| Part Ⅰ Summary of the related literaturesIn this section,the resource distribution,chemical composition,pharmacological activities and the metabolism Phyllanthus emblica L.were summarized through 268 references.It provides a reference for the in vivo metabilism and basic efficacy substances study of Phyllanthus emblica L.Part Ⅱ Experimental partChapter I Preparation and Content Determination of the tannin parts from Phyllanthus emblica L.1.Preparation and total tannin content determination of the tannin parts from Phyllanthus emblica L.25 batches of tannin parts were continuously prepared,one kilogram of raw herbs is used for each batch.About 165g of the tannins were abtained of each batch.The total tannin content is approximately 57.71%.2.Quantitation of multi-components in raw herbs and the tannin parts of Phyllanthus emblica L.An HPLC method was established for simultaneous determination of 9 components in raw herbs and the tannin parts of Phyllanthus emblica L.The regression equations and linear ranges of the nine components are as follows:Gallic acid:Y=2E+06X-30393,r=0.9999,linear range 0.094～1.872 μg.Punicalagin A:Y=3E+06X-66826,r=0.9999,linear range:0.058～1.39 μg.Punicalagin B:Y=2E+06X-77857,r=0.9999,linear range:0.10～2.51 μg.Methyl gallate:Y=3E+06X-55968,r=0.9999,linear range:0.047～1.49 μg.Geraniin:Y=741189X-29791,r=0.9999,linear range:0.12～3.84 μg.Corilagin:Y=844347X-75313,r=0.9999,linear range:0.28～8.80 μg.CHEBULINIC ACID:Y=4E+06X-87496,r=0.9999,linear range:0.055～1.7536 μg.Chebulagicacid:Y=886311X-15698,r=0.9999,linear range:0.042～1.34 μg.Ellagic acid:Y=1E+06X-6569.3,r=0.9999,linear range:0.0090～2.88 μg.The average content of gallic acid,Punicalagin B,Methyl gallate,Geraniin,corilagin,Chebulininc acid,Chebulagic acid and Ellagic acid in raw herbs are 2.50%,0.22%,0.18%,0.56%,0.68%,0.26%,0.90%,2.01%.The average content of gallic acid,Punicalagin A,Punicalagin B,Methyl gallate,Geraniin corilagin,Chebulininc acid,Chebulagic acid and Ellagic acid in the tannin parts of Phyllanthus emblica L.are 3.42%,0.26%,0.43%,0.45%,1.18%,2.72%,0.44%,1.15%,3.24%.The results show that after purification of the macroporous resin,the contents of the main components in the tannins of Phyllanthus emblica L.were higher than those in the herbs.It shows that the enrichment process established in our laboratory can well enrich the main components of Phyllanthus emblica L.3.Study on the quantitative analysis multi-components in Phyllanthus emblica L.A QAMS method was established for simultaneous determination of 9 major components in raw herbs and the tannin parts of Phyllanthus emblica L to explore the accuracy and feasibility of multiple-evaluation techniques in the determination of hydrolysable tannin content.The relative correction factors between gallic acid and Punicalagin A,Punicalagin B,Methyl gallate,corilagin,Chebulininc acid,Chebulagic acid and Ellagic acid were established.The retention time difference method,relative retention method,and linear regression method on accurate positioning of measured components were compared.The difference between the measured value of the external standard method and the QAMS calculated value were compared.In the linear ranges,the fi/s between gallic acid and Punicalagin A,Punicalagin B,Methyl gallate,Geraniin,corilagin,Chebulininc acid,Chebulagic acid and Ellagic acid were 1.45,0.93,1.53,0.37,0.42,2.01,0.45,0.63,The fi/s is accurate and reliable under different test conditions.Relative retention method can accurately locate the components to be measured.The relative retention method between gallic acid and Punicalagin A,Punicalagin B,Methyl gallate,Geraniin,corilagin,Chebulininc acid,Chebulagic acid and Ellagic acid were 0.71、0.55、0.47、0.41、0.36、0.26、0.25、0.20.There was no significant difference between external standard method and QAMS measurement in results.It shows that the established QAMS method is accurate and feasible and can be used for quality control of raw herbs and the tannin parts of Phyllanthus emblica L.Chapter Ⅱ Metabolism of the tannin parts of Phyllanthus emblica L.in SD rats and ICR mice1.Pharmacokinetics study of the tannin parts of Phyllanthus emblica L.in SD rats and ICR miceAn HPLC method was established to determine the concentration of three major components in mice serum.The method is highly specific and highly sensitive.SD rats and ICR mice were given intragastric administration of the tannin parts of Phyllanthus emblica L(0.1 g/10 g body weight).An HPLC method was used to determine the serum concentrations of three major components in mouse serum at different time points.Data were processed with DAS 2.0 software.The pharmacokinetic parameters were calculated and drug graphs were drawn.Comparison of serum pharmacokinetics was made,after oral administration of the tannin parts of Phyllanthus emblica