Research On The Composition And Polyphenols Isolation Of Pinus Koraiensis Seed Shell And Its Antioxidant And Antitumor Characteristics

Pinus koraiensis is widely distributed in Northeast China. Among variety of active ingredients, polyphenols are important antioxidant in Pinus koraiensis seed shell, and have tremendous development potential. The work in this paper analyzed the effective composition in the seed shell from Pinus koraiensis. Antioxidant activity and anticancer pathway of polyphenols in Pinus koraiensis seed shell were systematically studied in this paper. This systematic work provided theoretical foundation for exploitation and utilization of Pinus koraiensis seed shell resources on oxidative damage treatment and defence.Steam distillation and Gas Chromatography-Mass Spectrometer (GC-MS) were adopted to extract and analyze the volatile components and polyphenols composition in Pinus koraiensis seed shell. The whole active components of the seed shell were detected by spectrophotometry. The polyphenols were extracted by ultrasonic-assisted method, based on the results of single-factor trials, and the optimum extracting conditions of polyphenols from Pinus koraiensis seed shell were obtained by the response surface methodology. The seed shell polyphenols were purified by macroporous resin with the optimum conditions which obtained by static and dynamic tests. By establishing the in vitro models, the scavenging and inhibition activities of seed shell polyphenols on physiological and unphysiological free radicals were investigated. Furthermore the metal ion chelating capacity and the reducing power of the polyphenols from seed shell were also tested. By establishing the cell and tissue models in vitro, the inhibition of seed shell polyphenols for hemolysis and lipid peroxidation were studied. With the subacute aging model induced by D-galactose and the radiation model induced byγ-ray, the physiological indexes and oxidation-related enzymes activities were analyzed to investigate the antioxidant activity and mechanism of the seed shell polyphenols in depth. MTT [3-(4,5-Dimeth-ylthiazol-2-yl)-2,5-diphenyltetraz olium bromide] colorimetric determination, Colony formation and Agarose Gel Electrophoresis (AGE) assays were applied to investigate the effect of seed shell polyphenols on proliferation, differentiation and apoptosis of cancer cells, respectively. Cell immunostaining was operated to study the influence of the seed shell polyphenols on Bcl-2 and Bax gene expression in tumor cells. The activity of apoptosis-related enzyme caspase-9 in tumor cell was determined by spectrophotometry. Flow cytometer was applied to detect the effect of the seed shell polyphenols on tumor cell cycle, in order to explore the anticancer mechanism of Pinus koraiensis seed shell polyphenols. S180 and H22 tumor models (ascites tumor and solid tumor) in vivo were built with Kunming mice to study the anticancer function of seed shell polyphenols in vivo, and activities of the critical metabolism-related enzymes were investigated to explore the anticancer pathway of seed shell polyphenols in vivo.The research findings: 23 kinds of active components were analyzed in Pinus koraiensis seed shell by GC-MS method, out of these components, the phenolic substances were carvacrol, butylated hydroxytoluene and 6-tert-butyl-2, 4-dimethylph-enol et al. The optimal extraction conditions for seed shell polyphenols were that the extraction time was 2.8 h, the ratio of solvent to material was 1:26, the ethanol concentration was 43 %, and the extraction temperature was 80°C. The optimal purification conditions for seed shell polyphenols were that the resin type was AB-8, the sample concentration was 1.5 mg/mL, the sample flow rate was 2.0 BV/h, the eluant was 30 % and 70 % ethanol gradient elution and the flow rate was 1.5 BV/h. The antioxidant activity research results suggested that polyphenols from Pinus koraiensis seed shell could restrain hydroxyl and superoxide anion radicals effectively (physiological), the EC50 values were 0.391 and 4.37 mg/mL, respectively, the DPPH and ABTS radicals were also restrained effectively (unphysiological), the EC50 values were 0.023 and 0.46 mg/mL, respectively. The research findings demonstrated that Pinus koraiensis seed shell polyphenols could effectively inhibit lipid peroxidation of mice liver homogenate and yolk, the inhibition ratio was 91.50 % (7.0 mg/mL) and 95.44 % (1.5 mg/mL), respectively. Furthermore seed shell polyphenols could restrain oxidation hemolysis of red cell in mice.The antioxidant research results in vivo demonstrated that polyphenols from Pinus koraiensis seed shell could restrain subacute aging reaction in mice, the SOD activity and GSH content could be enhanced, while the MDA content was depressed (medium and high dose). The polyphenols also restrained and repaired the oxidation damage induced byγ-ray. The polyphenols could effectively restrain the proliferation of HT29, H460, SCG-7901 and MCF-7 cell, and the inhibition effect was strongest for HT29 cell. The polyphenols could induce differentiation and apoptosis of tumor cells, based on the case that the typical DNA Ladder in electrophoresis and decreasing colony formation units under the influence of the polyphenols.The expression of cancer gene and the tumor cell circle were impacted by Pinus koraiensis seed shell polyphenols. The research showed that the expressions of Bcl-2 and Bax gene in HT29 cell were depressed and enhanced respectively in time-dose dependence. Pinus koraiensis seed shell polyphenols could elongate G1 phase and shorten S phase in the cell cycle of HT29, implying that there was G1/S arrest in cell cycle under the effect of the polyphenols, and the arrest degree depended on the polyphenols dose. The in vivo anticancer research demonstrated that S180 and H22 solid tumor could be inhibited by the Pinus koraiensis seed shell polyphenols. The three doses in the trails were all significant for S180 solid tumor inhibition, the inhibition rates were 25.00 % (low dose), 31.82 % (medium dose) and 34.09 % (high dose). Medium and high doses were significant for H22 solid tumor inhibition, the inhibition rates were 26.32 % (medium dose) and 27.36 % (high dose). The activities of lactate dehydrogenase and aldolase in animal serum were decreased by the seed shell polyphenols, implying that there was relationship between the anticancer activities of seed shell polyphenols and glucose metabolism reduction in vivo.