| Hawthorn leaves extract is derived from the leaves of Crataegus pinnatifida Bge. var. major N.E.Br and Crataegus pinnatifida Bge. The principal active components of the extract are flavonoids, have been confirmed by various studies to possess a wide range of pharmacological action, especially in the prevention and treatment of cardiovascular diseases. In this paper, Hawthorn leaves flavonoids (HLF) are refined from Hawthorn leaves extract including vitexin-2"-O-rhamnoside (VOR) and vitexin-4"-O-glucoside (VOG) were loaded to bioadhesive microparticles in order to prolong gastrointestinal location time and enhance bioavailability. The preparations will also increase the low membrane permeability, avoid degradation in the gastrointestinal tract and elimination in the stomach, intestine and liver of HLF.The TLC and HPLC methods were developed for the qualitation and quantitation assay of HLF. The technical condition for purification of total flavones by polyamide resin was screened using the content and the transfer rate of total flavones as index. Results of preliminary trials indicated that concentration of HLF, pH and eluting rate affected the adsorption properties of polyamide. The purity of total flavones reached 90% by this optimum process. The VOG and VOR were isolated and purified by preparative liquid chromatography. The purity of VOG and VOR reached 99% for the quality control of HLF and its preparations.Preformulation study showed that HLF were soluble in water, and the solubility and oil/water partition coefficient of HLF were affected by pH owing to phenolic hydroxyl in the molecular structure. With the increase of pH value, their solubility and log P values were a slight increase and decrease, respectively. The stability study in the gastrointestinal tract showed HLF solution was stable at pH4.0-7.6. The VOG and VOR were incubated for 1 h and have become unstable, and their remaining concentrations increased and then decreased in the stomach contents. The significant degradations of VOG and VOR were more than 10% of drugs in the colon with 2h incubation. VOG and VOR were stable in other intestinal segments with 4h incubation. The isolated rat stomaches had no significant physical adsorption, a certain amount of uptake (in vitro absorption) and significant metabolism of HLF. The isolated intestinal mucosa and wall had no obvious absorption, uptake and metabolism of HLF.A high sensitivity, rapid and specific ultra performance liquid chromatography tandem mass spectrometry method has been developed for simultaneous determination of VOG and VOR in biological sample. The purposes of this study was to report the gastrointestinal absorption, hepatic and intestinal metabolic factors affecting bioavailability of HLF after intravenous, intraportal, intragastric, and intraduodenal administration of HLF and to identify the reason for the incomplete bioavailability of HLF. After oral administration of HLF to rats, the pharmacokinetic properties of VOG and VOR were complicated with the multi-peak phenomenons, which might result from the transform among several components, enterohepatic circulation, and multiple sites of absorption of the gastrointestinal tract. The pharmacokinetic behavior of VOG and VOR after different routes of administration to rats showed that the gastric availability, intestinal availability, and hepatic availability is 63.41%,5.25% and 63.86% respectively for VOG and 64.62%,6.99% and 75.34% respectively for VOR. HLF were poorly absorbed and lost by first-pass effects of passage through the gut wall and liver. The first-pass effect of the gut wall is greater than that of the liver and stomach, and determines the low bioavailability of HLF.The absorption mechanism and bioadhesive material on the absorption of HLF were systematically investigated based on the models of intestine loop. The results indicated that the absorption of the active ingredients all could be improved with the increased dose. Besides, the process of absorption accompanied the phenomenons of metabolism by enterobacteria, bile excretion, entero secretion and enterohepatic circulation. Several adhesive polymers were studied, among which sodium alginate (AN), carbomer 934 (CBM) and N-carboxymethylchitosan (CPCTS) were selected for preparing the bioadhesive microparticles of HLF.Regarding the appearance, particle size, entrapment efficieny (EE) and yield of formulation as index, HLF-loaded bioadhesive microparticles were investigated in detail. HLF bioadhesive microparticles were prepared by emulsification/evaporation techniques. The effects of process and formulation variables on the properties of microparticles (e.g. micrometric properties, release percent and bioadhesion) were investigated. The stirring rate mainly affected the diameter, EE and yield. The temperature of the solvent evaporation was the key factor, which affected all the properties of the microparticles. The time of solvent evaporation mainly affected the release of drug from microparticles. The ratio of continuous phase to dispersed phase affected all the properties of microparticles. The ratio of HLF to excipients affected EE and the release of drug from microparticles. The amount of emulsifier affected the diameter, yield and the release rate. By investigating the micrometric properties, bioadhesion and release rates of three batches of samples, it was found that the microparticles obtained had the advantages of good sphericity, with size ranging approximately from 1-200μm, repose and rest angle of the microparticlesθ<30℃, suitable bioadhesion and hygroscopicity. The EE of HLF-loaded carbopol bioadhesive microparticles (HLF-CBM-BP), HLF-loaded N-carboxymethylchitosan bioadhesive microparticles (HLF-CPCTS-BP) and HLF-loaded sodium alginate bioadhesive microparticles (HLF-AN-BP) were 73.23%,75.15% and 81.79%, respectively.The pharmacokinetics of YiXinTong pills (HLF-pills) and HLF-loaded bioadhesive microparticles in beagle dogs were investigated, respectively. The results showed the pharmacokinetic properties of VOG and VOR were complicated characteristics involved and multi-peak phenomenon observed. The pharmacokinetic behaviors of VOG and VOR were similar owing to their similar chemical structures (P>0.05). After oral administered a single dose of HLF-CBM-BP, HLF-CPCTS-BP and HLF-AN-BP, the AUC(0-∞) and Cmax of VOG and VOR were increased (P<0.05). The MRT0-∞of VOG and VOR of HLF-CBM-BP was 1.57-and 1.71-fold higher than that of HLF-pills. The V and CLof test product groups were also decreased. Based on the data of non-compartment model and HLF-pills as control, relative bioavailability (Fr) of HLF-CBM-BP, HLF-CPCTS-BP and HLF-AN-BP was 200.98%,157.82% and 169.82% respectively for VOG and 233.97%,184.66% and 173.35% respectively for VOR The result showed that bioadhesive microparticles prolonged gastrointestinal location time, promoted the absorption of HLF and increased its oral bioavailability.It was showed that the HLF-loaded bioadhesive microparticles were able to adhere closely to the mucosal surface and increase the oral bioavailability of HLF. Hope to give reliable scientific basis for highly efficient and long-acting preparations of HLF. |
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