| In recent years,phenols,tannins,terpenes,flavonoids have been isolated from Phyllanthus emblica.The researches of their remarkable antioxidant,antitumor,antiviral and hypoglycemic activities have become hot topics.Caco-2 cell monolayers is very well used in the investigation of the absorption of chemical compositions and extracts of Traditional Chinese Medicine.Based on our preliminary studies,we established the Caco-2 cell monolayers.Transport of corilagin,gallic acid and ellagic acid from Fructus Phyllanthi tannin fraction(PTF)in Caco-2 cell monolayers was studied.The mechanisms and rate-limiting factors of the transport were investigated.This experiment supported the prior in vivo absorption study.Further studies could be established to improve the in vivo absorption of Phyllanthus emblica and to improve its antitumor effect.XST capsules contains total saponins of Panax notognseng,which are widely used in the treatment of cardiovascular and cerebrovascular diseases.The mechanisms of XST capsules are closely related to the inhibition of platelet aggregation.We investigated the stability of platelet aggregation induced by ADP,thrombin and collagen.Quality control for XST capsules based on the bioassay of anti-platelet aggregation was established,which including the optimize of induction agent and standard control substance.This bioassay method could be used to further improve the quality control of XST capsules.This paper is divided into four chapters as below:Chapter ?:ReviewPhyllanthus emblica L.(Euphorbiaceae)is widely distributed in the southern provinces of China.The dry fruit of Phyllanthus emblica is named Fructus Phyllanthi,which has been used as food and medicine for a long time.As is said by Chen Cang-qi in his book "bencao shiyi" that:"The fruit of Phyllanthus emblica is called yuganzi because it tastes bitter and sweet." Traditional Chinese Medicine mainly use it to treat sore throat,cough.Tibetan Medicine employ it in the treatment of blood disease and gall bladder disease.As early as the Tang dynasty,the juice of Phyllanthus emblica is popular as a beverage.This chapter summarized the tradition use,phytochemistry research and pharmacological studies.Caco-2 cell monolayers is widely used in recent years for the absorption of drugs.This model is simple and reproducible,has been widely used to study the absorption characteristics of pure compounds and extracts and to preliminary screening of new drugs.This chapter summarizes the basic characteristics of Caco-2 cells model,transport pathways and application of oral absorption.Biological potency testing provides the information of biological activities that correlated with the clinical use.With the deep-going researches of chemical constituents and pharmacology,biological potency measurement has been used in Traditional Chinese Medicine.This chapter summarizes the basic meaning of biological potency,the selection of indicators and standard control material and summarizes the application of bioassay in the quality control of Traditional Chinese Medicine.Chapter ?:The transport of PTF on Caco-2 cell monolayers Part one:The establishment of Caco-2 cell monolayersThe Caco-2 cell monolayer model was establishment.Epithelial tight junction of the model was qualified.The TEER values of the monolayers increased over the first 12 days,and steadily for the last 9 days.The values were above 500 ?/cm 2 on day 21.The Papp value of sodium fluorescein,which is a poorly transported marker,Was 3.16±0.25×10-7cm/s which agreed with that published in the previous literature.The HBSS and FBS from different company were tested and those from Gibco were selected.When the TEER values were above 500 ?/cm2 and incubation for 3 hours,the monolayer model remained stable.Part two:Transport of PTF across Caco-2 cell monolayersThe Papp values of the three compounds were below 1.0×10-6cm/s,which may transported via passive diffusion.The absorption of corilagin and gallic acid were much lower than their efflux,and the uptake of both compounds were increased in the presence of inhibitors of P-gp and MRPs.The absorption of ellagic acid was decreased in the company of OATP and SGLT1 inhibitors.Moreover,the transport of corilagin,gallic acid and ellagic acid were enhanced by tight junction modulators.These observations indicated that the three compounds in PTF were transported via passive diffusion combined with protein mediated transport.P-gp and MRPs might get involved in the transport of corilagin and GA.The absorption of EA could be attributed to OATP and SGLT1 protein.Part three:The stability of gallic acid,corilagin and ellagic acid in HBSSGallic acid,corilagin and ellagic acid were instability in solutions of HBSS,but the stability of the three compounds in PTF were slightly higher than that of pure compounds.The instability may be due to the PH of HBSS,which showed weak alkaline and therefore result in acid-base reactions.So the samples should be kept dried before further analysis.Taking into account the stability data and adjusted the result is available.Part four:Metabolism