| The flower of Abelmoschus manihot (Linn.) Medicus (family Malvaceae), a traditional Chinese medicine (TCM), is recorded as "HuangKui" in Chinese and has a broad spectrum of applications as anti-inflammatory, antibacterial anticoagulant, antioxidant effects, diuresis effects and protective effects against myocardial and cerebral ischemia injury, and protective effects on renal cellular membrane cells. It is used to treat several diseases such as chronic renal disease, diabetic nephropathy, mouth ulcers, and burns. The extensive phytochemical and pharmacological investigations of A. manihot revealed the major bioactive compounds to be the flavonoids such as hyperoside, isoquercitrin, hibifolin, myricetin, gossypetin, quercetin and quercetin-3'-O-glucoside.'HuangKui Capsule', mainly composed of A. manihot flower extract, has acquired approval from State Food and Drug Administration (SFDA) as class III new drug for treating chronic glomerulonephritis for many years. Till now,'HuangKui Capsule'has become one of the most commonly used Chinese medicines and has played an important role in treating chronic glomerulonephritis in clinical application.With a wide range of clinical applications, it is important to analyze dynamic process of A. manihot in vivo. To get a lot of biological information, clarify the characteristics of absorption and metabolism in vivo and elucidate mechanism and process of flavonoids from A manihot, in situ intestinal recirculation, technology of microdialysis combined with ultra-performance liquid chromatography/quadrupole timeof-flight mass spectrometry (UPLC-Q-TOF-MS), in vitro metabolism of intestinal flora, A UPLC-Q-TOF-MS method including MSE technology and automated data processing MetaboLynxTM software using the MDF approach were performed in this paper. The main objectives of this researeh were to study and clarify the absorption and metabolism of flavonoids from A. manihot, in order to guide clinical medication and design of dosage forms, improve drug efficacy and safety, evaluate the medicinal value.First, a detailed review was given to the research progress of A. manihot including chemical constituents, pharmacological effects, microdialysis sampling technique, the conventional absorption model and metabolism of flavonoids, it is helpful to analyze dynamic process of A. manihot in vivo. To explore the mechanism of the absorption of flavonoids from A. manihot flowers, in situ intestinal recirculation was performed to study the effect of the absorption at different concentrations, at different intestinal regions. To evaluate the conditions of the absorption of six flavonoids from A. manihot flowers, the concentrations of A. manihot in the perfusion solution were determined by HPLC at predesigned time. And we have investigated the inhibitory effect of six flavonoids from A. manihot flowers on P-glycoprotein (P-gp) drug efflux pump. The results demonstrated that the absorption rates of flavonoids from A. manihot flowers is not significantly different (P> 0.05) at various drug concentrations, the absorption of flavonoids from A. manihot flowers is a first-order process with the passive diffusion mechanism. The absorption rates of each of flavonoids are significantly different. The absorption rate of flavonoid glycoside was lower than that of aglycone; the flavonoids from A. manihot flowers could be absorbed in whole intestinal segments. The best part of intestine to absorb hyperoside and myricetin are jejunum and duodenum, respectively. Verapamil could enhance the absorption of isoquercitrin, hyperoside, myricetin, quercetin-3'-O-glucoside by inhibiting the P-glycoprotein (P-gp) drug efflux pump; Cow Bile Salt could increase significantly the absorption of isoquercitrin> hibifolin^ quercetin-3'-O-glucoside^ myricetin hyperoside. SDS also could increase significantly the absorption of isoquercitrin^ hibifolin quercetin-3'-O-glucoside> hyperoside; Lecithin and EDTA were no significant absorption-promoting effect (P> 0.05). Some absorption enhancers could reduce the absorption of each component such as lecithin, EDTA; These absorption enhancers were not significantly different to quercetin, suggesting that the absorption mechanism of quercetin may be not related to the absorption enhancers.Intestinal flora was used to study the metabolism of flavonoids from A. manihot in vitro. The results showed that constituents and their metabolites in human incubated solution include hyperoside, isoquercitrin, hibifolin, myricetin, quercetin, hyperoside hydroxylation, two hyperoside acetylation, quercetin di-hydroxylation, quercetin methylation, myricetin acetylation, and a metabolites of quercetin after loss of oxygen while constituents and their metabolites in rat incubated solution are hyperoside, isoquercitrin, hibifolin, myricetin, quercetin-3'-O-glucoside, quercetin, two hyperoside hydroxylation, two hyperoside acetylation, quercetin methylation, myricetin acetylation, and a metabolites of quercetin after loss of oxygen. In addition, we find that these flavonoid glycoside in incubated solution samples were hydrolyzed into aglycone. Our results indicated that hydrolysis and hydroxylation were the major metabolic pathways of flavonoids from Abelmoschus manihot extract in intestinal bacteria.To find the target organ of flavonoids from Abelmoschus manihot flowers, the distribution process of flavonoids from Abelmoschus manihot in rats in vivo was studied by intragastric administration. The results showed that the distribution of flavonoids from Abelmoschus manihot in rats in vivo was mainly in kidney and liver; they have a higher concentration in liver and kidney tissues, which is consistent with treat chronic renal disease and diabetic nephropathy. To find the potential active components of flavonoids from Abelmoschus manihot flowers, microdialysis combining with UPLC-Q-TOF/MS was applied to simultaneously identify metabolites in blood and kidney dialysis after oral administration of Abelmoschus manihot extract. The results showed that unbound constituents in blood circulation of the rat include hyperoside, isoquercitrin, hibifolin, myricetin, quercetin-3'-O-glucoside, quercetin, quercetin monoglucuronide-1 and quercetin monoglucuronide-2 while unbound constituents in kidney are hyperoside, isoquercitrin, quercetin monoglucuronide-2, which might be the potential active components in vivo.It was found that Abelmoschus manihot extract could protect mice against PTZ-induced clonic convulsions and mortality. Abelmoschus manihot extract could also decrease immobility time in the FST in mice. Furthermore, the potential active components of Abelmoschus manihot extract in rat brain were identified by ultra performance liquid chromatography-mass spectrometer (UPLC-MS). Five parent components including isoquercitrin, hyperoside, hibifolin, quercetin-3'-O-glucoside, quercetin and three metabolites were detected in rat brain after administration of Abelmoschus manihot extract.UPLC-Q-TOF with collision energy (MSE) and automated data analysis software (MetabolynxTM) combined with mass defect filtering (MDF) together were applied for fast analysis of hyperoside metabolite in rat plasma, urine and bile after intraveneous administration. A total of 12 metabolites were identified in rat compared with blank samples. The results indicated that methylation, sulfation and glucuronidation were the major metabolic pathways of hyperoside, and parent compound and its metabolites in plasma include hyperoside, 3'-O-methyl-hyperoside, in urine are hyperoside,3'-O-methyl-hyperoside,4'-o-methyl-hyperoside, two hyperoside sulfate, methylated hyperoside monoglucuronide, hyperoside sulfate monoglucuronide, two methylated hyperoside sulfate, while in bile are 3'-O-methyl-hyperoside,4'-O-methyl-hyperoside, hyperoside sulfate, three methylated hyperoside momoglucuronide, two methylated hyperoside sulfate, two hyperoside glucuronide. The present study provided important information about the metabolism of hyperoside which will be helpful for fully understanding the mechanism of action of this compound, guiding clinical medication and improving drug efficacy and safety. |
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