Elsevier

Journal of Chromatography A

Volume 1217, Issue 38, 17 September 2010, Pages 5930-5934
Journal of Chromatography A

Isolation of a polysaccharide with anticancer activity from Auricularia polytricha using high-speed countercurrent chromatography with an aqueous two-phase system

https://doi.org/10.1016/j.chroma.2010.07.036Get rights and content

Abstract

Polysaccharides from a crude extract of Auricularia polytricha were separated by high-speed countercurrent chromatography (HSCCC). The separation was performed with an aqueous two-phase system of PEG1000–K2HPO4–KH2PO4–H2O (0.5:1.25:1.25:7.0, w/w). The crude sample (2.0 g) was successfully separated into three polysaccharide components of AAPS-1 (192 mg), AAPS-2 (137 mg), and AAPS-3 (98 mg) with molecular weights of 162, 259, and 483 kDa, respectively. These compounds were tested for growth inhibition of transplanted S180 sarcoma in mice. AAPS-2 had an inhibition rate of 40.4%. The structure of AAPS-2 was elucidated from partial hydrolysis, periodate oxidation, acetylation, methylation analysis, and NMR spectroscopy (1H, 13C). These results showed AAPS-2 is a polysaccharide with a backbone of (1  3)-linked-β-d-glucopyranosyl and (1  3, 6)-linked-β-d-glucopyranosyl residues in a 2:1 ratio, and has one terminal (1→)-β-d-glucopyranosyl at the O-6 position of (1→3, 6)-linked-β-d-glucopyranosyl of the main chain.

Introduction

Due to their interesting biological activities, mushrooms have recently become an attractive source material for the development of pharmaceutical products [1]. Many polysaccharides have been isolated from mushrooms, fungi, yeast, algae, lichens, and plants in recent years, and screened for biological activity [2], [3]. Most polysaccharides derived from plants are relatively nontoxic and do not cause significant side effects. These could allow development of an effective natural anticancer with few side effects. Auricularia polytricha, which belongs to the Auriculariaceae family, is widespread in many districts of China. Modern pharmacology research indicates that it has antioxidant, antitumor, and immunomodulatory activities [4], [5], [6]. Lentinan, a polysaccharide from the Shiitake mushroom (Lentinula edodes), has been demonstrated to have anticancer activity [7], [8], [9]. This inspired us to investigate the potential anticancer activity of A. polytricha. In this study, polysaccharides were isolated from A. polytricha using high-speed countercurrent chromatography (HSCCC) and an aqueous two-phase system. These systems have been successfully applied as the solvent in HSCCC to separate bioactive macromolecules such as polysaccharides and proteins [10], [11].

Section snippets

Reagents and materials

All chemical reagents were of analytical grade, and were purchased from Huadong Chemicals (Hangzhou, China). The polyethylene glycol (PEG) 1000 used had an average molecular weight of 950–1050 Da. Fresh A. polytricha were purchased at a local store in Hangzhou, China. Papain was provided by Javely Biological Products Co., Ltd. (Nanning, China).

Preparation of HSCCC sample solution

The fruiting body of A. polytricha (1.0 kg) was extracted three times with distilled water (15 L) at 90 °C for 3 h. After centrifugation (1700 × g, 10 min, 25 

Solvent system for separation of polysaccharide components

HSCCC is a liquid–liquid partition chromatography where the separation is based on the difference in partition coefficient (K) of solutes within a two-phase solvent system. To achieve efficient separation of polysaccharide components in a high molecular weight complex, it is essential to obtain a large difference of partition coefficient of the isolated components. TLC was used to check the partitioning of the polysaccharide components between the two phases of a series of solvent systems

Conclusion

Three polysaccharides (AAPS-1, AAPS-2 and AAPS-3) were separated from a crude extract of A. polytricha by HSCCC. Separation was achieved using a two-phase aqueous solvent system composed of PEG and a saline solution. AAPS-2 showed excellent inhibition against growth of S180 sarcoma in mice. This study demonstrates that HSCCC is an effective method for preparation of bioactive polysaccharides.

Acknowledgments

This work was supported by a grant (2007AA10Z342) from the National High Technology Research and Development Program of China (863 Program) and a grant (R507719) from the Natural Science Foundation of Zhejiang Province, China.

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