盤龍參中的兩個新二氫菲類

劉 靜，蘇雪會，李聰穎，文慶發(fā)，李延芳

四川大學化學工程學院制藥與生物工程系，成都610065

Introduction

The traditional Chinese medicine“Panlongcen”is derived from the dried root or whole plant of Spiranthes sinensis(Pers.)Ames，which is an Orchidacea plant distributed in South China and used as a folk medicine in the treatment of fever，cough，hemoptysis，vertigo，and so on［1］.Previous phytochemical investigations on S.sinensis have yielded homocyclotriucallane，dihydrophenanthrenes and sterols［2-6］.

In the course of our study on cytotoxic secondary metabolites from traditional Chinese medicine，we examined the constituents of S.sinensis and isolated two new dihydrophenanthrenes(Fig.1)from 95%aqueous ethanolic extract of the whole plants.The aim of this paper is to report the isolation and structures elucidation of new compounds.

Experimental

General Procedures

UV spectra were obtained on a UV-2100 UV spectrophotometer(RAYLEIGH Co.Beijing，China).The high resolution mass spectrometric analysis was performed by quadrupole time of flight(Q-TOF)premier spectrometer coupled with an ESI source(Micromass，Simonsway，Manchester，UK).The1H and13C NMR spectra were recorded on Bruker AM-400 spectrometers with TMS as internal standard.The chemical shifts were reported in ppm using CD3COCD3as solvent.Silica gel (200-300 mesh)for column chromatography and GF254(10-40 μ)for TLC were supplied by the Qingdao Marine Chemical Inc.China.Spots on TLC were monitored under UV lamp and by heating silica gel plates sprayed with 10%H2SO4in EtOH.Toyopearl HW-40F resin (TOSOH，Japan)and Sephadex LH-20(Merck，Germany)were used for column chromatography.

Plant Material

The whole plants of S.sinensis were purchased from a local herbal medicine store in Hebei Province，China，in August 2009.The plant was identified by comparison with the voucher specimens already deposited at the Herbarium of Institute of Botany，Chinese Academy of Sciences，Beijing(No:00804465，collected on August 27，1976).A voucher specimen(D201001)was deposited at the department of pharmaceutics＆bioengineering，Sichuan University.

Extraction and isolation

The dried whole plants of S.sinensis(5 kg)were powdered and extracted three times at room temperature with 95%EtOH(25 L，each time for 3 days).After concentration in vacuo，the concentrate was taken up in H2O and partitioned successively with PE，EtOAc and n-BuOH(each 3 L×3).The EtOAc-soluble fraction (69 g)was separated by open column chromatography (40×300 mm，300 g silica gel)using a step gradient of n-hexane-EtOAc-MeOH(20∶1∶0，2 L;10∶1∶0，2.8 L;5∶1∶0，2.8 L;3∶1∶0，2 L;2∶1∶0，2.8 L;1∶1∶0，2.8 L;1∶2∶0，2 L;1∶5∶0，2L;0∶1∶0，2.8 L;0∶0∶1，6.4 L).The flow rate was ca.50 mL/min.The effluent was combined to 10 fractions(Fr.A-J)according to TLC patterns(detection at 254 nm，365 nm and at daylight after staining with anisaldehyde-sulphuric acid reagent).The Fr.D(546 mg)were further fractionated on a Sephadex LH-20 column(18×700 mm，CH2Cl2-MeOH，1∶1)to afford 5 portions(Fr.D1-D5).The Fr.D3(413 mg)was further separated by HW-40F column(18×320 mm，CH2Cl2-MeOH，1∶1)and followed by preparative silica gel TLC(PE∶EtOAc=4∶1)afforded to compound 2(11.2 mg，Rf0.45). Then Fr.D5(36 mg)was further separated by HW-40F column(18×320 mm，CH2Cl2-MeOH，1∶1)and further PTLC(PE∶EtOAc=4∶1)to yield compound 1(14 mg，Rf0.53).

