| Purpose:Juglans mandshurica Maxim.,also known as hickory and walnut,belongs to Juglans of Juglandaceae.In the "Liaoning Province Traditional Chinese Medicine Standards",it is recorded that the root bark,bark and peel of Juglans mandshurica have anti-tumor effects,and the folks also describe oral administration of Juglans mandshurica decoction for the treatment of tumors.Studies on the anti-tumor effects of organic solvent extracts from different parts of Juglans mandshurica Maxim.have been reported in the literature,but there are few studies on the aqueous extracts of Juglans mandshurica.Our group has analyzed and identified more than 100 compounds from the stems,fruits,leaves and roots of Juglans mandshurica.In order to further clarify the components that can be absorbed into the body after oral administration of the decoction,based on the previous research results of the research group,the anti-tumor effect of the aqueous extract of Juglans mandshurica and its in vitro absorption and metabolism components were studied.This research will provide useful information for the research of the active constituents of Juglans mandshurica.Material and method:Material and method:1.Material1.1 The medicinal material samples studied in this thesis were picked from Lianshan Town,Pulandian,Dalian City,Liaoning Province,and freshly sliced and freeze-dried.1.2 Experimental animals SD rats,male,purchased from Liaoning Changsheng Biotechnology Co.,Ltd.The rats were kept in the SPF level environment of the air layer flow frame of the Animal Experimental Center of Liaoning University of Traditional Chinese Medicine.1.3 Caco-2 cells were purchased from Beijing Dingguo Changsheng Biotechnology Co.,Ltd.,from human colon cancer tissues.Human gastric cancer cells(HGC-27)were purchased from Shanghai Bogu Biotechnology Co.,Ltd.from human undifferentiated gastric cancer;well-differentiated human neuroblastoma cells(SH-SY5Y)were purchased from Shanghai Cell Bank.2.Methods2.1 Preparation and chemical composition of aqueous extract of Juglans mandshurica:The lyophilized powder of the water extract of the stems of the saplings was prepared by hot reflux and vacuum freeze-drying.The chemical constituents in the aqueous extract of Juglans mandshurica Maxim.were analyzed and identified by UHPLC-MS/MS.2.2 In vitro anti-tumor effect and toxicity study: The inhibitory effect of Juglans mandshurica aqueous extract on the proliferation of human gastric cancer cell(HGC-27)and highly differentiated human neuroblastoma cell(SH-SY5Y)was used as evaluation index respectively.MTT colorimetric method was used to evaluate the effect of Juglans mandshurica aqueous extract on the morphology and relative proliferation rate(RGR)of HGC-27 and SH-SY5 Y cells,and further to evaluate the selective inhibitory effect of Juglans mandshurica aqueous extract on tumor cells.2.3 Study on Absorption and Metabolism in Caco-2 Cell Model: MTT was used to evaluate the relative proliferation inhibition rate of Juglans mandshurica aqueous extract on Caco-2 cells,to judge the cytotoxicity,and to determine the dosage concentration.The aqueous extract of Juglans mandshurica was co-cultured with Caco-2 cells.A certain concentration of aqueous extract of Juglans mandshurica was applied to Caco-2monolayer cells,and cell disruption liquid was collected.The components absorbed and metabolized in Caco-2 cells were analyzed and identified by UHPLC-MS/MS technique.2.4 Study on Absorption and Metabolism of Rat Colitis Model in Vitro: Lactate dehydrogenase(LDH)was used to evaluate the activity of the rat’s extracorporeal intestinal overturning section,so as to determine the sampling time and administration concentration of the rat’s extracorporeal intestinal overturning capsule model.UHPLC-MS/MS was used to analyze and identify the components of absorption and metabolism of Juglans mandshurica aqueous extract in duodenum,jejunum and ileum.2.5 In vitro human liver microsome metabolism study of Gallic acid and Ellagic acid:Human liver microsomes were selected to simulate the human environment,and the metabolism of control drug phenacetin was used to determine the activity of