| Rotavirus is one of the most commonly virus which cause diarrhea. Theincidence and severity extent of rotavirus diarrhea are the first place in autumn andwinter diarrhea. People are susceptible to rotavirus and have temporary immunity. Sorotavirus diarrhea spreads all over the world. Rotavirus infects people with a wide agefrom newborns, adults to the elderly, especially more commonly in children under theage of 5. In recent years, scientific workers in many countries keep finding that RVcan result in multi-systemic diseases such as central nervous system, respiratorysystem and so on, the possibility that RV can cause intrauterine infection has beenraised. That rotavirus is the main reason which cause severe diarrhea in infants hasbecome a serious public health problem. This can result in morbidity and mortalitynot to be ignored both in developed and developing countries. Virus diarrhea hasbrought great impact on the children, families and society. At present, there are nospecific antiviral drugs for virus diarrhea, Chinese medicine appears a uniqueadvantage.Psidium guajava L had clear effect on virus diarrhea. Clinical practice proved that Psidium guajava L had a good effect on treating RV enteritis. Our completedstudy about Psidium guajava L treating virus diarrhea indicated Psidium guajava L.had a significant effect on RV enteritis. Experiment in vitro also proved that Psidiumguajava L. and its effective ingredient could enhance the tolerance of MA-104 cells torotavirus, reduce toxicity of rotavirus and damage shell and core of rotavirus particle.We focused on effective monomer extraction in the earlier studies. But simpleextraction of monomer may result in losses of some effective components oftraditional Chinese medicine. We overlooked characteristics of multi-target,multi-level treatment of Chinese medicine. So the study on medicine to treat virusdiarrhea may be more significant.The so-called Chinese medicine effective part is a class or classes of chemicalcomponents from a kind of Chinese medicine or Chinese medicine compoundwhose contents reach more than 50 percent, and this class or classes of chemicalcomponents are considered as effective components. Chinese medicine effectiveparts may play their roles from various aspects to treat diseases. So we try to screeneffective parts and study the effect of effective parts.This experiment includes three parts. The first part is based on the extraction oftotal saponin of Psidium guajava L. through basic principle of macroreticular resin.The contents of total saponin reach 55.3 percent. The second part is to observe theeffect of total saponin of Psidium guajava L. on rotavirus diarrhea in mice throughstool Na+, stool glucose, stool SIgA and negative rate of rotavirus antigen. The thirdpart is based on the part of immunologic mechanism according to comparison of thelevel of serum IL-2 and TNF-a of mice in every group.Chapter I Study on purification process of total saponin of Psidium guajavaL..with macroreticular resin Objective:To extract of total saponin of Psidium guajava L.Method:This experiment mainly depended on the basic principle of macroreticular resin.We extracted Psidium guajava L powder twice with 70% alcohol, then dried to gainextract in which the content of total saponin was 28.1 %. Then we further improvedthe content of total saponin throuth the principle of macroreticular resin. First topretreat macroreticular resin and optimize the technological parameters to determineadsorptive capacity, eluting dissolvent and quantity of eluting dissolvent.Results:1、Adsorptive capacity: 10 gram SA-1 macroreticular resin absorbed 30 millilitretrial-solution, we found that 10 gram SA-1 macroreticular resin can absorb 393.79milligram total saponin of Psidium guajava L. and the adsorption rate was 86.7%.2、Eluting dissolvent: We found that 3BV distilled water can fully wash awaywater-soluble impurities such as macromolecule carbohydrate. While using alcohol ofdifferent densities to elute total saponin, alcohol of low density could elute a centainamount of total saponin of Psidium guajava L. which increased with higher density.Total saponin of Psidium guajava L. mainly localized in eluent of 80 % alcohol.3、Quantity of eluting dissolvent: Extract from 4BV eluent of 80% alcoholaccounted 94.9%, total saponin accounted 96.1%. We determined 4BV for thequantity of eluting dissolvent.Conclusion:The content of totol saponin in extractum reached 28.1% by extracting twicePsidium guajava L. with 70% alcohol. The content of total saponin in extractumcould reach 55.3 % through the purification of SA-1 macroreticular resin. At last, wedetermined the method that 3BV distilled water washed away water-soluble