The Study Of Sapindus Alcoholic Extract On Non-alcoholic Fatty Liver Disease In Rats

BACKGROUNDLiver is an important organ in the body for metabolism, fatty liver disease (fatty liver disease) is a clinical syndrome of liver lipid excessive deposition, with the pathological manifestations of simple fatty liver, fatty liver, fatty liver fibrosis and cirrhosis. Liver has double blood-supply from hepatic vein and hepatic artery, but both are eventually through terminal venule and arteriole and hepatic sinusoid connected, complete with liver cells material exchange is collected in the hepatic vein, hepatic sinusoid is so main place of liver mass exchange. The liver sinusoidal endothelial cells (LSEC) and Kupffer cells are the main hepatic sinusoidal cells, and the metabolism and physiological and pathological changes of liver in closely related. If the LSEC were damaged, the fenestraes were out of control, such as persistently or excessively expand, so much CM rich in TG were uptaken by liver cells that caused lipid accumulation in liver cells, and causing acute fatty liver.Sapindus mukorossi Gaertn (SM) is a large deciduous trees, its fruit is also known as soap wash fruit or circle of soap.It is distributed in the south area of the Yangtze River and Taiwan Province, also in Japan, Korea, Indochina and India. SM is bitter in taste, cold in nature with a little of drug,and it has expectorant and insecticidal efficacy. In recent years, the chemical components and pharmacological effects of SM have been found, that has attracted more and more attention of researchers.SM as a Chinese herbal medicine in China has a long history of use. According to the research, SM was first recorded in "Chinese Materia Medica","Zhiwu Mingshi tukao" also with drawings, about its medicinal value in the "compendium of Materia Medica" were recorded in. Modern pharmacological researches have found, SM has many kinds of biological activity and medical value, including antitumor, antibacterial, antihypertensive, anti-inflammatory.lt has been reported that Sapindus saponins can play a role on regulating blood lipid in hyperlipidemia rat models, but the research on SM on prevention and treatment of fatty liver has not yet been reported.The pre-experimental results of this study suggested that, crude extract of SM has certain intervention effect in rats fatty liver model. On the basis of this effect,the study intends to conduct an indpeph research on non-alcoholic fatty liver of SM alcoholic extract.We were to detect the serum glutamic-pyruvic transaminase(ALT), aspartate aminotransferase (AST), serum triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL), gamma glutamyl transpeptidase (γ-GT), alkaline phosphatase (ALP), adiponectin (APN) of the rats, the LSEC and Kupffer cells were observed by scanning electron microscope and transmission electron microscope technology, to analyse the correlation between LSEC and enzymes in the pathogenesis of fatty liver. We aim to provide new ideas and new methods for prevention and treatment of fatty liver by further exploring the pathogenesis and drug targets of SM alcoholic extract.OBJECTIVE1. To identify macroscopically and microscopically the pericarp of Sapindus mukorossi for the screening of the drug active ingredients (or active site),and to provide a theoretical basis.2. Through acute toxicity test, to determine of median lethal dose (LD50) in mice with SM alcoholic extract, in order to ensure the safety and effectiveness of the study. 3. To study the correlation between LSEC and these enzymes in the pathogenesis of fatty liver through the detection of serum from experimental animal, by observing the changes of liver cells, fenestraes of LSEC, Kupffer cells.4. To verify the efficacy of SM alcoholic extract, in order to further explore the pathogenesis of fatty liver to provide reference data.METHODS1. Made medicine sampleThe paraffin sections and temporary water tablets were used to identify and analyse the microscopic structure of Sapindus mukorossi.2. Drug extract preparationTake SM peel, dried and crushed, for the first time with4times amount of85%ethanol for30min, reflux extraction2h in90℃, collecting extracted liquid; the second time with3times amount of85%ethanol reflux extraction1.5h.The extraction liquid was filtered after combined, concentrated to lg/ml, stored in4℃standby.3. Acute toxicity test60KM mice, were randomly divided into6groups. According to the conventional method of acute toxicity test of mice, the mice from1st groups to6th groups were respectively assigned to the500,1000,2000,3000,4000,5000mg/kg dose,by gavage2times daily. And we observed the mice if they would appear the symptoms such as unhairing, curling,concha pale or congestion, exophthalmos, teeter, muscle paralysis, difficulty in breathing, convulsions, coma, incontinence and other toxic reactions for14days of observation.4. Fatty liver animal model48SD rats were randomly divided into6groups:normal control group, model group, simvastatin group (7.2mg/kg), high dose (0.5g/kg), middle does (0.1g/kg) and low dose (0.05g/kg) of SM. Except the normal control group, the other groups were fed with high fat diet for6weeks, from4th to6th weeks,each rats were treated with different conditions as previously mentionde; At the 42nd days after5p.m. fasting, at the43rd days the blood and liver sample were collected.5. Measured indexes of serumAt the43rd day, measured the serum concentration of AST, ALT, TC, TG, HDL-C, LDL-C, γ-GT, ALP,APN.6. Preparation and observation sampleThe liver tissues were taken to make pathological sections for observation by optical microscope or were prepared for scanning electronic microscope or transmission electronic microscope.7. Statistical methodThe data were analyzed with SPSS13.0software.The LD50was calculated by Probit module; measurement data using analysis of One-Way ANOVA, grade data using K Independent Samples nonparametric test,showed the results as mean±standard deviation (X±s), P<0.05had statistical