| Bombyx batryticatus, an oriental crude drug firstly recorded in "Shen Nong’s Herbal Classic", is dried dead fungus-insect complex, which is dead and stiffened due to a Beauveria bassiana infection. As a valuable traditional Chinese medicinal material, it has been utilized for thousands of years. Bombyx batryticatus is salty and pungent, natured. It possesses muti-pharmacologic effects including anti-convulsion, anticoagulation, hypnogenesis, anti-fungus, anticancer and hypoglycemic and hypolipidemic effects. So it is widely used clinically alone or compatible with other drugs to treat exogenous fever, convulsion, epilepsy, headache, migraine, facial paralysis, hemiplegia, cough, asthma, exfoliative dermatitis, cutaneous pruritus, insomnia, thyroid adenoma, diabetes, urticaria, cerebral thrombosis, hyperlipidemia. With the deepening of research, Bomby batyaticatus’s application is more and more widely. The active constituents play an important role in its pharmacological effects. And flavonoids, beauvericin and polysaccharide, as mainly active component parts, are very helpful to the effects especially in antioxidant, anticancer aspects. The present study focus on extraction, determination, separation and purification, structural identification, pharmacologic actions about the three active constituents. The mainly results are as follows:1. Flavonoids was optimize-extracted by Central Composite Design of response surface methodology. The optimum program was like this:Bombyx batraticatus was extracted by80%ethanol after sifted with80item, pre-ultrasonic bath for20min,30:1liquid-to-solid ratio, for2.5h at100℃. The spectrophotometric method was established to determine flavonoids in Bombyx batraticatus, with qualified accuracy, stability and repeatability (RSD<2%) and recovery (95-105%). With the obtained optimum conditions, flavonoids content changes were investigated in silkworm body after infection of Beauveria bassiana within12days.2. With HPLC-MS/MS, the determination method of beauverrion in Bombyx batraticatus was constructed. The results showed beauvericin’s content was21.64μg/g. Methodology validation showed the LOD and LOQ were4μg/g and12μg/g. Repeatability (RSD=1.16%) and recoveries at3level (109±4.9%,97±3.8%,92±3.4%) were satisfied. Beauvericin content changes were investigated in silkworm body after infection of Beauveria bassiana within12days. 3. Polysaccharide of Bombyx batraticatus was separated and purified. The obtained crude polysaccharide was isolated and purified using DEAE Sepharose F. F. and gel-filtration chromatography (Sephacryl S-100-500) repeatedly. Two water-soluble homogeneous polysaccharides (BBPW-1, BBPW-2) were obtained. Lack of absorption at280nm and260nm by UV scanning indicated that they contained no protein and nucleic acid. IR spectra showed that the sugar characteristic groups were existed. HPLC producing a single symmetrical peak, indicated each of them was homogeneous and their molecular weight were3.67×106Da,2.0×103Da, respectively.4. The structure of homogeneous BBPW-2was studied. Their structures were investigated using chemical (Sugar analysis, Methylation analysis, partial acid hydrolysis, etc.), and spectroscopic methods (ID NMR:’H NMR,13C NMR, DEPT spectra;2D NMR:’H-’H COSY, TOCSY, HMQC, NOESY and HMBC spectra; etc.) to confirm the sugar composition, the number of sugar residues, the form of glycosyl linkage, the configuration of anomeric center, the sequence of sugar residues.Sugar analysis showed this polysaccharide was composed of mannose, glucose, and galactose in the ratio of1:0.74:0.59. Methylation analysis showed it contained2,6and1,3,6mannose linkage,1,6and terminal glucose linkage,1,6and2,6galactose linkage. Methylation analysis showed molar ration was1:0.75:0.47, which was accordance with the result of acetylation analysis. Methylation analysis showed BBPW-2contained1-,1,2,6-and1,3-mannose linkage,1,2,6-and1-glucose linkage and1,2-galactose linkage. NMR analysis further showed that its main chain was composed of (3-D-1,2,6linked glucosyl and (3-D-1,2,6linked mannosyl, side chain was composed of α-D-1,3mannose and α-1,2-D-galactose, and it also contains a minor terminal a-D-mannose and α-D-glucose. The structure was deducted as follows: 5. The effects of crude polysaccharide on immunologic function of normal mice and cyclophosphamide-induce immunosuppressed mice were investigated. It was found that polysaccharide of Bombyx batraticatus could improve immune organs’ weight, phagocytic function of abdominal macrophage, lymphocyte transformation rate, hemolysin antibody’s level in serum, the number of spleen plaque forming cells, which suggested that Bombyx batraticatus polysaccharide could promote mice immunologic function through many aspects.6. Crude flavonoids was obtained by combination of AB-8marcoporous resin and amilan and elution with60%ethanol. Its effects on proliferation of cancer cells and normal cells were tested. The results showed that crude flavonoids could obviously suppress cancer cells’ proliferation, and promote normal cells’ proliferation.7. Anticancer effect of three polysaccharides with different molecular weight were tested and it was found that they showed proliferation inhibition effect on both cancer cells and normal cells. Anticancer effect of four polysaccharides with different acidity obtained thought DEAE Sepharose F. F. were also tested and the result showed the anticancer effect of neutral polysaccharide eluted by water was more obvious than that of the other three acidic polysaccharides eluted by NaCl solution. All four polysaccharides could increase RAW264.7proliferation, and promote production of NO. Our results suggested that the anti-tumor activity could exert anti-tumor effects by increasing the production of NO by macrophages in vivo.8. Anticancer effect of two homogeneous polysaccharides (BBPW-1, BBPW-2) separated from Bombyx batraticatus was studied and anticancer mechanism of BBPW-2with clear structure characteristics was implored. Results demonstrated both of two showed evident inhibition effect on cancer cells and the effect of BBPW-1with larger molecular weight was more significant. BBPW-2showed long-term anticancer effect on human breast cancer cells MCF-7. Through cell cycle analysis and apoptosis analysis, we found that the mechanisms of the growth inhibitory effect of BBPW-2on Hela cells is mediated through inducing cell apoptosis and cell-cycle arrest at G0/G1, G2/M phase. |
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