liver microsomes and optimize incubation conditions.Metabolites of Gallic acid and Ellagic acid in the aqueous extract of Juglans mandshurica were identified by UHPLC-MS/MS analysis.Results:1.Preparation and chemical composition of Juglans mandshurica aqueous extract: The yield of lyophilized powder of Juglans mandshurica stem and branch aqueous extract was12.5%,and the calculated crude drug was 8 g·g-1.A total of 99 components in the aqueous extract of Juglans mandshurica were identified,including 53 tannins,33 organic acids,10 naphthane derivatives,2 amino acids and 1 flavone.2.In vitro anti-tumor effect and toxicity research: In the study of anti-tumor effect and toxicity in vitro,when the water extract of Juglans mandshurica with different concentrations is less than or equal to 0.1 mg·m L-1,there is no cytotoxicity to HGC-27 and SH-SY5 Y cells.When the concentration reached 0.5 mg·m L-1,the RGR of Juglans mandshurica aqueous extract to HGC-27 cells was 51.48%,which showed obvious cytotoxicity.At this time,there was no cytotoxicity to SH-SY5 Y cells.When the concentration reached 1.0 mg·m L-1,the RGR of Juglans mandshurica aqueous extract to HGC-27 cells was 45.45%,which showed obvious cytotoxicity.The RGR of SH-SY5 Y cells was 88.16%,and there was still no cytotoxicity.3.Study on Absorption and Metabolism in Caco-2 Cell Model: In the study of toxicity of Juglans mandshurica aqueous extract to Caco-2 cells,the relative proliferation inhibition rate of Caco-2 cells is > 75% and the cytotoxicity is ≤ 1 when the concentration is in the range of 0.2 ~ 2.0 mg·m L-1,which indicates that the aqueous extract of Juglans mandshurica has no toxic effect on Caco-2 cells within the concentration range,so the concentration of Juglans mandshurica aqueous extract is selected as 0.8 mg·m L-1 in the Caco-2 cell absorption model.Finally,two components absorbed by the aqueous extract of Juglans mandshurica in Caco-2 cell model were analyzed and identified,respectively citric acid and gallic acid,and one component was metabolized,presumably dimethoxybenzoic acid,and the metabolic pathways were dehydroxylation and methylation.4.Study on Absorption and Metabolism in Invert Intestinal Sac Model in Vitro: Taking LDH release as an index,the sampling time of the rat model of eversion of intestinal sac in vitro was determined to be 150 min,and the concentration of water extract of Juglans mandshurica was 0.005 g·m L-1(based on crude drug).The absorption components of Juglans mandshurica aqueous extract in duodenum,jejunum and ileum were analyzed and identified,including tannin(13),organic acid(16),naphthane(9),diarylheptane(2)and lignan(1).Eleven metabolic components were identified in duodenum,jejunum and ileum,of which 10 were glucuronic acid binding products and 1 was sulfated products.Its original composition includes 3 tannins,2 organic acids and 6 naphthalenes in the water extract of Juglans mandshurica.5.In vitro studies on human liver microsomal metabolism of Gallic acid and Ellagic acid:The established liver microsome incubation system can metabolize phenacetin to acetaminophen with a metabolic rate of about 74.89%,indicating that human liver microsomes have biological activity under the test conditions.Then,the metabolism of Gallic acid and Ellagic acid in the incubation system was studied.The results showed that no metabolites of the two were found,but it was found that the addition of Gallic acid and Ellagic acid could change the composition of the incubation system.Conclusion:1.Tannins,organic acids and naphthalenes were mainly found in the water extract of Juglans mandshurica.2.The water extract of Juglans mandshurica would possess selective inhibitory effects on the growth of HGC-27 cells.3.The main constituents,including tannins,organic acids and naphthalenes,in Juglans mandshurica water extract could be absorbed in Caco-2 Cell Model and in Invert Intestinal Sac Model.The metabolite was mainly glucuronic acid binding products.4.It is preliminarily estimated that Gallic acid and Ellagic acid have no metabolic reaction in the in vitro metabolic model. |
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