significance.RESULTS1. Microscopic identification of Sapindus mukorossi pericarpThe exocarp of S. mukorossi had cuticle at outermost layer.There was collenchyma outside and a number of lysigenous secretory cavities intersperse among the parenchyma with kinds of calcium oxalate crystals,stone cells,fiber bundles and collateral vascular bundles in the mesocarp. The endocarp was made up of flat lignified cells.2. Acute toxicity testWithin2weeks, the1st group(500mg/kg) showed normal, no death records;the2nd group (1000mg/kg) appeared symptoms of unhairing and activity weakened slightly,4died;the3rd group (2000mg/kg) and4th group(3000mg/kg) respectively died4and5with different degrees of hair removal, depression, lethargy, restlessness; there was a larger area of unhairing of5th group(4000mg/kg) with shortness of breath, uncoordinated, black and sticky stool,6died with the corpse bleeding;the6th group(5000mg/kg) all died in3days with the corpse dark, the gastrointestinal tract was swelling full of bubbles liquid, other organs were black.3. The body weight and liver indexCompared with the normal control group, the weigh of model group were reduced significantly, liver weight and liver index increased significantly (P<0.05or P<0.01);compared with model group, each group of rats liver wet weight and the liver index were decreased significantly (P<0.05or P<0.01).4. The results of serum biochemical testCompared with the normal control group,the serum TC, TG, LDL-C, AST, ALT, γ-GT, ALP of model group increased significantly (P<0.01), HDL-C, APN decreased significantly (P<0.05or P<0.01); compared with the model group, simvastatin group, SM high dose group (0.5g/kg) could reduce the serum TC, TG, LDL-C, AST, ALT and increased HDL-C and APN (P<0.05or P<0.01), but each SM dose group had no statistical significance to decreased γ-GT and ALP.5. The results of optical microscopeThe normal structure of hepatic lobules of normal control group;the liver cells of model group were irregular, a majority of them were swelling and containing a large number of lipid drop, even with ballooning degeneration, and the liver sinusoid was not clear,inflammatory cells was observed with necrosis; simvastatin group was part of liver cells swelling, with few vesicles containing lipid droplets; SM high dose group (0.5g/kg) had normal morphology of liver cells and liver sinusoid, a few liver cells swelling or contain lipids; SM middle dose group (0.1g/kg) was partial liver cells swelling and contain lipids,and the liver sinusoid narrowed; SM low dose group (0.05g/kg) partial liver cells swelling containing lipid droplets with visible focal necrosis.6. The results of scanning electron microscope①The structure of hepatic cord and liver sinusoid was clear of normal control group; model group was nearly all liver cells swelling and most containing lipids, liver sinusoid inconspicuously, collagen fiber hyperplasia, lipid droplets increased significantlyl in ipid-storing cells; simvastatin group was containing few lipid droplets in liver cells; SM high dose (0.5g/kg) was swelling slightly with vesicle lipid droplets in liver cells, and there was a few collagen fiber in local area; It could be found that lipid droplets and collagen fiber hyperplasia of SM middle dose group(0.1g/kg) and SM low dose group(0.05g/kg) were there obviously.②he LSEC fenestraes of normal control group with normal form (φ0.05μm to0.2μm) are arranged in cribriform uniform distribution in a cluster; The LSEC fenestraes of model group (φ0.3μm to1.2μm) obviously expanded; simvastatin group fenestraes (φ0.05μm to0.2μm) close to normal control group; SM high dose group fenestraes(φ0.1μm to0.5μm) expand slightly; SM middle dose group (φ0.1μm to0.8μm) and low dose group (φ0.2μm to1μm) fenestraes were cluttered, part of cribriform structure connected with each other into a large hole, microvilli below the LSEC exposed, but there was still some fenestraes keeping cribriform.7. The results of transmission electron microscopeThe structure of liver cell of normal control group was normal, the membrane was clear, the Disse gap and the sinusoidal were normal,kinds of normal organelle could be seen in the cytoplasm; the liver cells of model group were swelling with different sizes of lipid droplet vacuoles in cytoplasm, Disse gap was expanding, part of microvilli and collagen fiber were hyperplasia; simvastatin group liver cells contained a few lipid droplets; SM high dose group (0.5g/kg) contained only a few lipid droplet and the liver cells were a little swelling; SM middle dose group (0.1g/kg) and low dose group(0.05g/kg) contained more lipid droplets.CONCLUSION1.The fleshy part of the fruit is pericarp, but not aril; between the brown exocarp and membraniform endocarp, the tissue is the mesocarp of Sapindus mukorossi, a number of lysigenous secretory cavities in this parts is the source places to produce Sapindus saponins.2. Sapindus mukorossi can be a kind of duality drug, it has curative effect,but a little toxicity on the other hand. Acute toxicity test results of this study have shown, when the dose was more than1000mg/kg, the experimental animal appeared different degrees of poisoning reaction, even died within two weeks. It can be infered Sapindus saponins cause liver damage by lipid peroxidation, and the sulfhydryl lost in the tissue can lead to the liver demage presumably.3. By conventional serological detection and observation of liver pathological changes we can found, the levels of TC, TG, LDL, AST,ALT were obviously decreased while HDL and APN increased by SM higt dose,meanwhile the morphology of liver tisses as well, indicating SM alcoholic extract can ameliorate the levels of blood lipid and prevent fatty liver.4. By means of scanning electron microscopy and transmission electron microscopy,we can see the fenestraes of LSEC were improved.To some extent,we can indicate SM alcoholic extract has the function of protecting liver.According to this study,the effect of SM high dose (0.5g/kg)is the best.