impurities and 4BV 80% alcohol acted as eluting dissolvent.ChapterⅡResearch on the effect of total saponin of Psidium guajava L. onrotavirus diarrhea in miceObjective: To establish a mouse model of rotavirus diarrhea. To observe theeffect of total saponin on rotavirus diarrhea in mice according to some index such asgeneral performance, stool Na~+, stool glucose, stool SIgA and negative rate ofRotavirus antigen.Method: 84 mice were divided into seven groups: the high-dose saponin group,the middle-dose saponin group, the low-dose saponin group, the model group, thesmecta group, the decoction of Psidium guajava L. group and the normal controlgroup. There were 12 mice in every group. First detect stool of all mice which wasnegative with rotavirus group A diagnostic kit. All mice in other groups except thenormal control group were orally inoculated with l00ul 10~4TCID50 WA rotavirus. Tocollect the stool of mice after 24 hours and detect stool which was positive withrotavirus group A diagnostic kit. The mice in the normal control group were orallyinoculated with Sodium Chloride in an equivalent volume. The mice in the high,middle, low-dose saponin group, the smecta group and the decoction of Psidiumguajava L. group were orally inoculated with 0.5ml corresponding drugs. Meanwhile,the mice in the normal control group and the model group were orally inoculated withSodium Chloride in an equivalent volume once a day for three days 24 hours afterinfection. Three days later, to collect the stools of every mouse, pick eyeballs of themice for blood, execute the mice and take out of intestine tissue quickly which wasplaced in 10% formaldehyde. To conserve serum after centrifugalization. Then wedeteceted stool Na~+ using biochemical analyzer, deteceted stool glucose using glucoseoxidase method, deteceted stool SIgA using RIA, deteceted stool negative rate of rotavirus antigen using colloidal gold method and observed intestine tissuepathological examination.Statistics method:To use the SPSS13.0 software for statistical analysis. The levels of stool Na~+ andSIgA by One-way ANOVA to conduct significant analysis beween each group, theresults expressed by ((?)±s). The levels of stool sugar use the K Independent SamplesTest method to conduct significant analysis, the results expressed by ((?)±s) and theaverage rank. The rotavirus antigen negative rate use the K Independent Samples Testto conduct significant analysis, the results expressed by the average rank. If P≤0.05 ,there was significantly statistical difference.Results:1、The general performance: the condition of diarrher of the mice in the high,middle-dose saponin group obviously improved compared with the model group; thegeneral condition of the mice had no significant difference compared with the smectagroup and the decoction of Psidium guajava L.2、The level of stool Na~+: the level of stool Na~+ of the mice in the high, middleand low-dose saponin group, the smecta group, the decoction of Psidium guajava L.group had obviously degraded compared with the model group (P<0.01); the level ofstool Na~+ of the mice in the low-dose saponin group obviously higher than other 5groups (P<0.05) ; the level of stool Na~+ of the mice in the other 4 groups had nosignificant difference from the normal control group (P>0.05) ; the level of stool Na~+of the mice in the high, middle-dose saponin group had no significant difference fromthe smecta group and the decoction of Psidium guajava L. group (P>0.05) .3、The level of stool glucose: the level of stool glucose of the mice in the high,middle and low-dose saponin group, the smecta group, the decoction of Psidiumguajava L. group and the normal control group obviously degraded compared withthe model group (P<0.01) ;The smecta group had the best effect on the degree of decreasing stool glucose of rotavirus diarrhea mice, which followed by the high-dosesaponin group, the decoction of Psidium guajava L. group, the middle-dose saponingroup, finally the low-dose saponin group.4、The level of stool SIgA: the level of stool SIgA of the mice in the modelgroup was higher than that in the normal control group (P<0.05) . the level of stoolSIgA of the mice in the high, middle dose saponin group, the smecta group and thedecoction of Psidium guajava L. group significantly differed from the model group(P<0.01) ;the low-dose saponin group was obviously lower than other 5 groups(P<0.01) ; the high-dose saponin group was obviously higher than the smecta group(P<0.01) ; the level of stool SIgA of the mice in the low-dose saponin group had nosignificant difference from the model group (P>0.05) .5、The negative rate of rotavirus antigen: after three-day treatment, we detectedrotavirus antigen