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 Ito 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 IVwas 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）