Research On The Characteristics Component Active Ingredients In HepA Transplanted Tumor Bearing Mice Anti Tumor Mechanism And Safety Evaluation Of Clinacanthus Nutans(Burm. F.) Lindau

As a type of disease with relatively high incidence in the world, malignant tumor seriously endangers human health. Existing antitumor drugs have some problems such as poor therapeutic efficacy, low specificity and significant toxic effects. Therefore, it is still a hot research topic to select efficient and specific low-toxicity plant resources against tumor with dual purposes of food and medicine.Clinacanthus nutans Burm.f. Lindau(C. nutans), commonly known as “Youdun grass”, Sabah snake grass, Green Arrow, is a kind of perennial herb that belongs to the Acanthaceae family. They are mainly found in Indochina, the Malay Peninsula as well as Guangxi, Guangdong, Yunnan, Hainan and other regions of Mainland China. With the efficacy of both medicine and food, it is a type of wild vegetable used by the masses for preventing and treating malignant tumor. Aiming at the C. nutans from Malaysia, this paper intensively and systematically investigates issues concerning main antitumor constituents, antitumor efficacy/mechanism and food safety based on isolation, purification, structural identification, assessment of antitumor activity in vivo/vitro and regulatory mechanism for immune activation, thereby laying a foundation for developing natural, safe and effective C. nutans as antitumor food or dietary supplements.(1) Firstly, C. nutans is used as raw material for separating and purifying constituents by solvent extraction, column chromatography based on silica gel, Sephadex LH-20 column chromatography and recrystallization. Besides, six flavonol glycosides with anticancer activity and functions, including orientin, isovitexin, isoorientin, Vitexin, Schaftoside and Apigenin, are identified in some parts of n-butylalcohol after column chromatography, elution and separation by 30% ethanol with HPLC-MS, spectroscopies(e.g. 1HNMR, 13 CNMR, HBQC and COSY), GC-MS, IR and HPLC.(2) Subsequently, the expressions of differential genes are observed with high-through putting gene chips and expression profiles, in order to preliminarily screen pharmacological functions and anticancer mechanisms of CN30. The results suggest that a total amount of 773 genes are differentially expressed. Furthermore, approximately 500 differential genes are found to be related to tumor and immune regulation. To be exact, the expressions of 345 differential genes are downregulated(P<0.05), while the expressions of 430 differential genes are upregulated(P<0.05). According to the analysis results, 504 genes(i.e. target genes) related to diseases such as tumor and immune dysfunction are differentially expressed. Affected signal transduction pathways mainly include mitosis, Pi3k-Akt, WnT, p53, p450, NFkB, Janus kinase, transcription activators(JAK/STAT) and vitamin A acid and so on.(3) Next, the experimental results are verified by MTT, Western blotting, fluorescent immunohistochemistry, intracellular staining, flow cytometry analysis, enzyme-linked immunosorbent assay and determination of biochemical indicators and so on. Furthermore, antitumor mechanism in vivo of CN30(i.e. C. nutans extracts) is determined. According to the research data, flavone c-glycosides, as major active small-molecule antitumor substances of CN30, play great roles in inhibiting proliferation of HepA in vivo in transplanted tumor-bearing mice. They may upregulate the expressions of protein factors such as MHC class II, Foxp3, Casp 3, Bax, Bad, CD4+, IL-2 and IFN-? by activating CD8+ T cells, CTL cells, Th1, Th2 and Th17 cell subsets. Meanwhile, the expressions of protein factors, including Bcl 2, Bcl-xl, PI3 K and AKT, are downregulated. Furthermore, Western blot demonstrates that flavone c-glycosides extracted from Clinacanthus nutans may upregulate and downregulate the expressions of Casp 3/Bad and Bcl 2/IL4 respectively. Therefore, the major immunological antitumor mechanism of CN30 in vivo is considered to induce apoptosis of tumor cells and proliferation of antitumor cells through NF-?B signals and cell migration.(4) At last, edible safety of C. nutans extracts is systematically evaluated. The experimental mice are fed with C. nutans ethanol extract at different doses to observe their acute toxicity, subchronic toxicity and genetic toxicity. The results suggest that although no mice were dead in different dosage groups in acute toxicity test, the serum biochemical indices such as TBIL and ALT increased. Compared with the control group, serum total protein albumin / globulin ratio and serum aminotransferase(ALT and AST) significantly increased in each group, so did the liver weight. However, the C. nutans extracts did not show genetic toxicity. In a word, the traditional medicine/edible dose of C. nutans dose are general safe, whereas it is necessary to avoid eating a large amount of C. nutans, so as not to cause hepatic/renal toxicity and abnormality of serum biochemical indices.