| Background:Malignant tumor is one of the most serious diseases, which harmful to human life and health, and its incidence increasing year by year. According to the statistics released by the world health organization (WHO) and the American society of clinical oncology (ASCO), the number of newly diagnosed cancer patients more than10million, about6to7million patients died of cancer, accounting for12percent of total deaths in the whole world. Cancer is the primary cause of death in people developed country (22.3%of the total deaths), it is also the second cause of death in the developing country (accounting for9.5%of all deaths). According to the statistics released by China’s Ministry of Health, the incidence of malignant tumor increase year by year in china for the past few years. In2008, malignant tumor mortality has reached16.697per million in urban areas, it is27.12%of the total cause of death. In parts of the city and the countryside in china, malignant tumor has jump immediately to first death causes from the third place, become a leading killer to residents for the first time, more than the cerebral vascular disease and heart disease. The world health organization in18th international conference on cancer alliance report says the number of new patients with tumor by the current10million/year increased to15million/year in the coming20years, so the number of cancer deaths each year will also from6million to10million. It is difficult to measure the physical pain to patients with tumor, the economic burden to family and the damage to our society. Therefore, the WHO and China’s government has highlight the project of cancer treatment.In recent years, many countries are invested a lot of manpower and money resources to the anti-cancer drugs research, a lot of reliable efficacy drugs have been develop, such as cyclophosphamide,5-FU, cis-platinum. But these drugs are difficult to spread and for long application owing to their unsatisfactory effect or much toxic and side effects. With the rise interest of naturalize, getting natural anti-cancer drugs from the nature become the hot research areas, the practitioners and researchers have developed more than20kinds of plant medicine for cancer therapy, such as alkaloids (Vincristine, Camptothecin, Harringtonine, etc), lignans (derivatives of podophyllotoxin), macrocyclic compound (maytenin), diterpenoids (paclitaxel, oridonin), most of these drugs with small side effects and curative effects. So it is possible to search for new cancer drugs from natural plant.A lot of researches have uncovered increasing evidence that malfunction of the immune system plays a crucial role in triggering tumorigenesis and metastasis. Moreover, tumor responses to most chemotherapeutic drugs used in the clinic appear to strongly depend on activation of the immune response. Therefore, patients with increase to control the tumorigenesis and metastasis, not only it is a target the treatment effect for tumor, but strengthen body’s immune system. Traditional Chinese medicine emphasis improving the function of human body itself against disease, the mechanism is very close to anticancer with immune strengthen. This project aims to explore an anticancer herbal through immune cells differentiation.Alocasia cucullata is the root of Alocasia cucullata (Lour.) Schott (family Araceae), widely distributed Guangdong, Guangxi, Guizhou, Yunnan, Sichuan, etc in china. It has been compiled into Chinese Plant Mapping Database as one of the poisonous plant, Chinese medicine called JIEBO. Alocasia cucullata has clearing heat and detoxicating, detumescence analgesic effect, it is used to the treat multiple tumors, influenza, high fever, leptospirosis, snake bites, poison bee sting injury, chronic bronchitis, etc. The most medicinal effect is dictate only folk use and records, lack of system of the pharmacological studies deeply, so it is limited Alocasia cucullata further development and utilization.Objectives:To conduct pharmacodynamics of Alocasia cucullata, we prepared Alocasia cucullata water extraction in a similar way of the traditional Chinese medicine decocting. Firstly, we found that the high contents of polysaccharide in Alocasia cucullata component analysis, so further purification polysaccharide and find out the purification of polysaccharide process. And then establish mice breast cancer model for investigates it antitumor role, and test the melanoma bearing mice survival time, normal mice acute toxicological effect. With the cell model, we prove pointed it induce cell differentiation role, and find out and cell differentiation cells associated factors. It is not only scientific explanations the correctness of anticancer with Alocasia cucullata in folk, but also provides the scientific basis of high-efficiency, price moderate anti-cancer drugs developed. Methods:1The preparation of Alocasia cucullata water extraction and purification techniques of polysaccharide.