| Oviductus Ranae, one kind of traditional genuine regional drug in Changbai Mountain area, derived from the dry oviduct of female Rana temporaria chensinensis David. Oviductus Ranae has the effects such as nourishing yin and moistening lung,replenishing kidney and essence, strengthening the spleen and stomach and so forth.Modern pharmacological research also proves that Oviductus Ranae also has good efficacy on aspects of improving the immune function, anti-aging, anti-fatigue,relieving cough and asthma. The main effective constituents of Oviductus Ranae,1-methylhydantoin and steroidal constituents, were studied in this paper. The chemical composition, identification, quality control and pharmacokinetics of effective constituents of Oviductus Ranae were also investigated, which provides a basis for further study of quality control and clinical application of effective constituents of the medicine.1 Separation of main effective componentsThe main effective components, steroidal constituents, of Oviductus Ranae were separated by column chromatography combined with preparative high performance liquid chromatography in this paper and their chemical structure were identified by spectroscopic techniques. The separated steroidal constituents were assigned to stigmasterol, cholest-4,6-dien-3-ol, 7-dehydrocholesterol, and campesterol,respectively. The four compounds above were all separated from Oviductus Ranae for the first time.Cholesterol, 7-hydroxycholesterol and 7- ketocholesterol in Oviductus Ranae were prepared by high-speed countercurrent chromatography and their purity was 95.9%, 96.5% and 97.2%, respectively.2 Study on quality control2.1 TLC identification method of Oviductus RanaeA new thin layer chromatography(TLC) identification method of Oviductus Ranae was established in this study, which was superior to previous method. More reference substances,7-hydroxycholesterol and 7-ketocholesterol, were used in this method and the color development of those substances was visible by mean of 254 nm ultraviolet lamp and 10% sulfuric acid ethanol. Meanwhile, the resolution between each substance was good. The proposed method also has good specificity and was fast,accurate and of low cost. It can be used as a reference method of Oviductus Ranae identification and can make a complement to the absence of TLC identification inChinese pharmacopoeia.2.2 The establishment of fingerprint of Oviductus RanaeFingerprint of Oviductus Ranae was established by analyzing 14 batch of Oviductus Ranae from different places of production by way of HPLC-UV method in this paper. And the common peaks has increased to 33 compared to 12 of original method through method optimization. Meanwhile, Five common peaks of main effective components of Oviductus Ranae were identified and were assigned to1-methylhydantoin, cholesterol, 7-ketocholesterol, 7-dehydrocholesterol and stigmasterol, respectively. The proposed method has been improved significantly compared to existing fingerprint method. The above method has good specificity as well as good accuracy and was of great significance to the identification of Oviductus Ranae medicine. It can evaluate the quality of Oviductus Ranae completely and provides theoretical basis for quality control of Oviductus Ranae2.3 Determination of steroidal constituents in Oviductus Ranae by QAMS methodA high performance liquid chromatography method in which cholesterol was used as internal standard was developed to determine the relative retention time and relative correction factors between 7-hydroxycholesterol, 7-ketocholesterol,4-cholesten-3-one, stigmasterol, 7-dehydrocholesterol and cholesterol to and the contents of 7-hydroxycholesterol, 7-ketocholesterol, 4-cholesten-3-one, stigmasterol,7-dehydrocholesterol were calculated in the meantime. The quantitative analysis of multi-components(QAMS)of six steroidal constituents in Oviductus Ranae can be achieved by way of the proposed method. The contents of six steroidal constituents were determined by external standard method at the same time. Then results of the two method were compared together. It was found that there is no significant difference between the results of two methods. The proposed QAMS method was proved to be accurate and feasible according to methodological experiment and can be applied to determine the contents of steroidal constituents in Oviductus Ranae without using other standards in addition to cholesterol.3 Pharmacokinetics study3.1 Study on the stability of 1-methylhydantoin in gastrointestinal tractA rapid, simple and stable method was established to determine the stability of1-methylhydantoin in artificial gastric juice and intestinal juice. The determinationresults of stability of 1-methylhydantoin at the concentration of 3ug/mL、10ug/mL、15ug/mL in artificial gastric juice and intestinal juice within 24 h were compared. The results indicated that little 1-methylhydantoin was degraded and degradation was not more than 3% within 24 h. It can be predicted that 1-methylhydantoin was relatively stable in gastrointestinal tract after oral administration and can hardly be influenced by degradation. The result of the study can provide a evidence for pharmacokinetics study.3.2 Study on plasma concentration of 1-methylhydantoinPharmacokinetics of 1-methylhydantoin was investigated in this paper for the first time. The Pharmacokinetic parameters of 1-methylhydantoin was calculated and analyzed by DAS 2.0 software after intragastric administration at the dose of 20mg/kg,50mg/kg, 100mg/kg and tail vein injection administration at the dose of 3mg/kg. It was found that the absorption of 1-methylhydantoin was quick in vivo. Plasma peak concentration reached at about 1h after administration. Elimination of the substance was also fast with a half-life of about 1h. The determination result of biological availability was 18.84 ± 2.21% after calculation. The plasma concentration of1-methylhydantoin in rat was studied in this paper for the first time, which can provide foundation for further research of potential drug and was helpful to the clinic application of 1-methylhydantoin.3.3 Study on plasma protein binding rate of 1-methylhydantoinA new method was developed to determine the plasma protein binding rate of1-methylhydantoin for the first time. The determination was carried out at the concentration of 0.2ug/mL、5ug/mL and 50ug/mL. The plasma protein binding rate of1-methylhydantoin was 24.53%, which indicated that 1-methylhydantoin was substance with low plasma protein binding rate. This result was in accordance with the result of short half-life. The study also found that the plasma protein binding rate of 1-methylhydantoin was independent of determined concentration.3.4 Study on tissue distribution of 1-methylhydantoinAn efficient, fast and accurate method was developed to investigate the issue distribution of 1-methylhydantoin for the first time. Methodological experiment were carried out according to guidelines for the determination of biological products issued by SFDA and the specificity, precision, stability, accuracy and recovery results all met the requirement. The content of 1-methylhydantoin in heart, liver, spleen, lung,kidney, stomach, small intestine and brain were determined at 0.5h, 1h, 2h, 4h, 6h,12 h after intragastric administration at the dose of 50mg/kg. The result showed that1-methylhydantoin can distribute to each organs of rats rapidly at 0.5h. The concentration of 1-methylhydantoin in each organs reached to peak at 1h. The concentration of 1-methylhydantoin in each organs reduced by half at 3h. At 6h, only small amount of 1-methylhydantoin in each organs was remained, which showed that most of 1-methylhydantoin had been eliminated. 1-methylhydantoin in each organs can hardly be detected at 12 h, which indicated that 1-methylhydantoin had been eliminated completely. The results above showed that 1-methylhydantoin was not easy to accumulate in each organs of rats.3.5 Study on excretion of 1-methylhydantoinIt was found that total excretion of 1-methylhydantoin in rat urine and feces was19.222% of total dose after intragastric administration at the dose of 50mg/kg. About15.583% ± 4.483 of 1-methylhydantoin was excreted by urine and about 3.369 ±0.714% of the drug was excreted by feces, which indicated that 1-methylhydantoin was mainly excreted by urine. This may be related to the high water solubility of 1-methyl hydantoin. Only 19.222% of the drug was excreted as unchanged1-methylhydantoin, which showed that biological transformation of1-methylhydantoin that can change its own structure may exist in complex internal environment. |
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