The Effect Of Ultra-micro Qiweibaizhusan On Intestinal Microbiota In Diarrheal Mice With Dysbacteriosis

Gut microbes are closely associated with multiple human physiological functions, and play a significant role in the pharmacological effects of traditional Chinese medicine. This study is aim to investigate the microecological functions and their mechanism of ultra-micro powders of Qiweibaizhusan (QMBZS) on the mice diarrhea with dysbacteriosis. The intestinal dysbacteriosis in mice were induced by orally administrating with Gentamycin Sulfate injection and Cefradine Capsules, and the effects of traditional decoction of Qiweibaizhusan (BZS) and QMBZS (which dose is approximately equal to 1/2 dose of BZS) on the mice with diarrhea were respectively observed. A yeast strain which is associated with QMBZS was analyzed. This study will provide a scientific basis for the clinical application of QMBZS. The main results and conclusions are as follows.The intestinal microflora were extracted and analyzed by Biolog Microbial Identification System. AWCD (average well color development) in all groups were found to be stable after 72 h of cultivation. The orders of AWCD in each group were QMZBS> Normal>BZS> Model. The microbial diversity index, including Shannon index and Simpson index, were largest in the normal group by comparing to the other groups (P<0.01). Both of the Shannon evenness and Mclntosh index of QMZBS group were largest (P<0.05), while the Shannon evenness in this group was similar to the nomal group. Moreover, the results of clustering analysis and principal components analysis suggested that bacteriostasis of antibiotics was obvious, and some metabolism ways of intestinal microbial in mice were influenced. The metabolism diversities of intestinal microbial in mice were gradually regained balance due to the regulation of QMZBS and ZBS. Especially, the regulations of QMZBS on the diversity of intestinal microbial functions were stronger than that of ZBS.The intestinal microbial metagenomics in all groups were extracted, the 16S rDNA of microorganisms in intestinal contents were analyzed by PCR cloning, and the Gene library was further established. The results showed that Lactobacillus johnsonii, Enterococcus feacium, Clostridium sp., Blautia product, Anaerostipes sp., Staphylococcus saprophyticus subsp. and Uncultured bacterium were the control intestinal microbial flora, lactobacillus especially was the dominant intestinal microbial flora, which was accounting for 61.90% of the total bacterial DNA clone number. The proportion of lactobacillus in model group was significantly decreased by comparing with the control group, while they recovered in the QMZBS and ZBS respectively treated mice. Moreover, the proportion of lactobacillus in QMZBS group was very close to the normal group. According to calculating the flora diversity, it was found that species richness and dominance index of each group, all index of QMZBS group were approximately close to the control group. Moreover, QMZBS group was found to be clustered into one class with the normal group by establishing a clustering tree. In conclusion, the intestinal microbial diversity and gene library of QMZBS group is the most similar to the normal group, and the activity of QMZBS is better than that of the ZBS.To discuss the Lactobacilli diversity of intestinal microbiota in dysbacteria diarrheal mice is associated with activity of Qiweibaizhusan, the intestinal microbial metagenomics were extracted and then were PCR amplificated using specific primers. The diversity of PCR products were analyzed by using amplified rDNA restriction analysis (ARDRA). The results showed that the OTUs of lactobacilli of normal and QMZBS group were more than the ZBS group. The diversity, uniformity and dominance of Lactobacillus are recovered by treated with QMZBS and ZBS. The results of cluster analysis and principal component analysis also showed that QMZBS and ZBS play a certain role on the adjustment of intestinal Lactobacilli, and QMZBS has a superior effect to ZBS.After isolation of 43 yeast strains from mouse intestine, a yeast strain Y1 which was specifically promoted by QMZBS was identified by morphological, biochemical characteristics and molecular technology. The yeast colonies were milky white, homogeneous, form pseudohyphae, budding breeding for multiterminal, and could ferment glucose and D-xylose, but could not obtain amyloid substance. They could assimilate glucose, sucrose, D-xylose, fructose, sorbitol, without using of potassium nitrate and urea, and the urease test was negative. The homologies of 18SrDNA sequence between Yl (GenBank accession number:JQ217075) and Pichia burtonii (GenBank accession number: AB158656) was 99%,26SrDNA sequences of Y1 (GenBank accession number:KC192660) and Pichia burtonii (GenBank accession number:U45712.1) shared homology of 100%. A phylogenetic tree was constructed by comparing with the published 18SrDNA and 26SrDNA sequences of relative yeast species, and the Y1 was found to be the closest relative to Pichia burtonii with more than 99% sequence similarity. The strain Y1 is Pichia burtonii.