| Bombyx Batryticatus is a dried body that was dying from the larvae of the Bombycidae insect silkworm(Bombyx mori L.)infected with Beauveria bassiana(Bals.)Vuillant.It has a wide range of applications and has a significant medicinal effect.So far,2015 version ?Chinese Pharmacopoeia?and the mainstream medicinal material standard?76 medicinal product specifications?have only one kind of specifications for Bombyx Batryticatus standard.However,due to various Bombyx Batryticatus breeding techniques and complex market transactions,the quality of the Bombyx Batryticatus is uneven.The current standards cannot fully apply to the circulation of Bombyx Batryticatus.At present,the identification and quality control of Bombyx Batryticatus are cumbersome and needing expensive equipment,which is not conducive to the actual promotion and application in the Chinese herbal medicine market.Therefore,on the basis of literature review and market origin research,this paper analyzed the data of various indicators of commercially available Bombyx Batryticatus in order to formulate grade standard of Bombyx Batryticatus specification with strong applicability and facilitates market circulation.Systematic analysed the internal active components of Bombyx Batryticatus to provide a scientific basis for the quality control of silkworms.At the same time,in order to further expand the medicinal value of traditional Chinese medicinal materials,the possibility of preparing highly active ACEinhibitory peptides by enzymatic hydrolysis of Bombyx Batryticatus protein was explored,providing data foundation for in-depth study on the hypotensive effect of Bombyx Batryticatus.The research content and experimental results are as follows:1?Based on the literature review,the Bombyx Batryticatus samples were collected by the market investigation on Bozhou,Hehuachi,Yulin,and Anguo Chinese herbal medicines markets and origin investigation on Sichuan,Guangxi,Jiangsu and Zhejiang Provinces provinces.The length,weight,and diameter of the Bombyx Batryticatus were measured According to the Pharmacopoeia "Straight?stout" requirement for Bombyx Batryticatus and non-parametric K-S tests?correlation analysis were perform by the SPSS software on the collected data.The results showed that all data were in normal distribution,and the 99% confidence intervals for length,weight,and diameter were 3.13~3.93 cm,0.48~0.79 g,and 0.52~0.69 cm,respectively.The survey also found that silkworm gland ring was also an important parameter for judging the quality of the Bombyx Batryticatus.Cross-sectional conditions were related to length,weight and diameter data.The weight was the most significant correlative factor for the cross-sectional condition.According to 30% and 70% in the line of delineation of length,weight,diameter,Bombyx Batryticatus can be divided to three grades: top selected grade,second selected grade and popular grade.Top selected grade length> 3.8cm,diameter> 0.6cm,weight> 0.7g,85% section were bright black;second selected grade length 3.3cm ~ 3.8cm,diameter 0.5cm ~ 0.6cm,weight 0.5g ~0.7g,80% section were bright black;popular grade length <3.3cm,diameter <0.5cm,weight <0.5g.2?The pharmacologically active components such as ammonium oxalate,beauvericin,amino acids and proteins in three grades of Bombyx Batryticatus were studied,and the scientific criteria of the developed specification grade standard was verified.The ammonium oxalate content in top selected grade was 4.22%,which was higher than second selected grade(4.1%)and popular grade(2.7%);the beauscanin content in top selected grade was 571.06 mg/m L,which was higher than second selected grade(495.94 mg/m L)and popular grade(175.74 mg/m L).Thin layer chromatography(TLC)results showed that the total amount of amino acids in selected grade Bombyx Batryticatus was lower than popular grade Bombyx Batryticatus.Among them,there were more lysines in the top selected grade.However,the contents of threonine,glycine,leucine,Lphenylalanine,and other polar amino acids were lower than second selected grade and popular grade.Protein electrophoresis fingerprint analysis showed that the 35.3 k Da protein content in the top selected grade was higher than that of the second selected grade and popular grade,the 19.8 k Da protein content in popular grade was higher than top selected grade and second selected grade.The Bombyx Batryticatus proteins were analyzed by protein electrophoresis fingerprint analysis after salting out.The results showed that 15%-20% saturated ammonium sulfate solution treatment can enrich 35.3KDa protein,and 55%-60% saturated ammonium sulfate solution treatment can enrich 19.8KDa protein.3?The raw materials for the protein production from the Bombyx Batryticatus protein bromelain were studied.Combined with the results of single factor experiments,the enzymolysis time(min),enzyme ratio([E]/[S]),and initial p H had an effect on the ACE inhibition rate,which first increased and then decreased.The response surface methodology was used to optimize the process of Bromelain enzymatic hydrolysis of Bombyx Batryticatus protein.The optimal conditions were: enzymolysis time 100 min,[E]/[S] 3.71%,initial p H 6.0.The actual value of ACE inhibition rate was 87.44%,and the IC50 was 0.32 mg/m L.The fitting degree of the model was high and it had practical significance.The ACE inhibiting peptide was obtained by precipitating with 0.5% TCA and preliminary characterized.The ACE inhibiting peptide was separated by RP-HPLC with the presence of P1-P9 specific absorption peaks,P4 absorption peak showed significant inhibitory activity against ACE,IC50 was 0.12 mg/m L.In summary,after obtaining specific quantified data through literature review,market research and origin surveys,this paper established grade standard of Bombyx Batryticatus specification and studied active ingredients such as ammonium oxalate,beauvericin,amino acids and proteins in three levels of Bombyx Batryticatus classified under the standard.There were significant difference in the content and types of intrinsic active ingredients in each level Bombyx Batryticatus.At the same time,a highly active ACE-inhibitory peptide was prepared from the defatted Bombyx Batryticatus alcohol extract.The above results provide convenience for the circulation of the Bombyx Batryticatus market and safe use of drugs,and at the same time it expands the medicinal value of Bombyx Batryticatus medicinal materials. |
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