| Objective:This part is an investigation of the anti-inebriation and liver protection effects of a herbal composition of SRF against acute alcoholic intoxication in vivo. Methods:(1) Animal tests:Mouse experiments were designed to determine the effects of SRFE on acute alcoholic intoxication. Mice were divided into vehicle control group, SRFE control group, model control group, small, medium, and large doses of SRFE groups and positive control group. For SRFE groups, mice were intragastrically treated with low (0.06g/kg), medium (0.12g/kg) and high (0.24g/kg) doses of SRFE, respectively. For the positive control group, mice received0.40g/kg HWJZ tablet. After14consecutive days of drug administration,7.70g/kg ethanol was administered orally one hour subsequent to the final treatment with SRFE groups as well as model and HWJZ groups. The times to loss of righting reflex and the times to recovery of righting reflex were recorded immediately. Mice were euthanized12hours later and the livers were obtained for the assays of antioxidative enzymes. Rat experiments were designed to study the effects of SRFE on pharmacokinetics of ethanol. Drug administration protocols were identical with mouse experiment. The doses of SRFE were0.04(low-dose),0.08(medium-dose) and0.16(high-dose) g/kg, the doses of HWJZ and ethanol were0.25and4.50g/kg. Blood was obtained from the vena caudalis0,0.5,1,2and6hours after ethanol treatment, the serum was separated for detecting the concentrations of ethanol and acetaldehyde. Rats were euthanized12hours later, the stomachs, small intestinal mucosa, and livers were obtained for the assays of ethanol metabolic enzymes. Maximal tolerance dose tests:The mice were divided into vehicle control group and SRFC group. For SRFE group, mice were intragastrically treated SRFC with the maximum concentration (2g/mL) and the maximum volume (20mL/kg) twice in24hours. Animals were closely observed during the first48hours after administration, the weight, appearance, diet, behavior were recorde. Animals were continuously observed till the14th day, death and toxic symptoms were recorded.(2) Human tests:15healthy male adults were experienced in there parallel tests. The first one is a self-model control experiment in which the examinees drink alcohol without any drug, the second and third ones are double-blind experiments in which examinees take placebo or SRFC30min before they drinking. Heart rates were detected0,70,100min after drinking, blood were obtained20,50,70,100,120min after drinking and serum were separated for detecting the ethanol, acetaldehyde concentrations and ADH, ALDH contents. Results:(1) Animal tests: Compared with the model group, SRFE pretreated animals exhibited longer times to loss of righting reflex and shorter times to recovery of righting reflex (P<0.01, P<0.05), the inebriety rates were lower (P<0.05); the Cmax and AUC of serum ethanol were lower (P<0.05, P<0.01), Tmax values were not different, the Cmax and AUC of serum acetaldehyde were lower (P<0.01), Tmax values were shorter (P<0.05); ADH and ALDH levels in stomach, small intestine and liver were dose-dependently enhanced (P<0.01, P<0.05); the elevation of hepatic CYP2E1activity induced by ethanol was dose-dependently decreased (P<0.01), the inhibitory effects of ethanol on SOD, CAT, GPx, and GST were reversed (P<0.01, P<0.05) and MDA contents were reduced (P<0.01, P<0.05). Maximal tolerance dose tests:Compared with the blank group, the weights were drop, the consumptions of food and water were reduced, the independence activities were decreased, and the excrements turned darker in the SRFE treated group in the first24hours, while in the next24hours, all the conditions of the SRFE-treated mice recovered to the normal levels. Animals were continuously observed till the14th day, death and toxic symptoms of mice in both of the two groups showed no difference.(2) Human tests:Compared with the model group, the heart rates variation were decreased (P<0.01,P<0.05), the Cmax and AUC of serum ethanol and acetaldehyde were lower, Tmax values were not different, ADH and ALDH contents were enhanced (P<0.01, P<0.05) in drug treated group, but there is no change in the placebo group. Conclusion:SRF shows potent protective effects on acute alcoholic intoxication in rodent, hepatocyte and human models. These findings suggest that the anti-inebriation effects of SRF may involve inhibition of ethanol absorption, promotion of ethanol metabolism via ADH pathways, inhibition of ethanol metabolism via CYP2E1pathways, and enhancing hepatic anti-oxidative functions. SRFC shows an extremely high security in mice when the dosage is2600times larger than the recommended dose of human (0.03g/kg).Objective:This part is an investigation of the liver protection effects of SRF