Quality Analysis Of Venenum Bufonis And Study On Its Crude And Processed Fingerprints

Objective:Study on the component extraction technology, contents, processing technology, fingerprints of Venenum Bufonis which come from Shan Dong, He Nan, He Bei and so on. And research the changes of their fingerprint of the crude and processed. To build up the best method to extract and a method to determine the Venenum Bufonis contents Cinobufagin and resibufogenin. To establish the best wine processing method, the TLC fingerprint method and the HPLC fingerprint method of Venenum Bufonis. Compare with various processed samples and the crude to find out the differences by fingerprint methods, in order to offer scientific basis to standardize the processing technology, control the quality and for further develop and apply of Venenum Bufonis.Method:Measure the total extraction rate of Cinobufagin and Resibufogenin by HPLC to investigate the best extraction technology through selecting different solvents by heating reflux or ultrasonic extraction, designing single factor exploration of different ethanol concentration, and applying the L₉(3⁴) of orthogonal test.Measure the content of Cinobufagin and Resibufogenin by HPLC.Study the wine, milk and talcum powder as the processing excipient in single factor experiment. Select the wine as the suitable excipient. Then apply the L₉(3⁴) of orthogonal test and determine the total content of Cinobufagin and Resibufogenin by HPLC to build up the optimal processing technology.To analysis 12 groups Venenum Bufonis by TLC method.To measure 12 groups Venenum Bufonis by HPLC method and gradient elution, and analysis the fingerprint by the computer similarity evaluation system.Compare with various processed samples and the crude by TLC & HPLC fingerprint methods to find out the differences between before and after processed.Result:1. The optimal extraction technology of Cinobufagin and Resibufogenin of Venenum Bufonis is: add 50 times 80% ethanol and heating reflux twice, 90min per time.2. A new method for measuring the quality of Cinobufagin and Resibufogenin is established. 12 groups of Venenum Bufonis quantitative determination results show that, the contents circumscription of Cinobufagin is 0.937% to 6.520%. And the contents circumscription of Resibufogenin is 0.198% to 7.550%.3. The contents of Venenum Bufonis are both decline after wine, milk and talcum power processing. Compared with the milk processing and talcum power processing, the change of Venenum Bufonis component is more stable by wine processed. Choose wine as the processing excipient for further study. The results of the orthogonal test, the signal factor experiment of different ethanol concentration and the verification test show that the optimal processing technology is: add 2 times 55% ethanol (wine), heating processing in 60℃for 12 hours, aired and comminuted. Compare to the crude Venenum Bufonis, the quality of the effective component are decrease about 20% after wine processing.4. TLC fingerprint developing solvent composition is cyclohexane, dichloromethane and acetone. Proportion is 2:1:1. Developed two times and the spots were clear. There are 12 spots can be saw under the 365nm ultraviolet light and 9 spots can be seen under the visible light. Separation degree of the spots is great. Different origins of Venenum Bufonis samples have the same spots number. But the color and the size of the spots have difference according to different origin.5. HPLC fingerprints of 12 groups Venenum Bufonis consisted of 26 common characteristic peaks. It separate into two areas. One area is consisted of 9 common peaks. Its retention time range is 0-20 min. Different Venenum Bufonis origins have the similar peak appearance in this area. Another area is consisted of 17 common peaks. Its retention time range is 20-40 min. It is the mostly important characteristic area. The similarity degree of 12 groups samples both over 0.9, which has a great integer similarity degree.6. The TLC fingerprints of different processing samples have similar TLC action with the crude samples. The wine processing samples and the milk processing samples of the same group Venenum Bufonis both have the same spots numbers with the crude ones from different groups. But the color is weaker than the crude one. The spots of talcum powder processing samples are weakest, and some have taken great changed. It has the same phomonena in different groups. After different concentration ethanol processing of the same group, the samples have the same spot numbers with the crude ones, and the spot color is weaker than the crude ones. Each wine processing sample does not have evidence differences.7. The HPLC fingerprints of different processing samples have similar action with the crude samples. Both have the common peaks and the retain time and the relative retain time are identically with the crude ones. But there is great discrimination in the intension of the common peaks. The relative peak areas of the common peaks are great changed after the milk processing and talcum powder processing while which have less changed after the wine processing samples of the same origin. It has the same phomonena in different groups. After different concentration ethanol processing to the same origin group Venenum Bufonis, both the relative retain areas of common peaks are decreased except the No.6 peak.Conclusion:1. Compared to the extraction method of Venenum Bufonis contents in《Chinese Pharmacopoeia》, the new established extraction technology of this study has a higher extracting rate. This method can extract the Venenum Bufonis components more sufficient.2. Compared to the determination method of Venenum Bufonis contents in《Chinese Pharmacopoeia》, the new established determination method of this study is better.3. It makes the processing parameters into specific and identify. It offers basis for further study of the Venenum Bufonis processing.4. The TLC fingerprint can separate several components of the Venenum Bufonis. It can be used to identify Venenum Bufonis and evaluate its quality.5. The HPLC fingerprint can systematic analysis the hold components of Venenum Bufonis. It can be used to evaluate the Venenum Bufonis quality and offer abundant information to control its quality.6. Both the TLC and HPLC fingerprint methods can be used to evaluate the change between the processing and the crude of Venenum Bufonis. The results show that the content of most components are delined after processed. It is the likely reason that the toxicity become lower after wine processing.7. Preliminary establishment of quality standards of Venenum Bufonis processing.If it could be further comfirm, it would be offer a reference basis to the quality control of Venenum Bufonis processing in the vocation.