| Chapter one,Literature reviewThis chapter systematically summarizes the medicinal historical origins of silkworm excrement and clarifies the relationship between the effect of “expelling wind and eliminating dampness” of silkworm excrement and rheumatoid arthritis,and summarizes modern research progress of silkworm excrement,including the research on the chemical composition of resources,the pharmacological effects of silkworm excrement and the related application progress.It provides scientific basis for the development and rational utilization of silkworm excrement resources.Chapter two,Analysis and evaluation of chemical constituents in silkworm excrementTo clarify the composition and distribution characteristics of the resource chemical components in the silkworm excrement,this chapter takes the silkworm excrement from different producing areas and various instars as the research object.HPLC-PAD,UPLC-TQ/MS and other methods were used to determine the flavonoids,alkaloids,nucleosides,amino acids,polysaccharides,and chlorophyll in silkworm excrement.It provides basis for further research,and the results are as follows:1.The results of flavonoids analysis showed that the silkworm excrement mainly contains three kinds of flavonoids including isoquercitrin,astragalin and rutin.The results from different producing areas and various insars showed that the three kinds of flavonoids collected from Dali were significantly higher than those from other producing areas and the silkworm excrement collected from Chongqing showed a second content.In addition,it was found that the content of flavonoids in silkworm excrement was in the order of 3rd instar> 4th instar> 5th instar.2.The results of alkaloids analysis showed that the contents of alkaloids in silkworm excrement were abundant and the total content of 1-deoxynojirimycin and fagomine was above 0.1%.The content of alkaloids in silkworm excrement collected from Suzhou was the highest（1.63 mg/g）and the second one was collected from Zhenjiang.3.The results of nucleoside and amino acid analysis showed that there are 5 nucleosides and 14 amino acids in the silkworm excrement.The total content of nucleosides and amino acids collected from Suzhou were higher than those from others（2.61 mg/g）and the total content of nucleosides and amino acids collected from Guangxi were lower than those from others（0.58 mg/g）.The contents of L-leucine,L-proline,L-α-alanine,L-glutamic acid,and L-threonine were higher than other nucleosides and amino acids.4.The results of polysaccharides analysis showed that the content of polysaccharides in silkworm excrement was very rich and the content of crude polysaccharide was ab OTU 9%.In addition,the contents of polysaccharides collected from Suzhou,Tongxiang and Zhenjiang were similar and the content of polysaccharides collected from Dali was highest（11.85%）.Besides,the content of polysaccharides had a same tendency as well as the decrease of instars.5.The results of chlorophyll analysis showed that the total content of chlorophyll in silkworm excrement was ab OTU 0.2%.The content of chlorophyll a in all silkworm excrement samples was significantly higher than that of chlorophyll b.The highest chlorophyll content was found in the third instar silkworm excrement collected from Zhenjiang（2.62mg/g）and the content of chlorophyll in the silkworm excrement collected in Guangxi was significantly lower than that from other producing areas.Moreover,the contents of chlorophyll in the silkworm excrement collected from Dali,Tongxiang,and Chongqing were similar.As the instar increased,the content of chlorophyll in silkworm excrement decreased.Chapter three,Analysis and identification of bacterial communities in silkworm excrementThe Illumina MiSeq second-generation sequencing platform was used to determine the 16 S rDNA of the bacterial communities in silkworm excrement.And the alpha diversity analysis,beta diversity analysis,LefSe analysis,Metastats analysis,principal coordinate analysis,community metabolic function prediction,and other methods were used to compared the differences of bacterial communities in silkworm excrement before and after the drying process.In addition,the samples from different places and different instars were also compared to provide a new scientific basis for the quality evaluation and function indication of silkworm excrement.1.Effect of drying treatment on the structure of silkworm excrementThe fresh silkworm excrement and dried silkworm from Zhenjiang were selected to determine the 16 S rDNA-V4 region sequences and compared each other.As a result,it was found that the structure of the bacterial communities was significantly changed after low-temperature drying,in which the Citrobacter and Erwinia were significantly increased,Streptococcus,Cyanobacteria,Methyl bacilli,bacillus,and 03196G20 strains were significantly reduced,indicating that the silkworm excrement was better be properly processed when it was used for medicine and fresh products were not suitable for medicine.2.Influence of producing areas and instars on the structure of bacterial communities in silkworm excrementA comparative analysis of the differences in the composition of the bacterial communities and metabolic functions in silkworm excrement from different production areas and different instars.The results showed that the silkworm excrement mainly consists of Proteobacteria,Actinomycetes,and Thick-walled bacteria.It showed big differences in structure of bacterial communities in silkworm excrement from different producing areas,and the silkworm excrement from Guangxi was obviously different from other places.During different instars,the structure of bacterial communities were similar,while the Actinomycetes gradually decreased.These indicated that the structure of the bacterial communities in silkworm excrement changed with the growth of the age.Chapter four,Study on the effect of silkworm excrement on adjuvant arthritis in rats and its mechanism1.The content of 3 flavonoids in silkworm excrement was taken as the investigation factor.After orthogonal experiment,the optimum extraction process of flavonoids was gained,60% ethanol,1:12,2 extractions,1 h each time.Then,prepared the alcohol extract according to this condition.Finally,the composition of the water extracts and alcohol extracts of silkworm excrement was analyzed,and the ingredient composition of the drug was clearly defined.The content of polysaccharide in water was 43%,in addition,the contents of alkaloids and flavonoids were also been significantly improved compared with the original herbs.2.Adjuvant arthritis rats（AA）induced by Freund’s complete adjuvant were used as research objects to assess the efficacy of extracts in silkworm excrement from Suzhou against rheumatoid arthritis.After administration continuously for 21 days,we collected serum,synovial membrane and ankle joints of rats.The biochemical indicators were measured,the expression of signal protein in synovial tissues was analyzed by Western blotting and the pathological section was analyzed by ankle joints.The results showed that the extracts of silkworm excrement played a therapeutic role in rheumatoid arthritis through improving the oxidative stress of AA rats,inhibiting the abnormal increase of pro-inflammatory factors,relieving bone destruction and regulating NF-κB and MAPKs pathways.3.Based on metabonomics technology to study the effects of extracts of silkworm excrement on AA rats and selected serum and urine samples of experimental rats for analysis.A total of 33 endogenous metabolites were identified（10 in serum and 23 in urine）and 6 related metabolic pathways were constructed,namely nicotinic acid and nicotinamide metabolism;pentose and glucuronide conversion;glyoxylate and dicarboxylic acid metabolism;β-alanine metabolism;purine metabolism and glycolysis,glucose production.After drug intervention,these endogenous metabolites turned back to normal level some extent,indicating that the extracts from silkworm excrement can improve symptoms by regulating the metabolism of AA rats.4.Fresh feces from experimental rats were collected and 16 S rDNA sequencing technology was used to study the effect of extracts on intestinal flora in AA rats.The results showed that compared with the control group,the intestinal flora of AA rats significantly changed.At the genus level,S24-7,Bacteroides,Verrucomicrobiaceae,and Clostridium in the feces of the model rats were significantly increased,and Lactobacillus,Corynebacterium,Ruminococcus,and Adlercreutzia were significantly reduced.After drug intervention,these intestinal bacteria turned back to normal level some extent.These suggested that silkworm excrement has a certain role in improving the intestinal dysbacteriosis in AA rats. |
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