Anti-tumor Effects Of Betulin And Its Mechanism

Tumor is one of the major disease that threat people's health. Drug treatment usually has side effects as low-selectivity and drug-tolerance,so it is critical to develop new chemotherapy medicine from natural product.The bark of betula platyphylla suk contains betulinic acid and betulin. The study of betulin acid is more which focus on its anti-tumor and anti-HIV effects. Betulin is believed to be bactericidal, anti-virus, anti-UV, and a synthetic resin birchprecursor of betulin acid and its derivatives. The Objective of the study is to investigate the anti-tumor activity of betulin in vivo and in vitro and approach the possible mechanism to provide theory-support for the development of new chemotherapy medicine.1.Toxicity of betulin in vitroThe study used MTT method to detect the toxic action of betulin on Wish and HL-7702 cell lines in vitro. The result showed that: Toxic effect is not found in Wish and HL-7702 cell lines 48h after betulin is added(P>0.05); There is no toxic effect on both cell lines when the dose was below 448μg·ml-1 72h after betulin is added(P>0.05).2.Anti-tumor Activity of betulin in VitroThe study used MTT method to detect the anti-tumor action of betulin on HeLa,SMMC-7721,SGC-7901 cell lines. The result showed that:①Compared with CG, 56~448μg·ml-1 betulin had noticeable inbibitional effect on the proliferation of the above cancer cells 48h after betulin is added(P<0.05), the inhibition ratio increased while the dose increased. Betulin between 28~448μg·ml-1 had noticeable inbibitional effect on the proliferation of cancer cells 72h after betulin is added(P<0.05).②Noticeable difference is not found in negative control group compared with CG(P>0.05).3. Effect of differentiation on B16 cell linesThe study detected melanin in B16 cell lines by using spectrophotometer. The result showed that: betulin between 56~448μg·ml-1 promoted melanin production of B16 cell lines(P<0.05), so betulin may induce the differentiation of B16 cell lines.4. Effect on cell cycle and cell apoptosis of SMMC-7721 cell linesThe study used One-Fluorescence Dying method on Flow Cytometer to detect cell cycle and cell apoptosis of betulin on SMMC-7721cell lines. The result showed that:①48h after betulin is added , G0/G1 phase increased while S phase decreased. It indicated that betulin could delay 7721cell lines at G0/G1 phase, inhibit the synthesis of DNA, kill cells that had entered cell cycle, prohibit the normal operation of cell generation cycle.②There is dose-dependent way of cell apoptosis induced by betulin.5. Anti-tumor effect of betulin in VivoThe study built tumor bearing Kunming mice model by injecting 1×107·ml-1H22 tumor cells 0.2ml on the mice's left inguinal region. 50 Mice were randomly divided into 5 groups. Betulin groups were iged with 10.20.40mg·kg-1betulin respectively. Negative control group was treated with olive oil and positive control group was treated with 25mg·kg-1CYT. After 10 days, the mice were killed by snapping the spinal cord. The change of body weight,inhibition ratio,thymous index,spleen index,cardiac index,liver index,lung index,kidney index and testicle index were calculated. The result showed that:①There was no significant difference of body weight change between betulin groups and negative control group(P>0.05). Compare with NC group, the weight loss in CYT was of significant difference(P<0.05). It reflected that betulin has no effect on the growth of mice.②There was no significant difference of cardiac index,liver index,lung index,kidney index and testicle index between betulin groups and NC group(P>0.05). It reflected that betulin has no damage on the mice's major organ, so it is non toxic.③Compare with NC and CYT group, spleen index in three betulin groups increased and had significant difference compared with CYT group(P<0.05). MD and HD group had significant difference with NC group(P<0.05). Thymous index in MD group had significant difference compared with NC and CYT group(P<0.05). It reflected that betulin had certain stimulating effect on immune organ.④Tumor weight of three betulin groups decreased compared with NC group(P<0.05), especially MD group which had inhibition rate of 51.31%, close to CYT group. It suggested that betulin may inhibit the growth of tumor.6. Effect on T Lymphocyte TransformationThe study used MTT method to detect betulin's effect on T lymphocyte transformation of mice bearing H22. The result showed that: Tlymphocyte transformation rate of LD and MD group was higher than that of NC group, CYT group was lower than NC group. MD group had significant difference compared with NC and CYT(P<0.05). It reflected that MD betulin may promote T lymphocyte transformation and enhance immunity.7. Effect on NK and LAK activityThe study used MTT method to detect betulin's effect on NK and LAK activity of mice bearing H22. The result showed that:①When EC:TC=25:1, NK activity in LD group was higher than CYT group(P<0.05), When EC:TC=100:1,it is higher than NC group(P<0.05); NK activity in MD group was higher than NC and CYT group under three E/Ts(P<0.05);NK activity in HD group was higher than NC and CYT group when E:T=25:1.It reflected that betulin can enhance NK activity and non-specific immunity, especially MD group. When E:T=25:1, the killing activity was the highest.②When EC:TC=50:1, LAK activity in LD group was higher than NC group(P<0.05), When EC:TC=100:1,it is higher than NC and CYT group(P<0.05); NK activity in MD group was higher than NC and CYT group under three E/Ts(P<0.05);NK activity in HD group was higher than NC and CYT group when E:T=25:1and 50:1(P<0.05). It reflected that betulin can enhance LAK activity, when E:T=50:1, the killing activity was the highest.8. Effect on CTL activityThe study used MTT method to detect betulin's effect on CTL activity of mice bearing H22. The result showed that: There was no significant difference between three betulin groups and NC,CYT group under three E/Ts(P>0.05). It indicated that betulin had no effect of stimulating specific immunity .Conclusion: Betulin is of certain anti-tumor activity and low toxicity . It can inhibit the proliferation of tumor cell by changing cell cycle,inducing cell apoptosis,promoting differentiation,promoting T lymphocyte transformation and enhancing NK and LAK activity.