in the stool to observe the negative rate. The result indicated that:there were 9 mice whose rotavirus antigens were negative in the high-dose saponingroup(the negative rate of rotavirus antigen was 75%), there were 9 mice whoserotavirus antigens were negative in the middle-dose saponin group(the negative rateof rotavirus antigen was 75%), there were 7 mice whose rotavirus antigens werenegative in the low-dose saponin group(the negative rate of rotavirus antigen was58.3%), the rotavirus antigen of the mice in the model group were all positive(thenegative rate of rotavirus antigen was 0), there were 7 mice whose rotavirus antigenswere negative in the smecta group (the negative rate of rotavirus antigen was 58.3%),there were 8 mice whose rotavirus antigens were negative in the decoction of Psidiumguajava L. group (the negative rate of rotavirus antigen was 66.7%). The negative rateof rotavirus antigen in every group had significant difference (P=0.000)6、the result of intestine tissue pathological examination: total saponin ofhigh-dose and middle-dose could effectively protect mucous membrane of small intestine of mice of rotavirus diarrheaConclusion:Total saponin of Psidium guajava L.had a good effet on rotavirus diarrhea inmice.ChapterⅢThe effect of total saponin of Psidium guajava L.on serum IL-2 andTNF-αof mice of rotavirus diarrheaObjective: To observe the effect of total saponin of Psidium guajava L. onserum IL-2 and TNF-αof mice of rotavirus diarrhea for preliminarily exploring themechanisms.Method: 84 mice were divided into seven groups: the high-dose saponin group,the middle-dose saponin group, the low-dose saponin group, the model group, thesmecta group, the decoction of Psidium guajava L. group and the normal controlgroup. There were 12 mice in every group. First detect stool of all mice which wasnegative with rotavirus group A diagnostic kit. All mice in other groups except thenormal control group were orally inoculated with 100ul 10~4TCID50 WA rotavirus. Tocollect the stool of mice after 24hours and detect stool which was positive withrotavirus group A diagnostic kit. The mice in the normal control group were orallyinoculated with Sodium Chloride in an equivalent volume. The mice in the high,middle, low-dose saponin group, the smecta group and the decoction of Psidiumguajava L. group were orally inoculated with 0.5ml corresponding drugs. Meanwhile,the mice in the normal control group and the model group were orally inoculated withSodium Chloride in an equivalent volume once a day for three days 24hours afterinfection. Three days later, to pick eyeballs of the mice for blood, execute the miceand conserve serum after centrifugalization. Then we detected the level of serum IL-2and TNF-αin every group by RIA. Statistics method:To use the SPSS13.0 software for statistical analysis. To use One-way ANOVAto conduct significant analysis for the levels of serum IL-2 and TNF-αbeween eachgroup, the results expressed by ((?)±s). If P < 0.05, there was significant statisticaldifference.Results:1、The level of serum IL-2: the level of serum IL-2 of the mice in the high-dosesaponin group, the middle-dose saponin group and the decoction of Psidium guajavaL group were obviously higher than that in the model group (P<0.0l) ; the level ofserum IL-2 of the mice between the high-dose saponin group, the middle-dosesaponin group and the decoction of Psidium guajava L group had no significantdifference (P>0.05) ; the level of serum IL-2 of the mice in the the low-dose saponingroup, the smecta group and the normal control group had no significant differencefrom the model group (P>0.05 ) .2、The level of serum TNF-α: the level of serum TNF-αof the mice in the modelgroup was obviously higher than that in the normal control group (P<0.01) ; the levelof serum TNF-αof the mice in the high, middle and low-dose saponin group, thesmecta group, the decoction of Psidium guajava L group significantly differed fromthe model group; the level of serum TNF-αof the mice in the high, middle-dosesaponin group, the smecta group, the decoction of Psidium guajava L group and thenormal control group had no significant difference (P>0.05) ; the level of serumTNF-αof the mice in the high, middle-dose saponin group and the decoction ofPsidium guajava L group had no significant difference (P>0.05);. the level of serumTNF-αof the mice in the low-dose saponin group significantly differed from thedecoction of Psidium guajava L group (P<0.01) .Conclusion:Total saponin of Psidium guajava L. may indirectly develop antivial avtivity through regulating the level of serum IL-2 and TNF-αafter the mice were infected with rotavirus. |
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