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Asian Journal of Chemistry Asian Journal of Chemistry Asian Journal of Chemistry; Vol. 26, No. 12 (2014), 3574-3580 ASIAN JOURNAL OF CHEMISTRY http://dx.doi.org/10.14233/ajchem.2014.16500 Optimization of Extraction Process and Antibacterial Activity of Bletilla striata Polysaccharides 1 2 1 1,* 1,* QIAN LI , KUN LI , SHAN-SHAN HUANG , HOU-LI ZHANG and YUN-PENG DIAO 1College of Pharmacy, Dalian Medical University, Dalian, Liaoning, P.R, China 2College of Chemistry and Chemical Engineering, Liaoning Normal University, Dalian 116029, P.R. China *Corresponding authors: E-mail: [email protected]; [email protected] Received: 9 October 2013; Accepted: 21 January 2014; Published online: 5 June 2014; AJC-15297 The aim of the study was to optimize the optimal process conditions for extraction of Bletilla striata polysaccharides and its antibacterial activity in vitro. Conventional water extraction and ethanol precipition method with polysaccharide content as an index through single 4 factor investigation and L9(3 ) orthogonal table was used to optimize extraction conditions, respectively. The test tube double dilution method was chosen for preliminarily screening of the antibacterial activity and the determination of antibacterial mechanism was according to bacterial state changes in electron microscopy observation. The optimal extraction condition was obtained using cold soaking for 6 h prior to extraction, two times 3 h extraction technology, at 90 °C, with a extract-water ratio of 1:15, for purification using concentration of extract to 1:5 (w/v), addition of a 3-fold volume of 95 % ethanol, ethanol content of up to 80 % and polysaccharide content of 53.72 %, in addition, the MIC to S. aureus was 6.25 mg/mL and MBC could reach 12.50 mg/mL, the mechanism may be related with the permeability of cell membrane. The extraction and purification process for Bletilla striata polysaccharides established in this experiment is simpler, more scientific and suitable and Bletilla striata polysaccharides has a obvious inhibition against S. aureus. Keywords: Extraction, Bletilla striata, Polysaccharides, Antibacterial activity. INTRODUCTION polysaccharides from Bletilla striata and antibacterial activity in vitro. Bai Ji is a perennial herbaceous plant Bletilla striata (Thunb.) Reichb. f. of family Orchidaceae, which is also known EXPERIMENTAL as Xiao Bai Ji, Lian Ji Cao, etc1. Its medicinal part is dried ZHJH-CH09B clean bench (Shanghai Zhicheng Analytical tuber and its medicinal composition is Bletilla striata gum, Instrument Manufacturing Co., Ltd.); RT-08 herb pulverizer which contains polysaccharides, starches, glucose, volatile oils, (Beijing Huanya Tianyuan Machinery Technology Co., Ltd.); etc. It has less adverse reactions and has lung tonifying, hemo- JJ500 electronic high precision balance (G&G GmbH, in static, astringent and antibacterial actions2, which has all along USA), U-3010 UV-visible (Hitachi, in Japan); HH-8 digital been used as a traditional Chinese medicine clinically, mainly thermostat water bath (Changzhou Guohua Electric Appliance for the treatment of lung injury, hemoptysis, surgical wounds, Co., Ltd.); Rotary evaporator RE-52AA (Shanghai Yarong Bio- ulcers, pyogenic infections, etc.3,4. Meanwhile, Bletilla striata chemical Instrument Factory); TFFD-27 freeze dryer (Beijing gum can also be used as thickeners, emulsifiers and mois- Zhongyi Instruments Co., Ltd.); CT15RE high speed refrigerated touring agents in the petroleum, food, pharmaceutical and centrifuge (Hitachi, in Japan); ZD-85A dual-function air bath cosmetic industries, which has a bright prospect5. thermostat shaker (Jintan Jieruier Electric Appliance Co., Ltd.); Bletilla striata polysaccharides are a kind of sticky poly- Hitachi H-7500 transmission electron microscope (Hitachi, saccharides in the plant obtained by water extraction and in Japan); JSM-6360LV scanning electron microscope (JEOL, ethanol precipitation, their chemical components are glucose in Japan). and mannose, with a ratio of 1:4, it contents are extremely Bletilla striata decoction pieces (purchased from Dalian rich6, which account for about 55-60 % of the plant. Therefore, Baidu Medicine Co., Ltd., which was identified by Professor as one important direction for development of novel drugs, Diao Yun-peng from the Department of Pharmacognosy, College biologically active Bletilla striata polysaccharides have gained of Pharmacy, Dalian Medical University as the tubers of Bletilla increasing attention of scholars at home and abroad, in order striata (Thunb.) Reichb. f. of the family Orchidaceae); Anhy- to find a more reasonable and feasible production process. drous D-glucose reference substance (AR) (Panjin Tianyuan The present experiment studied the extraction process of total Pharmaceutical Co., Ltd.); 