Optimization of Conditions for Extracting Intracellular Polysaccharide with Antitumor Activity from

Optimization of Conditions for Extracting IntracellularPolysaccharide with Antitumor Activity from Abstract:The Box-Behnken design was used to determine the optimal time, temperature and solvent∶solid ratio for extracting intracellular polysaccharide from G. frondosa mycelium produced under submerged culture conditions. Application of response surface methodology predicted a maximum polysaccharide extraction yield of 7.28% using an extraction time of 2.24 h, an extraction temperature of 92.53 ℃, and a water∶mycelium ratio of 35.76∶1. Under these conditions, the actual yield was 7.15%. Both low (200 mg/kg/d) and high (500 mg/kg/d) doses of G. frondosa polysaccharide, and positive controls administered 20 mg/kg/d cyclophosphamide, significantly inhibited the growth of S180 tumors with inhibition values of 61.5%, 44.42% and 73.8%, respectively compared with negative controls administered physiological saline.Key words:Grifola frondosa;intracellular polysaccharide;Box-Behnken design;S180 tumorGrifola frondosa, also called Polyporus frondosus［1］, produces bioactive polysaccharide material that has been assigned anti-tumor［2］, anti-oxidative［3］, anti-viral and immunoen-hancing functions［4］. Although polysaccharide can be extracted from either the mushroom fruit bodies or the fungal mycelium, the latter is the preferred feedstock due to the high cost and long cultivation time associated with fruit body production. However, there are difficulties associated with extracting intracellular polysaccharide from mycelial biomass produced in submerged culture, and the nature of the extraction solvent, extraction temperature, extraction time, and the ratio of solvent to fungal biomass greatly affects the yield. We now describe the use of the Box-Behnken experimental design in response surface methodology (RSM) to optimize the conditions for extracting intracellular polysaccharide from G. frondosa mycelium produced under submerged culture conditions. We have also evaluated the inhibitory effect of the intracellular poly-saccharide on mouse S180 sarcoma.1 Materials and Methods1.1Materials1.1.1Fungal strainGrifola frondosa, strain GF270, was obtained from the Laboratory of Applied Mycology of Shandong Province.1.1.2Test miceKunming mice consisting of equal numbers of males and females (average weight, 18~22 g) were from the Experimental Animal Center of Medical College, Shandong University ［License no. SCXK (LU) 20030004］.1.1.3Tumor strainS180 tumor bearing mice were from the Institute of Pathology and Pharmacology, Shandong Academy of Medical Sciences.1.1.4Culture mediumMedium 1 for growing the fungal inoculum consisted of (w/v): 2% glucose, 1% sweet potato starch, 0.1% peptone, 0.1% yeast extract, 0.1% KH2PO 4，0.05% MnSO 4 and 2% agar，pH unadjusted.Medium 2 for submerged culture consisted of (w/v): 2% sucrose，1% corn powder，0.3% peptone，0.1% KH2PO 4 and 0.05% MgSO 4，pH unadjusted.LU Zhaoshuang, WANG Guangyuan, ZHAO Chen, et al1.2Methods1.2.1Growth of G. frondosa liquid spawn and fungal myceliumFlasks (500 mL) containing 200 mL of Medium 2 were sterilized at 121 ℃for 20 min, inoculated with five 0.5 cm2 discs of inoculum (grown on Medium 1 for 10 d at 28 ℃), and incubated at 28 ℃for 7 d to obtain liquid spawn.Fungal mycelium for polysaccharide extraction was grown on Medium 2 in a 20-L BIOTECH-X BS fermentor (Shanghai Baoxing Biotech Co., Ltd) inoculated with 5% (v/v) liquid spawn and incubated for 7 d at 28 ℃, 200 r/min and 0.5 vvm aeration.1.2.2Polysaccharide extractionMycelium was harvested by centrifugation at 6 000 g for 10 min, washed 3×with distilled water, then dried at 50 ℃prior to poly-saccharide extraction. Extraction was carried out using different extraction times, extraction temperatures and solvent∶mycelium ratios. Factors and levels of the Box-Behnken design were set with Minitab software (Table 1 in the Chinese version).1.2.3Polysaccharide determinationExtraction