Study Of Pharmacokinetics And Hypolipidemic, Hypoglycemic Effects Of Sanhuang Formula

Sanhuang Formula, which consists of Radix astragali, Coptis rhizome, Artemisia Scoparira, Pollen Typhae and Alisma orientalis, is a classic traditional Chinese medicine formula to replenish Qi, resolve phlegm, dissipate blood stasis. It is widely used in traditional Chinese medicine to prevent and treat central obesity, and this natural preparation has played a sporadic but important role in addressing obesity in the department of traditional Chinese medicine, Huashan Hospital, Fudan University.Metabolic syndrome (MS), namely insulin resistance (1R) syndrome, is a combination of medical disorders consisting of central obesity, hypertension, dyslipidemia, obesity, Type 2 diabetes mellitus and impaired glucose tolerance. MS often causes from IR.1R is a pathological condition that the sensitivity and/or reactivity of cells and tissues for insulin become sluggish. Though there are disadvantages for traditional chinese medicine in decreasing hyperglycemia, hypertension and hyperlipidemia, it has still some advantages, such as pretecting vascular endothelial cells. The better control effect would be obtained by integrating traditional chinese and western medicine.PPARs are ligand dependentting of transcription factors that regulate expression of target genes related to lipid and glucose metabolism. The PPARs play critical roles in the regulation of the adipocyte differentiation and lipid metabolism. P-TEFb, consisting of CDK9 and cyclin T1, involves in several specific differentiation processes of cells.First of all, an optimized protein precipitation method for the prepration of plasma samples and a rapid and sensitive liquid chromatography-electrospray ionization-mass spectrometry for the simultaneous determination of berberine, palmatine and jatrorrhizine was decribed. Plasma samples were pretreated by protein precipitation with the mixture of 3% formic acid and 50 ng/mL clozapine (internal standard) in acetonitrile followed by LC analysis using a C18 column and a mobile phase composed of 0.4% formic acid solution and 0.2% formic acid solution of methanol (60:40, v/v) operated at a flow rate of 0.4 ml/min. In addition, MS detection was performed on a Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer (San Jose, CA, USA) equipped with an ESI source in the positive ionization mode. The MS operating conditions were optimized as follows:the spray voltage was 3500 V, the source CID voltage was 10 eV, the heated capillary temperature was 500℃, the sheath gas (nitrogen) was 20 psi, the auxiliary gas (nitrogen) was 50 psi, the collision gas (argon) pressure was 1.0 mtorr (1 torr= 133.3 Pa), and the collision energy was 37 eV. Data acquisition, peak integration and calibration were performed by Analyst 1.5 software (San Jose, CA, USA). Quantification was obtained using the MRM mode of the transitions at m/z 336.2→320.1 for berberine, m/z 352.3→336.2 for palmatine, m/z 338.0→322.3 for jatrorrhizine, and m/z 327.1→270.1 for IS with a scan time of 0.3 sec per transition. The method was linear over the concentration range of 0.1-400.0 ng/mL for berberine, palmatine and jatrorrhizine. The method reported were found to be accurate and reproducible for quantifying the level of berberine, palmatine and jatrorrhizine, and can be used for the pharmacokinetic studies of Phase Ⅰ clinical trial and animal trial. Moreover, It was found that AUC0-t, Cmax, Vd and CL of berberine and palmatine in metabolic syndrome rats were with significant difference (P< 0.05) comparing with that in normal rats, which indicated that berberine and palmatine have higher uptake and slower elimination in the rats with metabolic syndrome.Secondly, Effects of hypo lip idem ic, hypoglycemic effects for Sanhuang Formula on MS rats with hyperlipidemia were systematically verificated. The results suggested that:(1)Sanhuang Formula markedly decreased blood glucose, TG, TC, LDL-C, ApoB and FFA contents of MS rats, while increased HDL-C and ApoAI levels. Sanhuang Formula increased the declined glycogen content in liver of MS rats, while decreased the augmented FFA and TG levels in MS tissues.(2) Sanhuang Formula significantly declined FFA, MDA and TNF-α contents in serum and adipose tissue of MS rats, while increased adiponectin level and total lipidase, SOD activities.(3) Berberine markedly up-regulated PPARa, PPARy, CDK9, cyclin T1 mRNA and protein expression in adipose tissue of diabetic rats. Berberine decreased adipose tissue to body weight ratio and adipocyte size, and increased adipocyte number.(4) Serum medicine of Sanhuang Formula promoted 3T3-L1 cells proliferation at low concentration, but inhibited proliferation at high concentration. It enhanced 3T3-L1 cells differentiation and degraded lipid accumulation in dose-dependent manner. And it up-regulated PPARa, PPARy, CDK9, cyclin T1 mRNA and protein expression in 3T3-L1 adipocytes.All in all, an optimized protein precipitation method for the prepration of plasma samples and a rapid and sensitive liquid chromatography-electrospray ionization-mass spectrometry for the simultaneous determination of berberine, palmatine and jatrorrhizine was described. Moreover, there was significant difference (P<0.05) for the pharmacokinetic parameters of berberine and palmatine in MS rats comparing with that in normal rats, which was important to future study (such as determinating the time to gain plasma sample of rats with better quality after oral administration) and clinical work. Last but not least, Sanhuang Formula had significant hypolipidemic and hypoglycemic effects on MS rats, and regulation of PPARs/P-TEFb signal transduction pathway was one of the mechanisms of hypolipidemic, hypoglycemic effects of Sanhuang Formula.