| Purpose:The white stiff silkworm,namely Bombyx Batryticatus mori.L(Abbreviated as BBm)is a kind of ethno drug which has been widely considered being antispasmodic and phlegm reducing.However,this ethno drug is easily to be polluted and degenerated without the treatment of Chinese Meteria Medica Processing(CMMP).Once the contaminated BBm is taken by mistake without processing,it will easily lead to patient poisoning.Therefore,the main purpose of this study is to exam whether the processing of heating can be used for detoxification by destroying toxic compounds and deducing the contents of some specific chemicals.However,this product is an animal herb that is rich in protein,polysaccharides,and oils.It is easily to be quality variations during storage.The contaminated BBm with mold,if not controlled and handled,can easily induce adverse reactions once taken by the patient.Not only that,even the normal silkworms that have not been degraded also induced many clinical reports of adverse reactions.However,the mechanism by which silkworms cause adverse reactions is not clear.In order to detoxify the deteriorating BBm and elucidate the toxic material basis,several questions need to be clarified in this study.What are the molds susceptible to infection by silkworms? Which toxins are produced by these molds? These toxins act on humans or experimental animals.What toxic reactions are caused when these toxins are thermally unstable,whether the destructive silkworms can be rapidly detoxified by heat-cooking methods,and so on.What is the toxic substance basis of undegraded BBm?What is the toxic dose situation? Whether these substances are also thermally unstable or not,and whether heat treatment can reduce their toxicity.Finally,toxic chemicals' contents between processed and unprocessed groups were compared and the detoxification mechanism of processing was clarified.Materials and Methods:In order to answer these questions mentioned aboce,the first study was the examination of the appearance traits of 12 batches of BBm collected from seven producing areas.Usingmoldy silkworm as a secondary fungal source,with solid culture,passaging purification and genetic identification methods to identify the fungal species of metamorphic BBm conducted.The identified single strains were subjected to large-scale solid or liquid culture.After the resulting cultured,the medium was extracted with organic reagents,and the chemical components were separated and purified,and spectral structure analysis was performed on the specific extraction sites with obvious toxicity.The identified toxins were used to demonstrate the magnitude of virulence and damage to tissues and cells through acute toxicity tests and cytotoxicity experiments in medical laboratory animals.The obtained chemical substances were processed by solidification or by liquids dissolved in specific reagents.The high-pressure liquid chromatography,liquid chromatography and mass spectrometry techniques were used to determine the changes in the content before and after heating,and the difference in fragmentation profiling were conducted by LC-MS/MS to characterization of the ions before and after heating.The metamorphic BBm were detoxified with specific excipients and heating methods.Thin-layer chromatographic analysis was used to characterize and contrast the lipophilic toxic components and the chemical constituents of the less polar bodies contained in the silkworm itself to find differential substances.The different extraction sites of normal BBm were screened for toxicity.Higher levels of toxic substances obtained in toxic sites were analyzed and identified.After the toxic substances are processed by heating,their content changes are measured to determine whether they are thermally unstable.In addition,the anti-convulsive effects of the silkworm and its various processed products were thoroughly examined to verify the ancient people's understanding of the production of the BBmr--the raw product was prevalent in confrontation of convulsion and the bran fried was prevalent in confrontation of nodule.Is it scientific?Finally,a preliminary study was conducted on the mechanism of action of 1-DNJ,a small molecule alkaloid that was specifically enriched in silkworms,against diabetic cardiomyopathy in mice.The db / db mice were used as a model to detect the changes of N-glycosylation during the initial stage of diabetic myocardium,and the differences of myocardial glycosylation before and after 1-DNJ treatment were determined.Hydrophilic chromatographic solid-phase extraction enrichment and LC-MS/MS identification techniqueswere used to characterize changes in protein glycosylation.At the same time,changes in N-glycan ? 1,6-fucosylation were analyzed by LCA lectin blotting and FITC-labeled lectin affinity histochemistry.Results:1.Successive fungi obtained from metamorphosed BBm were purified and genetically identified and found to be P.chrysogenum and A.flavus.2.Penicillium chrysogenum was expanded to obtain patulin,penicillic acid,cytochalasin B,and ergosterol from its liquid medium.The literature review verified that these substances are all toxic.The determination results before and after heating were processed showed that cytochalasin and ergosterol and other substances are thermally unstable,and heating can reduce their toxicity.3.The content of aflatoxin B1,B2,G1,G2 produced by Aspergillus flavus after treatment with certain excipients and heating methods was reduced.4.The chemical components beswaenin and cytochalasin obtained from the liquid culture medium of Beauveria bassiana are also thermally unstable.After the treatment with a specific heating method,the contents of these two compounds are reduced.5.Toxic screening of different polar parts of normal silkworm showed that the water extraction site was the most toxic.By further separating and purifying the chemical constituents of the water extraction site,the oxalate metabolite of Beauveria bassiana was found to have the most obvious toxicity and irritation to experimental animals,and its content in water extracts was high.These oxalates are also thermally unstable and can be heat-processed to properly reduce their content.6.Bombyx mori and its extracts from different processed products have some anticonvulsant effects,but this effect is less than that of the positive control drugs such as carbamazepine and alprazolam.Bombyx mori extract was more effective against the strangulation model of strychnine and penicillin ignited amygdala seizures,and was unable to resist the pentylenetetrazole-induced convulsions and electroconvulsions model.The results of molecular biology experiments showed that Bombyx mori extracts could reduce the expression of GRP78 and CAP3 and increase the expression of CHOP10.7.The 1-DNJ in silkworm can inhibit myocardial fibrosis in diabetic mice,and reduce theN-glycosylation and ?1,6-fucosylation of cardiac protein in db / db mice.Conclusion:1.When the metamorphosis of the BBm is obvious,it should be abandoned.2.When a small amount of metamorphic BBm is mixed with herbs,they should be picked out in time.If the surface and appearance of the silkworm are qualitatively inaccurate,the BBm should be treated in time.BBm is easily contaminated by Aspergillus flavus.In addition,molds such as Penicillium chrysogenum also contaminate silkworms.Through comparison and screening,the polluted BBm can be detoxified easily using ginger juice and ginger volatile oil,combined with wheat bran.3.Aspergillus flavus,Penicillium chrysogenum,and Beauveria bassiana.Some of the secondary metabolites produced by these fungal strains have a certain degree of thermal instability,which can be reduced by heating.4.Microwave heating processing method can play a role in high-temperature destruction of secondary metabolites of some fungi,and has a certain role in attenuating.5.Although Bombyx mori has some anticonvulsant effects,the effect of convulsions caused by fighting certain stimuli is not obvious.The results of this study are not completely consistent with the ancients' understanding of the production of the bombardment artillery,"the raw product was prevalent in confrontation of convulsion and the bran fried was prevalent in confrontation of nodule",which means that the understanding of the ancients still needs further experimental research to verify it.6.1-DNJ in silkworm has obvious hypoglycemic effect.The fibrosis of this compound against mouse diabetic cardiomyopathy is achieved by inhibiting N-glycosylation and ?1,6fucosylation of cardiac muscle protein,but The determination of the expression level of key glycosyltransferases was found to be achieved by reducing the concentration of substrate in the critical pathway... |
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