| Actinidia arguta Sieb.et Zucc is one of the most widely distributed wild fruit trees in China, it has the most abundant resource in the three provinces in northeast China. Actinidia is an ideal health food, because its fruit, seed, stem and root, are all useful in medical value and have healthy function. The fruit contains multifunctional compounds with various pharmacological activities, and the polysaccharide is one of the major bioactive of Actinidia arguta. Actinidia arguta polysaccharides, AAP-3b was extracted and purified from the fruit of Actinidia arguta. Infrared spectrum scanning results showed that AAP-3b is pectic polysaccharide, in which the content of (?-glycosidic bond is predominant, also has some a-glycosidic bond. Monosaccharide composition of AAP-3b was determined by PMP pr-ecolumn derivative HPLC, theresult showed that AAP-3b is composed of mannose, rhamnose, galacturonic acid, glucose, galactose and ara binose, mannose and galacturonic acid are the predominant sugars, mole percentage are 31.49% and 32.07% respectively. The molecular weight of AAP-3b was determined by gel permeation chromatography, the results showed that the molecular weight (Mw) of main component in AAP-3b is 7298 Da. In this study, we choose AAP-3b for the research object, study for its biological safety, resistance of liver cancer in vivo and in vitro activity and mechanism of action, it will not only lay the foundation for industrialization of Actinidia arguta but also provide theoretical basis of Actinidia arguta polysaccharides health care products development.In this paper, a detailed study was carried out for purified Actinidia arguta polysaccharide:safety evaluation, including acute toxicity test, bacterial reverse mutation test, chromosome aberration test, gene mutation test, micronucleus test,30 days feeding test. Pharmacological activities in vitro:FRAP and DPPH and ABTS method were used to study the antioxidant capacity of AAP-3b in vitro. Morphology observation, living cells count, MTT and colony formation test were used to study the growth inhibition of liver cancer HepG2 cell with AAP-3b. The influence of AAP-3b on HepG2 cell cycle and apoptosis induction was studied through flow cytometry instrument and the action mechanism was discussed. Anticancer activities in vivo:The tumor model was set up by inoculating HepG2 cell under the dermal of nude mouse, the influence of AAP-3b on tumor-burdened nude mice were studied through general condition check, blood cell number, liver and kidney function detection. The antitumor activity of AAP-3b to HepG2 cell in vivo and the action mechanism were studied through inhibition ration of AAP-3b on HepG2 solid tumor in mice, MTT method to detect different groups of tumor cell proliferation activity and immune index determination. The main results and conclusion are as follows:1. The results of acute toxicity experiment showed that AAP-3b belongs to actually non-toxic substances. The results of genotoxicity study showed that AAP-3b had no mutagenicity both in the absence and presence of S9 mix; could not cause structural chromosomal aberrations in cultured mammalian cells under the conditions of this study; unable to induce mutation in this test system; could not induce the increase of the incidence of micronucleated PCE in mice; the results of 30 days feeding test of AAP-3b showed that in the test scope dose, no adverse effect were found in the following:growth of rats, blood, biochemistry and pathological.2. FRAP, DPPH and ABTS methods were used to study the antioxidant capacity of AAP-3b in vitro, the results showed that radical scavenging ability of DPPH and ABTS, reduction ability of iron ion have dose correlation on a range of doses. HepG2 cell cultured in vitro was acted with AAP-3b, at the dose of 50 ?g/mL-500 ?g/mL, the time of 24 h-72 h, by inverted microscope observe cell morphology change, HepG2 cell obviously inhibited by AAP-3b and in a dose and time dependent manner. Count living cell under microscope and MTT assay showed that at the dose of 50 ?g/mL-500 ?g/mL, AAP-3b has good proliferation inhibition on HepG2 cell. The result of colony for mationtest was that HepG2 cell colony formation rate decrease gradually, colony formation inhibition rate increased with the dose of AAP-3b increasing. The influence of AAP-3b on HepG2 cell cycle was studied through flow cytometry instrument and the action mechanism was discussed. The result of FCM showed that HepG2 cell cycle were blocked in G2/M and presented a dose and time dependent manner at the dose of 50 ?g/mL-200 ?g/mL,24 h. The result of FCM also display that AAP-3b has good apoptosis induction of HepG2 cell, cells showed typical apoptosis changes and presented dose and time dependent manner at the dose of 50 ?g/mL-200 ?g/mL,48 h-72 h.3. Anticancer activity in vivo, with the extension of time during the treatment, tumor and body weight of each group has increased, but the tumor and body weight growth of AAP-3b and CP were more slowly than model control group; the nude mice of model control became depression and moved slowly over time, and the groups of dose and CP were better than model control group. After the treatment, there was no statistical difference (P>0.05) between the groups of dose, CP control and model control, but there was statistical difference (P<0.01) between the CP control group and high dose of AAP-3b group, indicated that CP mavbe has toxic effect on the bodv. After treated with AAP-3b. the general situation of tumor-burdened nude mice, the number of blood cells, the function of liver and kidney all showed no obvious effect, and no pathological changes, indicated that AAP-3b has no adverse effect on body. HepG2 tumor growth were inhibited by AAP-3b in vivo, the tumor inhibition rate of 50 and 200 mg/kg·bw were 16.76% and 43.06%. The result showed that AAP-3b has anti-cancer activity in vivo. Proliferation activity of tumor cell in each group was detected through MTT method, the result showed that AAP-3b has obvious activity of inhibition proliferation on HepG2 liver cancer cell. The HepG2 cell proliferation inhibition rate of 50 and 200 mg/kg·bw were 17.23% and 41.51% respectively. Reduce liver transplantation tumor cell proliferation activity maybe one of the mechanisms of AAP-3b anticancer in vivo. The result of immune index determination showed that compared with model control group, the spleen index significantly increased at dose of 200 mg/kg·bw AAP-3b (P< 0.01), indicated that antitumor mechanism of AAP-3b related to immune enhancement. The spleen index of CP group significantly decreased compared with model control group, indicated that cyclophosphamide has inhibitory effect on the immune system. Although the anticancer activity of AAP-3b was slightly weaker than cyclophosphamide in vivo, but it has no adverse effect on immune system, the impact of nude mice weight was weaker than CP, therefore Actinidia arguta polysaccharide AAP-3b was harmless natural bioactive substances.All the results indicated that (1) AAP-3b is biological safety substance. (2) AAP-3b has anti liver cancer activity. The mechanisms may due to:1) Inhibition proliferation of liver cancer cell.2) Blocking HepG2 cell cycle in G2/M, promoting good apoptosis induction of HepG2 cell.3) Scavenging free radicals, enhance the body's antioxidant activity.4) Increasing the spleen index, enhance the body's immunity. |
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