| Tumor is a killer for human health,and the conventionalchemical drugs for the treatment of tumor possess different grade of side effects on normal tissues and organs of body,bringing new healthy risks for patients.The immune system plays an important role in the development of tumor.However,the proliferation of tumor cells can not only lower the reactivity of immune cells,but also induce immune cell apoptosis and inhibit its antitumor activity.Therefore,screening safe and effective antitumor substances which could effectively enhance immune activity become a research hotspot.The polyphenols of P.koraiensis pinecones was selected as the research object in this study.PPP was separated using the ultrasonic-assisted extraction method,and the purification process was optimized using response surface methodology(RSM).By tracking the antitumor activity in vitro,40 %(v/v)ethanol eluate PPP-40 was selected as the most active component with the strongest antitumor activity.Here,we analyzed the main components of PPP-40,of which the antitumor mechanisms in vitro and in vivo were studied systematically and the repairing mechanisms of immune system in tumor mice was revealed.A ultrasonic-assisted procedure for the extraction of polyphenols from P.koraiensis pinecone.Optimized conditions for maximum yield were the ratio of liquid to material 20:1,the ethanol concentration 60 %,ultrasonic power 400 W and extraction time 60 min,and the content of polyphenols reached to 19.02±0.67 mg/g.The purification process of pinecone polyphenols was optimized using the way of AB-8 macroporous resin combining with the RSM,and the purity of primary separation product(PPP)was 27.93±1.40 %.After that,the D101 macroporous resin was engaged to further purify PPP,and the obtained PPP-40 possessed the highest the purity of 57.25±1.83 % and showed the strongest antitumor activity against human colon(LOVO)cells with EC50 0.21±0.03 mg/mL,which indicated that the methods of isolation and purification were effective to improve the antitumor activity of pinecones polyphenols.PPP-40 constituents were analyzed by UPLC-Q-TOF-MS.There were eleven kinds of polyphenols compounds in PPP-40: caffeic acid,7-methoxycoumarin-4-aceticacid,catechin,4,6,7-trimethoxy-5-methylcoumarin,lithospermic acid,taxifolin,7-xylose puerarin,rhamnetin,isorhamnetin,azaleatin and tamarixetin.The antitumor effect and mechanism of PPP-40 in vitro were studied.It was observed that PPP-40 can inhibite the clone formation of LOVO cells,induce the DNA damage and promote the apoptosis of LOVO cells.The specific apoptosis mechanism was clarified that PPP-40 leaded to the mitochondrial dysfunction through reducing mitochondrial membrane potential,decreasing ATP synthesis and increasing ROS levels in LOVO cells.Meanwhile,PPP-40 also induced Caspases-dependent apoptosis by promoting the release of mitochondrial cytochrome C and activating the Caspase-3,Caspase-8 and Caspase-9.The data of antitumor effect and mechanism of PPP-40 in vivo showed that the medium dose of PPP-40(150 mg/kg)possessed the highest inhibition rate of 48.29 % for Sarcoma 180(S180)-bearing mice.The results of the Tunel apoptosis rate and apoptotic proteins tests showed that PPP-40 up-regulated the related apoptotic proteins expressions of the mitochondria pathway(Bax and Caspase-3)and down-regulated the expression of Bcl-2 protein to induce S180 tumor cells apoptosis.The attack of tumor and the impact of tumor microenvironment can cause the dysfunction of body immune system and anti-oxidation defense system.Therefore,we further explored the effect of PPP-40 upon the immune system and antioxidant system in S180 mice.On the one hand,PPP-40 could increase significantly the spleen index of mice,promote T lymphocyte transformation,inhibit spleen cell cycle arrest(G0/G1)and apoptosis.On the other hand,PPP-40 could enhance the ratio of CD3+CD4+ and CD3+CD8+ in T lymphocyte subsetsand correspondingly increase the release of IL-2,IL-12 and TNF-α in spleen cells.The expression of apoptotic proteins in spleen tissue indicated that PPP-40 inhibited the expressions of Bax,Caspase-9 and Caspase-3 apoptotic proteins and promoted the expression of anti-apoptotic protein Bcl-2,blocking the mitochondrial apoptotic pathway of spleen cells.In addition,PPP-40 also could inhibit the apoptotic proteins expressions of death receptor-mediated apoptosis pathway(Fas,Caspase-8 and Caspase-3 proteins).The above results suggested that PPP-40 played an important role in repairing the immune system damage caused by tumor microenvironment while exerting antitumor effect.Finally,the expression of antioxidant enzymes and the ability of antioxidant synthesis in spleens and livers of S180 mice were analyzed,and it was found that PPP-40 could effectively regulate the redox imbalance caused by tumor microenvironment.This study is to reveal antitumor mechanisms of PPP-40 and has important theoretical and practical significance for antitumor development of natural polyphenols. |
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