| Mulberry is the fruit of urticales moraceae morus (Morus L.), and its medicinal value was record in the book of Compendium of Materia Medica. It is sweet and can carambola, nourish yin, tonify blood and calm soul. Mulberry fruit is considered as one kind of "drug homologous food" in China. According to statistics, in 2014, there were 0.83 million hectare mulberry plantations in China. However, the abundant mulberry fruit has not been fully utilized, just only uses for wine or vinegar in current stage... In addition, the extraction processes of anthocyanin, polysaccharides, polyphenols and other active substances from mulberry fruits were also studied. But these biological activities of antioxidation in mulberry fruit were mainly analyzed in vitro, even though there are some researches discussing about the effects of lowing blood glucose and lipid in mice by the extraction of mulberry fruit but the antitumor and antioxidation functions of anthocyanin in vivo are rarely reported. In order to utilize the mulberry fruit resources effectively, especially its medicinal functions, the extraction and purification processes of anthocyanin from mulberry fruit are studied. Meabwhile, the antioxidant activity and anti-tumor function of anthocyanin in vitro and in vivo are also investigated.This thesis consists of three parts:(1) The extraction and purification processes of anthocyanin from mulberry fruit; (2) The antioxidant activity of anthocyanin in vitro and in vivo; (3) The acute toxicity of anthocyanin from mulberry fruit and its inhibitory effect on tumor cell proliferation. The results are showed as follows:(1) Extraction:In order to determine the best extraction solvent of anthocyanin from mulberry fruit, distilled water, different concentrations of ethanol, different pH ethanol and acetone were used for testing. Meanwhile, mulberry fruit and the extract solvent-ratios were set as 1:40,1:70,1:100 and 1:130, and then treated under ultrasound (60W) for 30 minutes. The results showed that 75% ethanol containing 0.1%HCl, solid-liquid ratio of 1:100 resulted in a maximum absorbance value as 1.563±0.019 of anthocyanin crude extract.. In contrast, the damage of cell wall of mulberry fruit was larger which treated with ultrasonic rather than those treated with ethanol only by SEM.(2) Purification:Three kinds of macroporous adsorption resins, which included AB-8, HPD-100 and D101 were used in purification process. Meanwhile; the concentration, pH of crude extract and the elution concentration ethanol were all compared in this experiment. The results showed that the adsorption rates of AB-8, HPD-100 and D101 were 76.1%,77.9%and 77.1%, respectively; and the resolutions were 74.0%,43.1% and 45.3%. Based on above results, AB-8 was selected as the resin for purification.(3) Anthocyanin content in mulberry fruit:First, the anthocyanin extraction from mulberry fruit (MFAE) was concentrated, and then dried by freeze method, measurement of mulberry fruit anthocyanin the content of anthocyanin in mulberry fruit was analyzed by the pH differential method, and the results showed that the content of anthocyanin was 38.1% and color value was 171.2.(4) The antioxidant activity of MFAE in vitro was evaluated by the method of FRAP and ORAC, VC was set as positive control. The results showed that the reduction ability of MFAE was weaker than that of VC in same concentration at 31.35 g/mL. The, the absorbance value of the VC group was 0.73, while MFAE group was 0.36. However, the oxygen free radical scavenging capacity of MFAE group is about 6.6 times of the VC group.(5) The acute toxicity effects of MFAE on mice were also studied by feeding with MFAE 120 mg per day for every mouse, and saline as control. The body sign and weight of the mice were observed every day. After 14 days, the blood and serum of mice were used for detecting blood routine. In addition, liver and kidney functions, organs of mice were also analyzed. The results showed that there were no significant difference between treatment and control. Based on the "Acute toxicity evaluation standard (2003)", MFAE can be determined as non-toxic substance.(6) 4 groups of mice were fed with normal saline, VC, low dose and high dose of MFAE solution every day, respectively. On the 31th day, the oxidative damage model of mice was established by using the bromobenzene, which identify the antioxidant effect of MFAE by analyzing the contents of MDA and peroxidase (SOD and GSH-Px) activity in liver tissue and blood of mice. Based on the [evaluation criteria of antioxidant effect in the health food and evaluation of technical specifications (2003)], MFAE showed positive function in lowing lipid peroxidation.(7) The effect of MFAE on proliferation of breast cancer cell MCF-7 was measured by MTT method in static and dynamic conditions, respectively. Meanwhile, the cell morphology and cell proliferation inhibition rate were also calculated. The results showed that the change of cell morphology and cell shrinkage could be induced by MFAE. With time, the cell MCF-7 proliferation was significantly inhibited by MFAE.(8) MCF-7 was treated with different concentrations of MFAE solutions, and cell cycle was determined by method of flow cytometry after 24 hour. It indicated that the cell cycle was blocked in the G2 phase.(9) The lung metastasis model was established by injecting B16-F10 melanoma cells through the tail vein of KM mice, and then fed with saline, MFAE and injecting DITC, respectively. The mice body weight, lung weight and the number of metastatic foci were measured. The results showed that the weight of lung in MFAE group was significantly lower than that of the normal saline group, and the number of metastatic lesions was also less than that of the normal saline group, but there was no significant difference between these two treatments. |
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