The Establishment Of Ultnasonioassisted Extraction Of Pinus Koariensis Pinon Shell Polysacchairdes And The Study Of The Anit-tumor Bioacitvity

Polysaccharide, a kind of biomacromolecule, exists broadly in nature. Recentresearch on bioactivities of polysaccharides is mainly about anti-bacteria, anti-virus,anti-coagulation, anti-oxidation and anti-tumor.Pinus koraiensis is an economic forest in Northeast of China, thus the pinonshell resource is abundant. Accordingly, establishing the extraction process of Pinuskoraiensis Pinon Shell Polysaccharides (PPSP) is a critical step in development offorestry byproducts. The previous research in our lab suggested that PPSP possessesactivities of anti-virus, anti-oxidation and enhancing the viability of lymphocyte invitro. In this study, the ultrasonic-assisted extraction process was established andbioactivity of anti-tumor and the effect of immune regulation on tumor cell in vivowere evaluated.The ultrasonic-assisted extraction process was established through single factoroptimizations and the orthogonal optimization, and crude polysaccharides werepurified by ion-exchange chromatography, properties of polysaccharide fractionswere tested by chemical and physical methods finally. Results showed that theoptimal extraction process was as follow: extractive temperature:85℃, extractiveduration:45min, extractive times:2times, ratio of solid to liquid:1:6(P <0.001),the purification was54.41%under this condition; acidic fractions of PPSP1, PPSP2were obtained from PPSP by ion-exchange chromatography; those fractions allcontained polysaccharide.MTT was used to evaluate inhibiting effects on growth of tumor cells (Hela, HepG2, SW1116) and normal cell (293T) in vitro; on the H22solid tumor micemodel the study based to evaluate the effect of PPSP on mice weight and theanti-tumor activity in vivo. Results showed that tumor cell proliferations wereinhibited significantly by every fraction under every concentration in vitro (P <0.001), and inhibiting rates increased with the growth of polysaccharideconcentration and action time. Noticeably, the inhibiting effect on proliferation ofSW1116by PPSP2was of the most significance, which was95.88%under800mg/L and60h. On the contrary, although the proliferation was inhibited slightlyunder high concentrations, the proliferation of293T cell was enhanced by PPSP1and PPSP2under low polysaccharide concentrations (100mg/L、200mg/L),(P <0.001). H22solid tumor mice model was established. The growth of H22solid tumorwere inhibited significantly by PPSP (P <0.001), and the inhibition rate of high-dosegroup was the highest:75.63%. PPSP had no significant effect on normal miceweight (P﹥0.05), but could reduce weight loss of tumor-bearing mice significantly(P <0.001).Elisa was used to evaluate the effect of PPSP on protein level of IL-2, TNF-α, IFN-γin tumor-bearing mice spleen, thymus and serum. Results showed that IL-2, TNF-α, IFN-γwere induced in tumor-bearing mice spleen, thymus and serum by PPSP, and thoseprotein levels of high-dose group mice were higher than other group.This study suggested that: the optimal extraction process was as follow:extractive temperature:85℃, extractive duration:45min, extractive times:2times,ratio of solid to liquid:1:6(P <0.001); PPSP inhibits proliferation of tumor cell invitro as well as in vivo, and reduces the weight loss of tumor-bearing mice incomparison to positive control group; PPSP acts as the immunoregulator against thetumor cell in vivo.