Study Of Liposoluble Compounds In Cinobufacini Injection And Primary Hepatic Metabolism In Vitro

BackgroundCinobufacini injection is a boiling-water extract of the dried toad skin (Bufo bufo gargarizans Cantor) skin, which is widely used for pathopyretic ulcer, stasis and edema in china. It is called a most effective injection of traditional chinese medicine, showing potent inhibiting effect on kinds of malignant tumor. Single cinobufacini is good for inhibiting the growth of mid-term or later-term tumor, improving the quality of patients’life with less side effect. When comes to the associated drug in therapy, cinobufacini also shows more potent antitumor effect, strengthening curative effect and alleviating side effect. According some research finding around cinobufacini injection, four main active constituent are described. One of the constituent is indole alkaloids, including bufotenine, dehydroxylation bufotenine, bufothionine and dehydroxylation bufothionine. Another constituent part is antitumor bufadienolids, including bufalin, bufotalin, cinobufagin and telocinobufagin. The third part is mixed peptides with molecular weight ranging from700to5000dalton. Peptides is considered to show analgesic effect and inhibit tumor in some degree. The last part is nucleic acids, including adenine, guanine, thymine, and uracil.Most recent research are focused on the bufadienolids of toad skin and Senso. But few is on the liposoluble components in cinobufacini injection. Considering the unknown antitumor constituent in cinobufacini injection, we make a systemic study on the chemical constituent of liposoluble components in cinobufacini injection and do some pharmacodynamics in vitro with preliminary investigation. Meanwhile, we analysis the metabolites from the incubation system with eight bufadienolides. Our destination is to interpret the pharmacological foundation of antitumor in cinobufacini injection.Aims1To do further research on extraction, separation and identification of liposolublecomponents in cinobufacini injection by advanced phytochemistry, interpreting the pharmacological foundation of antitumor.2Screening antitumor constituent in vitro depending on the isolated bufadienolids, finding the connection between its’chemical structure and its antitumor activity.3Doing incubation experiment on animals’liver microsome, depending on eight isolated bufadienolids to elucidate the metabolic pattern between bufadienolids and its metabolites as well as to discriminate the difference in different kinds of animals’microsome incubation system.Methods1Using silica gel, reverse phase C18silica gel, sephadex LH-20, preparation liquid chromatography and some other modern chromatographic technique to separate main chemical constituent and then identify isolated constituents by HPLC-MS and NMR data.2Using HPLC-Solarix FT-ICR-MS online analysis and reported document to derive the chemical compounds in liposoluble parts of cinobufacini injection with the fragmentation pattern of known compounds.3Comparing the bufadienolides’antitumor activity by MTT colorimetry to study their structure-cytotoxic activity relationships.4Searching possible metabolic pathway by incubating with animals’microsome in vitro and detecting metabolism by UHPLC-LTQ-Orbitrap MS.Results121compounds are isolated from cinobufacini injection and18compounds are identified finally.2In the antitumor activity study of ten bufadienolides, we find decrease in antitumor activity when oxygen ring exists on C14-C15. When acetyl is on, the activity differs with the oxygen ring. We also see it potent antitumor activity when compounds contain much hydroxyl and carboxide.333compounds are derived by accurate LC-MS data, clarifing it’s liposoluble compounds basically.4Metabolic pattern and it’s metabolic difference is found preliminary in the metabolites according to the result from microsome incubation system in vitro.Conclusions1We clarified the compounds of liposoluble components in cinobufacini injection basically and did a preliminary study on the structure-activity relationship between bufadienolides and tumor cells.2We elucidated the possible metabolic pathway of bufadienolides in animals’liver microsome in vitro and compared the metabolic diversity between different kinds of animals’liver microsome.