| Objective: In order to select the most suitable pretreatment methods, culture media, dilution gradient and p H value for the soil microorganisms, samples from the root mud of Haikou Dongzhai port were isolated and purified in this research. Methods: Here, pretreatment methods including gradient dilution, air drying and differential centrifugation method were used to isolate the fungi and actinomycetes. In the media preparation, different ratio of carbon source,nitrogen source and trace element were prepared. These samples were diluted from 10-1 to 10-5concentration range using geometric dilution method. The medias were prepared to be p H value3 and 7 respectively. In the last step, the visual formand TLC were applied to remove the duplicate strains. Results: In our research, 84 strains of fungi and 77 strains of actinomycetes were obtained. Withinthese fungi, 72 strains were got from gradient dilution method, 4 strains were got from dry method, 1 strain was got from Dispersion and Differential Centrifugation(DDC). Therefore, the optimal treatment method was the gradient dilution method. Under different dilution gradient, we got 60 strains from 10-1 concentration condition, 6 strains from10-2 concentration condition, 3 strains from 10-3 concentration condition, 5 strains from 10-4concentration condition, 3 strains from 10-5 concentration condition. Therefore, the most suitable condition for isolatingthese fungi was 10-1 concentration condition. Under different p H value, 29 strains were obtained under acidic condition, and 43 strains were obtained under neutral condition. So neutral condition was the best condition to separate these fungi. Under different culture conditions, 23 strains were got from PDA culture medium, 13 strains were got from WJ culture medium, 13 strains were got from MY culture medium, 13 strains were got from GYP culture medium, 10 strains were got from MD culture medium. Thus PDA culture medium was the most suitable medium to separate these fungi. In 84 strains of actinomycetes, 82 strains were from gradient dilution method and 2 strains were from dry method. Therefore, the gradient dilution method was the bestmethod. Under different dilution gradient, we got 64 strains from10-1 concentration condition, 19 strains from 10-2 concentration condition, 1 strain from 10-3concentration condition. So the optimal dilution gradient was 10-1 concentration condition.Under different p H value, 6 strains were obtained under acidic condition, and 78 strains were obtained under neutral condition. Therefore, the neutral condition was the bestcondition. Under different culture conditions, 13 strains were got from PDA culture medium, 37 strains were got from WJ culture medium, 17 strains were got from MY culture medium, 9 strains were got fromGYP culture medium, 6 strains were got from MD culture medium. So the WJ culture medium was the most suitable culture medium. Conclusion: For the microbes which researched in our project, the most suitable methods to isolate and purify them were gradient dilution method,neutral condition and dilution gradient, WJ culture medium for actinomycetes strain and PDA culture medium for fungus.Objective: The aim of this study was to establish bromocriptine induced puerperal hypogalactia rat model and investigate the lactagogue effect of the aquatic extraction from lance asiabell root(ALAR), including the maternal lactation yield, offspring body weight increase, serum prolactin(PRL) level and histopathological changes in mammary/pituitary glands. Methods:Polygamous breeding was performed in 96 adult SD rats with gender ratio 3:1(Female/male) for 18 days, then male rats were removed,female rats were kept in individual cages. 56 female rats with similar parturition time(differences among were less than 24 hour) were included in subsequent experiments.The litter size was adjusted to eight for every maternal rat, and then the animals were randomly assigned according to the weight into control group, bromocriptine model group and ALAR groups(ALAR-Guizhou, ALAR-Anhui, ALAR-Hunan and ALAR-Shandong) and Ruquan granule(positive control) group. All animals except for those in control group received 0.5mg/kg/d bromocriptine starting from two days postpartum, ALAR groups and positive control group also received corresponding co-treatments(ALAR: 15 g/kg/d, Ruquan granule: 2.6 g/kg/d) for seven days. At the eighth day of experiment, ELISA was used to detect the serum levels of PRL in the rats of all the groups, mammary glands and pituitary glands were processed for histology and subjected to hematoxylin and eosin(HE) staining, and observed under optical microscope for histopathologcal changes. The daily maternal 3h lactation yield and offspring body weight increase were calculated. SPSS 19.0 was utilized for the statistical analysis. Results: 1. Significant lower lactation yield, offspring body weight increase and PRL levels were observed in the rats of bromocriptine model group(P<0.01), indicating successful establishment of puerperal hypogalactia rat model. Meanwhile, ALAR treatments remarkably increased the above parameters at the dose tested(P<0.05), demonstrating the lactagogue effects of ALARs. No statistical difference were observed among ALARs from the four origins(P>0.05). 2.Histopathology: Hyperplastic, lactating mammary gland were observed in control rats,with obvious lobules, plentiful acinar cells, dilated ducts, columnar duct epithelium and enlarged lumen filled with eosinophilic secretion. Multiple colostrum lobules were present. On the other hand, mammary glands in model group rats were mainly consist of fat tissue without clear lobules. Scarce and scattered acini tissues were observed; narrow ducts with little secretion were present. ALARs treatment resulted in similar histopathological results as the control group. Regarding to the pituitary glands, normal lactotropes were observed in control group rat pituitary glands, with round or elliptical nucleus, uniform nuclear chromatin and large amount of uniform sized, evenly distributed granules. In the model group rat pituitary glands, condensed nuclear chromatin was observed, along with small amount of ununiformly sized,irregularly distributed granules. A few abnormal granules were also present. No significant histological changes were observed in ALARs-treated rat pituitary glands comparing to control group. Conclusions: The bromocriptine-induced puerperal hypogalactia rat model was established, in which ALARs could exert lactagogue effect. The underlying mechanism is associated with the promotion of PRL secretion. |
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