Study Of Quality Standard And Fingerprint Of Grifola Frondosa Beta-glucan For Injection

Grifola frondosa(GF) is one kind of medicinal and edible fungus with special flavor and rich nutrient. Polysaccharides purified from GF mycelium can enhance immunity of human body. The main drug, grifola frondosa beta-glucan(GFG), is extracted from GF mycelium. Grifola frondosa beta-glucan for infection(GFGI) is made from GFG, which was freeze-dried after being added mannitol as an excipient. GFGI is designed to be used for the assistant therapy of malignant tumor and immunodeficiency disease.Based on the study of basic physical and chemical properties of GFGI, identification, content and content uniformity, molecular weight (Mw ) and influence factors on Mw, degradation products analyses of GFGI are studied systematically, and the Quality Standard Draft of GFGI is established in this paper. According to the technical requirement of traditional chinese medicine(TCM) injection fingerprinting made by State Food and Drug Administration(SFDA), the medicinal material,intermediate and preparation of GFGI are also studied systematically using high performance gel permeation chromatography (HPGPC), and RP-HPLC method respectively. Two graphs named fingerprint (1) and fingerprint (2), which can reflect the fingerprint characteristic of active ingredient GFGI and other chemical ingredients, have established in the paper. Detection standard drafts of the fingerprint of GFGI medicinal material, intermediate and preparation are drawn up elementally.In the study of quality standard, the HPGPC method is set up to the determination of content and content uniformity of GFGI in the paper. This method is proved to be simple, reproducible. The concentrations of GFGI between 0.2-5.0 mg·mL-1 have good linear relationship with peak areas and peak heights in the method. And it can eliminate the interference of mannitol. At the same time, GFGI content and it's content uniformity determination are studied by sulfuric acid-phenol method. It is found that the sulfuric acid-phenol method based on glucose which containing formulated mannitol as standard curve is suitable to GFGI determination. It can eliminate the interference of mannitol and has good accuracy and reproducibility, but the operation is tedious. Methods for determination of GFGI Mw,and it's Mw width, influence of different chromatograph conditions on Mw determination are set up. The result showed mobile phase can influence behaviors of GFGI and Mw determination in a great degree. The Mw of GFGI are 822410,177520,29112 and 25066 dalton determined by HPGPC using pure water,0.02%NaN3 solution,0.1mol·L-1NaNO3 solution and 0.1mol·L-1Na2SO4 solution as mobile phase respectively. When using 0.02%NaN3 as mobile phase, chromatographic peak have a good symmetry,GFGI can separate with excipient feasibly. The deviation of Mw of GFGI are above 10% between Shodex OHpak SB-804HQ and TSK G4000PWxl columns determined by HPGPC using 0.02%NaN3 solution as mobile phase. Concentration and volume of sample and temperature have obvious influence on Mw determination of GFGI, and ratios of excipient to GFG have little influence on Mw determination of GFGI. Determination of Mw and Mw width had little difference between Aglient GPC software and home-made longzhida software. In addition, the HPGPC method is set up to determination of the degradation of GFGI. After being destroyed in the high temperature of 105℃some minor degradations, for example some oligosaccharides can be detected using HPGPC method, which is produced from the glicosidic backbone is cleaved.In the study of the fingerprinting chromatography of GFGI, 7 peaks is detected by RID and similarity of 10 batches of sample is over 95% in fingerprint (1). In fingerprint (2), 16 peaks are detected by DAD and similarity of 10 batches of sample is over 90%. The studies of the two graphs include the sample preparation, chromatography condition optimization, method precision , repeatability and reproducibility. While studying the fingerprints of the intermediate and the preparation, the same method is used. The result showed that the analytical method using fingerprint is feasible. It can reflect the characteristic relativity of active ingredient in medicinal material, intermediate and preparation. It can be used for quality control of the preparation.