| Chronic fatigue syndrome(CFS)is a type of disease with symptoms of extreme fatigue,abnormal sleep,muscle pain,depression,and anxiety.It is characterized by the sustained fatigue that is hard to be relieved through rest for more than six months.The prevalence of this disease is relatively high and it mainly affects the high-educated population.At present,CFS has been found in adolescents,reaching 0.19%to 0.6%.However,the effective treatment methods for the disease are still not available and there are only some methods to relieve certain symptoms such as Western medicine(antidepressants,painkillers,etc.)and traditional Chinese medicine(acupuncture,moxibustion,Chinese herbal decoctions,etc.)or psychological counseling and exercise.Black sesame is the dried and mature seed of Sesamum indicum L.which has excellent medicinal value and health care function.The studies have shown that the food protein can be degraded by various enzymes in the digestive system of human body,and absorbed finally by the body in the form of peptides.The Sesame protein can be transformed into bioactive peptides of small molecular through various biological enzymes.In the present thesis,based on preparing food of sesame bioactive peptide,the effect against chronic fatigue syndrome and the underlying mechanism of sesame bioactive peptide were studied.It is mainly composed of the following three parts.In the first part,the conditions of enzyme hydrolyzing sesame protein with pepsin,trypsin,and alkaline protease were optimized to provide the best process technology for the preparation of sesame bioactive peptides.After removing the seed coats of black sesame seeds by alkali water solution,it was degreased by pressing and extracting with petroleum ether.The defatted sesame powder was then extracted by alkali and precipitated by acid to obtain sesame protein,and the conditions were as followed:ratio of ultrapure water and defatted sesame powder was 25:1,temperature was 75?,p H was 10,extract time was 290 min,the extract solution was adjusted to p H=4.5 for precipitation.Then,using the degree of hydrolysis as the index,single-factor experiments were firstly performed to investigate the effects of temperature(T),p H,substrate concentration,enzymatic hydrolysis time(t),enzyme amount on the hydrolysis of sesame protein by the pepsin,trypsin and alkaline protease.On this basis,the routine factor was screened with Regular two-level factorial design of Design Expert 11 to select the three significant factors,and then the Central Composite Design(CCD)was used to optimize the response surface with three factors and five levels,and finally enzymatic hydrolysis conditions with the highest degree was predicted according to the multiple regression equation.The optimization results was finally obtained by verification.In consideration of the actual production and efficiency,the final enzymatic hydrolysis process conditions were selected as:the conditions of pepsin were p H=1,T=37?,enzyme amount 62500 u/mg,substrate concentration 5%,hydrolysis time 3 h;the conditions of trypsin were p H=10,T=30?,enzyme amount100000 u/mg,substrate concentration 3%,hydrolysis time 3 h;the conditions of alkaline protease were p H=12,temperature 50?,substrate concentration 1%,enzyme amount 10000 u/g,hydrolysis time 3 h.Simulating the order of enzymatic digestion of food in the human body,the optimized enzymolysis conditions were used to sequentially hydrolyze the sesame protein with pepsin-trypsin-alkaline protease for getting a three-enzyme hydrolysis solution.Under the conditions of pump speed 8 and needle passing frequency 8,sesame bioactive peptide was obtained by spray drying.In the second part,the vitro activities of sesame bioactive peptides were studied.The mouse spleen lymphocytes were used to prepare cell membrane chromatography of 5-hydroxytryptamine 1A receptor(5-HT1AR)in vitro.The effect of stimulating 5-HT1AR was detected by High performance liquid chromatography(HPLC).The results showed that the hydrolysate of pepsin,trypsin and alkaline protease may contain ingredients that stimulate 5-HT1AR.The hydrolysate of three-enzyme may have better agonistic activity,because the retention time was longer,which shows that its ability binding to the 5-HT1AR receptor on the 5-HT1AR cell membrane chromatography was stronger.In addition,four compounds(+)-sesamelin,(+)-sesamesin,samin and pinoresinol,isolated from black sesame seeds by our laboratory,were screened.Among them,(+)-sesamesin and pinoresinol have agonistic effects on 5-HT1AR,and(+)-sesamesin and samin have no effects.In the third part,the vivo activity of sesame bioactive peptides were studied.A mouse chronic fatigue model was established by exhaustive swimming,reversing day and night,and crowding.The three-enzyme hydrolysate were intragastrically administrated in the high,middle and low dose groups.The anti-fatigue effect of the three-enzyme hydrolysate was evaluated by exhaustive swimming time and weight-loading swimming time,and its immunomodulatory effect was studied by the immune organ index,mouse spleen lymphocyte proliferation test and the distribution of splenic lymphocyte subsets.The CD4~+and CD8~+subgroups of mouse spleen lymphocytes were detected by flow cytometry,the relative expression levels of 5-HT1AR m RNA in mouse hippocampus and IL-10,IL-17 and IL-6 m RNA in brain tissue were determined by RT-PCR.The mechanism of action of sesame bioactive peptide on improving the state of chronic fatigue mice was preliminarily studied.The results showed the modeling method is feasible,due to exhaustive swimming causing fatigue,crowded condition causing irritability,and the reversal of day and night causing mental fatigue by sleep deprivation for mice.The food intake of model mice were reduced,and weight gain slowed down.Compared with the blank group,the spleen index of the model group decreased significantly(p<0.05),and thymus index showed a slight decreased trend;compared with the model group,the spleen index of the mice in the high,medium and low dose groups increased significantly,and the weight-bearing swimming time and exhausted swimming time of the mice in each dose group increased significantly(p<0.05).These results showed that sesame bioactive peptide could improve the fatigue,and its mechanism might be ascribed to improve the immune ability of mice.The proliferation ability of splenic lymphocytes in low dose group increased significantly(p<0.05).The distribution of splenic lymphocyte subsets showed that the percentage of CD4~+T lymphocytes could be increased and the percentage of CD8~+T lymphocytes could be decreased by each dose group compared with the model group,thus increasing their proportion of CD4~+/CD8~+cells,which might be related with the improvement of the immune ability in mice.Compared with the blank group,the expression level of 5-HT1AR m RNA in the hippocampus of the model group was increased,but there was no significant difference;compared with the blank group,the expression level of 5-HT1AR m RNA in hippocampus in high and middle dose groups were increased significantly(p<0.05),but there was no significant difference in low dose group.The results showed that the chronic fatigue model had no effect on the expression of 5-HT1AR m RNA in mouse hippocampus,while sesame bioactive peptide had a significant increase in the expression of 5-HT1AR m RNA comparing with normal mice.Compared with the blank group,the IL-10 m RNA and IL-17 m RNA expression levels were decreased in the model group,the high and low dose groups;but there was no significant difference among them.Compared with the blank group,the IL-6 m RNA expression level has a decreasing trend in the model group;compared with the model group,the IL-6 m RNA expression level has an increasing trend in the high,medium and low dose groups,but there is no significant difference.These results indicated that there is no central inflammation in the chronic fatigue mouse model under the experimental conditions. |
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