| Stroke,which is also known as cerebral apoplexy,is a common barely uncurable disease.People can be easily susceptible and disabled.Even after treatment,people still must face bad therapeutic effects and high mortality.It has done harm to our health and life very seriously.Not only patients lose their life qualities,but also their families and the society have to suffer in huge burdens.Pueraria is commonly used for curing stroke as a kind of traditional Chinese medicine.There are four kinds of listed intravenous injections which mainly contain puerarin at present.They have marked effect in treating ischemic cerebrovascular disease.The mechanism of action includes several related aspects,such as expansion of cerebrovascular,removing of free radicals,antioxidant,inhibiting of the inflammatory response,and so on.On the other hand,modern pharmacological studies have also shown that puerarin can protect brain,so can the remaining components of total flavonoids.They can significantly reduce the infarction volume of MCAO model rats,reduce the brain edema and malondialdehyde,and improve the activity of superoxide dismutase.Ge ketone Tongluo capsule has been listed now.However,since puerarin injection was listed in 1993,a variety of adverse reactions have often occured,in which the worst case can even cause death.And difficulty in taking medicine,terrible drug compliance and low bioavailability are shown in Ge ketone Tongluo capsule's application.The above defects limit the clinical application of puerarin injection and Ge ketone Tongluo capsule.It has been proved that the nasal delivery system is an effective way to cure encephalopathy in both traditional Chinese medicine theory and modern medical research.Our subject is based on the "Pathway from nose to brain" theory of traditional Chinese medicine.According to early studies,the subject chooses the nasal delivery system,and it has done research on pharmacokinetic behavior differences of puerarin flavonoids and puerarin in different delivery ways,relying on microdialysis sampling technique.The research content is below:1 Extraction and purification research of puerarin flavonoidsThe subject studies puerarin flavonoids' extraction by orthogonal test,choosing puerarin and total flavonoids extraction rate as indicators,so that the selected extraction process is convenient and practical,and the extraction rate of puerarin flavonoids is high.Through selecting the alcohol precipitation process of aqueous extract of Pueraria,the purity of puerarin flavonoids in extract is further improved.Then select the macroporous adsorption resin precipitation process in single factor method,involving the following parameters,including resin type,sample solution concentration,sample volume,water volume,ethanol concentration of eluent,eluent volume,diameter ratio,sample velocity,water velocity,ethanol velocity,and so on.So the purity of puerarin and total flavonoids moves forward a single step.Through amplification experiments,the purity of puerarin and flavonoids are stable,the resin precipitation process is proved practically.Using mass spectrometry analysis,I come to the preliminary conclusion that there are five main ingredients in the extract,containing 3'-hydroxy puerarin,puerarin,puerarin wood indican,3'-methoxy-puerarin and Daidzin.Then the nerve cells of cerebral cortex of Kunming mice were cultured.The anti-apoptosis effect was evaluated by the expression of Bcl-2 mRNA.Compraed with Puerarin group,the expression of Bcl-2 mRNA was highly improved when the cells were treated by extract before hypoxia test was carried out.The extract may contain some compounds which can enhance the expression of Bcl-2 mRNA.2 Microdialysis probe recovery rate research of puerarin in vivo and in vitroVia the probe clearance experiment,it confirms that no adsorption exists between puerarin and probe membrane material;by incremental method experiment and reducing method experiment,probe recovery rate in vitro and pass rate are determined,and there is a significant difference between them;the factors affecting probe recovery rate in vitro and pass rate consist of liquid mixing speed,perfusate additives and probe membrane length.Based on the conclusions above,the probe recovery of blood and olfactory bulb in vivo is calculated by zero net flux method,respectively 17.53%and 29.13%.Establish the HPLC-MS/MS determination method of puerarin in dialysate and naringin.The chromatographic conditions include C18 chromatographic column(Agilent ZORBAX Eclipse Plus C18 column(3.5?m,4.6×10 mm,USA)),the mobile phase were methanol and water,and the mass spectrometry conditions were described as follows:the mass spectrometer was operated in negative ionization mode using selective reaction monitoring(SRM)to measure puerarin and naringin.The spray voltage was 3,500 V.Sheath gas was 10 psi.The capillary temperature was 300?.The collision-induced dissociation voltage was 25 V for puerarin and 30 V for naringin.The transitions(precursor to product)monitored were m/z 415?295 for puerarin and m/z 579?295 for naringin.The dwell time was 100 ms for each transition.There is a good linear relationship of puerarin in 0.002-0.111?g/mL and 0.111-8.9?g/mL.The regression equations are respectively C=0.23816ratio-0.0001(r=0.998)and C=0.25562ratio-0.01582(r=0.999).Precision and stability are both qualified.The loq is at the lowest point of the standard curve.3 Pharmacokinetic study of puerarin via different administration routes in