Separation And Purification Of The Flavonoids From Mulberry Leaves And The Estimation Of Its Hypolipidemic Effects

It is well known that high serum total cholesterol (TC), triacylglycerol (TG) and low density lipoprotein cholesterol (LDL-C) levels are primary risk factors for vessel diseases, and high serum levels of high density lipoprotein cholesterol (HDL-C) confer a protective benefit against its development. Associated with the alteration of lifestyle, dylipidemia has become one of important risk factors threatening health of Chinese people. The epidemiologic data shows that the prevalence of dyslipidemia in Chinese adults aged 18 and over is 18.6%, which means the number of dyslipidemic patients has been reached 160 million. Therefor, it is very important to pay more attention to earlier comprehensive prevention and control of hyperlipidemia. Mulberry (Morns alba L.) leaves has been long used in Chinese traditional medicine to protect the liver, improve eyesight, facilitate discharge of urine, lower blood pressure and keep weight. In our prior study, when we were interest in the effect of the mulberry leaves extracter on sugar metabolism and antioxidation in diabetic rats, we also found that it was the scavenger of lipid free radicals, indicating a lipid lowering activity of the flavonoids from mulberry leaves (MTF). This character of MTF is further certificated in this study.To extract MTF, an orthogonal table has been used to find the best procedure formicrowave-assisted extraction (MAE) of flavonoids. The advantages of MAE have been explored by comparing with the commonly used extraction method. The best condition for procedure is extracting 20 min at 60 °C with 12 folds of 70% ethanol. The ethanol concentration was significant among all factors on the test of extraction. The flabonoids contents in mulberry leaves is 2.87% by MAE, 55% more than commonly used extraction method.After extraction, the MTF have been purified by macroporous resins. The D101, AB-8 and NKA-9 macroporous adsorbents were investigated to separate and purify MTF, evaluating by adsorption capacity, elution ratio and product purity. Among the three types of macroporous adsorbents, the NKA-9 type shows better property with the adsorption capacity of 43.4 mgg*1 and the elution ratio of 98.2%. The purity of MTF product can reach 58.2% in the technological conditions flowing: extract sample concentration of 20 mg-ml"1, 6 times washing water and elution reagent of 70% ethanol. The adsorbent can be used 4 times repeatedly.Moreover, to establish a kinetic model for this separation process, the curves of dynamic adsorption-elution process have been plotted, and the kinetic equation has been present as jy=?(l — e**).hi this study, liquid chromatography-mass spectrometry (HPLC-MS) method has been chosen to investigate the chemical constituents of MTF. Seven flavonois were detected: Quercetin-3-O-glucoside, Quercetin-3-O-rutinoside (rutin), Quercetin-3-0-(6 "-acetyl)-glucoside, Kaempferol-3 -0-rutinoside, Kaempferol-3 -0-glucopyranoside, Kaempferol-3-O-(6"-acetyl)-glucoside and Luteolin-7-O-rutinoside. And it's worth to point out that a new constituent from mulberry leaves was separated, whose structure requires further identified.Following extraction, separation and identification of MTF, the hypolipidemic effect of MTF has been estimated in two animal models, triton WR-1339 induced hyperlipidemic mice and high fat diet induced hyperlipidemic rats. In mice pretreated with triton WR-1339 (400 mg-kg"1), the beneficial effects of MTF on serum lipid levels are more significant at 12 h post MTF administration than at 6 h. The levels ofTG, TC and LDL-C have been remarkably reduced to 388.0, 256.6 and 189.2 mglOO ml"1 in MTF (30 mg-kg"1) treatment mice, compared with 540.1, 463.7 and 298.8 mg-100 ml"1 in only triton WR-1339 treatment group. The ratios HDL-C/TC and HDL-C/LDL-C have been increased to 0.42 and 0.57 post MTF (30 mg-kg"1) administration, whereas these two ratios presented low levels of 0.33 and 0.52 in model group. The similar effects could be obtained from the rats fed high fat diet. Four weeks later, the serum lipid levels reached 93.5 mglOO ml"1 in TG, 108.2 mglOO ml"1 in TC, 62.0 mglOO ml"1 in LDL-C and 87.6 mglOO ml"1 in HDL-C in rats fed high fat diet, whereas the TG, TC, LDL-C and HDL-C content were remained at 67.2, 84.0, 47.5 and 101.0 mg-100 ml"1 in rats treated with MTF (20 mg-kg"1-d"1) as well as high fat diet. The body weight was not changed by MTF administration, compared with control. But the hepatosomatic indices (HSI) were decreased to 3.2 by MTF treatment, compared to 4.1 in model group. These results indicate the serum lipid lowing activity of MTF in mice and rats, with dose dependent.