Two new coumarin glucosides biosynthesized by transgenic

hairy roots of Polygonum multiflorum

Rong Min Yu a,*, Liang Bin Zhou a, Chun Yan Yan a,Guo Yan Duan a, Yu Zhao b

a College of Pharmacy, Jinan University, Guangzhou 510632, Chinab Department of Traditional Chinese Medicines and Natural Drug Research,

College of Pharmaceutical Sciences, Zhejiang University, 310031 Hangzhou, China

Received 27 August 2007

Abstract

The glycosylation of hydroxylcoumarin was investigated by using suspension cultures of hairy roots of Polygonum multiflorum.

Two new coumarin glucosides (3 and 4) were biosysthesized by regioselectively glycosylation of corresponding substrates (1 and 2)

in the system. The structures of two products were identified as 7-hydroxy-4-methylcoumarin 5-O-b-D-glucopyranoside and 7-

hydroxy-3,4-dimethylcoumarin 5-O-b-D-glucopyranoside on the ground of chemical and spectroscopic methods, respectively.

# 2007 Rong Min Yu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Keywords: Polygonum multiflorum; Hairy roots; Glycosylation; 7-Hydroxy-4-methylcoumarin 5-O-b-D-glucopyranoside; 7-Hydroxy-3,4-

dimethylcoumarin 5-O-b-D-glucopyranoside

Glycosylation, an important reaction in plant secondary metabolism, regioselectively and stereoselectively

biotransforms xenobiotics. Glycosides, products of glycosylation, differentiate much in the aspects of physico-

chemical property and bioactivity, such as enhancement of solubility and stability. Coumarins, one of the most

important secondary metabolites in the plant kingdom, have been reported to show various biological and

pharmacological activities. However, the pharmaceutical application of most coumarins is confined by their low water

solubility. Since their glycosides are more soluble in water and blood plasma, it is relatively easy to transport in

biological systems and play significant roles in pharmaceutical utilization by means of biotransformation of

coumarins. Hairy roots of Polygonum multiflorum, induced by Ri plasmid in Agrobacterium rhizogenes, are chosen to

be the biotransformation system, with characteristics of fast growth, stable genetic traits, high content of bioactive

constituents and so on [1].

Suspension cultures of P. multiflorum hairy roots were induced and cultured by our research group as described

previously [1,2]. Substrates 1 (100 mg) and 2 (100 mg), both were synthesized by our research group (purity >98%),

were administered to 20 flasks, respectively, which contain suspension cultures of P. multiflorum hairy roots. Co-

culture was proceeded at 25 8C for 4 days on a rotary shaker (110 rpm) in the dark. The dried cultures were extracted

with methanol for five times at 50 8C. The extract was combined and evaporated to dryness in vacuo. The residue was

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Chinese Chemical Letters 19 (2008) 76–78

* Corresponding author.

E-mail address: tyrm@jnu.edu.cn (R.M. Yu).

1001-8417/$ – see front matter # 2007 Rong Min Yu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

doi:10.1016/j.cclet.2007.10.047

chromatographed on repeated silica gel columns using petroleum ether–ethyl acetate and ethyl acetate–methanol as

solvent systems to afford 30 mg of 3 and 35 mg of 4, respectively.

Compound 3 was isolated as colorless needle crystals (CH3OH), mp 294–295 8C and assigned the molecular

formula C16H18O9 by HR-MS ([M]+ m/z 354.0942, calcd. 354.0945). And the base peak ([M–glc]+ at m/z 192,

aglycon) in the EI-MS indicated that product 3 should be a molecular which is a sugar attached to a phenolic group of

substrate 1. In the 1H and 13C NMR spectra of 3 (Table 1), the coupling pattern of the sugar proton signals and the

chemical shifts of the sugar carbon signals indicated that the sugar component in 3 was b-glucose. Correlations were

observed in the HMBC spectrum between the proton signal at d 4.92 (H-10) and the carbon resonance at d 157.30 (C-5),

and between the proton signal at d 10.58 (7-OH), and the carbon resonance at d 96.82 (C-8) and 99.20 (C-6). These

results indicated that the b-D-glucopyranosyl residue was attached to 5-OH of 5,7-dihydroxy-4-methylcoumarin. Each

signal in the NMR spectra of 3 was assigned by HMBC analysis and comparison with similar compounds [3,4]. Thus,

compound 3 was identified as 7-hydroxy-4-methylcoumarin 5-O-b-D-glucopyranoside (Fig. 1), which is a new

compound.

Compound 4 was isolated as white amorphous powder (CH3OH), mp 301–302 8C and assigned the molecular

formula C17H20O9 by HR-MS ([M]+ m/z 368.1103, calcd. 368.1102). The base peak ([M–glc]+ at m/z 206, aglycon) in

the EI-MS suggested that product 4 should be a molecular which is a sugar attached to a phenolic group of substrate 2.

In the 1H and 13C NMR spectra of 4, the coupling pattern of the sugar proton signals and the chemical shifts of the

sugar carbon signals demonstrated that the sugar component in 4 was b-glucose. A correlation was observed in the

HMBC spectrum between the proton signal at d 4.91 (H-10) and the carbon resonance at d 156.73 (C-5). The result

R.M. Yu et al. / Chinese Chemical Letters 19 (2008) 76–78 77

Table 11H NMR (400 MHz) and 13C NMR (100 MHz) data of compounds 3 and 4 (in DMSO-d6, d ppm)

No. 3 4

dH (J = Hz) dC dH (J = Hz) dC

2 161.38 160.99

3 5.95 (s, 1H) 110.37 2.02 (s, 3H, –CH3) 116.33

4 2.55 (s, 3H, –CH3) 154.70 2.56 (s, 3H, –CH3) 148.50

5 157.30 156.73

6 6.52 (d, 1H, 2.3) 99.20 6.53 (d, 1H, 2.3) 96.49

7 10.58 (s, 1H, –OH) 159.89 10.39 (s, 1H, –OH) 160.03

8 6.37 (d, 1H, 2.3) 96.82 6.35 (d, 1H, 2.1) 99.48

9 156.16 154.17

10 103.50 104.03

10 4.92 (d, 1H, 7.2) 101.26 4.91 (d, 1H, 7.2) 101.44

20 3.30–3.72 (m) 73.39 3.29–3.70 (m) 73.42

30 3.30–3.72 (m) 76.93 3.29–3.70 (m) 76.96

40 3.30–3.72 (m) 69.64 3.29–3.70 (m) 69.65

50 3.30–3.72 (m) 77.32 3.29–3.70 (m) 77.30

60 3.30–3.72 (m) 60.71 3.29–3.70 (m) 60.70

12.90 (3-CH3)

23.85(4-CH3) 19.53 (4-CH3)

Fig. 1. The biosynthesis pathway of compounds 3 and 4 and their key HMBC (H C). 1, 3: R = H; 2, 4: R = –CH3.

showed that the b-D-glucopyranosyl residue was attached to 5-OH of substrate 2. Each signal in the NMR spectra of 4was assigned by HMBC analyses and comparison with product 3. Thus, compound 4 was identified as 7-hydroxy-3,4-

dimethylcoumarin 5-O-b-D-glucopyranoside (Fig. 1) [3–4], which is also a new compound.

Acknowledgments

This research was financially supported by Natural Sciences Foundation of Guangdong (No. 04010461). We thank

Dr. Dongbo Yu of University of Texas Southwestern Medical Center, USA, for his check in our manuscript.

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