and medicated serum against acute alcoholic intoxication in vitro. Methods:(1) SRFE tests:Rat primary hepatocytes were divided into vehicle control group, SRFE control group, model control group, and SRFE treated groups. In the SRFE treated groups, cells were treated with different doses of SRFE (1,10,100and1000μg/mL) for12hours or with1000μg/mL doses of SRFE for different times (3,6,12and24h), then100mmol/L ethanol was administered with SRFE groups as well as model group. The culture medium and cells were collected after6hours’incubation, the LDH and GOT activity in the medium, the ADH as well as SOD, CAT, GPx activities and the GSH and MDA contents in the hepatocyte were detected. Meanwhile, the MTT assay was used for detecting the ethanol-induced cell inhibition rate.Serum pharmaceutical chemical studies:Rats were orally given2.4g/kg SRFC,40min later, blood was obtained from carotis and serum was prepared for the qualitative and quantitative HPLC analysis.(2) Medicated serum tests:the animals were divided into vehicle control group and the SRFC group, the blank and medicated serum were prepared. Rat primary hepatocytes were divided into vehicle control group, medicated serum control group, model control group, blank serum control group and medicated serum treated groups. In the medicated serum treated groups, cells were treated with different doses of medicated serum (5%,10%and20%) for24h or with20%doses of medicated serum for different times (12,24and48h), then100mmol/L ethanol was administered with medicated serum groups as well as model control group. The culture medium and cells were collected after6hours’incubation, the LDH and GOT activity in the medium, the ADH as well as SOD, CAT, GPx activities and the GSH and MDA contents in the hepatocyte were detected. Results:(1) SRFE tests:All the indexes were dose and time dependence changed in the SRFE treated groups. Compared with the model group, LDH and GOT activities in culture medium were reduced (P<0.01, P<0.05), ethanol-induced cell inhibition rates were reduced (P<0.01, P<0.05), ADH, SOD, CAT, and GPx activities in primary hepatocytes were enhanced (P<0.01, P<0.05), GSH contents were increased (P<0.01), and MDA contents were decreased (P<0.01).(2) Medicated serum tests:Compared with the model group, LDH and GOT activities in culture medium were reduced (P<0.05), ethanol-induced cell inhibition rates were reduced (P<0.01), SOD, CAT, and GPx activities and GSH contents in primary hepatocytes were enhanced (P<0.05), and MDA contents were decreased (P<0.01), but the ADH activities were unaltered; in the medicated serum control group, LDH and GOT activities and cell inhibition rate were constant, while the hepatocellular antioxidative function were enhanced, in the blank serum control group, none of them were changed in the in vitro tests. Conclusion:Both SRFE and medicated serum show potent protective effects on acute alcoholic hepatocyte damage. These findings sμggest that the anti-inebriation effects of SRF may involve increase of the hepatocyte vitality and function, promotion of ethanol metabolism via ADH pathways, and enhancing hepatic anti-oxidative functions.Objective:To find the active ingredients in SRFC, and provide a theoretical basis for its quality control. Methods:(1) The fingerprint study of SRFC:0.1g SRFC and50mL methanol were mixed and ultrasonic extract for30min, after filtering, the extract was separation by a C18column. The mobile phases consisted of0.2%phosphoric acid (A) and acetonitrile (B). The system was run with the following gradient program:9:1(5min)â†'7:3(15min)â†'7:3(30min)â†'9:1(40min)â†'9:1(50min). The flow rate was kept at1mL/min. Absorbance was measured at250nm. The compounds and the respective concentrations in SRFC were detected.(2) HPLC study of medicated serum:Rats were orally given2.4g/kg SRFC,40minutes later, blood was obtained from carotis and serum was prepared for the qualitative and quantitative HPLC analysis. Results:(1) The fingerprint study of SRFC:More than30 compounds in SRFC were well separated, the major compounds and the respective concentrations in SRFC were:puerarin (633.040μg/mL), rutin (6.232μg/mL), quercetin (62.585μg/mL), kaempferol (10.213μg/mL), schisandrin (131.265μg/mL), and y-schizandrin (0.560μg/mL).(2) HPLC study of medicated serum:We got5components from the medicated serum samples, two of them were identified as puerarin (40.73μg/mL) and Schisandrin, the rest ones may be the secondary metabolites. Conclusion:The components that can be detected both in SRFC and the blood may be the substance foundation of SRFC on acute alcoholic intoxication. The active ingredients may be puerarin (which belongs to flavonoids) and schisandrin (which belongs to lignans). |
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