Isolation Of Constituents From Angelica Pubescens And Corydalis Yanhusuo Guided By Antitumor Activity And Investigation On Their Effects

PART I:BACKGROUDCancer is a kind of disease that extremely threatens human health. Treatment by drugs is one ofthe main methods on cancer. Nowaday, trends in anticancer drugs are personalized usage of chemotherapeutic agents and discovery on molecular targeted ones. However, those roles are limited in clinic by severe side effects and huge costs. It's an important task for us to find out some drugs that possess higher anticancer activity and lower toxicity and are even cheaper. Traditional Chinese medicine (TCM) may be a useful model for scientific research because of its standardized system of therapies and long-time practices. Chinese medicine provides a rich pool of novel and efficacious agents for treating a variety of cancer. So, isolation and identification of new effective constituents of Chinese medicine and investigation its mechanism using molecular biochemistry methods are ascendant aspects of drug-development in our country.PART II ISOLATION OF ANTITUMOR-ACTIVE CONSTITUENTS FROM ANGELICAPUBESCENS AND INVESTIGATION ON THEIR EFFECTSObjective To isolate the antitumor-active constituents from Angelica pubescens guided byactivity and investigate their effects on tumor in vitro.Methods The effective extract of Angelica pubescens was separated by using several types ofcolumn chromtograpys and recrystallization to obtain the compounds, which structures weredetermined by spectroscopic methods, as well as comparing of their spectral data with those ofknown compounds. MTT assay was applied to detect the inhibitory effect of osthol. MTT andclonogenic assay were used to investigate radiosensitivity of osthol on human gastric cancer celllines MKN28 and MKN45. Apoptosis, cell cycle phases and ROS levels of MKN28 treated byosthol combining with radiotherapy were inspected by flow cytometery (FCM). And Realtime-PCR method was used to detect the mRNA expressions of BRCA1 after exposure to OSTand RT.Results1. Four compounds were isolated from the EtOH extract of this plant: osthol, columbianadin,lignoceric acid and columbianetin acetate. Among them, osthol is the main compound of thisherb to display antitumor activity. 2. Osthol has a significant cytotoxic effect on ten kinds of human cancer cells in vitro, and IC50 numerical values of OST are between 17.35 and 34.40μg/ml.3. The enhanced radiosensitivity of gastric cancer cells MKN28 but not MKN45 has been observed. The sensitivity enhancement ratio (SER) at 37% survival level is 1.137 for 24h pretreatment of OST at dose of 10μg/ml.4. Pretreatment of OST can increase apoptosis, cell cycle arrest at G₀/G₁ and intracellular ROS production induced by RT. And at the very early time after RT, OST can downregulate the expression of BRCA1 mRNA in MKN28.Conclusions Osthol which is the main antitumor-active compound of Angelica pubescens has a significant cytotoxic effect in vitro and can sensitize MKN28 to radiotherapy. This sensitization may be associated with increasing apoptosis, cell cycle arrest at G₀/G₁ and intracellular ROS production and downregulating the expression of BRCA1 mRNA by OST.PART III ISOLATION OF ANTITUMOR-ACTIVE PARTS FROM CORYDALISYANHUSUO AND INVESTIGATION ON THEIR EFFECTSObjective To isolate the antitumor-active parts from Corydalis yanhusuo guided by activityand investigate their effects on tumor in vitro.Methods The effective extract of Corydalis yanhusuo was separated by systematic solventextraction to obtain the parts. MTT assay was applied to detect the inhibitory effect of totalalkaloid fraction. Apoptosis and cell cycle phases of HepG2 treated by the total alkaloid wereinspected by flow cytometery (FCM). And the expression profile of microRNA in HepG2 wasdetermined by quantitative real-time PCR.Results1. The total alkaloid which was isolated from the EtOH extract of this plant is the main fraction displaying antitumor activity.2. The total alkaloid fraction of C. yanhusuo has a significant cytotoxic effect on ten kinds of human cancer cells in vitro, and IC50 numerical values of it are between 18.39 and 40.24μg/ml.3. The result of FCM in the range of 25μg/ml, 50μg/ml and 100μg/ml for 24h or 48h on HepG2 cells showed higher apoptosis rates. The ratio of S phase was significantly increased after exposure to the total alkaloid fraction for 24h or 48h compared with the control group. After exposure to the alkaloid for 24h or 48h, the expression of 15 cancer related microRNAs in HepG2 was altered, among which the up-regulation of let-7a and down-regulation of miR-221, mir-222 was the most significant.Conclusions The total alkaloid fraction which is the main antitumor-active compound of C. yanhusuo has a significant cytotoxic effect in vitro. The mechanism is probably related with its regulatory effects on cell cycle, apoptosis and expression of miRNA in cancer cells.