| Abelmoschi Corolla, a commonly traditional Chinese medicine, is the flower of Abelmoschus manihot (Linn.) Medicus and has been widely used for the treatment of chronic renal disease, oral ulcers and burn in China for centuries.’HuangKui Capsules’(HKC), the ethanol extraction of Abelmoschi Corolla has become the most common Chinese medicinal product used for the treatment of chronic glomerulonephritis, diabetic nephropathy and nephrotic syndrome in China. Pharmacological studies revealed that Abelmoschi Corolla also have anti-inflammatory, antibacterial, anticoagulant, antiplatelet aggregation, reducing blood lipid, oxy-radical inhibition, prompting the elimination of immune complexes and protecting renal function. At present, the enterprise assure the product quality of HKC mainly by measuring the amounts of hyperoside, isoquercitrin, hibifolin and quercetin-3’-O-glucoside, but its use has been limited because of the shortage of reference substances. Meanwhile, the species of non-flavonoids in HKC are still unknown; whether the non-flavonoids have pharmacodynamic, have influences on pharmacodynamic, pharmacokinetics and toxicology to flavonoids still unclear. In this paper researches will be conducted from three perspectives of quality standard improvement, influences on pharmacodynamic and pharmacokinetics to flavonoids, metabolism of main ingredient. The main objectives of this researeh were helpful to guide the design of new drugs to improve the efficacy and safety of HKC in clinical, and lay the foundation to refine preparation into total flavonoids.Firstly, a detailed review was given to the research progress of Abelmoschi Corolla and its preparation HKC including chemical constituents, pharmacological effects, current quality standards, the process in vivo study, the mechanisms and effects of Abelmoschus manihot preparations in treating chronic kidney disease, common nephritis model, it is helpful to improve the current current quality standards and analyze the effects of non-flavoinoid constituents in Huangkui Capsule.To improve the current quality standards of HKC, the hyperiode was used as the internal reference substance, quantitative assay of multicomponents by single-marker (QAMS) was applied to calculate the relative correlation factors (RCF) of rutin, isoquercitrin, hibifolin, myricetin, quercetin-3’-O-glycoside, quercetin and determinate the seven bioactive components in HKC by UPLC. Sulfuric acid method was employed to determinate of saccharide and UPLC-TQ-MS was employed to identificate and determinate the type and content of the nucleoside and amino acids. Finally, a quality standard draft about simultaneous monitoring the amounts of flavonoids and non-flavonoids content was drawn up.Nephritis rats induced by Adriamycin were established to study the pharmacodynamic in treating chronic nephritis rats by different fractions extracted by different solvents (95% ethanol and water) and separated by polyamide column from Abelmoschi Corolla. Biochemical indicators such as blood urea nitrogen, serum creatinine, total protein and renal MDA, SOD, GSH, renal tissue biopsy and immunohistochemistry were measured in model and different administration groups, the results showed that total flavonoids of Abelmoschi Corolla have good therapeutic effect to adriamycin-induced nephritis rats, macromolecules and small molecular constituents didn’t have any therapeutic effects to nephritis rats which suggested that the active ingredient in HKC are flavonoids.A selective and sensitive UPLC-MS/MS method was established to determine the plasma concentrations of the 7 compounds. Sprague-Dawley rats were allocated to four groups which orally administered flavonoid fraction (FFA), FFA combined with macromolecular fraction (FFA-MF), FFA combined with small molecule fraction (FFA-SF) and FFA combined with MF-SF (FFA-MF-SF) with approximately the same dose of FFA. At different time points, the concentration of rutin (1), hyperoside (2), isoquercitrin (3), hibifolin (4), myricetin (5), quercetin-3’-O-glucose (6), quercetin (7) in rat plasma were determined and main pharmacokinetic parameters including T1/2, Tmax, AUC and Cmax were calculated using the DAS 2.0 software package. The statistical analysis was performed using the Student’s t-test with P<0.05 as the level of significance. The results showed that flavonoids almost had similar pharmacokinetics profile that were rapidly absorbed, reached the peak concentration at 30-60 min in group FFA, but the pharmacokinetic profiles and parameters of these flavonoids changed when co-administered with non-flavonoid components. It was found that AUC of five flavonoids but not hibifolin and quercetin in group FFA-SF and group FFA-MF-SF increased (P<0.05) in comparison with group FFA while the tendency was not observed in group FFA-MF. Moreover, seven flavonoids had varying degrees of differences in the pharmacokinetics parameters such as Cmax, Tmax and T1/2 (P<0.05) in group FFA-MF, FFA-SF and FFA-MF-SF by comparison with group FFA. The non-flavonoid components could improve the bioavailability and delay the elimination of some flavonoids in rat.Intestinal flora was used to study the metabolism of isoquercitrin in vitro. Ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) and the MetaboLynxTM software combined with mass defect filtering together were applied to identity the metabolites of isoquercitrin by the intestinal mixture bacteria and ninety six isolated strains from human feces. The human incubated samples collected 72 h in the anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC-Q-TOF/MS within 10 min. A total of parent competent and 5 metabolites were identified by eight isolated strains including Bacillus sp.17, Veillonella sp.23,32 and Bacteroides sp.40,41,56,75,88 in vitro. The results indicated that quercetin, acetylated isoquercitrin, dehydroxylated isoquercitrin, and hydroxylated quercetin, hydroxymethylated quercetin were the major metabolites of isoquercitrin. Furthermore, a possible metabolism pathway on the biotransformation of isoquercitrin is established in intestinal flora. This study will be helpful for understanding the metabolic rout of isoquercitrin and the role of different intestinal bacteria on the metabolism of natural compounds.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 myricetin metabolite in rat plasma, urine, feces and bile after intraveneous administration. A total of 5 metabolites were identified in rat plasma compared with blank samples. The results indicated that methylation; hydroxylation-methylation; methylation-sulfation were the major metabolic pathways of myricetin. The present study provided important information about the metabolism of myricetin 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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