vivo.Implant the blood probe in the jugular vein of male SD rats,brain probe in the olfactory bulb.The dose of puerarin is 7 mg/kg,respectively through intravenous injection(i.v.),intravenous drip(i.v.gtt)and nasal delivery(i.n.).Conduct microdialysis sampling every 20min,and determine drug concentration in the dialysate via HPLC-MS/MS.The area under the concentration-time curves(0-5h)was calculated using non-compartmental methods by the program Kinetica 4.4.Blood dynamics results show that maximum drug concentrations of blood(Cmax)are respectively 30.89±10.69 ?g/ml(i.v.),9.31±3.99 ?g/ml(i.v.gtt),3.82±1.03 ?g/ml(i.n.),AUC0-5h are 1524.63±584.05 ?g/ml-min(i.v.),1037.18±501.70 ?g/ml·min(i.v.gtt),623.12±170.86 ?g/ml-min(i.n.).There is an obvious difference between nasal delivery group(i.n.)and the intravenous groups(P<0.05).Compared with intravenous administration,intranasal administration has lower bioavailability but gets longer mean residence time(MRT).Olfactory bulb dynamics results show that maximum drug concentrations of olfactory bulb(Cmax)are respectively 0.29±0.07 ?g/ml(i.v.),0.0523 ?g/ml(i.v.gtt),0.50±0.16?g/ml(i.n.),AUC0-5h are 28.44±6.89 ?g/ml·min(i.v.),8.30±4.85 ?g/ml·min(i.v.gtt),86.84±23.50?g/ml-min(i.n.).There is an obvious difference between group intravenous injection(i.v.)and group intravenous drip(i.v.gtt)(P<0.05),between group nasal delivery(i.n.)and the other two groups(P<0.05).Nasal delivery(i.n.)has significantly longer t1/2 and MRT in olfactory bulb,its brain targeting index is 7.5 times as much as intravenous injection(i.v.),17.4 times intravenous drip(i.v.gtt).It suggests that nasal delivery(i.n.)can not only reduce blood drug concentration,but also obviously increase brain drug concentration and brain targeting.4 Pharmacokinetic research of puerarin in different drug delivery ways of rats MCAO model in vivoTo simulate clinical patients and medication of puerarin,make male SD rats middle cerebral artery occlusion models.Do a research on pharmacokinetic behavior of puerarin in different drug delivery ways under the pathological state.Blood dynamics results show that maximum drug concentrations of blood(Cmax)are respectively 13.84±2.45?g/ml(i.v.gtt),4.36±1.06 ?g/ml(i.n.),AUC0-5h are 1416.68±249.74?g/ml·min(i.v.gtt),533.48±136.75 ?g/ml-min(i.n.).The absolute bioavailability of nasal delivery(i.n.)is 37.66%,but t1/2 and MRT have significantly extended because of the absorption process of nasal delivery(i.n.).Blood AUC of intravenous drip(i.v.gtt)in MCAO rats is higher than normal rats,which may be caused by rapid clearance in normal rats;there is no significant difference between MCAO rats and normal rats in blood AUC of nasal delivery(i.n.),we speculate that stroke causes no changes in nasal mucosa.Olfactory bulb dynamics results show that maximum drug concentrations of olfactory bulb(Cmax)are respectively 0.19±0.12 ?g/ml(i.v.gtt),1.54±0.43 ?g/ml(i.n.),AUC0-5h are 24.50±16.74 ?g/ml·min(i.v.gtt),255.96±87.74 ?g/ml-min(i.n.).Nasal delivery(i.n.)has its advantage in obviously higher level of Cmax,AUC,t1/2,its brain targeting index is 27 times as much as intravenous drip(i.v.gtt).Comparing to normal rats,olfactory bulb AUC of MCAO rats of intravenous drip(i.v.gtt)and nasal delivery(i.n.)significantly increases,the trend of the curves were changed as well.It is possibly affected by the blood-brain barrier damage and cerebral ischemia on olfactory mucosa and olfactory nerve.5 Pharmacokinetic research of puerarin flavonoids in different drug delivery ways of rats MCAO model in vivoMake male SD rats MCAO models.The dose of homemade pueraria extract is 20 mg/kg,respectively through intravenous drip(i.v.gtt)and nasal delivery(i.n.).Conduct microdialysis sampling,and determine drug concentration in the dialysate via HPLC-MS/MS.The area under the concentration-time curves(0-5h)was calculated using non-compartmental methods by the program Kinetica 4.4.Blood dynamics results show that maximum drug concentrations of blood(Cmax)are respectively 14.96±3.97 ?g/ml(i.v.gtt),0.75±0.30?g/ml(i.n.),AUC0-5h are 1707.02±457.88?g/ml-min(i.v.gtt),134.72±37.61 ?g/ml-min(i.n.).The absolute bioavailability of nasal delivery(i.n.)is 7.89%,but t1/2 and MRT are significantly longer than intravenous drip(i.v.gtt)because of the absorption process of nasal delivery(i.n.).There is no significant difference between pueraria extract and puerarin in blood AUC of intravenous drip(i.v.gtt),which prompts that the rest of flavonoids don't affect the metabolism of puerarin under the drug concentration;blood AUC of nasal delivery(i.n.)in extract group is 25%as much as in puerarin group,possibly resulting from the competition of a variety of ingredients in the extract,while absorbed through the nasal mucosa.Olfactory bulb dynamics results show that maximum drug concentrations of olfactory bulb(Cmax)are respectively 0.060±0.03 pg/ml(i.v.gtt),0.37±0.11 ?g/ml(i.n.),AUC0-5h are 7.38±4.65 ?g/ml·min(i.v.gtt),58.60±14.48 ?g/ml·min(i.n.).DTIs are 0.43%and 43.50%.Olfactory bulb AUC of intravenous drip(i.v.gtt)in extract group is 30%as much as in puerarin group,possibly resulting from the competition of flavonoids through the blood-brain barrier.So does the process of nasal delivery(i.n.).DTI in extract group and puerarin group of nasal delivery(i.n.)are approximate,respectively 47.98%and 43.50%,resulting in further proof of the competition among flavonoids through the blood-brain barrier. |
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