A new biflavonoid from Selaginella uncinata (Desv.) Spring

Abstract A new biflavonoid, 5,7,4',5''-tetrahydroxy-7''-metroxy-[3-O-4'''] biflavone along with four known biflavonoids and one known phenolic acid was isolated from the herb of Selaginella uncinata(Desv.) Spring. Their structures were elucidated by spectroscopic methods.
Keywords Selaginella uncinata；5,7,4',5''-tetrahydroxy-7''-metroxy-[3-O-4'''] biflavone

1. INTRODUCTION
Selaginella uncinata (Desv.) Spring as a medicinal plant is used to treat acute infectious jaundice hepatitis, cholecystitis, enteritis and so on. In previous investigations of the plant, two kinds of chromone glycoside (uncinoside A and uncinoside B) have been separated from S. uncinata , which have good anti-tumor and anti-viral activity^[1]. Our preliminary study found that ethyl acetate extract exhibited good anti-HSV-1 virus and Cox B3 virus activity ^[2]. Through activity-guided fractionation, we isolated a new biflavonoid, named 5,7,4',5''-tetrahydroxy-7''- metroxy-[3-O-4'''] biflavone(IV), together with five known compounds：7,4',7',4^'''- tetra-O-methylamento-flavone(I)^[3]，7, 7'', 4^'''-trim-O-methyl amentoflavone(II)^[4]，7, 7''-di-O-methylamentoflavone(III)^[4]，amentoflavone(V)^[5], shikimic acid(VI)^[6], and all the structures of these compounds were elucidated by spectral analysis.

2. EXPERIMENTAL
The air dried aerial parts of S. uncinata (10kg) was extracted three times with 95% ethanol at room temperature. The combined EtOH extract was evaporated in vacuo to afford a gummy residue (520g). The extracts were suspended in water and successively partitioned with petroleum ether, EtOAc and n-BuOH, respectively. The EtOAc extract (50g) was repeatedly subjected to column chromatography to yield compound I to VI.

3. RESULTS AND DISCUSSION
Compound IV was obtained as yellow powder, its molecular formula was established as C₃₁H₂₀O₁₀ (m/z 552.1022, calculated 552.1051) by HRESIMS. The IR (KBr, n, cm^-1) spectrum showed the bands at 3409 (hydroxyl group), 1656 (conjugated carbonyl group), 1603, 1500 (aromatic ring). The ¹H NMR and ¹³C NMR signals of compound IV (Table 1) were very similar to those of delicaflavone except for the existence of the one methoxyl signals [d 3.86(3H, s)] instead of hydroxyl. All the above information suggested that compound IV was a derivative of delicaflavone. The methoxyl position was determined by analyzing the difference of proton signals between compound IV and delicaflavone ^[7], that is d value of H-6'' shifted from d 6.38 to d 6.19, and d value of H-8'' shifted from d 6.75 to d 6.47, respectively. Which showed the methoxyl group was located at C-7''. This was further confirmed by HMBC spectrum, in which the signals at d_H 6.75 and 6.38 along with signals of methoxyl group at d_H 3.86 displayed correlation to d_C 165.74. Therefore the structure of compound IV was elucidated as 5,7,4',5''-tetrahydroxy-7''- metroxy-[3-O-4'''] biflavone.

Table 1 NMR spectral data for compound IV in DMSO

Figure 1 HMBC correlations of compound IV

ACKNOWLEDGMENT
This work was supported by the National Natural Science Foundation of China (No. 20772047，20472024), Natural Science Foundation of Guangdong Province, China (039213）