| Zingerone,4-(4-Hydroxy-3-methoxyphenyl)-2-butanone,one of the major pungent compound present in ginger,has showed many pharmacological properties. Recent pharmacological studies have indicated that zingerone possess a wide spectrum of biological and pharmacological effects including anti-oxidant, lowering cholesterol,anti-digestive,tract ulcer,calm,the analgesia,to allay a fever,antibacterial activities and so on.It could prevente the cardiovascular and the liver from harms and has excellent application prospect as medicine.So it worth to develop into a new drug.Compared to the extensive research of the pharmacological activities of zingerone,however,little is known of its pharmacokinetic and disposition properties in animals and humans. For example,research data in the aspects of its oral bioavailability,the method on either assay for the determination of zingerone or its pharmacokinetics,as well as the urinary,fecal and biliary excretion,are still very limited.Pharmacokinetics study plays an important role in new drug development. thus this thesis concerned with a study of the pharmacokinetics of zingerone in rats,so as to support the metabolism and pharmacokinetic study in human and to offer the reference for clinical dosag and dosage form changing.This thesis is made up of five parts:(1)In the present study,the zingerone was separated from the ginger by silica gel column chromatography and the content of zingerone was determined by GC/MS;(2)The acute toxicity of zingerone in mice was studied after oral administration;(3)Studied on the absorption of zingerone with in vivo recirculating perfusion device in rats;(4)Studied the characteristics of pharmacokinetics,tissue distribution,urinary,fecal and biliary excretion and banding rates of plasma protein of zingerone in rats;(5)examined the effect on rat's hepatic microsome P450 enzyme and the inhibitory effect of zingerone on the microsome P450 enzyme.This study would hopefully improve our understanding of the pharmacokinetics and the mechanism of disposition of zingerone in rats and to support future drug development of this compound in its oral formulations.1.Preparatin of the zingeroneThe preparation procedure of zingerone was developed by the silical gel column.The dried ginger was powdered and extracted with 95%EtOH for three times under reflux.After filtration,the three filtrates were pooled and concentrated to a certain volumne of solution to get the extracts.The extracts was partitioned following Petroleum ether(60-90℃),Ethyl acetate and n-Butanol solvents.The ethyl acetate fration was concentrated under vacuum and get the ethyl acetate extracts.Purification of Ethyl acetate was accomplished by column chromatograph,TLC and recrystallization.To date,a slightly yellow crystal have been isolated from the extracts.The crystal was examined by GC/MS and all the data were in accord with the recorded.Thus the crystal was identified as zingerone and the purification was 98%.The zingerone separated from ginger by silica gel coulmn chromatography contained high quantity of zingerone,can be used to investigate drug.2.The acute toxicity of the zingeroneThe acute toxicity reaction and mortality in mice were observed after oral administration of the zionerone(separated from the dried ginger by silica gel column chromatography).The oral half lethal dose(LD50) of the zingerone in mice was calculated by Bliss method,and 95%confidence intervals of LD50 were computed.The results showed that the half lethal dose(LD50) of zingerone in mice by oral administration was 657.55mg/kg,and 95%confidence intervals of LD50 were 471.30mg/kg -882.29mg/kg.The acute toxicity reaction and mortality in mice were showed that the oral administration of zingerone was wide for the medicine margin for safety,conforms to the low poisonous standard.3.Studies on the absorption kinetics of zingerone in intestines of ratsThe absorption kinetics and absorption site were investigated by using the in situ intestine perfusion method in rats.The concentration of zingerone in the intestine perfusate was determined by HPLC-UV. The sample was chromatographed on a DiamonsilC18 cloumn(150×4.6mm I.D.,5μm).The mobile phase consisted of methanol-water(47:53,v/v) was run at flow rate of 0.5mL/min for different biological matrix.Ultraviolet detection wavelength was set at 280 nm and the column temperature was kept at 30℃.The chromatographic system used provided good separation of the compound without interfering peaks from endogenous substances.The assay was then used to determine the concentration of zingerone in the intestine perfusates.The results showed that the absorption rate constants(K) of zingerone were 0.0121,0.0124 and 0.0118min-1 at the concentration of 25.0,50.0 and 100.0μg/mL,respectively;K of zingerone were 0.0064,0.0032,0.0035和0.0026 min-1 at