β1.1Extraction:All the fresh root of Alocasia cucullata were rinsed well and choped finely, Baked in the oven at60℃, crushed and screened.1000g Alocasia cucullata powder extracted with boiling water for two times,4h once, filtrated and combined filtrate, enriched to1000mL, dried under vacuum, weighed, calculated and stored for subsequent use.1.2The components of the polysaccharide:Hydrolyzed Alocasia cucullata water extraction, neutralized it with BaCO3. Take D-fructose, D-glucose, L-rhamnose, D-xylose, D-galactose as control article, lactose do reference substance, the TLC conditions:n-butyl alcohol-acetone-H2O for developing solvent, alpha naphthol-sulfuric acid as colour reagent, analysis with silica G thin layer plate.1.3Preliminary purification of Alocasia cucullata polysaccharide.Crude polysaccharides were prepared by hydrogen peroxide decoloration, alcohol precipitation, and deproteinization, washing with ethyl ether and acetone, repeatedly. Alocasia cucullata polysaccharides were purificated by DEAE-52and Sephadex G-200column chromatography, then freeze-drying and stored for subsequent use.1.4Purity and physical&chemical charactersTo identification purity of polysaccharide, the content of proteins were determined by Coomassiae billiant blue, and determination of the physical and chemical properties, such as solubility.2Anti-tumor effects of Alocasia cucullata and acute toxicity experiments in mice. 2.1Alocasia cucullata water extracts suppress breast cancer growth in miceSeventy BALB/c mice were randomly divided into seven major group,(1) Normal mice administrated with negative control group (N-Nctrl);(2) Model group treated with negative control group (Nctrl);(3) Model group treated with positive control group (Pctrl);(4) Model group treated with Alocasia cucullata at low doses (A.c-L)(5) Model group treated with Alocasia cucullata at middle doses (A.c-M);(6) Model group treated with Alocasia cucullata at high doses (A.c-H);(7) Normal mice administrated with Alocasia cucullata group (N-A.c).4T1cells were subcutaneous injection in right maxillas of mice in group (1),(2),(3),(4),(5) at the density of1.0×106cells per mouse. Simultaneously, the adaptometer begin timing. Seven days later, the first group, second group, third group and sixth group mice were fed by ig with80,160,320,160mg/mL Alocasia cucullata, respectively, the fifth group and seventh group fed by ig with distilled water, the fourth group with lentinan.21days later, the tumor growth in different groups were observed, and measured the tumor volume, tumors weight, spleen index and the thymus index. Hematoxylin-eosin staining was performed to observe the morphologic changes of tumor tissue. Serum levels of IL-2, IL-10, IFN-γ, TNF-α, TGF-1β were measured with ELISA, The normal BALB/c spleen cell and macrophage activity were determined by MTT.2.2The effect of Alocasia cucullata on the survival time of melanin bearing mice.Twenty healthy C57BL/6J mice were randomly divided into two groups, ten mice of each group. B16cells were subcutaneous injection in mice right axilla at the density of1.0×10’cells per mL,0.1mL per mouse. Simultaneously, the adaptometer begin timing. Seven days later, conditioned mice stomachs were perfused with Alocasia cucullata, control group which were treated by distilled water. And set the observation time limit of60days, observe the survival time of tumor-burdened mice. Alocasia cucullata would neither stimulate nor inhibit4T1cells growth in vitro. The role of Alocasia cucullata induces normal mice splenic cell activity evaluated by MTT, the results indicated it was no significant difference between conditioned group and control group while Alocasia cucullata concentration is200μg/mL, it was significant difference when the concentration increase to500or1000μg/mL. Alocasia cucullata induce splenic cell activity in concentration dependent manners, it also have promoting multiplication effect on mice spleen cells. Alocasia cucullata would longer survival time of tumor-bearing mice, The median survival for condition group was34(30.60,55.39) day, which was significant longer than the control group (p=0.033).160.4g/kg.d Alocasia cucullata on mice in acute toxicology experiment, the value is481.2times as people clinical dose, which indicated Alocasia cucullata is a low toxic drug.2.3The acute toxicity experiment of Alocasia cucullata.Choosing the40Kunming mice (bisexual each half, weighting18to22), randomly divided into2groups,20mice of each group. All mice were administered in a volume of40mL/kg body weight and two times a day. Then mice were routinely feed, the appearance, activity, breathing, secretion and defecation were observed. The mice were killed by the method of cervical dislocation after the fifteenth day and examined pathological changes of heart, liver, spleen, lung and kidney.3Alocasia cucullata induce differentiation of THP-1cell.3.1Induce differentiation of THP-1cells.THP-1cell were seeded in6-well plates, blank control cells cultured with RPMI-1640complete medium, experimental group cell treated with1000、2000mg/L Alocasia cucullata,10ng/mL PMA treated cells as positive control, morphological changes were observed after72hour. Then cells were washed once with PBS, adherent cells were collected by scraper, well-distributed and cell counted and calculated cells adherent and inhibition ratio. Besides, the conditioned THP-1cells were stained with CD14and CD11b antibody, fixed with paraformaldehyde. The proportion of cells expressing and the fluorescence intensity of surface markers were measured by flow cytomerty.3.2Cell cycle, cytokines and chemokines assay of differentiated THP-1cells.Conditioned THP-1cells was collected, the supernatant aliquot was taken away. The cells were washed twice with PBS and fixed by80%ice-cold ethanol. Suspend the cells in PBS contained0.1%Triton,25μg/mL propidium iodide, The PI fluorescence of individual nuclei was measured using a flow cytometer. Moreover, TNF-a and IL-1β in the supernatants of the samples were determined by sandwich ELISA kit according to the instructions of the manufacturer. The other cytokines and chemokines in cell supernatants were analysed by human cytokine array panel array Kit and human chemokine array kit.4Statistical analysesAll statistical analysis were carried out using the SPSS13.0statistical software package, and all data were expressed as mean±S.D. Student’s t-test was used for statistical comparisons two groups. If the variances between groups were homogenous (Levene’stest), groups were subjected to the multiple comparisons