Preparation Of Arctigenin And Its Anti-tumor Mechanism

Malignant tumour is one of the major diseases severely threatening the health of human beings. In average, 1 out of 8 deaths is due to cancer globally. Currently, most anti-neoplastic drugs have a therapeutic effect on the treatment of tumor, but there are still some shortages including expensiveness and side effects. Hence, it is a huge challenge we are confronting to look for new, effective and low-toxic anti-neoplastic drugs. Traditional Chinese medicine has a long history in tumor treatment, and moreover, Chinese medical herbs contain various monomers which possess strong anti-tumour effect. It is a significant subject for global medical circle on how to extract them for reasonable development and utilization. Researches on modern pharmacology prove that, Arctium lappa L., a kind of traditional Chinese medicine, possesses a variety of biological activities, e.g., anti-inflammatory, anti-virus and anti-tumor. The main active ingredient, Arctiin(ARC), is of low bioavailability in human body and can only play its therapeutic effect after being converted into Arctigenin(ARG) through enzymolysis of intestinal microflora and absorbed into the bloodstream. At present, ARG for pharmaceutical researches are mostly self-prepared in laboratories and the preparing methods more or less have some shortcomings, including low extracting efficiency, complicated extracting steps and high requirements on laboratory equipment. In our study, we extracted and purified ARG from Arctium lappa L. powder by microbial fermentation method and silica gel column chromatography method, and successfully established a simple and high-efficient ARG preparation method. Besides, it is reported that ARG could inhibit the growth of multiple malignant tumor cells, but people know little about its molecular mechanism for anti-tumor effect. In our study, the anti-tumor effect of ARG and the mechanism were studied in vivo and in vitro.Preparation of ARG: In our study, in order to convert Arctiin(ARC) into ARG-the active ingredient in Arctium lappa L, we use microbial fermentation method to process Arctium lappa L.. Aspergillus awamori var.and Trichoderma reesei were chosen as the fermentative strains, which could produce enzyme contributed to the conversion of ARC. Through the research on multiple fermentative factors including carbon source, fermentation time, p H, liquid volume, inoculation size, solid-to-liquid ratio of the substrate and carbon-nitrogen ratio, an optimal fermentation technology were put forward, which could achieve 97.8% of ARC conversion and 95.7% of dissolution rate and lower loss rate down to 4.3%. Finally we purified Arctium lappa L. fermentation extracts by silica gel column chromatography. ARG purified product is white crystal, with pureness possibly above 99%.Study on the anti-tumor mechanism of ARG in vitro: MTT method is employed to test the cytotoxicity of ARG to tumour cell lines and normal cell lines in vitro. The results show that, ARG has a broad spectrum anti-neoplastic activity, without a significant inhibitory effect on normal cells. We chose human hepatocellular carcinoma to further investigate the anti-tumour mechanism of ARG. According to the results of detection of mitochondrial transmembrane potential, Annexin V-FITC/PI double stain, transmission electron microscope(TEM) and Hoechst33342 / immunofluorescence staining, ARG could induce the apoptosis in Hep G2 and SMMC7721 cells. Furthermore, we also tested apoptosis signal pathway and apoptosis-related factors through q PCR, flow cytometry and Western blot. We found that, ARG could firstly lead to a dysfunction of mitochondria, simultaneously, cause an abnormality in the metabolic process in which NADPH and cytochrome P450 emzyme are involved, so as to result in an excessive ROS production in Hep G2 cells. Then, JNK and p38 MAPK signal pathways were activated by ROS and they both promoted the activation of intrinsic apoptotic pathway in Hep G2 cells. Meanwhile, p38 participated in activating the extrinsic apoptotic pathway and up-regulating p53 expression. Finally, both of two apoptotic pathways contributed to the caspase cascade activation, mediating apoptosis in Hep G2 cells. In addition, ARG could also inhibit nuclear translocation of NF-κB and the activation of PI3K/Akt signal pathway in Hep G2 cells. After all, the above results show that, ARG, a kind of “multi-target” compound, can induce apoptosis through regulating multiple signal transduction in tumor cells.Study on the anti-tumor mechanism of ARG in vivo: In the study of anti-tumor effect of ARG in vivo, we found that ARG could significantly inhibit the growth of human hepatocellular carcinoma xenografts in nude mice, and 20 mg/Kg ARG could achieve 44.55% of inhibition rate to Hep G2 xenograft. Moreover, it hardly damaged their main visceral organs including livers, lungs and kidneys. We found through further study that, the inhibition effect of ARG on the growth of tumor tissues in vivo was achieved through inducing apoptosis of tumor cells. The major mechanism is due to induce cell apoptosis through up-regulating p-JNK, p-p38, Bax and TNF-α expressions. The study on anti-tumor of ARG in vivo demonstrated that ARG not only could inhibit the growth of tumors, but also possessed the feature of low toxicity. The study results have laid an experimental foundation for the further development of ARG.In summary, we have created and established a new, simple and high-efficient method for extracting ARG through study, which was applicable for extracting and preparing ARG in laboratories, with multiple advantages, e.g., low cost, high extracting purity and low pollution. On this basis, we have discussed profoundly the anti-tumor effect of ARG and its mechanism in vivo and in vitro. The study result shows that, ARG has good anti-tumor effects both in vivo and in vitro and its mechanism is to inhibit the viability of tumor cells mainly through inducing apoptosis of tumor cells. Simultaneously, we also found through study that, ARG hardly played inhibitory effect on normal tissue cells in vitro, with low toxicity in animals. Therefore, ARG is a kind of anti-tumor compound with great potential for development. Our study has laid an experimental foundation for the further development and utilization of ARG.