Fitoterapia 81 (2010) 274–275

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Fitoterapia

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A new flavonol glycoside from Millettia speciosa

Ting Yin a,b, Hong Liang a,b,⁎, Bin Wang b, Yuying Zhao a,b

a State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, PR Chinab Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, PR China

a r t i c l e i n f o

⁎ Corresponding author. Department of NaturalPharmaceutical Sciences, Peking University Health S100191, PR China. Tel.: +86 10 82801592.

E-mail address: nmechem@bjmu.edu.cn (H. Liang)

0367-326X/$ – see front matter © 2009 Elsevier B.V.doi:10.1016/j.fitote.2009.09.013

a b s t r a c t

Article history:Received 11 November 2008Accepted in revised form 22 September 2009Available online 3 October 2009

A new flavonol triglycoside, millettiaspecoside D, was isolated from the caulis of Millettiaspeciosa Champ. Its structure was elucidated on the basis of spectroscopic analysis.

© 2009 Elsevier B.V. All rights reserved.

Keywords:Millettia speciosa Champ.FlavonoidsMillettiaspecoside D

1. Introduction

Millettia speciosaChamp. belongs to the Leguminosae familyand is distributed in Hainan Province, China. M. speciosa wasrecorded to be used as ‘ji-xue-teng’ regionally in literature [1].The roots of the plant are applied for the treatment of pain ornumbness of the wrists, knees or other joints and irregularmenstruation. Among its previous chemical studies, twooleanane-type saponins, two known pterocarpans [2], and sixphenolic glycosides have been isolated [3]. As a part of ourfurther investigation on this species, a newflavonol triglycoside(1) was isolated from the plant by column chromatograph andprep-HPLC. This paper deals with the isolation and structuralelucidation of the compound.

2. Experimental

2.1. Generals

UV: Jasco MD-1501. HR-ESI-MS: Bruker Apex II FT-ICR.NMR: Bruker DRX-500. Semi-preparative HPLC: Waters 600series.

Medicines, School ofcience Center, Beijing

.

All rights reserved.

2.2. Plant

The caulis of M. speciosa were collected in HainanProvince, China, in 2003, and was taxonomically identifiedby Prof. Hubiao Chen of the Department of Natural Medicines,School of Pharmaceutical Sciences, Peking University HealthScience Center, where a voucher specimen is deposited.

2.3. Extraction and isolation

Air-dried powdered caulis of M. speciosa (9.2 kg) wasextracted consecutively with EtOH at room temperature. Thecombined extracts were concentrated in vacuo to yield a darkresidue which was suspended in water and partitionedwith EtOAc and n-BuOH. The n-BuOH extract (159.0 g) wassubjected to column chromatography on Diaion D101 andeluted with water and 10%, 30%, 50%, 70% and 95% EtOH,successively. The 30% EtOH eluting extract (28.8 g) wasrechromatographed on a silica gel column eluting with gra-dient mixtures of CHCl3–MeOH (85:15 to 7:3, v/v) giving sixfractions. Fr. 3 (2.34 g) was separated with Sephadex LH-20eluting with MeOH–H2O (1:9 to 4:6) and then purified bysemi-preparative reverse-phase HPLC with mobile phase ofMeCN-0.5% phosphate (22:78) to yield compound 1 (9.5 mg).

Compound 1 (Fig. 1), millettiaspecoside D, yellow amor-phous powder. UV λmax (MeOH) nm: 253, 353; positiveHRESIMS m/z: 779.2024 [M+Na]+, 774.2462 [M+NH4]+,

Fig. 1. The key HMBC and NOESY correlations of compound 1.

275T. Yin et al. / Fitoterapia 81 (2010) 274–275

757.2175 [M+H]+ (Calcd for C33H40O20: 756.2113); 1H and13C NMR data see Table 1.

3. Results and discussion

Compound 1 was obtained as a yellow amorphouspowder. The molecular formula of 1 was established to beC33H40O20 based on HR-ESI-MS datam/z 779.2024 [M+Na]+,774.2462 [M+NH4]+, and 757.2175 [M+H]+. The UV spec-trum showed absorption maximum at 253 and 353 nm, in-dicating that 1 was a flavonoid glycoside. Acid hydrolysis of 1resulted in a release of apiose, galactose and rhamnose, whichwas identified by HPTLC comparison of the hydrolyzate withan authentic sample [4]. The configuration of the apiose,galactose and rhamnose residues in 1 was assigned to be β-,β-, and α-, based on the coupling constant of the anomericproton δ 5.53 (1H, d, J=7.5 Hz), 5.30 (1H, s), and 4.41 (1H, s).