least significant differences (LSD) test. In case of no homogeneity variances, differences were evaluated by welch and the groups were subjected to the multiple comparisons Dunnett’s T3test. Graft survival was evaluated by the Kaplan-Meier method. Statistical significance was set at p<0.05. Results:1The preparation of Alocasia cucullata water extraction and purification techniques of polysaccharide.We got249.3gram Alocasia cucullata water extraction, the extract percentage was24.93%. The Alocasia cucullata polysaccharide was constituted by D-glucose and D-galactose. The purified Alocasia cucullata polysaccharide is white solid, easily dissolved in water, insoluble in methanol, alcohol, acetone, n-butyl alcohol and other organic solvents. Alocasia cucullata polysaccharide is non starch polysaccharide, without ionized monosaccharide, polyphenols, uronic acid, protein and nucleic acid.2Alocasia cucullata water extracts suppress breast cancer growth in mice.Tumor volume in an ascending order is as follows:Ac.-H, Pctrl, A.c-M, A.c-L, Nctrl, it is statistically significant between the groups in difference conditions and difference time points. Results of hematoxylin eosin staining showed that, tumor cell grows well in the control group with fewer apoptosis cell, which density increase obviously, Ac.-H group and Pctrl group tumor cellular outlines vaguely remain, arranging loosely. IL-2, TNF alpha, INF-y contents of tumor-bearing mice serum were higher in Ac.-H group as compared with the control group and the difference was significant (p<0.01), but there were no obvious difference in TGF-β1content (p=0.075). IL-2, TNF alpha, INF-y contents of tumor-bearing mice serum were higher in Ac.-H group as compared with the control group and the difference was significant (p<0.01), but there were no obvious difference in TGF-β1content (p-0.075). In normal mice serum, IL-2, TNF alpha were higher in Ac.-M group than N-Nctrl group and the difference was significant (p<0.05), but it is no significant difference in IL-10, INF-γ and TGF-β1(p>0.05). Alocasia cucullata would neither stimulate nor inhibit4T1cells growth in vitro. The role of Alocasia cucullata induces normal mice splenic cell activity evaluated by MTT, the results indicated it was no significant difference between conditioned group and control group while Alocasia cucullata concentration is200μg/mL, it was significant difference when the concentration increase to500or1000μg/mL. Alocasia cucullata induce splenic cell activity in concentration dependent manners, it also have an promoting multiplication effect on mice spleen cells. Alocasia cucullata would longer survival time of tumor-bearing mice, The median survival for condition group was34(30.60,55.39) day, which was significant longer than the control group (p=0.033).50.14g/kg.d Alocasia cucullata on mice in acute toxicology experiment, the value is300.84times as people clinical dose, which indicated Alocasia cucullata is a low toxic drug.3Alocasia cucullata induce differentiation of THP-1cell.THP-1cells grew as a single-cell suspension, and only a few cells spread onto the grass substrate. It showed obvious differentiation after induced by Alocasia cucullata for72h. Treated THP-1cells adhered to the glass substrate. Since the adherent cells could not be detach from the dish with EDTA and trypsin treatment. But some suspension cells are still present even prolong the inducing time. And the number of cells suspended is inversely proportional to the concentration of Alocasia cucullata, and the inhibition rate to the cell is proportional to the concentration. The expression of the CD11b and CD14increased in treated THP-1cells. The flow cytometry histogram showed twin peaks in1000mg/L Alocasia cucullata treated THP-1cells, consistent with two forms observated under the microscope, it is indicated not all THP-1cell differentiation. The percentage of CD14and CD11b positive cells is57.40%,39.65%, which was significant difference from the control group (p<0.01), and cell fluorescence intensity increase significantly compared with the control group (p<0.01). Alocasia cucullata induced IL-1β and TNF-a production in THP-1cells. Cell cycle analysis by flow cytometry demonstrated that THP-1cells treated with2000,4000mg/L Alocasia cucullata resulted in sub-G1phase cell ratio increasing to (22.3±4.1)%,(60.5±0.7)%, which was significantly higher than that of the control group (p<0.01). The chemokine array kit assay results show that, compared with the normal group, RANTES, IL-8, MIP-la,β, GRO expression was up-regulated in Alocasia cucullata group. The cytokines array kit assay results show that MIF, MIP-1α IL-8、GRO α、 IL-1ra、RANTES、TNF-a expression was increased in Alocasia cucullata treated THP-1cell.Conclusion:1Alocasia cucullata water extraction in the mainly contain polysaccharide. Through the decolouring, take off the protein, dialysis and column chromatography can be purified to a high purity of polysaccharides. The extract has no acute toxicological effect on mice at a maximum concentration.2The results showed that Alocasia cucullata can inhibit the growth of tumors in mice, but weak to cancer cell in vitro. This effect is due to it enhancing activity of spleen and macrophage’s cells, up-regulating the expression of IL-2, TNF alpha, INF-γ in serum. What is more, it also lengthens melanoma of the mice survival. It indicated Alocasia cucullata enable host cells to generate antitumor immune response by stimulating and enhancing immunological function response.3Alocasia cucullata induce differentiation of THP-1cells, mainly embodies in induced cells produce immune related factors, such as TNF alpha andIL-1β, making the cell block in sub-G1phase. |
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