| Traditional Chinese Formula is an important part of traditional medicine theories.However, due to the complex composition and lacking of modern technology analysisof Traditional Chinese Formula, it becomes the obstacles of the development and theinheritance of Traditional Chinese medicine. Therefore, it is extremely significant tostudy the Traditional Chinese Formula systematically through modern analyticaltechniques to promote the globalization and modernization of Traditional Chinesemedicine. We studied the chemical constituents and the transport process in vitro intwo classic Chinese Formula Gancaofuzi decoction and Wutou decoction for thetreatment of rheumatism. The main research content includes several aspects asfollows：1. Chemical profiling of Gancaofuzi decoction by UPLC-Q-TOF-MS.We established the method for separation of Gancaofuzi decotion at the positiveand negative ion mode separately by UPLC-Q-TOF (ultra performance liquidchromatography coupled with quadrupole time-of-flight mass spectrometry),72compounds was detected. We identified the compounds by the precise molecularweight (with error less than15ppm) and the pieces obtained from MSEsecondary ionmass spectrometry according to the regular fractures in the references and the existingstandards.17flavones and flavone glycosides,13triterpene saponins,25alkaloids,4phenolic acids and1lactone were identified. The others were not identified. Flavones,flavone glycosides and triterpene saponins were derived from R. glycyrrhizae.Alkaloids were derived from processing aconite. Phenolic acids and lactone belongedto R. cinnamomi and A. macrocephalae Rhizoma separately. The results above laid thefoundation for the research in vitro transoport later.2. Study on the flavonoids in the Gancaofuzi decoction.We detected the quantity changes of the flavonoids extracted in single herbextract, herb-pair extract and Gancaofuzi decoction by the way of UVspectrophotometer and HPLC-MS. We mainly focused the changes of flavonoids in R.glycyrrhizae. When R. glycyrrhizae was decocting together with A. macrocephalaeRhizoma, the total quantity of flavonoids and the content of liquiritin in R. glycyrrhizae both increased. It is indicated that A. macrocephalae Rhizoma canimprove the dissolution of flavonoids in R. glycyrrhizae which is confirmed by thedecrease of flavonoids in the dregs of the herb-pair. The rich alkaloids in processingaconite and the flavonoids became precipitate and led to the reduction of flavonoids inR. glycyrrhizae. Changes in the dregs of processing aconite and R. glycyrrhizae wereconsisting with that. When R. glycyrrhizae was decocting together with R. cinnamomithe content of liquiritin in R. glycyrrhizae decreased. Peak areas of compounds in theGancaofuzi decoction showed an increasing trend comparing with the single herbdecoction and herb-pair decoction. It is easy to clarify that the compatibility ofChinese medicinal herbs.3. Study of purification process and antioxidant of flavonoid constituents inGancaofuzi decoction.AB-8, DM130macroporous resin and polyamide resin were applied to screen ofthe best conditions for attaining the most pure and the highest recovery of flavonoidconstituents in Gancaofuzi decoction. The optimum approach was to use DM130macroporous resin combined with polyamideresin which could obtain the flavonoidconstituents at the purity of47.9%and the recovery of47.0%. As we known, freeradicals contributed to the occurrence and development of rheumatic diseases. So westudied the antioxidant ability of flavonoid constituents gained from the formerapproach in R. glycyrrhizae, herb-pairs containing R. glycyrrhizae and Gancaofuzidecoction. The research methods included the evaluation of DPPH, superoxide anion, OH and FRAP. It was observed that the antioxidant capacity of flavonoidconstituents in Gancaofuzi decoction was better than Gancaofuzi decoction. Theantioxidant capacity of flavonoid constituents in herb-pair R. cinnamomi and R.glycyrrhizae was better than other herb-pairs. It is possible that the phenolic acidscontributed to the function of antioxidant which implies the importance role of R.glycyrrhizae in Gancaofuzi decoction.4. Study of intestinal transport of the flavonoids in Gancaofuzi decoction in Caco-2monolayersWe established the Caco-2cell monolayer which is simulated with intestinalepithelial cells to study the flavonoids in Gancaofuzi decoction. Three flavonoidsincluding liquiritin, liquiritin