The Pharmacology And Pharmacokinetics Study Of γ-oryzanol Combined With Berberine

The World Health Organization (WHO) said that high cholesterol levels contribute to56%of coronary heart disease worldwide and claims more than4million lives per year.Generally, bile acid sequestrants, statins, fibrates and nicotinic acids are efficacious antihyperlipidaemic drugs currently available, mainly fibrates and statins. But statins will elevate the liver transaminase and casuse pain, as well as myositis. What’s worse, it will result in serious myositis, rhabdomyolysis and acute renal failure when combined with firbrates. In addition, at the beginning, patients will feel gastrointestinal dicomfor and suffer from pruritus, urticaria, skin rashes, hair loss, headache, insomnia and hyposexuality accasionally, and will lead to liver and kdney damage for a long-term use. And both of statins and fibrates are expensive--a severe economic burben for patients who need a long-term medication. Therefore, it’s urgen to develop a safe and effective regimen for dyslipidemia.Berberine (BBR) has been used for thousands of years as a traditional herbal medicine to treat gastro-intestinal infection. Oryzanol (OZ) has been used for the treatment of menopause syndrome, neurological disorder and nerve disorders. Interestingly, BBR combined with OZ has an effective role in lowering plasma total cholesterol (TC), total glycerin (TG) and apo-B levels clinically. Moreover, they are proved to be safe and effective through chronic clinical treatment of byperlipidemia. The cholesterol-lowering mechanism of BBR is different from the other drugs:it is reported that BBR increases LDL-receptor expression by mRNA stabilization through a post-transcriptional regulatory mechanism. And it was reported that OZ can elevate LDL-R and hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase mRNA expressions and prevent cholesterol absoption by reductions in aortic fatty streak formation. Importantly, it can reduce the dosage of BBR and OZ respectively when use together, which help to reduce side effects and improve living quality. Meanwhile, OZ could promote the absorption of BBR, enhance the bioavailability of BBR.In the present work, ultraviolet-visible (UV-Vis) analysis, high performance liquid chromatography coupled with tandem mass spectrometric (LC/MS/MS) method were conducted to study the pharmacy, pharmacodynamics and pharmacokinetics of BBR and OZ. First, we established a simple but reproducible and reliable simultaneous determination of the subjects for the quality control. Then, a hyperlipidemia animal model was established to study the lipie-lowering effect after combining them together. Meanwhile, to make the synergistic effect mechanism clear, we studied the pharmacokinetics of BBR, OZ and BBR-OZ. Besides, OZ’s low solubility leads to a poor absorption and bioavailabilty of OZ. Thus, we also studied the solubilization of OZ under combined use of Tween80(TW-80) both in vitro and vivo.Objections:1. Quantitative determination. To determine the contents of BBR and OZ and to provide an experimental basis for further study on the agent, a method of quantitative determination was established.2. Pharmacodynamic study. To choose the best formulation of BBR and OZ for hyperlipidemic, a hyperlipidemia animal model was established.3. Pharmacokinetic study. To investigate the pharmacokinetic parameters’changes after giving BBR and OZ alone or together, such as absorption, distribution and elimination and to study the mechanism of drug-interaction through analyzing the pharmacokinetic mutual influence between OZ and BBR (berberine), a LC/MS/MS method was performed.4. Solubilization study of OZ. To evaluate the solubilization behavior of OZ under combined use of TW-80(serial concentration), we stimulated the in vitro situation of stomach and intestines by0.1mol·L-1HCl and phosphate buffer solution (PBS, pH=6.8), respectively. The solubilizing effect of Tween80is determined by the absorbance (A). In vivo, we conducted a LC/MS/MS method to analysis the pharmacokinetics of OZ with or without1%TW80to further comfirm the solubilizing effect of TW-80on OZ.Methods:1. Quantitative determination. According to "Chinese Pharmacopoeia"(version2010), ministerial standard of Ministry of Health and related references, we applied UV-vis to determine the contents of BBR and OZ. In the work, the measuring wavelength of OZ was304nm and the reference wavelength was369nm. Meanwhile, the wavelength of determination of BBR was430nm. The contents of BBR and OZ were calculated by the standard curves of the two agents.2. Pharmacodynamic study. After1week of accommodation,96adult male healthy Wistar rats were then divided into2groups by weight randomly; group1,8rats were fed with normal diet (ND) until sacrifice, serving as normal-diet controls, while group2, the other32rats were fed with a high-fat and high-cholesterol (HFHC) diet for continuous4weeks to induce hyperlipidemia animal model. Blood samples were taken (following fasting for12h) before, after, and at the indicated days during the treatment from the eye socket vein of the rats. Serum LDL-C, total cholesterol (TC), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) levels were assayed by Automatic Biochemical Analyzer.Then group2were