study of gallic acid,ellagic acid and corilagin Caco-2 cell monolayersLC-MSn method was established for the determination of metabolize of gallic acid,corilagin and ellagic acid in Caco-2 cell monolayers.Urolithin A was detected from the transport samples of corilagin and ellagic acid.The low absorption of corilagin and ellagic acid may partly due to the metabolize activities during the transport.Chapter ?:Anti-platelet aggregation bioassay based quality control for XST capsulesPart one:Variability analysis of optical aggregometry measurement of platelet aggregationLBY-NJ4 aggregometer is used to study the variability and variation amplitude of platelet aggregation.Quality control fluid was used to test the variability caused by instrument,quality control platelet rich plasma(PRP)was used to test the variability of platelet aggregation under different inducers.The results showed that the instrument itself and the normal operation cause of variability is small.For the platelet aggregation induced by ADP and collagen,three gathered parameter are suitable for pharmacodynamic evaluation and clinical course;For the induction of thrombin,maximum aggregation rate and the area under the curve is stable for efficacy evaluation.Part two:Anti-platelet aggregation bioassay based quality control for XST capsulesThe in vitro anti-platelet aggregation effect was observed to detect the bioactivity of XST capsules.Thrombin was employed as platelet aggregation inducer and XST lyophilized powder was employed as standard control substance.XST capsules showed a significant inhibitory effect on thrombin-induced platelet aggregation in a dose-dependent manner.Parallel line assay was used to establish the bioassay of XST capsules.The established bioassay method had a good repeatability(RSD=2.92%)and could be used as one of the complementary quality control methods for XST capsules.Chapter ?:Conclusion and discussionThe TEER values of the monolayers increased over the first 12 days,and steadily for the last 9 days.The values were above 500 ?/cm 2 on day 21.The Papp value of sodium fluorescein,which is a poorly transported marker,tested across the monolayers was 3.16±0.25×10-7 cm/s which agreed with that published in the previous literatureHBSS and FBS from Gibco were selected in the culture of the Caco-2 cell monolayers;when the TEER values were greater than 500 ?/cm2,the Caco-2 cells model remain stable within 3 hours.For corilagin the retention time was 6.61 min,the mass transition used was m/z 633.1?301.0(collision energy,-35 eV),the linear equation was Y=23.705X-26.106(r=0.9999)with a good linearity over the range from 0.65 ng/mL to 653 ng/mL,the limit of detection(LOD,signal-to-noise ratio?3)was 1.35 ng/mL and the limit of quantitation(LOQ,signal-to-noise ratio?10)was 1.94 ng/mL.For GA the retention time was 4.14 min,the mass transition used was m/z 169.0?125.0(collision energy,-10 eV),the linear equation was Y=28.769X+22.697(r=0.9999)with a good linearity over the range from 1.01 ng/mL to 252 ng/mL,the LOD was 0.08 ng/mL and the LOQ was 1.64 ng/mL.For EA the retention time was 8.39min,the mass transition used was m/z 301.0?301.0(collision energy,?10 eV),the linear equation was Y=111.21X+130.99(r=0.9982)with a good linearity over the range from 1.98 ng/mL to 989 ng/mL,the LOD was 0.17 ng/mL and the LOQ was 0.97 ng/mL.When the concentration of PTF,gallic acid,corilagin and ellagic acid was 1 mg/mL,45,16 and 36 ?g/mL,respectively,no significant effect on growth of Caco-2 cells were observed in4h.The apparent permeability coefficient(Papp)values of the three compounds were below 1.0×10-6 cm/s.The absorption of corilagin and GA were much lower than their efflux,and the uptake of both compounds were increased in the presence of inhibitors of P-gp and MRPs.The absorption of EA was decreased in the company of OATP and SGLT1 inhibitors.Gallic acid and corilagin were extremely unstable(9.45-24.97)in HBSS,when mixed with other compounds,their stability increased(>49.95%),when existed in extracts,their stability increased too(>52.22%).Ellagic acid is relatively stable,and when existed in the extract,the concentration of which increased about 20%.LC-MSn method was used,urolithin A was identified,which may explained the poor absorption of ellagic acid.ADP,collagen and Thrombin-induced platelet aggregation were studied,maximum aggregation rates of which were most stable with RSDs less than 4%.XST capsules inhibited ADP,collagen and Thrombin-induced platelet aggregation in a dose-dependent manner.Thrombin was chosen as the best agent to induce platelet aggregation,and to determine the anti-platelet aggregation bioassay of XST capsules.XST lyophilized powder was chosen as the best standard control substance,and to determine the anti-platelet aggregation bioassay of XST capsules.The established bioassay method had a good repeatability RSD=2.92%,when BS2000 software was used to analysis the results,the potency of XST capsules is 92.04 U/mL,FL=3.89%,confidence interval range was 88.53-95.69 U/mL?... |
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