A New Isoflavone from Smilax glabra

Abstract

A new isoflavone, 7,6’-dihydroxy-3’-methoxyisoflavone, has been isolated from the roots of Smilax glabra. The structure was determined by 2D-NMR techniques.

Introduction

Smilax glabra Roxb. (Liliaceae), which was called ”Tu Fu Ling”, is widely distributed in east and southwest regions of China. In traditional chinese medicine, the rhizome of S. glabra has been used clinically to prevent leptospirosis, and to treat syphilis, acute bacterial dysentery, acute and chronic nephritis, etc. [1].

Several chemical constituents have been isolated from this plant in our laboratory [2,3,4,5]. In the present work, silica gel chromatography of the EtOAc part of the EtOH extracts of the roots of S. glabra has led to the isolation of a new isoflavone. It was elucidated as 7,6’-dihydroxy-3’-methoxy-isoflavone (1) based on MS and ¹H-and ¹³C-NMR spectroscopy, including ¹H-¹H COSY, COLOC and NOEDS techniques. In this paper we report the structural elucidation of the new isoflavone 1.

Results and Discussions

Compound 1, pale yellow needles (acetone), mp 250~252. Gave positive Mg-HCl (reddish) and FeCl₃ (greenish-brown) colour tests. In the ¹H-NMR spectrum, the singlet was observed at δ 7.7(s, 1H). In the UV spectrum, the maximum absorption was at 255 nm. Thus, 1 was deduced to be an isoflavone which was also confirmed by the ¹³C-NMR(DEPT) spectra (see

Table 1. Correlated ¹³C-and ¹H-Data and COSY for compound 1.

Atomic no.	δC	CHn	δH	Mult.	J(Hz)	COLOC (C to H)	1H-1H COSY
2	153.4	CH	7.7	s		H-2	H-2’, CH3
3	125.0	C				H-2,H-2’
4	175.0	C				H-5, H-2,H-8
5	127.6	CH	7.4	d	8.7	H-5	H-6
6	115.5	CH	6.3	m		H-8,OH-7	H-5, H-8
7	162.9	C				H-5, H-8
8	102.4	CH	6.1	d	2.0	OH-7, H-8,H-6	H-5,H-6
9	157.7	C				H-5, H-2, H-8
10	117.0	C				H-6, H-8
1’	123.0	C				H-2’,H-2,H-5’
2’	120.1	CH	6.5	d	2.0	H-2’,H-4’	H-2, H-4’
3’	147.8	C				CH3, H-2’,H-4’
4’	116.8	CH	6.3	m		H-2’,H-5’	CH3 ,H-2’
5’	112.2	CH	6.3	m		H-4’	H-2’
6’	146.3	C				H-2’,H-5’
OMe	55.9	CH3	3.8	s		CH3	H-2, H-4’

).

The EI-MS of 1 exhibited [M]⁺ at m/z 284.0672 for C₁₆H₁₂O₅ (Calc. 284.0679) which is in accord with an isoflavone containing two hydroxyl groups and a methoxy group. In order to determine the substituted positions, 2D-NMR techniques were used. The COLOC spectrum showed a correlation between H-2 and C-9, H-8 and C-9, C-7. This correlation confirmed that the hydroxyl group in ring A was at the C-7 position.

Furthermore, the ion m/z 148 observed in the EIMS was derived from ring B and suggested that it carried one hydroxyl group and a methoxy group. In the ¹H-NMR, there are two groups of peaks of B ring, δ6.5(1H, d, J=2Hz) and δ6.3(2H, m). The position of methoxy group at C-3’ was deduced from the NOEDS of 1 that showed an enhancement of the peaks of methoxy and H-2 on irradiation of H-2’. In the ¹H-¹H-COSY, the correlations of H-2 and H-2’, H-2 and MeO were shown. The position of the hydroxyl group at C-6’ was confirmed by ¹H-¹H-COSY which displayed the correlation between methoxy and H-4’.

Figure 1. Atomic numbering of compound 1.

Figure 1. Atomic numbering of compound 1.

The structure of 1 was fully in accordance with its NMR spectra including COLOC, DEPT and ¹H-¹H-COSY spectra. Thus, 1 was deduced to be 7,6’-dihydroxy, 3’-methoxy isoflavone.

Experimental Section

Melting point was uncorrected. The IR spectrum was measured on a Necolet IR-2000, ¹HNMR and ¹³CNMR spectra were recorded on an ACF-300, DMSO-d6 as solvent and TMS as int. standard. MS was recorded on a Finnigan FTMS-2000.

The dried roots of Smilax glabra were extracted with 95% EtOH under reflux. After evaporating the solvent, the crude extract was extracted with Et₂O and EtOAc, respectively to give 30g of EtOAc extract. Successive column chromatography using a CHCl3-MeOH solvent system with increasing polarity, and repeated column chromatography of series (CHCl₃-MeOH, 20:1) afforded compound 1.

Compound 1 is pale yellow crystal, mp 250-252 °C. IR λ_max 3420, 1640 cm^-1. UV (MeOH) λ_max 227, 255, 290, 308 nm. EIMS m/z 284 (M⁺), 269, 253, 241, 213, 148, 137, 105. For ¹HNMR, ¹³CNMR, ¹H-¹H COSY, COLOC data, see Table 1.