Table 11H and 13C NMR data for 1 (500 MHz and 125 MHz, DMSO-d6, J in Hz, δ inppm).

C δH δC C δH δC

2 – 155.4 Gal-1 5.53 d (7.5) 99.13 – 133.4 2 3.76 a 74.74 – 177.2 3 3.59 a 73.55 – 156.2 4 3.34 a 68.16 6.18 d (1.5) 98.6 5 3.53 a 73.37 – 164.2 6 3.55 a 65.0

3.22dd (8.5, 5.0)8 6.39 d (1.5) 93.4 Api-1 5.30 s 108.79 – 161.2 2 3.79 s 76.110 – 103.8 3 – 79.11′ – 122.6 4 3.82 d (9.0) 73.8

3.48 d (9.0)2′ 7.51 d (1.5) 115.3 5 3.44 d (11.0) 64.3

3.37 d (11.0)3′ – 145.9 Rha-1 4.41 s 99.94′ – 149.9 2 3.35 a 70.35′ 6.96 d (7.0) 111.3 3 3.29 dd (9.0,3.0) 70.56′ 7.83 dd (1.5, 7.0) 121.8 4 3.07 t (9) 71.8OCH3 3.85 s 55.5 5 3.58 a 68.35-OH 8.19 br. s – 6 1.04 d (6.0) 17.7

a Overlapped signals.

The 1H NMR spectrum of 1 exhibited 1,3,4-trisubstitutedbenzene signals at δ 7.83 (1H, dd, J=1.5, 7.0 Hz, H-6′), 7.51(1H, d, J=1.5 Hz, H-2′), and 6.96 (1H, d, J=7.0 Hz, H-5′);and meta-coupled aromatic proton signals at δ 6.18 (1H, d,J=1.5 Hz, H-6), and 6.39 (1H, d, J=1.5 Hz, H-8). The 1HNMR spectrum also showed a hydroxyl proton signal at δ 8.99(1H, s), one methoxyl at δ 3.85 (3H, s). 13C NMR spectraexhibited thirty-three carbon signals, including fifteen aro-matic carbon signals for the aglycone, seventeen signals forthe sugar moieties and one methoxyl at δ 55.5. The threeanomeric carbon signals at δ 99.1, 99.9 and 108.7, accompa-nied by a methyl carbon at δ 17.7, confirmed that 1 was aflavonoid triglycoside. The carbon signals of aglycone weresimilar to tamarixetin [5], except for an upfield shift of2.6 ppm (C-3) and a downfield shift of 7.9 ppm (C-2), whichsuggested that the sugar moiety was linked to C-3 of theaglycone.

One and two-dimensional NMR techniques (1H NMR, 13CNMR, 1H-1H COSY, HMQC, HMBC and NOESY) permittedassignments of all 1H and 13C signals of 1 (Table 1). TheNOESY spectrum (Fig. 1) of 1 exhibited a correlation betweenδ 3.85 (OMe) and δ 6.96 (H-5′), confirming the methoxylgroup attached at C-4′. In the HMBC (Fig. 1), the anomericproton signal of the rhamnose at δ 4.41 correlated with thecarbon signal at δ 65.0 (C-6 of the galactose), the protonsignal at δ 3.76 (H-2 of the galactose) correlated with theanomeric carbon signal of the apiose at δ 108.7, and theanomeric proton signal at δ 5.53 of the galactose correlatedwith the C-3 of the aglycone at δ 133.4. These indicated thatthe rhamnosyl attached to C-6 of the galactosyl, the apiosylattached to C-2 of the galactosyl, and the galactosyl attachedto C-3 of the aglycone. Thus, the structure of 1 was de-termined to be tamarixetin-3-O-{β-apiofuranosyl-(1→2)-[α-rhamnopyranosyl-(1→6)]}-β-galactopyranoside, namedmillettiaspecoside D.

Acknowledgements

This work was financially supported by the NationalNatural Science Foundation of China (20432030) and by theProgram for Changjiang Scholar and Innovative Team inUniversity (985-2-063-112).

References

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