Extraction, Composition Identification And Anti Tumor Pathways For S180 Tumor Bearing Mice Of Polyphenols In Picea Koraiensis

The morbidity and mortality of cancer rising year by year, and the radiotherapy and chemotherapy often are given to treatment cancer at present. The radiotherapy and chemotherapy can obviously control cancer, but it seriously damage to human body simultaneously. Therefore, it is scientific research and the development trend in the future that develop the novel anti-tumor drugs. Many studies have shown that polyphenols have anti-tumor effect. So polyphenols can be raw materials as new drugs for anti-tumor. This paper takes polyphenols in Picea koraiensis(PKP) as research object, optimizing process conditions of ultrasonic-assisted extraction, microwave-assisted extraction and macroporous adsorption resin purification, identified composition of PKP. Then we analyze anti-tumor effect in vitro and anti-tumor pathways in vivo. The main content of this paper as follows:1.The optimal conditions of extracting PKP were obtained by single factors and response surface methods. The optimal conditions of ultrasonic-assisted extraction method were that ratio of liquid to solid 1:33, ultrasonic power 301W, ethanol concentration 40%, extraction temperature 60℃, extraction time 2.4h, and the yield of PKP was 18.92%. The optimal conditions of microwave-assisted method were that ratio of liquid to solid 1:31, microwave power 295W, extraction time 46s, ethanol concentration 40%, and the yield of PKP was 17.38%.2.The optimal purification conditions of PKP were obtained by single factors and respon-se surface methods using D-101 macroporous adsorption resin, which were sample volume 25 mL, diameter-length ratio 1:25, eluent concentration 70%, elution velocity 2.0mL/min. Purified PKP was analyzed by HPLC. The results showed that the main composition of PKP were p-coumaric acid, salicylic acid, isoquercitrin and catechin, whose content of 31.91%、24.85%、 2.54% and 1.52%, respectively.3.The results of MTT test showed that PKP had the highest antiproliferative activities on S180 tumor cell, followed by A549 tumor cell, but had the lower activities on A375, SY5Y, Hepg 2 and Skov3 tumor cell. The results of cell morphological observation showed that PKP had obviously inhibitory effect on S180 tumor cell with a certain degree of concentration-response relationship. DNA damage, the results show that the red-skinned spruce polyphenols on S180 and A549 tumor cell DNA damage obviously, and showed obviously concentration-response relationship, including stronger effect on S180 tumor cell DNA damage. Single cell gel electrophoresis was used to detect the DNA molecular damage of S180 and A549 tumor cell. The results showed that PKP had obviously damage effect on S180 and A549 tumor cell with significant concentration-response relationship, and the damage effect on S180 was higher than A549. The effect of PKP treatment on S180 and A549 tumor cell cycles were measured by using flow cytometer. The results showed that PKP could efficiently block S180 and A549 at the stage of G0/G1, which couldn’t synthesis DNA. This indicated that PKP could induce apoptosis of S180 and A549 tumor cell.4.It was used to study antitumor ability of PKP that S180 tumor bearing mice in the state of radiation damage. The results showed that PKP had significant effect on solid tumor of S180 tumor bearing mice. Low, moderate and high dose of PKP could inhibit S180 solid tumor by 54.15%,42.24% and 23.00%, respectively. PKP treatment could also significantly enhance immunity ability of S180 tumor bearing mice. Then CTX had higher antitumor ability than high dose of PKP, but CTX couldn’t enhance immunity ability.5.The results of HE staining showed that CTX and high dose of PKP could obviously restrain proliferation of tumor cell, and the CTX’s effect was higher than high dose of PKP. It was analyzed by immunohistochemical method that the expression of Bax, Bcl-2, p53, β-catenin, Cyclin D1 and NF-κB in solid tumor of S180 tumor bearing mice, and we further analyzed antitumor pathways of PKP. The results showed that CTX and high dose of PKP could significantly increase the expression of Bax and p53, decrease the expression of Bcl-2, (3-catenin, Cyclin D1 and NF-κB. It demonstrated that PKP could restrain Wnt/β-catenin pathway and NF-κB pathway, and NF-κB pathway had cross effect with other pathways.