Pharmacokinetics And Neurological Activity Of Pueraria Flavonoids In Vivo And Its Mechanism Of Enhancing Hypoglycemic Activity

Radix Pueraria (Chinese name Ge Gen), the roots of Pueraria lobata (Wild.) Ohwi., has been widely used as antipyretic and spasmolytic agents for the treatment of colds、 influenza. measles diarrhea. acute dysentery. neck and upper back stiffness. cardiovascular and cerebrovascular diseases in traditional Chinese medicine for centuries. The main bioactive constituents of Radix Pueraria are isoflavones (the total content in the herb can reach approximately 12%), including puerarin. daidzin and daidzein et al. Pharmacological studies demonstrated that puerariae flavonoids possess significant biological activitie, such as expanding coronary artery and cerebrovascular. improving microcirculation and antioxidant activities, and also exhibiting protective effects against cerebral ischemia-reperfusion injury and neurological disorders. Puerariae flavonoids preparations is mainly used for the treatment of cardiovascular and cerebrovascular diseases in clinic.At present, researches about puerariae flavonoids were mainly focused on puerarin and daidzein, two main active puerariae flavonoids. The other active compounds and effective substances of puerariae flavonoids still need to be further studied.In the first part of this study, the substance basis and mechanism of neuro protective effect of Puerariae flavonoids were discussed from the pharmacokinetic and pharmacodynamic perspective. The main contents and results are indicated as follows:1 Development of an UFLC-MS/MS method for determination of five Puerariae flavonoids in biological samples. A highly sensitive and reliable ultra fast liquid chromatography-tandem mass spectrometric (UFLC-MS/MS) method was developed for the determination of puerarin (PU). daidzein (DA).3’- methoxy puerarin (MPU).3’-hydroxy puerarin (HPU) and daidzein-8-c-celery glycosylation glucoside (DAC) in rat plasma and brain microdialysate. The assay was linear over the tested concentration ranges in rat plasma and brain microdialysate. The limit of quantification (LOQs) of DAC. PU. MPU. HPU and DA were 10.00.1.00.2.00.10.00 and 5.00 ng/mL and the limit of detection (LODs) were 5.00、0.50、1.00.5.00 and 2.00 ng/mL, respectively, when 60 μL of dailysate sample was used for assay. In plasma, the LODs for PU、MPU、 HPU、DA and DAC were 5.00.1.00.5.00.10.00 and 10.00 ng/mL and the LOQs were 1.00,0.50,2.00,5.00 and 2.00 ng/mL, respectively. Their extraction recoveries (n=3) from plasma were ranged from 75% to 101%. The method showed excellent reproducibility with intra-day (n=5) and inter-day (n=3) precision less than 13.87% and accuracy bias less than 12.45% in plasma and microdialysate.2 The pharmacokinetic studies of Puerariae flavonoids in rat plasma and cerebralspinal fluid. The pharmacokinetics properties of five active components of RPIF in rat plasma and brain were investigated using microdialysis and UFLC-MS/MS methods. After administration, the average Co values of PU、MPU、DA、DAC and HPU in plasma were estimated as 6.5313.72、1.54、15.84 and 86.07 μg/mL and PU、MPU、 DA and DAC reached to their mean highest drug concentrations of 521.52、415.00、74.34 and 380.03 ng/mL in CSF at about 0.5-0.8 h, respectively. The average t1/2 of the five analytes were within 1 h. Their penetration index (AUCbrain/AUCplasma) was found to be about 9.58、7.25、11.96 and 4.21% for PU、MPU、DA and DAC respectively.Therefore, the results suggested that PU、MPU、DA、DAC can quickly pass through the blood-CSF barrier and PU and MPU may play more important roles in exhibiting neuro-activities attributing to the higher concerntrations in brain.3 The pharmacokinetic and pharmacodynamic studies of Puerariae flavonoids in rat striatum. The pharmacokinetic properties and the effect on the level of neurotransmitter of RPIF in rat striatal extracellular fluid were investigated using microdialysis and UFLC-MS/MS methods to further investigate the neuro activity of RPIF. The concerntration of seven neurotransmitters, dopamine (DOA). glutamic acid (Glu). 5-hydroxytryptamine (5-HT). homovanilic acid (HVA).5-hydroxy indole acetic acid (5-HIAA). acetylcholine (Ach) and y-amino acid butyric acid (GABA) were determined. After intravenous administration of RPIF (80 mg/kg), PU exhibited the highest concentration in rat striatum among the four analytes, with Cmax at 2.19μg/mL and AUC0-t, at 2.14 μg·h/mLThe Cmax of HPU and DAC were 1.30 and 1.02 μg/mL, respectively. And their AUC0-t were 1.40 and 1.11 μg·h/mL.The concentration of MPU was the lowest with Cmax at 0.65μg/mL、AUC0-t, at 1.02 μg·h/mL and t1/2 of 1.2 h. The t1/2 of PU、HPU and DAC were about 0.3 h. The Cmax and AUC0-t, of PU、MPU、HPU and DAC increased accordingly when the dose of RPIF was increased. The peak time (Tmax) became shorter.The content of DA in the striatum was very low, which was undetectable through the experiments.Through the determination of neurotransmitters in rat striatum, it has been found that the levels of DOA in normal rat striatum could be reduced by intravenous administration of RPIF at 80 mg/kg. The concentration of DOA decreased from 81 ng/mL to 45 ng/mL, while