liquids were centrifuged (6 000 g, 10 min), supernatants (0.5 mL) were mixed with 5 vols of 95% ethanol, and left to stand at 4 ℃for 24 h to allow complete precipitation. After removal of the precipitate, total sugar in the solvent phase was determined by the anthrone-sulfuric acid colorimetric method［5］. Polysaccharide content was calculated from the differential value before and after ethanol precipitation. Polysaccharide yield (%) = poly-saccharide content/mycelia dry weight ×100.1.2.4Animal testing1.2.4.1Preparation of tumor bearing mice modelEach of the test mice were subjected to subaxillary inoculation with 0.2 mL of a tumor cell suspension in physiological saline (5×106 cells/mL) prepared after 6 days intraperitoneal passage［6］.1.2.4.2Animal treatment groupsPolysaccharide was extracted under the optimal conditions determined by the Box-Behnken experiment. Four volumes of 95% ethanol were added to the extraction liquid, the precipitated polysaccharide was collected by centrifugation (6 000 g for 10 min), dried at 50 ℃and re-dissolved in distilled water prior to intraperitoneal administration into the test mice. Mice were randomly divided into four groups (six per group) and, 24 h after inoculation with the tumor cell suspension, were treated daily for 10 d as follows: 1) Low dose polysaccharide group: 200 mg/kg/d; 2) High dose polysaccharide group: 500 mg/kg/d; 3) Cyclophosphamide group (positive control): 20 mg/kg/d; 4) Negative control group phy-siological saline: 10 mL/kg/d. Mice were sacrificed 24 h after the final treatment and the tumors were removed and weighed. Tumor inhibition rate (%) = (tumor weight of the negative control group - tumor weight of the treatmentgroup)/tumor weight of the negative control group ×100.2 Results2.1Extraction of intracellular polysaccharideFifteen combinations were included in the Box-Behnken experimental design and the data are shown in Table 2 (see the Chinese version). The quadratic model was obtained from the data using Minitab software. The polysaccharide extraction yield (Y%) in response to extraction time (A), temperature (B) and ratio of water tomycelium (C) is derived from the following regression equation：Y=7.0367＋0.2663A＋0.4387B＋0.5525C－0.3346A2－0.9596B2－0.4921C2－0.1375A*B－0.1300 A*C＋0.1450 B*CTable 3 shows the analysis of variance applicable to the polysaccharide extraction yield (see the Chinese version). Interaction within factors had no significant effect on the polysaccharide extraction yield, but the linear and quadratic terms of each test parameter had a significant effect on extraction yield (P＜0.01). The equation had a coefficient value (R-Sq) of 97.14%, indicating the high reliability of this model.Response surface plots obtained from the regression equation are shown in Figs. 1~3 (see the Chinese version). Extraction yields increased with higher extraction times and water∶mycelium ratios. Increasing the extraction temperature was also beneficial although yields declined slightly at temperatures above 93 ℃. The response value was optimized using software according to the regression equation, and the predicted maximum polysaccharide extraction yield was 7.28% with an extraction time of 2.24 h, extraction temperature of 92.53 ℃, and a water∶mycelium ratio of 35.76∶1. In order to confirm the model, three replicate extractions using these optimal conditions were performed and the response values were 7.18%, 7.16% and 7.11%, respectively. The 7.15% average was very close to the predicted value.2.2Inhibitory effect of intracellular polysaccharide on tumor growthBoth low and high doses of G. frondosa polysaccharide significantly inhibited the growth of S180 tumors with inhibition values of 61.5% (P＜0.01) and 44.42% (P＜0.01), respectively. Tumor growth was inhibited 73.75%（P＜0.01）in the cyclophosphamide positive control (see Table 4 in the Chinese version).3 DiscussionNumerous procedures have been used to extract intracellular polysaccharide from Grifola frondosa including acid-, alkali-, salt-, hot