apioside and liquiritigenin were detected in the transportprocess. Comparing the results of the mixture of two herbs decoction and theherb-pairs, it is showed that the changes in the content of R.glycyrrhizae in the decoction influenced the transport of the above three flavonoids. A. macrocephalaeRhizoma could improve the bioavailability of the three flavonoids according to thefunction of increasing the flavonoids in R. glycyrrhizae when boied together. Thealkaloids in processing aconite increased the bioavailability of the three flavonoids.While in the Gancaofuzi decoction, the improvement of the bioavailability of theflavonoids was even higher than the R. glycyrrhizae decoction and the mixture of eachone decoction. The results confirmed the necessary of the Chinese Formula.Different ratios of flavonoid constituents, saponins constituents and alkaloidsconstituents in Gancofuzi decoction were studied in the Caco-2cell monolayers. Thesaponins constituents can promote the bioavailability of the three flavonoids in tworatios. The alkaloids constituents can increase the bioavailability of the threeflavonoids by reducing the efflux in the transport process. The three constituentsmixed together also showed the function of improving of the bioavailability of thethree flavonoids.Three main alkaloids in processing aconite were mixed with the flavonoidsconstituents separately in the transport process. BM exhibited to increase thebioavailability of the three flavonoids, while BH and BA did not. The flavonoidconstituents can improve the bioavailability of BM, BH and BA separately and furtherpromote pharmacodynamics.We also mixed the main standard to investigate the interactions in the three kindsof constituents. Five alkaloids increased the bioavailability of liquiritin, whileGlycyrrhizic acid decreased the bioavailability of liquiritin and liquiritigenin. Themixture of the three kinds standard improved the bioavailability of liquiritin, too.5. Study of intestinal transport of the alkaloids in the Wu-tou decoction with itscombination taboo by Caco-2Cell Culture System with UPLC/MS.The research in this part was to investigate the effect of Fritillariae CirrhosaeBulbus, Fritillariae Thunbergii Bulbus, Pinelliae Rhizoma in different ratios withWu-tou decoction detecting the therapeutic effects in Wu-tou decoction of main7alkaloids including benzoylaconitine (BA), benzoylmesaconitine (BM),benzoylhypaconitine (BH), hypaconitine (HA), fuziline (FU), niaolin (NE) anddeoxyaconitine (DA) through UPLC-MS. The permeability of aconitum alkaloidsextract through a Caco-2cell monolayer was analyzed in the absences and presencesof Fritillariae Cirrhosae Bulbus, Fritillariae Thunbergii Bulbus, and PinelliaeRhizoma, respectively. The results showed that Pinelliae Rhizoma could reduce the absorption of the alkaloids and increase the excretion of the alkaloids, which wouldattenuate the therapeutic effects of Wu-tou decoction. Therefore, Pinelliae Rhizoma isan incompatible herb of Wu-tou decoction because of the inhibition of the absorptionof alkaloids in the intestine. And that Fritillariae Cirrhosae Bulbus and FritillariaeThunbergii Bulbus showed the effects to improve the permeability of the alkaloids inWu-tou decoction. These effects of these two herbs were similar, but the former wasstronger than the latter, which most likely is due to the fact that the compositions ofthese two traditional Chinese medicines are similar. The in vitro data suggests that thecompounds such as fritillary presented in alkaloids in the formula maybe improve thetherapeutic function caused by the increased bioavailability of alkaloids in intestine.6. Study of intestinal transport of the alkaloids in the Wu-tou decoction with PinelliaeRhizoma and Pinelliae Praeparatum Rhizoma by Caco-2Cell Culture System withUPLC/MS.Pinelliae Rhizoma and Pinelliae Praeparatumm Rhizoma were both detected todecrease the bioavailability of alkaloids in Wu-tou decoction. We added verapamiland MK-571to the Q, QX11and QF11so as to investigate the mechanism. Throughcomparing the differences in the transport, the influence of QX11and QF11on theefflux was not as obvious as Q. It is indicated that some products in PinelliaeRhizoma and Pinelliae Praeparatum Rhizoma may compete with P-gp inhibitor sites.Therefore the role of verapamil weakened, resulting the bioavailability of alkaloids inWu-tou decoction reduced. |
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