Preparation Of Morusin In The Branch Bark Of Mulberry And Its Inhibitory Effects On Transplanted H₂₂ Hepatocarcinoma In Mice

In this experiment, we used the branches of cultivated mulberry Husang-32（Morusmulticaulis Perr.） as experimental material, and the anhydrous ethanol as the extractionsolution to get the crude extract from the branch bark. The ethanolic extract wassuccessively further purified by macroporous resin, Sephadex LH-20andsemi-preparative reversed-phase high performance liquidchromatography （RP-HPLC）.The high purity monomer was identified as morusin by high performance liquidchromatographic diode array detection （HPLC-DAD） and its UV spectrum. The contentof morusin in samples was quantitative analysised by the linear equation for morusinstandards, within the scope of0.05to1.00μg has a good linear relationship to itsabsorbance value （R2=0.9996）. The methods used in this study for determiningmorusin exhibited good repeatability （RSD6.02%） and recovery （100.62%）. The resultsshowed that the content of morusin was almost no difference between the root andbranch bark in Husang-32and the leaves was not detected. But the morusin in branchbarks of Yanbianqiuyu was about3-fold higher than in their roots. The analytical resultsrevealed a great diversity （about20-fold） in levels of morusin of the100differentcultivated species of mulberry. The morusin content of Husang-32was200.22μg/g. Thehighest concent of morusin was Husang-13（585.81μg/g） and the lowest was Yin-3（29.64μg/g）. Therefore, the method of HPLC-DAD to identificate and analysis morusinin branch bark of mulberry is fast, reliable and applicable. The results in our studyprovide reliable data for researching and developping the morusin as active compoundwith anti-tumor, anti-bacterial, anti-HIV in the future. Also it can lay the groundworkfor development and utilization of mulberry branch.In view of this, we detected the gene expression to disscus the mechanism thatinhibit the hepatoma in mouse model and the anti-tumor molecular mechanism ofmorusin through molecular biology methods. The H₂₂bearing mice were divided into5 groups: the model group, morusin low （10mg/kg d）, medium （20mg/kg d）, high （40mg/kg d） dose groups and5-fluorouracil （5-FU） positive control group. Ten days afterdrug injection, the tumor, spleen and liver were dissected, the inhibition rate of thetumor weight and the growing rate of the liver coefficient, the spleen coefficient and thegaining in weight calculated the pathology sections of liver tumor observed byimmune-chemical SP method. In comparison with model group, the tumor inhibitor rateof low, middle high dose morusin groups and5-fluorouracil positive control groupshowed an inhibition rate of13.84%,26.21%,34.43%and47.21%, respectively. Thegrowing rate of liver conclusion, spleen coefficient and the rate of weight gainingreflected the morusin from mulberry had not the obvious action to the liver, spleen andthe gaining in weight.QPCR assay was used to detect the tumor gene expression to study the anti-tumormolecular mechanism of morusin. In comparison with model group, the expression ofP53, Survivin, CyclinB1and Caspase-3in morusin3groups and5-fluorouracilchemotherapy treatment group were increased, and the difference was significant（P<0.05）. The expression of Caspase-3in low dose group of morusin was about4-foldhigher than the model group. The middle and high dose group of morusin were higherthan the model group3.6and3.0fold, respectively. Caspase-3expression negativecorrected with the concentration of morusin. The expression of NF-κB in morusintreatment groups and5-fluorouracil group were lower than that of the model group. Theexpression of NF-κB in high dose group of morusin down to36.5%compared to themodel group. Three doses of the down-regulation of the gene expression of NF-κB havea significant level （P<0.01）. According to the above results，the anti-tumor effect ofmorusin is through the mechanism of apoptosis, raised expression levels of Caspase-3and reduced expression levels of NF-κB to promote apoptosis of tumor cells.