Study On The Extraction Technolo And Metabolism Of The Active Components In The Leaves Of F. Virens VAR Sublanceolata

F.virens var sublanceolata?FVS?,also known as big banyan tree and Huangjue tree,is one of the banyan plants in the mulberry family.It was first produced in southwest and Southern China.The results showed that banyan leaves contain triterpene glycosides,flavone glycosides,acid resins and tannins.Flavonoids have significant pharmacological effects,such as antibacterial,anti-inflammatory,antihypertensive,anti-oxidation,anti mutation,anticancer,sedative,heat clearing and detoxification.All kinds of flavonoids are widely studied and widely used in medicine.Chinese herbal medicine preparation and national Chinese herbal medicine collection record the medicinal value of the FVS leaves,a banyan plant in the mulberry family,which can dispel wind and dredge collaterals,promote blood circulation and reduce swelling,relieve itching and reduce sores.FVS leaves are rich in resources,have many pharmacological activities,and have certain development and research value.The development of Ginkgo flavone is a very successful case.There are many methods for the extraction of plant flavonoids,such as reflux extraction,ultrasonic extraction,microwave extraction,supercritical extraction,enzymolysis,mechanochemical extraction and infrared and far infrared extraction.High performance liquid chromatography is the most classical method to determine the content.Once the method is established,it can determine the content of related substances stably and accurately.Therefore,this paper will study and analyze the FVS leaves from four parts:optimize the extraction process of total flavonoids from FVS leaves,establish an HPLC method for the determination of related substances in FVS leaves extract,establish a method for the determination of related substances in plasma samples,and study the pharmacokinetic characteristics of FVS leaves extract after intragastric administration in rats.Part ?:Optimization of extraction technology of Flavonoids from FVS leavesObjective:Optimization of extraction technology of total flavonoids from FVS leaves.Method:In this study,reflux extraction method was used;first,the parameters of each experimental condition,ethanol volume fraction,material liquid ratio,water bath temperature and extraction time were individually selected one by one,then orthogonal test was used to further optimize the extraction conditions,and finally verify the optimized process.RU was used as the control substance for quantitative analysis of total flavonoids in FVS leaves.The content of total flavonoids in the sample to be tested was calculated by measuring the absorbance after the color experiment of NaNO₂-Al?NO₃?₃-NaOH.Results:The optimized extraction process of total flavonoids from FVS leaves was as follows:the ratio of material to solvent was 1g:50ml,the volume percent concentration of ethanol was 60%,the temperature of water bath was 95?,and the extraction time was 2 hours.The results showed that under the best extraction conditions,the extraction rate of total flavonoids in FVS leaves was 3.74%,with good reproducibility.Part ? Determination of chlorogenic acid and rutin in the extract of FVS leavesObjective:To establish an HPLC method for determination of CGA and RU in the FVS leaves.Method:Water e2695-2998?PAD?HPLC analyzer was used.Separation of analytes by ODS2 column.The column temperature box is set at 25±2?.The samples to be tested are separated and determined by gradient elution at a flow rate of 1.0 mL·min^-1.Mobile phase A is organic phase?100%methanol?.Mobile phase B is inorganic phase?pH 3.0 phosphoric acid solution?.All mobile phases were filtered before use,and then degassed by ultrasound.When the sample is determined,the sample is injected with an automatic sampler,and the injection volume is 20?L.The single running time of the sample was 72minutes.Results:All substances in the sample are well separated,and the separation degree of chlorogenic acid and rutin was up to the requirement without interference of other substances.The value of the correlation coefficient of the linear regression equation of chlorogenic acid and rutin is more than 0.9998.LOQ and LOD of CGA and RU were lower than 0.06?g·mL^-11 and 0.02?g·mL^-11 respectively.In the determination of low,medium and high concentration samples,the RSD values of intraday precision were less than 1.45%,1.89%and 0.73%respectively,and that of intraday precision were less than 1.68%,1.82%and 1.34%respectively.The results show that the precision of this method is good.The accuracy is expressed by relative recovery?RR?,which is between99.10%and 104.7%.Conclusion:The established HPLC-PAD method is suitable for the separation and determination of chlorogenic acid and rutin from FVS leaves extracts.Part ? Establishment of a method for the determination of Coumarin-3-carboxylic acid analogue?C3AA?in plasma of ratsObjective:To establish a HPLC method for the determination of C3AA in plasma of rats after intragastric administration of 60%ethanol extract.Method:Water e2695-2998?PAD?HPLC analyzer was used.FA as internal standard.Coumarin-3-carboxylic acid?C3A?was used as the substitute of quantitative C3AA.ODS2 column was used to separate analytes.The column temperature box is set at 30±2?.The samples to be tested are separated and determined by gradient elution at a flow rate of 1.0mL·min^-1.Mobile phase A is organic phase?100%methanol?.Mobile phase B is inorganic phase?pH 3.0 phosphoric acid solution?.All mobile phases were filtered before use,and then degassed by ultrasound.When the sample is determined,the sample is injected with an automatic sampler,and the injection volume is 20?L.The single running time of the sample was72 minutes.Results:The separation of all substances in the sample is good,the separation degree of FA and C3A meets the requirements,and there is no interference of other substances.The r value of C3A is equal to 0.9990,which has a good linear relationship.The LOQ of C3A is equal to 0.05?g·mL^-11 and the LOD is 0.02?g·mL^-1.In the determination of low,medium and high concentration samples,the RSD values of intraday precision were 2.86%?2.30%and 1.25%respectively,and that of interday precision were 3.58%?2.73%and 1.84%respectively.The results show that the precision of this method is good.The accuracy is expressed by relative recovery?RR?,which is between 99.05%and 105.0%.Conclusion:HPLC-PAD method can be used to separate and determine C3AA in plasma of rats after intragastric administration of 60%ethanol extract.Part ? The preliminary study on pharmacokinetics of 60% ethanol extract from FVS leavesObjective:Preliminary study on pharmacokinetics of FVS leaf extract.Method:the concentration data of C3AA in the plasma measured by 60%ethanol extract of rats were sorted out and fitted to get the concentration time curve of C3AA.PK solver 2.0 software was applied to reckon the concentration data of C3AA in the plasma and get the relevant pharmacokinetic parameters.Results:the change of C3AA concentration in plasma with time could be seen from the curve of blood concentration time.There are two peaks in the curve of blood concentration and time.The pharmacokinetic parameters t_1/2/2 was 1.89±0.03 h,T _maxax was 0.39±0.14 h,C _maxwas 1.81±0.10?g·mL^-1,AUC _0-t-t was 7.88±0.24?g·mL^-1·h,MRT was3.23±0.14 h,Vz/F was 0.43±0.03(mg·kg^-1)·(?g·mL^-1)^-11 and Cl/F was0.16±0.01(mg·kg^-1)·(?g·mL^-1)^-1·h^-1.Conclusion:the absorption of C3AA in the blood of rats after intragastric administration can be reflected by the concentration time curve and pharmacokinetic parameters.