Study On The Extraction And Purification Processes Of Mulberroside A From Mulberry Branch Bark And Its Activities

According to the pharmacopoeias of past ages, Ramulus Mori has many medicinal effects and be one of the traditional Chinese medicinal materials. China is the major country in sericulture industry, up to ten million of mulberry branches are produced which are due to summer cutting and winter reshearing. Its output is far greater than the demand as Chinese herbal medicines, so the excess parties are burned as firewood, causing a great waste of resources and a lot of environmental pollution. Modern botanical studies have shown that:the bark of mulberry twig is rich in mulberroside A which has anti-inflammatory, analgesiccough, anti-tumor, anti-virus, protect the brain and so on. It has broad application prospects, but the current findings are lacking system. In this thesis, the bark of mulberry branch had been researched, including the extraction, separation, transformation, induction and antioxidant activity of mulberroside A,which is one of stilbene compounds. The main research content and results are as follows:1 Study on Production Process of mulberroside AIn this study, we presented a rapid method of qualitative and quantitative research of mulberroside A in the bark of mulberry branch by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). There were two conditions when used TLC. First, the silica gel plate must be used. Second, the mobile phase is mixed solution of chloroform-95% ethanol (2:1). The conditions of HPLC were as following:A Spherisob C18 column (150mm* 4.6mm,5μm) was used and the mixture of methanol-water (20:80, V/V) was used as the mobile phase, the flow rate was 1mL/min and detection wavelength at 330nm.. Response surface methodology (RSM) was applied to optimize the extraction conditions. Based on the signal factor test, extraction temperature、extraction time and liquid-to-solid ratio were chosen as casual factors, and a three-factor and three-level Box-Behnken central composite design leading to a set of 15 combinations of these variables was performed to attain the highest weighed value of yields of mulberroside A. The optimal extraction conditions were determined as follows:extraction solvent was Running water, extraction temperature 29℃, extraction time 120 min, and ratio of liquid to solid 23:1 mL/g.The best experimental parameters of mulberroside A separated by D101 and polyamide resins were investigated through static and dynamic adsorption experiments. The results showed that:The optimum conditions on static adsorption of D101 resin was that the adsorption time was 1h, the concentration of the mulberroside A was 1.2106 mg/mL and the eluting reagent was 40% EtOH. The optimum conditions on dynamic adsorption was that the adsorption velocity was 18 BV/h, the concentration of the mulberroside A was 2.9141 mg/mL and the eluting reagent was 40% EtOH. The optimum conditions on static adsorption of polyamide resin was that the adsorption time was 1h, the concentration of the mulberroside A was 0.9617mg/mL and the eluting reagent was 40% EtOH. The optimum conditions on dynamic adsorption was that the adsorption velocity was 18 BV/h, the concentration of the mulberroside A was 2.4284mg/mL and the eluting reagent was 40% EtOH. Based on these research, a mixed method was adopted to separate mulberroside A. Through this method, The purity of mulberroside A was up to 76.9%.2 The content of mulberroside A in the bark of mulberry branch of different mulberry varieties was comparedUsed the detection and extraction methods which were established by the above experiments, the content of mulberroside A in several mulberry branch which were widely grown in Chongqing of China was determined. The result showed that different varieties of mulberry have different flavonoids content, the order as follows:Jialing 20(4.64%)> Jialing 16(2.73%)> Husang 32(2.63%)＞YunSang 1(2.54%)>Xiqing 1(2.36%)>Xiqing 4(1.90%)> Yu 2(0.79%)> 7920(0.69%)> Guiyou 62(0.39%)＞Xinong6071(0.18%).3 Determination on antioxidation and antityrosinase activity of mulberroside A In this experiment, vitamin C, vitamin E, and arbutin as the control, the free radical scavenging and reducing power of mulberroside A. resveratrol and oxyresveratrol had be measured. The results showed that:the order of DPPH radical scavenging activity as follows:vitamin C＞vitamin E＞oxyresveratrol>＞Resveratrol＞mulberroside A＞arbutin; the order of clear the ABTS free radical activity as follows:resveratrol＞oxyresveratrol＞arbutin＞mulberroside A＞vitamin C＞vitamin E; the order of total reducing power activity as follows:vitamin C＞resveratrol> oxyresveratrol＞vitamin E＞arbutin＞mulberroside A. Arbutin as the control, we compared the inhibit tyrosinase activity between mulberroside A and oxyresveratrol. The results of the tests indicated that they were having antityrosinase activity. Oxyresveratrol was the best one. Its antityrosinase activity was 2.8 times more than mulberroside A and 9.3 times more than arbutin.4 Transformation of mulberroside AThe effect of reaction time and enzyme-substrate mass percent on the Enzyme hydrolysis mulberroside A into oxyresveratrol was investigated by single factor experiments. The results showed that:the best hydrolysis time was 20min, the best enzyme-substrate mass percent was 4%.