Pharmacodynamics And Material Basis Studies Of Fructus Mori Against Ethanol-induced Hepatic Injury

Fructus Mori(FM), the fruit of Morus alba L., is teastes delicious and has a variety of nutrients and is one of the first materials approved for usage both as food and medicine by the State Ministry of Health in 1993. Based on Traditional Chinese Medicine(TCM) and “Yin-Yang Balance” theories, FM is thought to have nourishing yin for blood, tranquilizing excitement, tonifying liver & kidney, benefiting hair & eye, prolonging one’s life, lightening one’s skin, and lowering one’s blood pressure activities. Modern medical research illustrate that FM can stimulate the growth of hematopoietic cell, prevent the occurrence of angiosclerosis, and regulate blood sugar and blood fats levels. Moveover, FM has been proven to have anti-mutagenic, anti-viral, and anti-xidant properties.Some articles have report the potential hepatic protection effects of FM against alcohol / carbon tetrachloride(CCl4) introduced liver injury. Our previous study has also found that FM has activated the activity of alcohol dehydrogenase(ADH), which shows a potential adjuvant therapeutic effect to alcoholic liver damage(ALD). The incidence rate and fatality rate of ALD increased rapidly in recent years, and seriously threat people’s health, which made badly in need of specific drug for ALD treatment. However, whether or not does FM have the positive effects on promoting alcohol metabolism and excretion, and protecting liver from alcohol damage are not clear yet. So, our research has focused on this issue to find out what efficacious components in FM exert hepatic protection against alcohol, and how does it play the role.The pharmacodynamics and pharmacodynamic material basis research of FM were focused on the following four aspects. Firstly, different FM extracts were screened based on the ADH activation degree, and FructusMori polysaccharides(FMPS) was selected for further research. Secondly, the evaluation research of FMPS against ALD was conducted in vivo. Thirdly, preliminary characterization of FMPS was carried out. Fourthly, optimization of enzyme assisted extraction of FMPS was carried on by using response surface design method. The detailed results are as follows:1. The screening study of FM extracts on the basis of ADH activationA sensitive, selective and rapid external ADH activity screening model was developed and validated for evaluation of three different FM extracts, namely ethanol extract(FME), methanol extract(FMM), and water extract(FMW). The statistical analysis results showed that all these 3 different FM extracts(FME, FMM, FMW) could up-regulate the ADH activity, the activation rate was 89.34 ± 2.26%(n = 6, RSD = 2.54%), 59.56 ± 1.06%(n = 6, RSD = 1.79%), 135.4 ± 2.96%(n = 6, RSD = 2.19%), respectively, with a significant difference degree(P < 0.05). Finally, The FMW was selected for further research.The FMW was purified through degreasing, bleaching, deproteinization process, and FMPS was obtained with saccharide content 67.96 ± 2.46%. Compared with FMW, FMPS exhibited more powerful capacity in up-regulation of ADH activity in the some concentration(P < 0.05). Moreover, the activation rate was proportional to the concentration of FMPS in the range of 25.24 μg·m L-1 to 176.68 μg·m L-1 with obvious dose-response relationship.2. The in vivo study of FMPS resistance to ALDTwo different sensitive and selective ALD models, namely acute and subacute model, were established to evaluate the hepatic protection effects of FMPS. Using the established models, the pharmacodynamics of FMPS inrats was studied after intragastric administration of different dosage of FMPS(low, middle, high dose, respectively).In the acute ALD model, all three doses of FMPS could resistant to the ethanol induced increase of serum alanine amino transferase(ALT), aspartate amino transferase(AST), and liver methane dicarboxylic aldehyde(MDA). The high dose FMPS group showed a great significant effect(P < 0.01). Compared with the model group, the ALT, AST and MDA values in the high dose FMPS group was reduced by 31.71%(P < 0.01), 40.90%(P < 0.01), and 30.10%(P < 0.01), respectively. Meanwhile, each FMPS group could resistant to the ethanol induced decrease of liver glutathione(GSH), superoxide dismutase(SOD), and glutathione peroxidase(GSH-Px). The high dose FMPS group showed a great significant effect(P < 0.01). Compared with the model group, the GSH, SOD and GSH-Px values in the high dose FMPS group was increased by 73.22%(P < 0.01), 40.90%(P < 0.01), and 20.23%(P < 0.01), respectively.In the subacute ALD model, all 3 doses of FMPS could also resistant to the ethanol induced increase of serum ALT, AST, and liver MDA. The high dose FMPS group showed a significant effect(P < 0.05). Compared with the model group, the ALT, AST and MDA values in the high dose FMPS group was reduced by 45.23%(P < 0.01), 54.38%(P < 0.01), and 22.22%(P < 0.05), respectively. Meanwhile, each FMPS group could resistant to the ethanol induced decrease of liver GSH, SOD, and GSH-Px. The high dose FMPS group showed a significant effect(P < 0.05). Compared with the model group, the GSH, SOD and GSH-Px values in the high dose FMPS group was increased by 52.45%(P < 0.05), 19.65%(P < 0.05), 20.63%(P < 0.01), respectively.3. Chemical characterization of FMPSIn order to further clarify the pharmacodyamic material basis of FMPS, both chemical method and instrument method were adopted for the structure analysis of FMPS. Composition analysis indicated that FMPS contains carbohydrate(67.96%), uronic acid(18.51%), protein(1.63%), sulfate radical(6.21%). Monosaccharide analysis showed that FMPS was a heteropolysaccharide mainly consisted of mannose(Man, 50.54%), glucose(Glc, 21.53%), galacose(Gal, 18.13%), galacturonic acid(Gal A, 1.51%), glucuronic acid(Glc A, 2.41%), xylose(Xyl, 2.22%), rhamnose(Rha, 2.01%), arabinose(Ara, 1.50%) and L-fucose(Fuc, 0.61%).4. Optimization of enzyme assisted extraction of FMPSIn this section, basing on FMPS extraction yield, antioxidant activity, ADH activation, the conditions for extraction of FMPS from FM were optimized by using enzyme assisted extraction, single-factor test, and the response surface methodology(RSM). A central composite design(CCD) was adopted to investigate the effects of four independent variables including enzyme amount(%), enzyme treated time(min), treated temperature(?C), and liquid-solid radio on the responses, FMPS yield. The optimized conditions of the extraction were as follows: enzyme amount 0.40%, enzyme treated time 38 min, treated temperature 58?C and liquid-solid radio 11.00. Under these conditions, the mean experimental value of extraction yield(16.16 ± 0.14%) corresponded well with the predicted values and increased 160% than none enzyme treated ones.