randomly divided into11subgroups based on TC levels:HFHC diet control (HFHC diet), positive control (HFHC diet and simvastatin), BBR Ⅰ (0.01g·kg-1), BBR Ⅱ(0.03g·kg-1), BBR Ⅲ(0.1g·kg-1), OZ Ⅰ (0.01g·kg-1), OZ Ⅱ(0.03g/kg), OZ Ⅲ(0.1g·kg-1), Compoud I (BBR0.025g·kg-1+OZ0.005g·kg-1), CompoudⅡ(BBR0.02g·kg-1+OZ0.01g·kg-1), CompoudⅢ(BBR0.015g·kg-1+OZ0.015g·kg-1). The HFHC diet control and ND were ig normal saline every day, while others were administrated agents for continuous4weeks. Then collected blood to test the serum lipid and analyzed the hyperlipidemic effect of BBR, OZ and BBR-OZ.3. Pharmacokinetic study.24Sprague-Dawley rats, weighing250-320g, acclimatized to the situation for one week and before the study, they were fasted but free to waster for12h. Then rats were randomly into4groups:OZ, BBR, BBR-OZ and OZ-TW80, which were administration orally a dosage of100mg·kg-1. Blood samples (300μL) were collected to a heparinized eppendorf tube from the rat eye socket vein prior to dosing and subsequently at2,5,10,20,30,60,120,180,240,360,480and720min following dosage OZ and BBR-OZ and5,10,20,30,60,120,180,240,360,480,720and1440min following dosage BBR. Following sample preparation, the obtained plasma were kept at-80℃until study. The pharmacokinetic parameters were analyzed by Drug Analysis System (DAS, Version2.1.1).4. Solubilization study of OZ. OZ was dissolved with10ml0.1mol·L-1HCl or PBS that containing serial concentration of TW-80. After ultrasonic dissolving for30min, the solutions were filtered and tested on UV-Vis. Place cuvette into UV-Vis spectrophotometer and take a background scan (280-400nm) using0.1mol·L-1HCl or PBS containing different concentrations of Tween80as background. Then measure optical absorbance at329nrn. The solubilizing effect of Tween80is determined by the absorbance (A).Results:1. Quantitative determination. The linear range of OZ was2.93～52.8, mg·L-1, R2=0.9999and BBR was2.1-37.8mg·L-1, R2=1.0000. The mean recovery was98.69%,2.36%(n=6);101.79%,2.01%(n=6), respectively.2. Pharmacodynamic study. After giving HFHC diet to rats for continuous4weeks, TC and LDL-C levels increased remarkably in the HFHC diet control group when compared to ND (P<0.05), while HDL-C/TC (H/T) level decreased significantly (P<0.05). The results suggested that HFHC diet can establish hypercholecholesterolemic model. Following administration agents for4weeks, lipid levels were much better between BBR Ⅱ and Ⅲ, OZ Ⅱ and Ⅲ, but there were no significant difference between group Ⅱ and Ⅲ. Overall, the dosage in BBR Ⅱ and OZ Ⅱ were better than other groups with a remarkable efficacy and moderate dosage. In addition, there were hyperlipidemic effect of the3compound groups and were no difference with the positive group. But referring to dosage, it was lower in the compound groups than in the sigle groups. Compound Ⅰ, the dosage of BBR was5/6of single group, OZ was1/6; compound Ⅱ, BBR and OZ were reduced to a half of the single group; and compound Ⅲ, BBR was only1/3of the single group, OZ was2/3. The results revealed that3compound groups have obvious hyperlipidemic effect.3. Pharmacokinetic study. LC/MS/MS was performed in multiple-ion monitoring (MRM) mode using target ions at m/z193->121for FA and m/z336->292for BBR. The linear range was3.082-400.32ng·mL-1(Y=0.096262X+0.027720, R2=0.9957) for FA and5.008-127.2ng-mL-1(Y=5.441408X-0.096780, R2=0.9981) for BBR, respectively. The disposition process of OZ (FA) and BBR in rats could be described by open two-compartment model. Compared to OZ and BBR group, significant differences were found on the pharmacokinetic parameters of FA and BBR in BBR-OZ group. FA was distributed faster and eliminated slower but the absorption was significantly increased, while BBR was distributed slower and eliminated faster but the absorption was increased. There is a pharmacokinetic mutual influence between OZ and BBR, which influence the absorption, distribution, metabolism and elimination between them.4. Solubilization study of OZ. The solubility of OZ was greatly increased with increasing concentration of Tween80from0%-1%. The pharmacokinetic study revealed that TW-80disable to inhence the absorption of OZ in vivo, but it can extent Tmax and increase AUC0-∞significantly, suggesting that TW-80was able to inhence the bioavailability of OZ by extenting its absorption and distribution.Conclusion:Double wavelength UV-vis can be used to simultaneously determine the contents of BBR and OZ. Additionaly, BBR combined with OZ can synergistic lower the serum lipid level with a smaller dosage, which will reduce side-effect theoretically. Futhermore, to investigate the mechanism of synergistic lipid-lowering effect of BBR combined with OZ, pharmacokinetic study was adopted to analyze the pharmacokinetic changes after giving BBR and OZ alone or together. Results showed that it can influence the absorption, distribution, metabolism and elimination between them after combination, revealing that a promoted absorption mutually may responsible for the synergistic lipid-lowering effect. Simultaneously, we found that TW-80can significantly upgrate the solubility of OZ in vitro and extend the absorption and distribution of OZ in vivo, resulting in a improved bioavailability of OZ. Therefore, TW-80could be solubilizer for OZ.