Isolation And Extraction Of Five Natural Total Alkaloids And Experimental Study Of Their Antitumor Activity In Vitro

Cancer seriously threat to life and health of human beings. Surgery, chemotherapy and radiotherapy are the three major cancer treatment methods, including chemotherapy in cancer multidisciplinary, comprehensive treatment of multiple means to play a important role. Chemotherapy, however, to some extent still has its limitations, such as specific inhibition of tumor cells is not so obvious and toxicity. Chinese medicine as traditional medicine in China, in the treatment of malignant tumors has a unique advantage. Large number of clinical practice shows that Chinese medicine in improving the clinical efficacy of cancer and improve quality of life, playing an increasingly important role. Most traditional Chinese medicine derived from plants, with the upsurge return to nature, from the traditional Chinese medicine (traditional herbal medicine) in the excavation and development of high efficiency, low toxicity, low-cost, broad spectrum of natural antitumor drugs have become the research hotspot. Study found that Chinese herbs contain many ingredients in varying degrees of anti-tumor effects, such as alkaloids (vinblastine, camptothecin), diterpenoids (paclitaxel, oridonin). It has confimed that Chinese Coptis, Evodia and flavescens have the anti-tumor effect and their aklaloids as their major effective components. Extraction and purification of modern means of drug escalation, from natural herbal anti-tumor effect in the search for active ingredients and the active site, and on this basis, the development of cancer drugs that has tremendous potential and development prospects.[Objective]1.Comparison of three different extraction:aqueous extraction, ethanol extraction, acid extraction method to Evodia, Coptis, flavescens, Corydalis, Uncaria contained active site-the separation of alkaloids extracted; draw the corresponding optimum extraction method.2.Using HPLC to determinate the effective ingredients contained in the five natural effective part of alkaloids.[Methods]Three diffenent extraction of aqueous extraction, ethanol extraction, acid extraction were used to isolate and extract the total alkaloids of Evodia, berberine, flavescens, Corydalis, Uncaria. The effective part of their active ingredients contained in the corresponding levels were determinated using by HPLC(high performance liquid chromatography).[Results]1. Three kinds of extraction of natural anti-tumor active components of the five total alkolids content of biological comparison: Ethanol extraction> acid extraction>aqueous extraction.2. The comparison of corresponding effective ingredients in active sites extracted from five natural total alkolids measured by HPLC:①corydalis alkaloids (Tetrahydropalmatine/total corydalis alkaloids):ethanol extraction> acid extraction> aqueous extraction.②berberine alkaloids (berberine/total alkaloids):ethanol extraction> acid extraction> aqueous extraction.③Evodia alkaloids (rutaecarpine/total alkaloids):acid extraction> aqueous extraction> ethanol extraction.④Flavescens alkaloids:ethanol extraction> acid extraction> aqueous extraction Oxymatrine/alkaloids:ethanol extraction> acid extraction> aqueous extraction Matrine/alkaloids:aqueous extraction> acid extraction> ethanol extraction⑤Uncaria alkaloids:not determined precisely contained Rhynchophylline, isorhynchophylline content, considering the result of pre-separation and extraction reagents and temperature, has a certain influence on its structural stability, leading to structural change, it is not can be measured.[Objective]To investigate the anti-tumor effect of five natural total alkaloids of Evodia, Coptis, flavescens, Corydalis and Uncaria extracted from three kinds extraction of aqueous extraction, ethanol extraction, acid extraction and their corresponding active ingredients contained in total alkaloids on human gastric cancer cell lines in vitro, in order to compare the strongest anti-proliferation activity of five total alkaloids. And compaere the antitumor activity of the corresponding active ingredient alkaloids and effect part of total alkaloids on human gastric cancer cell lines.