| Objective::Spatholobi Caulis is the dried rattan of Spatholobus suberectus Dunn(Leguminosae),as a traditional Chinese herbal medicine,is mainly distributed in guangxi,Guangdong and Yunnan provinces of China.It has traditional efficacy of enrichment the blood,promoting blood circulation and activating collaterals.Modern pharmacological research show that the Spatholobi Caulis has function of antithrombotic,anti-tumor,lipid-lowering,anti-inflammatory,anti-oxidation,anti-virus,antibacterial and so on.Spatholobi Caulis varieties used in the market is relatively complex,in addition to the"Chinese pharmacopoeia" contains,having other varieties of plants,their chemical composition is different,at the same time because of the quality of wild and cultivated without distinction,harvesting time,different processing methods,storage transportation,make medicinal materials quality is uneven,lead to the quality of Spatholobi Caulis is difficult to control,restricted the product internationalization.Spatholobi Caulis has no content determination item in Chinese Pharmacopoeia(2015).To ensure quality control of Spatholobi Caulis,the study established the quality control mode,fingerprint in combination with the more contents determination,so that the Spatholobi Caulis medicinal materials quality control,at the same time also ensure to Spatholobi Caulis for the dosage and proprietary Chinese medicine is safe and effective.In recent years,the zebra fish,with its convenient,fast,low cost,high throughput screening become research hot spot,so this experiment based on zebra fish model validation of Spatholobi Caulis antithrombotic activity,screening the antithrombotic activity of parts at the same time,provide scientific basis for its clinical application.Method:(1)To establish a HPLC method for the fingerprint analysis of Spatholobi Caulis using HPLC method and pattern recognition research(similarity evaluation,cluster analysis and principal component analysis).(2)To establish a HPLC method for simultaneous determination of protocatechuic acid,catechol,L-epicatechin,liquiritigenin,formononetin and medicarpinin Spatholobi Caulis,and to offer reference for improvement of the standard of the drug.(3)To analysis of the chemical composition of Spatholobi Caulis with the UPLC-Q-TOF/MSE method combined with UNIFI data,lay the foundation for the research of its efficacy material base.(4)To screening the antithrombotic activity parts of Spatholobi Caulis using the zebra fish thrombosis model induced by adrenaline hydrochloride,provide theoretical data for its clinical application.Results:(1)The HPLC fingerprint of 17 batches of Spatholobi Caulis medicinal materials and 4 batch of Spatholobi Caulis mixed was established,and there were significant differences between them.Spatholobi Caulis HPLC fingerprint was identified 35 common peaks,identified six common peaks through the standard recognition.And using similarity evaluation,cluster analysis,principal component to analysis the 17 batches Spatholobi Caulis.According to the results,17 batches the herbs of 4 regions have different quality.The different origins of medicinal materials have bigger difference,the same origin of medicinal materials also has differences,but the overall similarity is higher,compared with the standard map,each batch of similarity in the range of 0.816-0.968,similarity above 0.9 medicinal accounted for 64.70%.The results of cluster analysis and principal component analysis provided that S5 producing area in guangxi,Yunnan origin of S11,S16,Vietnam origin S15 quality is better.(2)The study established a HPLC method for simultaneous determination of protocatechuic acid,catechol,L-epicatechin,liquiritigenin,formononetin and medicarpinin,and determinated the six chemical composition content of 17 batches Spatholobi Caulis,The content ranges of protocatechuic acid,catechol,L-epicatechin,liquiritigenin,formononetin and medicarpinin Spatholobi Caulis were 0.250-0.898 mg·g-1,1.447-5.662 mg·g-1,1.759-11.925 mg·g-1,0.035-0.134 mg·g-1,0.218-0.535 mg·g-1 and 0-0.585 mg·g-1,respectively.Methodology investigation show that the method precision,repeatability,stability and recovery tests are in accordance with Chinese Pharmacopoeial can offer reference for improvement of the standard of the drug.(3)A total of 17 compounds were identified from Spatholobi Caulis with the UPLC-Q-TOF/MSE method combined with UNIFI data,and speculated the pyrolysis rule of the flavonoids in Spatholobi Caulis.(4)Adrenaline hydrochloride induced zebra fish thrombus model is verified by the experiments,and using the model to screen the antithrombotic active site of Spatholobi Caulis,the results showed that the water extract of Spatholobi Caulis,ethanol extracts,ethyl acetateextract,dichloromethane extracts under certain concentration have certain antithrombotic activity,but the water extract from ethanol extracts,ethyl acetate extract of dichloromethane extracts of thrombosis inhibition rate is bigger,stronger antithrombotic activity,When the concentration of exposed to 20 μg/mL,water extract group antithrombotic rate is as high as 82.96%.which may be the water extract of tannins or glycosides compounds have stronger antithrombotic activity.Conclusion:This study established a quality control model which HPLC fingerprint combined with six kinds of chemical composition determination of Spatholobi Caulis,and it comprehensive and effective evaluated the Spatholobi Caulis quality and provided scientific data for Chinese pharmacopoeia to contain the drug.A total of 17 compounds were identified from Spatholobi Caulis with the UPLC-Q-TOF/MSE method combined with UNIFI data,and speculated the pyrolysis rule of the flavonoids in Spatholobi Caulis.Adrenaline hydrochloride induced zebra fish thrombus model is verified by the experiments,and using the model to screen the antithrombotic active site of Spatholobi Caulis,providing providing scientific data for the clinical application of Spatholobi Caulis and providing other antithrombotic drugs screening with new ideas,new methods. |
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