L,the tannin parts were absorbed very quickly in both groups of animals.After 5 minutes 3 components can be detected in the blood.The maximum plasma concentration was reached before 1 h.The maximum plasma concentration(Cmax)trends were Gallic Acid>Ellagic acid>Corilagin.The pharmacokinetic parameters of the two groups of animals were compared,and it was found that the absorption of the tannin parts of Phyllanthus emblica L.was slightly increased in SD rats group.There was no significant difference in absorption between SD rats and ICR mice.2.Distribution study of the tannin parts of Phyllanthus emblica L.in SD rats and ICR muceA HPLC method was established for the determination of three major components(gallic acid,corilagin,Ellagic acid)in the seven tissues(liver,heart,spleen,lung,kidney,stomach,and intestine)of mice.After examining this method,it is characterized by high specificity,high sensitivity and stable recovery.The standard curves are drawn and the linear ranges are given.30 min after oral administration of the tannin parts of Phyllanthus emblica L,each component has been rapidly distributed in each tissue in the two groups.The concentrations of three components have peaked at about 2 h.The concentrations of the three components in each tissue range from high to low:liver≥ stomach≥ intestine≥ lung>kidney>heart>spleen.The contents of all five components were the highest in the liver and stomach tissues,followed by the intestine and lung tissues.The contents of the three components in the heart and kidney tissues were low,and almost undetectable in the spleen.It shows that it has obvious targeting in liver,stomach,lung and intestine tissues.This is consistent with previous pharmacological experiments that the tannin part of Phyllanthus emblica L has good anti-liver cancer,lung cancer,and gastric cancer effects.Chapter Ⅲ Metabolism of the tannin parts of Phyllanthus emblica L.in Normal and Tumor-bearing Mice1.Pharmacokinetics study of the tannin parts of Phyllanthus emblica L.in Normal and Tumor-bearing MiceAn HPLC method was established to determine the concentration of five major components in mice serum.The method is highly specific and highly sensitive.Normal and tumor-bearing mice were given intragastric administration of the tannin parts of Phyllanthus emblica L(0.1 g/10 g body weight).An HPLC method was established to determine the serum concentrations of five major components in mouse serum at different time points.Data were processed with DAS 2.0 software.The pharmacokinetic parameters were calculate and drug graphs were draw.After oral administration of the tannin parts of Phyllanthus emblica L,the tannin parts were absorbed very quickly in both groups of animals.After 5 minutes 5 components can be detected into the blood.The maximum plasma concentration was reached before 2 h.Cmax trends were Gallic Acid>Ellagic acid>Chebulagic acid>Corilagin>Geraniin.However,there were significant differences in Cmax of the five components(gallic acid,geranin,corilagin,ellaric acid,and ellagic acid)between normal and model mice.In normal mouse Cmax were 10.75、1.89、2.95、3.74、8.68 mg/L,in tumor-bearing mice Cmax were 13.86、4.69、6.29、8.03、10.84 mg/L.It showed that the absorption of tannins increased significantly in pathological animals.In particular,the absorption of the three components(geranin,corilagin,and Chebulagic acid)is 2 times higher than that of normal mice.2.Distribution study of the tannin parts of Phyllanthus emblica L.in Normal and Tumor-bearing MiceA HPLC method was established for the determination of five major components(gallic acid,geranin,corilagin,Chebulagic acid,Ellagic acid)in the seven tissues(liver,heart,spleen,lung,kidney,stomach,and intestine)of mice.After examining this method,it is characterized by high specificity,high sensitivity and stable recovery.The standard curves are drawn and the linear ranges are given.30 min after oral administration of the tannin parts of Phyllanthus emblica L,each component has been rapidly distributed in each tissue in normal mice.The concentrations of five components have peaked at about 2 h.The concentrations of the five components in each tissue range from high to low:liver≥ stomach≥ intestine≥ lung>kidney>heart>spleen.The contents of all five components were the highest in the liver and stomach tissues,followed by the intestine and lung tissues.The contents of the five components in the heart and kidney tissues were low,and almost no detectable in the spleen.10 min after oral administration of the tannin parts of Phyllanthus emblica L,each component has been rapidly distributed in each tissue in model mice.The concentrations of five components have peaked at about 2 h.The concentrations of the five components in each tissue range from high to low:liver≥ lung≥ intestine≥ stomach>kidney>heart>spleen.The contents of all five components were the highest in the liver and lung tissues,followed by the intestine and stomach tissues.The contents of the five components in the heart and kidney tissues were low,and almost no detectable in the spleen.This is consistent with previous pharmacological experiments that the tannin part of Phyllanthus emblica L has good anti-liver cancer,lung cancer,and gastric cancer effects.Compared with normal mice,it was found that the contents of the five components in the tumor-bearing mice at different time points were significantly higher than those in the normal mice tissues.And the contents of five components in liver,lung,stomach and intestine increased more significantly.It shows that it has obvious targeting in liver,lung,stomach and intestine tissues.Chapter Ⅳ Analysis of metabolic components in normal and tumor bearing mice by UPLC-MSn1.Analysis of chemical components of medicinal herbs and the tannin parts of Phyllanthus emblica L.A UPLC-MSn method was used to analyze the chemical constituents in the medicinal herbs and the tannin parts of Phyllanthus emblica L.94 and 129 components were detected in the herbs and the tannin parts of Phyllanthus emblica L.respectively.Among them,tannins were 35 and 52 respectively,mucic acid and its lipids were 16 and 19 respectively,flavonoids were 14 and 20 respectively,phenolic acids and glycosides were 15 and 19 respectively,and aliphatic compounds were 14 and 18 respectively.It can be seen that the number of tannin compounds significantly increased in the tannin parts.It shows that the purification process of in the tannin parts of Phyllanthus emblica established in our laboratory can well enrich the tannin components of Phyllanthus emblica L.2.Metabolism Components and Their In vivo Metabolic Pathways in the tannin parts of Phyllanthus emblica150,266 components were detected in normal and tumor-bearing mice serum samples after Orally Administered.The prototype components in normal mice and tumor-bearing mice were 67,118 respectively.Among them,there were 26 and 36 tannin compounds in normal mice and tumor-bearing mice respectively,12 and 18 phenolic acids respectively,10 and 22 flavonoids respectively,9,26 mucic acids respectively,9 and 14 fatty acids respectively,and 1,2 pentacyclic triterpenoid in normal mice and tumor-bearing mice respectively.There were 83 and 109 metabolites in normal and tumor-bearing mice,respectively.Among them,there were 14 and 26 tannin metabolites in normal mice and tumor-bearing mice respectively,18 and 19 phenolic acids metabolites respectively,19 and 23 flavonoids metabolites respectively,15,22 mucic acids metabolites respectively,17 and 19 fatty acids metabolites in normal mice and tumor-bearing mice respectively.Compared with normal mice,there were significantly more prototypical and metabolic components in the tumor-bearing mice.65 prototype components and 70 metabolic components are consistent in normal mice and tumor-bearing mice.After analyzing the metabolic components,it was found that the metabolic pathways of the tannin parts of Phyllanthus emblica in the normal and tumor-bearing mice were almost the same.It was summarized by compound classification:The main metabolic pathways of tannin compounds in vivo are:hydrolysis,sulfation,deoxygenation,hydroxylation,glucuronidation,methylation.The main metabolic pathways of phenolic compounds in vivo are:glucuronidation,sulfation,deoxygenation,and methylation.The main metabolic pathways of flavonoids in vivo are hydrolysis,glucuronidation,sulfonation,methylation and hydroxylation.The main metabolic pathways of mucic acid compounds and their glycosides were deoxygenation,methylation,glucuronidation,and hydrolysis.The main metabolic pathway of fatty acid compounds in the body is:deoxygenation.Part Ⅲ Full text summary1.Four kilograms of Phyllanthus emblica L.were continuously prepared and the total tannin content and main monomer contents were determined.2.A method for the simultaneous determination of main monomer contents in Phyllanthus emblica L.was established by QAMS.3.The metabolic characteristics of Phyllanthus emblica L.in normal rats and normal mice were compared.It was found that there was no significant difference in the pharmacokinetics and tissue distribution of Phyllanthus emblica L.in two groups of animals.4.The metabolic characteristics of Phyllanthus emblica L.in normal mice and tumor-bearing mice were compared.The pharmacokinetics and tissue distribution of Phyllanthus emblica L.in tumor-bearing mice were significantly different.5.UPLC-MSn method was used to analyze the in vivo metabolic prototypical components and metabolic components of Phyllanthus emblica L.,and the in vivo metabolic pathways of five major types of components were summarized. |
PDF Full Text Request