21 at δ 1.27 was indicative for the common 20S-configuration hydroxyl group ((20S)-and (20R)-20hydroxycholestane: δ 1.28 and 1.13, respectively) [7]. The observation of NOESY correlations between H 3 -21 and H-12β and between H-17 and H-14 was in agreement with the α-orientation of H-17 and the R stereochemistry of C-20, which features normally exhibited by a steroid skeleton [8]. Following detailed consideration of molecular models, a (20S)-20,25-dihydroxy-4-cholesten-3-one structure was established for the new steroid 1, which was named stachsterol (Figure 1). The new steroid 1, a polyoxygenated 3-oxo-4-ene sterol, was found to have cytotoxic activity against human Hela cell lines in vitro with an IC 50 value 2.5 µg/mL. Literature reportes indicate that 3-oxo-4-ene sterols (OH-substituted at C 1~15 hydrocarbon chain) have been utilized in the treatment of conditions associated with rapidly growing cells and cancers such as melanoma [9] and except for the estrogens, many steroid hormones have the 3-oxo-4-ene (∆ 4 )-structure [10]. In addition to 1, the three known ecdysteroids 20-hydroxyecdysone (2) [11], 20-hydroxyecdysone-20,22-monoacetonide (3) [12] and polypodine B-20,22-monoacetonide (4) [13], were isolated from Stachyurus himalaicus var. himalaicus. Their structures were identified by comparison of their spectroscopic data (FABMS, 1 H-and 13 C-NMR) with those reported in the literature. Ecdysteroids were discovered decades ago as hormones of arthropods; they have effects on moulting and development. There are several plants containing ecdysteroids, the so-called phytoecdysteroids. These substances have proved to be extremely interesting because of their capability for enhancing the biosynthesis of protein in the organism of experimental animals in a manner similar to the steranabols, but without at the same time exhibiting androgenic, thymolytic and other side effects. Ecdysteroids are even used for doping purposes. Since there is a great demand for compounds with this type of action at present, much research has and is being carried out to identify ecdysteroid-containing specimens [14]. This is the first time that ecdysteroids were found from the genus Stachyurus and we hope to find more ecdysteroids from the members of Stachyurus.

General
Optical rotations [α] D were measured on a Jasco-20 MC Polarimeter. IR spectra were taken on a Nicolet AVATAR-360 spectrophotometer, ν max in cm -1 . UV was determined on a Shimadzu UV-2401PC spectrophotometer, λ max in nm. Commercial Silica gel plates (QingDao Marine Chemical Group Co.) were used for TLC. The chromatograms were sprayed with 10% H 2 SO 4 and heated at 80°C to detect the spots. 1 H-, 13 C-and 2D NMR were recorded on a Bruker AV 300 (300 MHz for 1 H and 75 MHz for 13 C) and AV-500 spectrometer (500 MHz for 1 H and 125 MHz for 13 C), respectively; chemical shifts are given in ppm as δ values relative to TMS as internal standard. ESI /MS were obtained on a Finnigan-MAT (San José, CA, USA) TSQ-700 triple-stage quadrupole instrument.

Plant Material
Twigs and leaves of Stachyurus himalaicus var. himalaicus were collected in May 2002 from Wenshan County of Yunnan Province, P. R. China and identified by Prof. Hu Zhi-Hao, Yunnan University. A voucher specimen (YN 0205) has been deposited at Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, Yunnan University.