[Methods]1. The preparation of five total alkaloids:aqueous extraction,70% ethanol extraction,1% acetic acid extraction were prepared the active site-alkaloids of Evodia, berberine, flavescens and Uncaria.2. Cell proliferation inhibition test:Using the tetrazolium blue (MTT) to study the antitumor activities of five effect sites-total alkaloids and the natural active ingredients on human gastric cancer cells BGC-823, MKN-45 in vitro. Survival rate= (experimental group calculated OD)/(control group OD)×100%, Inhibition rate= (1-experimental OD/ control OD)×100%.3. Morphological observation:different concentrations of Evodia total alkaloids, berberine total alkaloids, Flavescens total alkaloids, Uncaria total alkaloids and corydalis total alkaloids to treat on human gastric cancer cell line BGC-823 and MKN-45 after observed under inverted microscope Morphological changes of treated cells, and photographed.[Results]1. The inhibition effect of the effective part of Evodia alkaloids, berberine alkaloids, matrine alkaloids, corydalis alkaloids, Uncaria alkaloids on human gastric cancer cell line MKN-45, BGC-823 were obviously observed and with the increase in drug concentration, inhibition rate increased in a dose dependent relationship.2. The comparison of inhibite rate of five alkaloids between the 2μg/ml-200μg/ml human gastric cancer cell line MKN-45:Evodia alkaloids aqueous> aqueous berberine alkaloids> ethanol uncaria alkaloids> ethanol matrine alkloids> ethanol corydalis alkaloids①Evodia alkaloids:aqueous extraction>ethanol extraction>acid extraction;②berberine alkaloids:aquesous extraction>ethanol extraction>acid extraction;③Flavescens alkaloids:ethanol extraction>aqueous extraction>acid extraction;④Corydalis alkaloids:ethanol extraction>aqueous extraction>acid extraction;⑤Uncaris alkloidst:ethanol extraction>aqueous extraction>acid extraction; The comparison of inhibition rate of five natural effective part of the active component of alkaloids in 2μg/ml-200μg/ml concetration on human gastric cancer cell line MKN-45: Aqueous extract Evodia alkaloids> rutaecarpine; aqueous berberine alkaloids> berberine; Ethanol total alkaloids ethanol matrine> matrine.3. Five alkaloids between 6.25μg/ml-200μg/ml concentration on human gastric cancer cell line BGC-823 after 48,72 hours, with time and drug concentration, inhibition rate increased gradually in a time-dose dependent manner in a certain dose range.4. Evodia alkaloids from aqueous extraction, Berberine alkaloids from aqueous extraction, Alkaloids ethanol, ethanol total alkaloids, Uncaria alkaloids in the role of ethanol on gastric cancer BGC-823 cells 48 hours, cell growth was inhibited, and the aqueous berberine alkaloids of IC50=20.34μg/ml, different drug concentrations were significant differences between5. Morphologic changes:under normal circumstances, human gastric cancer cell line BGC-823 and MKN-45 were polygonal, spindle, adhered to the wall. The role of alkaloids in five, the cells became round and shrunken, reduce the number of adherent cells, cell swelling, the size of inconsistency, the cell gap widened.[Conclusion]1. Evodia alkaloids, berberine alkaloids, Flavescens alkaloids, Uncaria alkaloids, corydalis alkaloids in vitro have a strong inhibition of proliferation on human gastric cancer cell line BGC-823 and MKN-45 in vitro, and with the dose increased the inhibiton increased. Evodia alkaloids from aqueous extraction and berberine alkaloids from aqueous extraction have a stronger inhibitory effect on gastric cancer cells.2. Evodia alkaloids from aqueous extraction, Berberine alkaloids from aqueous extraction, Flavescens alkaloids from ethanol extraction, Corydalis alkaloids from ethanol extraction, and Uncaria alkloids from ethanol extraction on human gastric cancer cells BGC-823 inhibition, IC50:25.76μg/30.34μg/ml,68.51μg/ml,34.50μg/ml,33.10μg/ml.3. The inhibition the proliferation of aqueous extraction Evodia alkaloids and berberine alkaloids than the corresponding active ingredients.