95% ethanol, concentrated sulfuric Vol. 26, No. 12 (2014) Optimization of Extraction Process and Antibacterial Activity of Bletilla striata Polysaccharides 3575 acid, phenol, anhydrous ethanol, acetone, etc. (the above reagents which was dried to constant weight, was accurately weighed were all of analytical grade) (Tianjin Kermel Chemical Reagent and extracted twice under reflux at 90 °C by addition of Co., Ltd.); Nutrient broth (Beijing Land Bridge Technology distilled water with a ratio of 1:15, each time lasted 3 h, under Co., Ltd.); Agar (Sigma, USA), Cefoperazone sodium for the same conditions as described above. Bletilla striata was injection (General Pharm. Factory, Harbin Pharmaceutical soaked at room temperature for 1 h, 6 h and 12 h, respectively Group Co., Ltd.). and polysaccharide content was separately determined. S. aureus (ATCC25923) was provided by the Department Investigation of extraction temperature during the of Pharmacology, Liaoning University of Traditional Chinese extraction of Bletilla striata polysaccharides: 5 g of Bletilla Medicine. striata powder, which was dried to constant weight, was Determination of Bletilla striata polysaccharides extraction accurately weighed and refluxed twice by addition of distilled technology water with a ratio of 1:15 at different temperatures of 70, 80 and 90 °C, respectively, each time lasted 3h, the two filtrates Plotting of standard curve7: 50 mg of glucose, which were combined and polysaccharide content was separately was dried to constant weight, was accurately weighed, placed determined. into a 50 mL volumetric flask, dissolved in water and diluted Optimization of orthogonal test during the extraction to the mark to obtain 1 mg/mL glucose stock solution. 1, 2, 3, of Bletilla striata polysaccharides: The present experiment 4, 5 and 6 mL of the solution were accurately pipetted, used the conventional water extraction and ethanol preci- respectively, placed into 50 mL amber volumetric flasks, pitation method to extract Bletilla striata polysaccharides. The diluted to the mark with water and shaken well to obtain a results of preliminary experiments, factors influencing the series of glucose standard solutions with the concentrations extraction effect of Bletilla striata polysaccharides were deter- of 20, 40, 60, 80, 100 and 120 µg/mL, respectively. 1 mL of mined to be: solvent volume (a), extraction time (b) and extrac- each glucose standard solution was accurately pipetted and 1 tion times (c), each factor had three levels and test was arranged mL of distilled water was served as the blank control, the above according to L (34) orthogonal table. A total of 9 portions of were separately placed into 10 mL stoppered test tubes, added 9 5 g of Bletilla striata powder were used in the test, which were with 1 mL of 5 % phenol reagent and shaken vigorously, then extracted under reflux at 90 °C, comprehensive scoring was quickly added with 6 mL of concentrated sulfuric acid, mixed performed with extraction yield and polysaccharide content well and allowed to stand at room temperature for 0.5 h, as the indices and optimal process was screened out. followed by measurement of absorbance at 490 nm. Standard curve was plotted with the absorbance value as the ordinate Determination of Bletilla striata polysaccharides purity and the concentration (µg/mL) of standard glucose solution Influence of concentration multiple on extraction yield as the abscissa8. when added with the same volume of ethanol during the Preparation process of Bletilla striata polysaccharides: purification: To remove impurities and maximize the retention The process for extraction of Bletilla striata polysaccharides of active components, purification process is necessary after was as follows: crude material was rinsed, dried, pulverized the water extraction. At first, the concentration multiple of and then dried to constant weight. 5 g of Bletilla striata powder water extract was investigated. Bletilla striata water extract was accurately weighed, added with 75 mL of distilled water was prepared according to the optimal extraction process and extracted under reflux at 90 °C for 3 h twice, then filtered, optimized as above. The same amounts of water extracts were the two filtrates were combined and then concentrated under separately concentrated under reduced pressure, concentration reduced pressure to a volume of 30 mL. The concentrated multiples were 2, 3, 4, 5 and 6 folds, respectively. The concen- solution was then precipitated with 95 % ethanol, ethanol trated water extracts were then added with equivalent amount volume was three times that of the concentrated solution so of ethanol and mixed, followed by ethanol precipitation. The that the ethanol content could reach 80 % and allowed to stand ethanol precipitates
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