Construction And Application Of Quality Evaluation Method Of Ranae Oviductus Based On Anti-aging Biological Potency

Objective:At present,the quality evaluation methods of Ranae Oviductus（RO）can only be based on empirical judgment.There is no quality evaluation method of RO in Chinese Pharmacopoeia.Its components are complex,the material basis of efficacy is not clear,and it is difficult to correlate efficacy and components.It is difficult to achieve a breakthrough in the quality control of RO by taking the composition as the index alone.This study intends to establish the quality control and evaluation model and method of RO based on literature research and anti-aging biological potency,so as to supplement and improve the current quality control and evaluation system of RO.Methods:（1）By consulting the historical materials of RO recorded in domestic dynasties,this paper systematically combs the evolution process of RO based protozoa records and the development law of RO efficacy,and clarifies the species,functions and curative effect of RO based protozoa.（2）Taking the acute aging model induced by H₂O₂and spontaneous replicative aging model of MRC-5 cell as the research object,the cell viability was detected by CCK-8 method,the optimal dosage was selected,and then the effect of RO pretreatment on aging MRC-5 cell was investigated SA-β-gal on the positive rate of staining.The changes of cell cycle were analyzed by flow cytometry.The effects of RO on the levels of superoxide dismutase（SOD）,malondialdehyde（MDA）and glutathione peroxidase（GSH-Px）in aging MRC-5 cells were detected by kits.The expressions of protein aging related proteins p16,p53,Id-1 and p21 were detected by Western blot.（3）The subacute aging rat model was established by subcutaneous injection of high-dose D-galactose（D-gal）for 8 weeks,and the anti-aging effect of RO was evaluated based on the model.The experiment was divided into blank group,model group,positive control group(Vc 200mg·kg^-1)and low,medium and high dose groups of RO(crude drug doses 100,200 and 400mg·kg^-1).The effect of different doses of RO on cognitive ability of aging mice was evaluated by behavioral experiment.On this basis,the effects of serum biochemical indexes SOD,MDA and GSH-Px were detected.The morphological changes of liver,heart,brain,kidney and lung were observed by H&E staining.At the same time,the expression levels of p53 and p21 in hippocampus of mice in each group were detected by immunohistochemical staining,and the expression levels of p53 and p21 protein in brain were detected by Western blot.（4）Taking the relative proliferation rate as the evaluation index,the"qualitative reaction parallel line method"was used to establish the biological potency determination method of RO based on anti-aging activity.The methodological investigation was carried out to determine the biological titer and confidence limit of 12 batches of RO.At the same time,the content of 1-methylhydantoin,swelling degree and total protein of RO from various producing areas were measured,the correlation difference with biological potency was calculated,and the correlation between each index and anti-aging biological potency was evaluated.Results:（1）The name of RO transliterated from Manchu in the early Qing Dynasty,referring to Cambaroides.By the middle and late Qing Dynasty,the name of Rana dybowskii Günther as Hasima was gradually accepted by Manchu and Han cultures.The character description of R.dybowskii,a primitive RO,began in Shengjing Tongzhi.Combined with its morphological description and production area distribution,it is now R.dybowskii.As far as the efficacy of RO is concerned,since the Manchu and Qing Dynasties,RO has been mainly used to“tonify the kidney”,which is used by doctors and people to nourish and delaying senescence.It is not the“treatment of childhood tuberculosis,thin and chancre”contained in mountain clam.（2）The detection of cell viability showed that H₂O₂could cause aging damage to MRC-5 cells,and the degree of damage increased in a dose-dependent and time-dependent manner when the concentration of H₂O₂was 50μM.After incubation for 24 h,the maximum injury degree was basically reached,and the proportion of Ki67 positive cells was significantly lower than that in the control group（P<0.01）.With Vc as the positive control,RO could antagonize acute aging and replicative aging injury after intervening in MRC-5 cell culture,and the effect increased in a dose-dependent manner,reaching 250μg/ml was the best（P<0.01）.RO pretreatment can reduce the acute aging cells and replicative aging cells SA-β-gal activity.Compared with the acute model group,the S-phase of cell growth period pretreated with RO and Vc increased significantly（P<0.05）,indicating that RO can slow down the aging of MRC-5cells by increasing the rapid cell growth period（S-phase）.The test kit showed that RO could effectively increase the activity of SOD（P<0.05）,the activity of GSH-Px（P<0.01）and reduce the level of MDA（P<0.01）.The expression of aging related proteins was detected by Western blot.With the increase of RO drug concentration,the expression of Id-1 protein increased in a concentration dependent manner（P<0.01）,and the expression of p16,p21 and p53 protein decreased in a concentration dependent manner（P<0.01）.（3）The subacute aging model of mice was constructed by D-gal.compared with the blank control group,the aging symptoms of mice in the model group were obvious.It was confirmed by Morris water maze experiment,open field experiment and tail suspension experiment that the aging mice had significant differences in behavior from the blank group,while the aging symptoms of mice in each administration group were significantly improved.RO can increase the activities of serum SOD and GSH-Px induced by D-gal and reduce the level of MDA in a dose-dependent manner.The results of H&E staining showed that compared with the control group,the tissue damage of liver,heart,brain,kidney and lung in the model group mice was serious,and the morphology and arrangement of the tissue sections of mice in the medium and high dose RO groups were significantly improved.It can be seen that RO effectively improved the organ tissue damage caused by aging of mice stimulated by D-gal.Immunohistochemical staining of mouse hippocampus showed that the expression levels of p53 and p21 protein in hippocampus of model group were increased.After RO was given to model mice,the expression levels of p53 and p21 proteins in mouse hippocampus decreased.The expression levels of p53 and p21 protein in the brain tissue of mice in each group were detected by Western blot.The expression levels of p53 and p21 protein in the brain tissue of mice in the model group were significantly higher than those in the control group;Compared with the model group,the expression levels of p53 and p21 protein in the brain of mice in medium and high dose RO group were down regulated.（4）The detection method of RO anti-aging biological titer was established,and the RSD value met the requirements.12batches of samples at home and abroad were tested.The anti-aging activity potency of RO medicinal materials from different producing areas ranged from 218.03 to 1152.04 U·mg^-1.The potency of RO medicinal materials produced in Jingyu,Huadian and other areas were better than those from other producing areas.In terms of quality distribution,there was an obvious distribution trend of gradual attenuation from the hinterland of Changbai Mountain to the surrounding areas.There were significant differences in 1-methylhydantoin,swelling degree and total protein content of RO from different producing areas.Pearson correlation analysis showed that the total protein content of RO was significantly positively correlated with the anti-aging titer index（r=0.931）.Conclusion:Based on literature research,this study clarified the historical evolution of RO,corrected the basic origin of RO,and determined its traditional effect of Tonifying the kidney and anti-aging.Based on the acute aging model of MRC-5 cells,the spontaneous replicative aging model and the aging model of mice induced by D-gal,it is confirmed that RO has the direct effect of anti-aging.Based on the above research,the anti-aging biological titer detection scheme of RO is established,which can clearly distinguish the quality differences of RO,reflect its“main functions”as much as possible,make up for the defects of the current quality control of RO,and provide new ideas and methods for the quality control and quality evaluation of other kidney tonifying herbs.