Physicochemical Characterization and Antitumor Activity of Water-Soluble Polysaccharides from Bupleurum chinense

Abstract

Bupleurum chinense belongs to the genus Bupleurum spp. and is one of the most important plant medicines in China used for over a thousand years. B. chinense is widely distributed in the northern region of China and is customarily called “Bei chaihu.” The clinical practice from ancient plant pharmacy experts showed that B. chinense possesses many pharmacological functions, such as balancing different organs and energies within the body, strengthening the action of the digestive tract, improving liver and circulatory system function, and relieving liver tension. In addition, contemporary pharmacological research indicated that B. chinense can effectively inhibit hepatoma and protect and promote hepatocyte regeneration. However, the active ingredients in B. chinense and possible mechanisms of its antihepatoma action are still unclear. The crude polysaccharide from the roots of Bupleurum chinense was extracted by hot water and ethanol precipitation with a yield of 12.4%. After deproteination by a combination of proteinase and the Sevag method, the supernatant was collected, dialyzed, and lyophilized to obtain water-soluble B. chinense polysaccharides (BCPs) 1 . BCPs appeared as a pale yellow powder and had a negative response to the Bradford assay 2 . In addition, no absorption at either 280 or 260 nm was detected by UV spectrophotometer, which indicated the absence of protein and nucleic acid. Results from phenol–sulfuric acid assay showed that BCPs contained 93.7% carbohydrate 3 and 5.4% uronic acid 4 . According to GC analysis, BCPs are composed of mannose, galactose, glucose, and galacturonic acid with the molar ratio 25:7.4:10:6.7 5 . We tested in vitro growth inhibition and cytotoxicity of BCPs against three tumor cell lines and two nontumorous cell lines. BCPs exhibited a significant cytotoxicity against H22 with the IC50 value of 103.3 g/mL. The IC50 values against B16 and S-180 were 289.3 and 371.2 g/mL, which showed that the inhibition effects of BCPs against B16 and S-180 were significantly lower than that against H22. The above results demonstrated that BCPs could specifically inhibit proliferation of tumor cells and had a discriminate antiproliferative activity against different types of tumor cells. In addition, BCPs exhibited extremely lower cytotoxicity to mouse embryonic fibroblast cell (NIH 3T3) and mouse macrophage cell (RAW 264.7) with high IC50 values over 1700 g/mL, implying that BCPs had no direct cytotoxicity to noncancerous cells and was safer than other chemotherapeutic drugs, having no negative side effects in inhibiting proliferation of nontumorous cells. We further evaluated the in vivo antitumor activity of BCPs on H22 solid tumor-bearing mice. Due to the fast growth of H22 tumor cells, the transplanted tumor model mice gradually exhibited a series of weakness symptoms such as loss of appetite and reduced body weight with dull hairs. After oral treatment with BCPs (50, 100, and 200 mg/kg), the growth of H22 tumors in the model mice was significantly suppressed compared with the negative control group (p < 0.05), and the tumor inhibition ratios were 22.7, 35.1, and 51.1% at concentrations of 50, 100, and 200 mg/kg body weight, respectively. Furthermore, the body weights of the BCP-treated group were increased significantly when compared with the negative control group and the CTX-treated group during the 15-day experimental period (data not shown).