water-, enzyme-based, and microwave-treatment methods Relatively higher yields have been obtained using alkali extraction but it is difficult to separate the liquid and solid phases due to the high viscosity of the extracted product. Yields using hot water extraction are slightly lower but the extraction process is relatively straightforward［7］, and we have therefore used hot water as the extraction solvent in the present study. RSM uses a statistical approach to explore the relationships between several variables and the expected value of a response variable. Usually, the main objective is to find thecombination that gives a global maximum (or minimum). In this study, we used RSM to obtain the optimal extraction time, temperature and ratio of water to solid for obtaining intracellular polysaccharide from G. frondosa mycelia, and have confirmed that the predicted values corresponded to the response value.G. frondosa polysaccharide exhibited a significant inhibitory effect on mouse S180 tumors. DU et al［8］reported that G. frondosa polysaccharide inhibited the growth of S180 tumors by 47.92%, which is very close to the value (44.42%) obtained with high doses (500 mg/kg/d) of polysaccharide in this study.It is generally recognized that the anti-tumor function of G. frondosa polysaccharide is related to its immuno-enhancing properties. G. frondosa polysaccharide significantly increased humoral and cellular immunity in Kunming mice［9］, activated T-lymphocytes, macrophages and natural killer cells, and increased the secretion of cell factors such as interferon-γ（IFN-γ）, tumor necrosis factor-α（TNF-α）and interleukin cell factor-2（IL-2）［10］. Furthermore, our study showed that the polysaccharide had very low side effects although slight increases in weight and dietary intake were observed compared with the positive and negative controls. Clinical tests have revealed that G. frondosa polysaccharide was a safe and effective adjunct therapy for the treatment of advanced m alignant tumors［11］. However, further research is required to provide a better understanding of the mechanism(s) involved in tumor inhibition.References:[1]XU JT. Chinese medical fungi［M］. Beijing: Peking Medical College and Peking Union Medical College Publishing House, 1997:707-717. (in Chinese)［2］LEE BC, BAE JT, PYO HB, et al. Biological activities of the polysaccharides produced from submerged culture of edible Basidiomycete Grifola frondosa［J］. Enzyme Microb Technol, 2003, 32(5): 574-581.［3］MAU JL, LIN HC, SONG SF. Antioxidant properties of several specialty mushrooms［J］. Food Res Intl, 2002, 35(6):519-526.［4］XING ZT, ZHOU CY, PAN YJ, et al. Recent chemical and pharmacological studies on poly-saccharide constituent of Grifola frondosa［J］. Acta Edulis Fungi, 1999, 6(3):54-58.［5］SONG AR, WANG GY, ZHAO C, et al. Utilization of carbon and nitrogen sources in Ganoderma applanatum［J］. Edible Fungi, 2006, 4:16-17. (in Chinese).［6］DING FQ. Course of pharmacological experiment［M］. Beijing: People’s Medical Publishing House, 1996:8.［7］SHI ML, SONG AR, LU HT, et al. Comparison of polysaccharide extracted from the fermentation fluid of Grifola frondosa by different treatments［J］. Edible Fungi of China, 2002, 22(3):45-46.(in Chinese)［8］DU JW, YANG MF, SU YY, et al. Experimental study of Grifola frondosa polysaccharide on mice with S180 sarcoma［J］. Shandong Journal of Traditional Chinese Medicine, 2006, 25(3):391-393.(in Chinese)［9］WANG QJ, YANG MF, SU YY, et al. Inhibition effect of polysaccharide from submerged culture products of Grifola frondosa on S180 tumor bearing mice［J］. Edible Fungi, 2003, 1:24-25.(in Chinese)［10］NORIKO K, NACHI H, HIROAKI N. A polysaccharide, extract from Grifola frondosa, induces Th-1 dominant responses in carcinoma-bearing BALB/c mice［J］. Jpn J Pharmacol, 2002, 90(4): 357-360.［11］LIU A, ZANG LH, SUN QJ. Clinical observation of effect on Grifola frondosa amylose against tumor［J］. Journal of Shandong Institute of Light Industry, 2008, 22(2):43-45. (in Chinese with English abstract) 59。