duodenum,jejunum,ileum and colon,respectively.Concentration of zingerone has no significant effect on its absorption kinetics.The best absorption segment in intestine was duodenum,ileum,jejunum and colon by turns. In the perfusate concentration range from 25.0μg/mL to 100.0μg/mL,the intestinal absorption mechanism of zingerone was conform to be passive trasport mechanism and fit to first order kinetics.4.The pharmacokinetics study of zingerone in ratsIn order to elucidate the absorption and pharmacokinetic profile of zingerone,a rapid,sensitive,and reliable analytical HPLC-UV method was developed and fully validated to determine zingerone in biological samples. After a single step ethyl ether-incluced liquid-liquid extraction from plasma, zingerone and the internal standard vanillin were subjected to HPLC analysis using ultraviolet detector.The samples were chromatographed on a DiamonsilC18 cloumn(150×4.6mm I.D.,5μm).The mobile phase consisted of methanol-water (47:53,v/v) was run at flow rate of 0.5mL/min for different biological matrix. Ultraviolet detection wavelength was set at 280 nm and the column temperature was kept at 30℃.The chromatographic system used provided good separation of the compound without interfering peaks from endogenous substances.The assay was shown to be good linear over the plasma concentration range from 0.05-20.0μg/mL of zingerone with the mean correlation coefficient 0.9959(r=0.9959)when applying the weighted least-squares method.The lower limit of quantitation of zingerone was 10ng/mL.The intra- and inter-day precision(relative standard deviation) was generally good(<10%) at low, medium and high concentrations.The overall recovery of the analytes was over 80%.These results indicate that the method was efficinet with a simple preparation procedure and a short running time for zingerone.With its high selectivity,acceptable accuracy,precision and sensitivity,this validated HPLC-UV method was successfully used to support the pharmacokinetics study of zingerone.The assay was then used to determine the pharmacokinetic and absolute bioavailability of zingerone in rats.Pharmacokinetic and bioavailability of zingerone were studied following a single oral or intravenous administration in rats.Individual zingerone concentration-time data were fitted by conpartmental models,to calculate the pharmacokinetic parameters.Follwing a single intravenous injection with dose of 4mg/kg of zingerone to rats,the boold samples were collected at different time points after dosing. All the collected boold samples were centrifuged to obtain plasma and the concentration of zingerone in plasma were determined by HPLC-UV method described as above.The pharmacokinetic parameter calculations and pharmacokinetic modeling were carried out with the PK Solution 2.0(Summit Research Services,USA) pharmacokinetics software.Following intravenous administration,zingerone was eliminated rapidly.The results showed that the plasma concentration-time data was fitted to an open two-compartment model in rats after iv administration and the elimination of zingerone exhibited first order kinetic characteristics.The area under the curve AUC0→∞ was 80617.7±8155.9(min×ng/mL),the terminal t1/2 was 26.55±16.02(min),and the Vd was11981.2±6659.2(mL/kg),respectively.When the 32mg/kg dose of drug was given by oral administration to rats, the boold samples were collected at different time points after dosing.All the collected boold samples were centrifuged to obtain plasma and the concentration of zingerone in plasma were determined by HPLC-UV method described as above.The pharmacokinetic parameter calculations and pharmacokinetic modeling were carried out with the PK Solution 2.0(Summit Research Services,USA) pharmacokinetics software.The results showed that the concentration-time curve was best fitted to an open two-compartment model in rats after oral administration and the elimination of zingerone exhibited first order kinetic characteristics.The area under the curve AUC0→∞ was 465340±95132.4(min×ng/mL).The maximum plasma concentration Cmax was 4560.6±1577.8(ng/ml) and the absorption t1/2ka was 40.923±4.868(min), distribution t1/2α was 14.38±3.48(min),respectively.The oral absolute bioavailability of zingerone was 72.2%.For the first time,a simple and reliable HPLC-UV method to determine zingerone levels in rat plasma was established and validated,which had been successfully applied to the pharmacokinetics studies.The developed assay involved simple sample preparation procedures,and showed acceptable precision,accuracy,linearity,stability and specificity.To the best of our knowledge,this method meets the request need in the present pharmacokinetic studies of zingerone.5.The tissue distribution study of zingerone in ratsTissue distribution of zingerone in rats after oral or intravenous