Fitoterapia 77 (2006) 456–459www.elsevier.com/locate/fitote

Short report

β-Glucosidase inhibitory activities of phenylpropanoid glycosides,vanicoside A and B from Polygonum sachalinense rhizome

Y. Kawai a,⁎, H. Kumagai a, H. Kurihara a, K. Yamazaki a, R. Sawano b, N. Inoue a

a Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japanb Daiichi Pharmaceutical Co., Ltd., Chuo-ku, Tokyo 103-8234, Japan

Received 21 December 2004; accepted 11 May 2006Available online 24 May 2006

Abstract

The phenylpropanoid glycosides, vanicoside A and B, isolated from rhizomes of giant knotweed (Polygonum sachalinense)showed β-glucosidase inhibitory activity, with IC50 values of 59.8 and 48.3 μg/ml (59.9 and 50.5 μM), respectively. In contrast, p-coumaric acid and ferulic acid, corresponding to phenylpropanoyl moieties of vanicosides, exhibited very little inhibition.© 2006 Elsevier B.V. All rights reserved.

Keywords: Polygonum sachalinense; Vanicoside A and B; β-Glucosidase inhibitory activity

1. Plant

Polygonum sachalinense F. Schmidt ex Maxim. (Polygonaceae) [1] rhizomes were collected in Hakodate,Hokkaido, Japan in July 2001. The voucher specimens are deposited in the laboratory of the Faculty of FisheriesSciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan.

2. Uses in traditional medicine

The rhizomes of the P. sachalinense, as well as its closely related species P. cuspidatum Siebold and Zucc. (Japaneseknotweed), have been used in China as an herbal folk medicine, emmenagogue, hydragogue and an aperient agent andin Japan as analgesic and for haemostatic purposes [2,3].

3. Previously isolated classes of constituents

Anthraquinones [4,5].

⁎ Corresponding author. Tel.: +81 138 40 5583; fax: +81 138 40 5573.E-mail address: kawai@fish.hokudai.ac.jp (Y. Kawai).

0367-326X/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.fitote.2006.05.008

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4. Tested materials

Vanicoside A (1) and B (2) were isolated according to the procedure of Kumagai et al. [6]. The yields of 1 and 2were25 mg and 54 mg, respectively, from 1 kg of rhizomes (Fig. 1).

For comparison, p-coumaric acid (3, ICN Biochemicals, Aurora, OH, USA) and ferulic acid (4, Sigma Chemical,St. Louis, MO, USA), corresponding to phenylpropanoyl moieties of vanicosides, were also tested (Fig. 2).

5. Studied activity

β-Glucosidase inhibitory activity, p-nitrophenyl-β-D-glucopyranoside was used as a substrate. p-Nitrophenol, p-nitrophenyl-β-D-glucopyranoside, vanicoside A and B in the sample mixture could be successfully distinguished byHPLC, using a LiChrospher 100 RP-18(e) column (250×5 mm i.d., Merck, Darmstadt, Germany), 0.7 ml/ml of flowrate (eluent MeOH–water mixtures with 5% acetic acid, 280 nm).

The assay was done as follows: 10 mM p-nitrophenyl-β-D-glucopyranoside (10 μl) and various concentrationsof vanicoside A and B in MeOH (10 μl) were added in 10 mM phosphate buffer (200 μl, pH 7.0) so that thevanicoside concentrations finally reached 0.09 to 90 μg/ml. The mixture was preincubated at 37 °C for 3 min.Then, β-glucosidase (1 unit/ml) in 10 mM phosphate buffer (pH 7.0, 4 μl) (from almonds, Oriental Yeast, Tokyo,Japan) were added to the sample mixture and incubated at 37 °C for 90 min. After incubation, distilled water(150 μl) was added and heated at 100 °C for 3 min to stop the reaction. The β-glucosidase inhibitory activitywas evaluated as inhibitory ratio of the production of p-nitrophenol from substrate. For comparison, activities of

Fig. 1. Compounds 1 and 2.

Fig. 2. Compounds 3 and 4.

458 Y. Kawai et al. / Fitoterapia 77 (2006) 456–459

p-coumaric acid and ferulic acid were colorimetrically assayed using a spectrophotometer (U-2000, Hitachi,Tokyo, Japan). The 50% inhibitory concentration (IC50) was determined from a plot of percent inhibition vs thetest substance concentration using linear regression analysis. Statistical analysis was performed by Student's t-test.

6. Results

Vanicoside A and B showed β-glucosidase inhibitory activities with an IC50 of 59.8±2.49 μg/ml (59.9±2.49 μM)and 48.3±1.39 μg/ml (50.5±1.45 μM), respectively. The vanicoside B had a significantly higher activity thanvanicoside A. In contrast, p-coumaric acid and ferulic acid exhibited very little inhibition. Also they did not show asynergistic action in their mixture. The inhibitory activities (IC50 values) for the vanicosides and their relatedsubstances are shown in Table 1.

7. Conclusions

Vanicoside A and B in the traditional herbal plant, P. sachalinense, have β-glucosidase inhibitory activities. Thehigher activity of vanicoside B suggests that the acetyl moiety in sucrose might induce decreased inhibitory activity ofvanicoside. The phenylpropanoyl moiety of vanicosides showed little β-glucosidase inhibition. Consequently, themolecular structures of vanicosides should contribute to the inhibition of β-glucosidase activity. It has been reportedthat vanicoside A and B have inhibitory activity on protein kinase C with an IC50 of 44 and 32 μg/ml, respectively [7].Further researches for other enzyme inhibitory activities are required to develop a novel beneficial utilization ofvanicoside A and B.

Table 1β-Glucosidase inhibitory activity of vanicoside A and B (1 and 2) and their related phenylpropanoic acids 3 and 4

Compounds Inhibition (%)1 Tested concentration (μg/ml) IC50 [μg/ml (mM)]2

1 64.1±0.17 0.09, 0.45, 2.25, 11.25, 45, 90 59.8±2.49a (0.060±0.0025)a

2 78.9±0.46 0.09, 0.45, 2.25, 11.25, 45, 90 48.3±1.39b (0.051±0.0015)b

3 81.4±2.38 800, 1200, 1600 1133.7±87.8c (6.90±0.53)c

4 65.6±3.46 800, 1200, 1600 1306.0±73.2c (6.72±0.38)c

3 + 4 (1:1 w/w) 81.6±1.33 800, 1200, 1600 1137.9±47.6c (6.40±0.27)c

All assays were done in triplicate and the values are expressed as the mean±standard deviation.1Inhibitory activity (%) at the highest concentration in each assay was exhibited.2Different characters indicate significant difference between results in the column (P<0.05).

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References

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[2] Okuda T. Encyclopedia of Natural Medicine. Tokyo: Hirokawa Publishing; 1986. p. 155. (in Japanese).[3] Mizuno M, Yoneda K. Herbal folk medicines in Japan. Tokyo: Shinnippon-Hoki Publishing; 1995. p. 128. (in Japanese).[4] Inoue M, Nishimura H, Li HH, Mizutani J. J Chem Ecol 1992;18:1833.[5] Umek A, Bohnic P. Acta Pharm Jugosl 1983;33:51.[6] Kumagai H, Kawai Y, Sawano R, Kurihara H, Yamazaki K, Inoue N. Z Naturforsch 2005;60c:39.[7] Zimmermann ML, Sneden AT. J Nat Prod 1994;57:236.