Effects Of Ultra-micro Qiweibaizhusan On Molecular Diversity Of Intestinal Microbes

Objective To study the micro-ecological mechanism of curative effect of ultra-micro Qiweibaizhusan, and to provide the experimental evidence for the application of ultra-micro Qiweibaizhusan.Methods70Kunming mice were randomly divided into7groups:the control group, the model group, the traditional Qiweibaizhusan decoction group, the whole,1/2,1/4and1/8volume of ultra-micro qiweibaizhusan groups. Except for the mice in control group, all the mice were fed with complex antibiotic composing of ampicillin, cephradine and lincomycin to simulate diarrhea with dysbacteriosis. And then, all the mice were fed drugs by gavage3days. In the whole process, the mice in control group were given sterile water as a comparison. After that, the efficacy was judged by faeces, the amount of E.coli, lactobacillus and bifidobacterium in the intestinal tract.The faecal samples were collected from intestinal tracts of mice. These samples were washed by acetone, and then centrifuged in different speed. The cells were lysed by lysozyme and SDS, treated by CTAB, and then extracted with phenol/chloroform. The metagenome DNA of bacteria was determined by UV spectrophotometer, agarose gel electrophoresis. The16S rDNA was amplified from these DNA samples through a set of bacterial universal primers. The PCR products were analyzed by Amplifed Ribosomal DNA Restriction Analysis.The dysbacteria mice were fed with ultra-micro Qiweibaizhusan by gavage, and the metagenome DNA of intestinal flora was extracted, then the cluster tree was built by unweighted pair group method with arithmetic through ARDRA and DPS data processing system, and the diversity and similarity of each group were calculated. The effects of ultra-micro Qiweibaizhusan on molecular diversity of intestinal microbial in mice were analysised by these dates.Results After being fed with Qiweibaizhusan3days, the diarrhea mice in traditional Qiweibaizhusan group, the whole and1/2volume of ultra-micro Qiweibaizhusan group were cured. The amount of E.coli and the dose of ultra-micro Qiweibaizhusan had obviously positive correlations (R2=0.8326). The number of lactobacillus and bifidobacterium had a significant change between pretherapy and post-treatment (p<0.01). The growth of intestinal lactobacillus and bifidobacterrium could be stimulated by ultra-micro Qiweibaishusan (p<0.05). And the quantities of intestinal lactobacillus and bifidobacterium in the group treated with1/2amount of ultra-micro Qiweibaishusan were far more than which in the other ultra-micro Qiweibaishusan group mice (p<0.01).The metagenome DNA of intestinal flora in mice extracted by improved CTAB method was about23kb, the OD260/OD280of DNA was between1.8and2.0, the PCR products were about1.4kb and can be applied to ARDRA for further analysis.The endonuclease digested bands of16S rDNA from intestinal flora in dysbacteriosis mice cured by ultra-micro Qiweibaizhusan increased from3to5, lower than the control group (n=7). Being fed with1/2dose of ultra-micro Qiweibaizhusan by gavage after3days, the diversity of intestinal microbes in mice increased from0.7324to1.2207, the same as which treated by traditional Qiweibaizhusan decoction. Compared with the control group, the similarity of intestinal flora increased from40%to67%, higher than the traditional decoction group (50%).Conclusion A metagenome DNA extracting method of intestinal flora in mice for molecular diversity analysis based on PCR technology was established. Both the traditional and ultra-decoction of Qiweibaizhusan could regulate intestinal microflora in mice, and restore the diversity of intestinal microbes to cure diarrhea with dysbacteriosis caused by antibiotics. The therapeutic effect of1/2dose of ultra-micro Qiweibaizhusan could reach the curative level of traditional Qiweibaizhusan decoction. Thus, ultra-micro Chinese medicine can save medicinal material.