Isolation and Characterization of Batatasin III and 3,4'- Dihydroxy-5-methoxybibenzyl: A Pair of Positional Isomers from Sunipia scariosa

Purpose : To isolate and characterize chemical compounds of biological importance from the whole plant of Sunipia scariosa. Methods : The whole plant of Sunipia scariosa was extracted with methanol (MeOH) and chromatographed on silica gel and sephadex LH-20 to afford the pure isolates. High perfomance liquild chromatography (HPLC) was used for further purification of the isolated compounds. Characterization of the isolated compounds was achieved by 1H and 13C nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). Results : Batatasin III (3,3’-dihydroxy-5-methoxybibenzyl) and  3,4’-dihydroxy-5-methoxybibenzyl, a pair of positional isomers, were isolated from the whole plant of Sunipia scariosa. The yields of the two isomers were 60 and 40 %, respectively, from the mixture of two  compounds. Conclusion : Batatasin III and 3,4’-dihydroxy-5-methoxybibenzyl, a pair of positional isomers were successfully isolated from the whole plant of Sunipia scariosa for the first time. Keywords : Sunipia scariosa, Batatasin III, 3,4’-Dihydroxy-5-methoxybibenzyl, Isomers


INTRODUCTION
Bibenzyls are regarded as important secondary metabolites occurring exclusively in the plants of Orchidaceae, which is distributed in both tropical and subtropical regions of the world, and comprises of about 700 genus and 20000 species [1]. In the plants of some genera, such as Dendrobium, Bulbophyllum, Pholidota and Bletilla, bibenzyls are dominant compounds and exhibit diverse biological activities such as antitumor, anti-inflammatory and platelet antiaggregation activities [2][3][4][5][6][7][8][9][10].
Sunipia scariosa is an Orchidaceae plant mainly distributed in India, Nepal, Sikkim, Bhutan, Burma, Thailand, Laos, Vietnam and China [14]. Previously, there has been no report on the chemical study of the plant in the literature. In the course of our search for new bioactive compounds from the whole plant of S. scariosa, we study the plant and isolated batatasin III and 3,4'-dihydroxy-5-methoxybibenzyl, a pair of positional isomers from the plant.

Extraction
The dried powdered whole plant of S. scariosa (0.25 kg) was soaked with MeOH for 24 h and extracted for 6 times. The liquid extract was concentrated to dryness under reduced pressure in a rotary evaporator, yielding the dry crude extract.

Isolation of compounds
Silica gel (200-300 mesh, Qingdao Marine Chemical Co., China), and Sephadex LH-20 (25-100 μm, Pharmacia Fine Chemical Co. Ltd.) were used for column chromatography (CC), and silica gel GF254 was used for TLC (Qingdao Marine Chemical Co., China). Solvents were of industrial purity and distilled prior to use, but MeOH used for semi-preparative HPLC was of chromatography grade and purchased from Beijing Chemical Factory, China. Semipreparative HPLC was carried out using a system composed of a Waters 600 pump, with an Agilent 1100 detector and Sunfire C18 reversed phase column (10×150 mm, detected at UV of 254 nm).

Identification of compounds
Identification of compounds was carried out by MS and 1 H-, 13 C-NMR spectra. MS were determined on an API Qstar Pulsa LC/TOF mass spectrometer, and NMR spectra were measured on a Bruker DRX-500 spectrometer with CD 3 OD as solvent.

RESULTS
The MeOH extract from the whole plant of S. scariosa was subjected to column chromatography on silica gel and Sephadex LH-20 to afford the mixture of two isomers, which was further purified by HPLC to obtain batatasin III and 3,4'-dihydroxy-5-methoxybibenzyl, a pair of positional isomers. Comparison with the data shown in literature [16], led to the compound being identified as batatasin III.

DISCUSSION
The plants of Orchidaceae usually contain aromatics such as phenanthrenes and bibenzyls, as characteristic compounds. Some of the isolated compounds have been found to show significant anti-tumor, anti-inflammatory and platelet anti-aggregation activities. Batatasin III, and 3,4'-dihydroxy-5-methoxybibenzyl, two positional isomers, have been isolated from Orchidaceae species, but both are rarely obtained from a plant simultaneously.
In the present study, the MeOH extract from the whole plant of S. scariosa was subjected to column chromatography on silica gel and Sephadex LH-20 to afford the mixture of two isomers. Detailed analysis of the 1 H and 13 C NMR showed that it was a dimeric bibenzyl or two similar bibenzyls in one structure. The use of HPLC, with the aid of a solvent system comprised of methanol/water (9:11, v/v) successfully led to the differentiation of two peaks. Subsequent preparative isolation by HPLC afforded two compounds 3,4'-dihydroxy-5methoxybibenzyl and batatasin III.

Batatasin
III and 3,4'-dihydroxy-5methoxybibenzyl, a pair of positional isomers have been simultaneously isolated from the extract of S. scariosa for the first time. The compounds can be used for further research in pharmacological studies and as reference substances.