administration was studied.Rats were serially euthanized by spondylopathy luxation at 5 min,30 min,60 min,90 min and 180 min after a single i.v. administration(4mk/kg).Heart,liver,spleen,lung,kidney,stomach, intestines,pancreatic gland,brain,skin,muscle,fat,testis and prostate were sampled.Each tissue was taken out and made into homogenates preparation with physiological saline.The concentrations of tissue homagenate samples were measured by HPLC-UV method.The sample was chromatographed on a Oiamonsil C18 cloumn(150×4.6mm I.D.,5μm).The mobile phase consisted of methanol-water (47:53,v/v) was run at flow rate of 0.5mL/min for different biological matrix. Ultraviolet detection wavelength was set at 280 nm and the column temperature was kept at 30℃.The chromatographic system used provided good separation of the compound without interfering peaks from endogenous substances.Results showed that zingerone was distributed to the entire organism rapidly after iv adminitration and the concentration of zingerone in plasma fell more quicker than in tissue.Five minutes after iv administration,zingerone was found in all of these organs.The highest concentration of zingerone was found in the liver and the pancreatic gland with the next highest concentration in spleen, kidney and heart.But at 30 minutes after dosing,the concentration of zingerone in fat was increased.The tissue concentration obviously declined with the lapse of time.180 minutes after injection,it could not find zingerone in tissues except the pancreatic gland and the fat.It showed that zingerone was not accumulated in rats after a single iv administration.Rats were killed by exsanguination at 15 min,45 min,90min,180min and 360min after a single oral administration(32mk/kg).Heart,liver,spleen,lung, kidney,stomach,intestines,pancreatic gland,brain,skin,muscle,fat, testis and prostate were sampled.Each tissue was taken out and made into homogenates preparation with physiological saline.The concentrations of tissue homagenate samples were determined by HPLC-UV method described as above. Results showed that zingerone was distributed to the entire organism rapidly after oral adminitration and the concentration of zingerone in plasma fell more quicker than in tissue.15 min after oral administration,all of these organs contain zingerone.The highest concentration of zingerone was found in the liver and the stomach with the next highest concentration in intestines, spleen and pancreatic gland.But at 45 minutes after dosing,the concentration of zingerone in pancreatic gland was increased.The tissue concentration obviously declined with the lapse of time.360 minutes after injection,it could not find zingerone in tissues except the pancreatic gland and the fat. It showed that zingerone was not accumulated in rats after a single oral administration.For the first time study on the tissue distribution of zingerone in rat. In summary,zingerone was rapidly,extensively distributed to the various rat tissues both after i.v.administration and oral administration,and an accumulation in liver was found.The concentration of zingerone in the tissue were higher than that in plasma.The zingerone concentration were high in the liver,pancreatic gland and spleen.After oral dosing,concentrations in stomach and small intestine were much higher than those non-gut tissues.The concentration existed in brain and testes showing a good penetration into the blood-brain barrier and the blood-testes barrier,which may be consistent with its high lipophilicity.6.Urinary,fecal and biliary excretion of zingerone in rats after oral administrationExcretion of zingerone in rat urine,bile and feces after oral administration was studied.The experiment design of excretion in urine and feces:Put five rats into metabolic cages,collected the blank urine and feces samples prior to experiment.Then the rats were given zingerone and urine and feces samples were collected every 6 hours for 72 hours;recorded the volumes of urine.0.2 mL of urine samples were extracted by ethyl ether.The feces were ground and 0.1g were extracted by ethyl ether.The experiment design of excretion in bile:zingerone was oral administration to bile duct-cannulated rats;collected the bile every 2 hours for 24 hours and recorded the volumes of bile.0.2mL of bile samples were extracted by ethyl ether.The concentration of zingerone were determined by HPLC-UV method,and then the accumulative excretion ammount and cumulative excretion percentage were calculated.The sample was chromatographed on a DiamonsilC18 cloumn(15O×4.6mm I.D.,5μm).The mobile phase consisted of methanol-water(47:53,v/v) was run at flow rate of 0.5mL/min for different biological matrix.Ultraviolet detection wavelength was set at 280 nm and the column temperature was kept at 30℃.The chromatographic system used provided good separation