the concentration of its metabolite HVA, reduced from 2320 ng/mL to 1810 ng/mL and HVA/DOA ratio increased. The level of GLu was not changed after dose. The basal level of GABA in striatum is 154 ng/mL, it decreased at 1.5 h after dose firstly, then increased to 197 ng/mL, then, it showed another downward trend. The level of 5-HT was not changed after dose, while the concentration of its metabolite, 5-HIAA decreased from 529 ng/mL to 331 ng/mL at 2 h after dose. Therefore, 5-HIAA/5-HT ratio reduced. The Basal level of Ach in striatum is 32 ng/mL and there was no significant change after dose.The level of DOA in striatum was not changed by intravenous administration of RPIF at 160 mg/kg, while the level of HVA do increase. The level of Glu in striatum was reduced significantly from 1558 ng/mL to the lowest level 301 ng/mL at 2 h after dose of RPIF at 160 mg/kg. No obvious changes of GABA concerntration was observed. The ratio of Glu/GABA decreased. The concentration of 5-HT showed no significant change after dose, while the level of its metabolite,5-HIAA, increased. The level of Ach exhibited a fluctuating uprising tendency. The results suggested that the effect of RPIF on the neurotransmitter levels was not consistent at different doses.The results of this part studies determine the pharmacokinetic profiles of five active compounds from RPIF, PU、MPU、DA、DAC and HPU in rat plasma, cerebrospinal fluid and the striatum; reveal the blood brain barrier penetration properties of the five conpounds, and also the effect of RPIF on the neurotransmitter levels in straium. It could provide guidance for application of RPIF in treating cerebral vascular disease.The second part of this study aims to reveal the mechanism of enhancement by Radix Pueraria flavonoids on the hyperglycemic effects of Cortex Mori extract in rats. 1-Deoxynojirimycin (DNJ), the main active ingredient of Cortex Mori, is recognized as a potenta-glycosidase inhibitor. Our previous studies have shown that the hypoglycemic effect of Cortex Mori extract (CME) was significantly improved when giving CME in combination with Radix Pueraria flavonoids (RPIF). In the present study, the effects of RPIF on pharmacokinetics and intestinal permeability of DNJ were investigated,to explore the mechanism of this synergistic effect. The main research contents and results are indicated as follows:1 The effects of RPIF on hyperglycemic activity of CME. The hyperglycemic activity of CME、RPIF、CME-RPIF were evaluated through oral glucose tolerance test in rats. The results showed that administration of CME-RPIF result in potent inhibitions of glucose responses. The incremental area under the curve (AUC) of blood glucose level after co-administration of CME、RPIF、CME-RPIF、water with starch solution, were 7.52、7.68、5.16 and 8.71 h/mmol*L respectively. CME-RPIF significantly lower the AUC of blood glucose level compared with rat administered with water, the reduction reaches 40.8%, while the AUC of CME and RPIF group decreased at 13.6% and 11.8%, respectively. The results suggest that hypoglycemic activity of CME was significantly improved when co-administered with RPIF.2 Development of an LC-MS/MS method for determination of DNJ in biological samples. A highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of DNJ in rat plasma and urine. The assay was linear over the selected concentration ranges, with limits of detection and quantification of 5 and 10 ng/mL, respectively. The precision (RSD< 16.09%) and accuracy (Bias,< 13.37) were satisfied and extraction recovery ranged from 80.3-96.9%.3 The effect of RPIF on the pharmacokinetics and intestinal absorption profiles of DNJ from CME in rat. The role of RPIF on pharmacokinetics and intestinal absorption profiles of DNJ from CME was investigated using in vivo rat model、in situ rat intestinal perfusion model and in vitro Caco-2 cell monalayer model. Four groups of rats received a single oral dose of either CME or CME-RPF at DNJ equivalent doses of 20 and 40 mg/kg, respectively. Compared to the data when dosing with CME alone, the Cmax of DNJ were decreased from 5.78 to 2.94 mg/m and 10.66 to 5.35 mg/ml; Tmax were delayed from 0.40 to 0.55 h and 0.35 to 0.50 h (po0.05) after dosing with CME-RPF at DNJ doses of 20 and 40 mg/kg, respectively. In addition, the urinary recovery of DNJ over the first 4 h after dosing significantly decreased from 48.76% to 33.86%. Effective permeability (Peff) of DNJ was decreased from 7.53x10-3 cm/min to 3.52 x 10-3 cm/min when RPIF was added to CME, when it was evaluated using the rat intestinal perfusion model. And RPIF could significantly inhibited the absorption of 10 μg/mL DNJ on Caco-2 cells. Apparent permeability coefficient (Papp) of DNJ was decreased from 2.7×10-4 to 1.0×10-4 cm/min by RPIF.All the above results demonstrate that RPF was able to suspend and delay the absorption of DNJ, but did not affect the total amount of DNJ in the body. The resulting higher concentration of DNJ in the small intestine produced a relatively stronger effect of depressing the elevation of the postprandial blood glucose level. These findings support the important role of RPIF in the application of CME on blood glucose control.