Results and Discussion

Compound 1 was obtained as a yellow amorphous powder.Its molecular formula was established as C17H14O3according to the molecular ion at m/z 267.1022［M+ H］+by HR-ESI-MS(calcd.for C17H15O3，［M+H］+267.1021).UV(MeOH)λmax:226，278，298 nm which are strikingly similar to those of 9，10-dihydrophenanthrene derivatives［7］.The1H NMR spectrum (Table 1)indicated one methoxyl group［δH3.77 (3H，s)］，two o-coupled aromatic protons［δH7.36 (1H，d，J=9.2 Hz)，8.42(1H，d，J=8.8 Hz)］，two m-coupled aromatic protons［δH6.49(1H，d，J=2.4 Hz)，6.45(1H，d，J=2.4 Hz)］.Two methylene groups attributable to the 9-and 10-protons of a dihydrophenanthrene at 2.96(2H，m)and 2.79(2H，m) are exhibited［8］.The13C NMR spectrum(Table 1) displayed the presence of 12 aromatic carbons［three bearing an oxygen atom(δC153.1，155.3，158.9)］，two methylene carbons(δC30.1，25.6)，and one methoxyl carbon(δC54.4).Characteristic1H and13C NMR resonances for a furan structure also showed in HSQC spectrum［δH6.98(1H，d，J=2.0 Hz)/δC105.2，7.79(1H，d，J=2.0 Hz)/δC144.8］.

Comparison of the NMR data of 1 and orchinol，one 9，10-dihydrophenanthrene isolated from this plant previously［3］，suggested that they had the same skeleton and similar substitution pattern，with the exception of the hydroxyl group and furan ring in 1 instead of a methoxyl and a hydroxyl group in orchinol.The HMBC correlations of H-2＇(δH7.79)and the H-3＇(δH6.98)protons with the C-8(δC125.5)and C-7(δC153.1)confirmed that furan ring should be connected to C-7(oxygenated)and C-8 of the aromatic ring.The cross peaks of H-3(δH6.49)/C-4(δC155.3)，C-2 (δC158.9);H-1(δH6.45)/C-2(δC158.9)，C-10 (δC30.1);and CH3O(δH3.77)/C-2(δC158.9)in HMBC spectrum aided in establishing the location of the hydroxyl and methoxyl group on the dihydrophenanthrene skeleton.From the foregoing data the structure of 1 was assigned as 4-hydroxy-2-methoxy-8-furano［4＇，5＇∶7，8］-9，10-dihydrophenanthrene.

Compound 2，a yellow amorphous solid，has the molecular formula C20H20O3as determined by HR-ESI-MS m/z 309.1486(calcd.for C20H21O3，［M+H］+309.1491).UV(MeOH)λmax:228，266，297 nm which are strikingly similar to those of 9，10-dihydrophenanthrene derivatives［7］.The1H and13C NMR data of 2(Table 1)indicated that it is also a 9，10-dihydrophenanthrene derivative.Comparison of the NMR data of 2 and 1 indicated that they had the same skeleton and substitution pattern.Extensive analysis of its 1D and 2D NMR data revealed that it appeared a 2，2-dimethyl pyran ring［δH1.39(6H，s)，5.78 and 6.71 (1H each，d，J=10.0 Hz)and δC27.0，74.6，119.2，130.5］.The long-range HMBC correlations of H-3＇/C-8 and H-4＇/C-7，8 helped to confirm the location of 2，2-dimethyl pyran ring.From the above analysis，compound 2 was given the structure 4-hydroxy-2-methoxy-8-{2＇，2＇-dimethylpyrano［5＇，6＇∶7，8］}-9，10-dihydrophenanthrene.

Table 1 1H and13C NMR spectroscopic data of compounds 1 and 2(in CD3COCD3，δ in ppm)

Acknowledgements

Financial support by the ChengDa special research scholarships of China for Jing Liu and NMR measurements afforded by the Centre of Testing＆Analysis，Sichuan University are gratefully acknowledged.

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