of the compound without interfering peaks from endogenous substances.It was shown that after a single oral administration,the accumulative excretion ammount of zingerone were(10.57±2.56)%in bile,(27.72±2.12)% in urine and(12.98±4.09)%in feces,respectively.The concentration of the unchanged durg was high in urine,bile and feces after oral administration 32mg/kg zingerone in rat.It showed that zingerone excreted basically by the unchanged durg from the urine,feces and the bile, which indicates an extensive metabolism of it in rats.7.In vitro banding rates of zingerone to rat and human plasma proteins Plasma protein binding was studied by equilibriun dialysis method(72h) at 4℃with final concentration of 0.5,5 and 10μg/mL.After the dialysis,the concentration of zingerone was determined by HPLC-UV method.The sample was chromatographed on a DiamonsilC18 cloumn(150×4.6mm I.D.,5μm).The mobile phase consisted of methanol-water(47:53,v/v) was run at flow rate of 0.5mL/min for different biological matrix.Ultraviolet detection wavelength was set at 280 nm and the column temperature was kept at 30℃.The chromatographic system used provided good separation of the compound without interfering peaks from endogenous substances.The binding ratio was calculated as percent of total concentration.The bingding percentage of zingerone in rat and human were(56.30±5.11)%and (55.44±3.56)%,respectively.The results of banding rates of zingerone to rats and human plasma protein indiced that the mean extent of binding to plasma protein in rat and human appeared to be independent of concentration uesd.The mean bingding percentage of the drug was 55%for all specises examined.In summary,this study demonstrated that zingerone is not highly bound to plasma proteins of rat and human. 8.The effect of zingerone on hepatic microsome P450 enzyme content in rats Rats were euthanized by spondylopathy luxation after oral administration of physiological saline(5mL/kg/d,3d),zingerone(32mg/kg/d,3d), respectively.The microsomes were prepared from rat livers by calcium precipitation method and the content of P450 enzyme in microsomes were determined by ultraviolet spectrophotometer.The results showed that the enzyme content in microsomes was not statistically significantly affected by zingerone.9.Enzyme Kinetics of zingerone metabolism in Rat Liver MicrosomeThe metabolic kinetics of zingerone,the effects of selective CYP450 inhibitors on the metabolism of zingerone were investigated in rat liver microsomes.This study established a HPLC-UV method for the determination of zingerone, and calculated its enzyme kinetics in rat microsome.Rat liver microsome was was used to perform enzyme kinetic studies and calculated enzyme kinetics parameters with substrate depletion method in the rat microsome.Rat liver microsomes were prepared using calcium precipitation method.The concentration of zingerone in the incubation samples at different time points was determined by HPLC-UV method.The sample was chromatographed on a DiamonsilC18 cloumn(150×4.6mm I.D.,5μm).The mobile phase consisted of methanol-water(47:53,v/v) was run at flow rate of 0.5mL/min for different biological matrix.Ultraviolet detection wavelength was set at 280 nm and the column temperature was kept at 30℃.The chromatographic system used provided good separation of the compound without interfering peaks from endogenous substances.Various selective CYP inhibitors were used to investigate their inhibitory effects on the metabolism of zingerone and the principal CYP isoform involved in zingerone metabolicly ring.Metabolism of zingerone was significantly inhibited by quinidine,but furafylline,sulfaphenazole,tranylcypromineand and ketoconazole showed little inhibitory effect on the metabolism of zingerone.Diethyldithiocarbamate inhibited the metabolism of zingerone only in high dose.This indicated that CYP2D6 was considerable involved in the metabolism of zingerone,for the metabolism of zingerone was significantly inhibited by quinidine,the CYP2D6 inhibitor. In a word,the HPLC-UV assay may be a constant,sensitive,accurate and specific method to determine zingerone in biological samples.The pharmacokinetics conforms to the open two comparement model with the first order elimination after oral or intravenous adminstration.Zingerone was absorpted and eliminated at a rapid rate and widely distributed in rats.It distributed rapidly in the blood-rich tissue such as liver,kidney and spleen. The plasma protein binding ratio was found to be 55%.About 51%of unchanged zingerone was excreted in feces,bile and urine.Zingerone was mainly excrisised through kidney with other excrisising process as bile excretion method,ect.The results will provide the theretical basis for pharmacokinetics course of zingerone in human.
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