| Backgroud:Chemical liver injury has become the second-largest liver injury incentives after the viral hepatitis. There are about 500 to 1,000 kinds of Chinese and Western medicines in clinical application as well as variety of chemicals, can cause of liver disease. According to information reported at home and abroad,20% of fulminant hepatic failure caused by chemical substances. In recent years. chemical liver injury has been growing as a serious threat to People's health. The study occurrence of hepatitis mechanism and to find effective treatment measures are the most popular in domestic and foreign scholars, a large number of studies have shown that many domestic experts and scholars to study Chinese medicine focus to our traditional. Study of Chinese medicine is the treatment of hepatitis is a hot research field in China. But there is still no effective clinical Prevention drugs, thus it is very importamce to enhance the protection of liver injury and carry out chemical protection work.Doctors prevalently use anti-viral drugs, liver protection drugs and immunomodulatory drugs to treat hepatitis in western medicine. These drugs commonly contain varieties interferon, vidarabine, interferon-inducing agent polyinosinic, acyclovir, levamisole, transfer factor, coenzyme Q, thymosin, cianidanol, glycyrrhizin, oleanolic acid, cucurbitacin B, cucurbitacin E, bifendate, vitamins and so on. Clinical results show that many of these drugs have good control of virus replication and restore normal liver function, protect liver cells and so on with efficacy of rapid. Disadvantage is unstable long-term efficacy, easily repeated, low rate of HBsAg darkening, expensive and the certain side effects.Knowledge of traditional Chinese medicine for liver injury common is that the cause is eating disorders, emotional disorders, or upright weakness and so on. Jin shi etal Presume that cause of this disease that is mainly exogenous heat cult poison and eating properly, and with the physically weak related. mainly pathomechanisms is hot and humid cult poison accumulating the liver and spleen, liver and gallbladder loss thinning, stomach is not healthy, qi stagnation, disharmony caused by context. Lian yue xiang etal presume that the etiology and pathogenesis of this disease for the hot and humid outside invasion or Immunotoxin infection, depression in the spleen and stomach, resulting in heat fumigation, loss of health movement spleen, liver failure catharsis, Liver and gallbladder fluid spill. Sun jiang qiao etal presume that pathogenesis of this disease are mostly hot and humid toxic silt, qi stagnation, blood stasis, loss of health movement spleen,weak of liver and kidney. Chinese medieine has fewer side effects and long-term medication characteristics, In recent years, treatment of Chinese medicine In hepatitis achieved good effect.Alkaloid is present in living organisms (mainly plants) in a class of nitrogen-containing organic compounds, most have complex ring structures, nitrogen and more included in the ring, mostly alkaline, with acid Salt, have significant biological activity is an important Chinese herbal medicine active ingredients.Alkaloid is first studied a class of biologically active natural organic compounds. Today there has been isolated from more than 10,000 species, of which more than 80 species have been used in clinic. Berberine in Coptis for antibacterial anti-inflammatory, ephedra ephedrine used in asthma, Rauwolfia reserpine used in the buck, the camptothecin Camptotheca acuminata and vincristine in periwinkle with In the anti-tumor.Many polysaccharide has been carried out assaying, Ephedrine, matrine, oxymatrine, colchicine, nicotine, drugs, quinoa base, papaverine, alkaline phenolic ipecac, lobelia base, quinine, cinchonine, Corydalis base, berberine, morphine, Codeine, tetrandrine, tetrahydropalmatine evodiamine, aconitine, strychnine, strychnine, vinblastine, vincristine, arecoline, betel second base, scopolamine, anisodamine, Mulan base, dauricine, sinomenine, Caulis base, Lotus's base, methysergide, reserpine, taxol, mother of pearl base etal. Many of them to apply and obtain a better clinical efficacy, such as ephedrine, colchicine, matrine, morphine, ergometrine, Tetrahydropalmatine anisodamine and so on. Many alkaloids have been reported the literature has a protective effect on the liver, For example:Crude Extract of Rubus total alkaloids of acute liver injury in rat liver enzymes CYP2E1 and CYP3A1 mRNA expression, Rock berberine alkaloids extract play the role on improve immunological liver injury, Bidens extract on experimental liver injury in mice, Anti-oxidative alkali Oxymatrine B and hepatitis C virus effect, Tetrahydropalmatine on carbon tetrachloride induced liver injury. For example: raspberry leaf crude total alkaloids of acute liver injury in rat liver enzymes CYP2E1 and CYP3A1 mRNA expression. Berberine alkaloids rock extracts can improve the immunological liver injury in mice. Bidens extract on experimental liver injury in mice. Alkali resistant to oxidation Oxymatrine hepatitis B and C virus. Tetrahydropalmatine on carbon tetrachloride induced liver injury.Hongbeiyegen as dicotyledon herbal plants of Euphorbia root of the red mountains and the Stick, the alias name of the red silver bullets, red Luoqun, Red Hat, top, red back mother, red bone white zygomatic root. Mainly distributed in central and southeastern China, southern China. Its sweet, cool, the root may be taken four seasons, set out in the "Guangxi Chinese herbal medicine." Shrubs or Joe the twigs hairy, raw slopes, wasteland bushes. Hongbeiyegen has eddects of clearing and detoxifying, expelling wind and dampness, stasis, bleeding, asthma, itching and other insecticidal effect. Tradition used to treat colitis diarrhea. Dysentery, jaundice, hepatitis, urinary tract infections and stones, nephritis, dysuria, hematuria, vaginal bleeding, vaginal discharge, eczema, urticaria, sore carbuncle swollen drugs, eventually becoming sore heal sickness. Also used to treat various types of hepatitis, the liver is the common folk Liver drug, but its study is not yet in-depth coverage at home and abroad.Modern medical research shows that Hongbeiyegen contains a large number of alkaloids and phenolic substances. It has a effect on cough, expectorant, anti acetylcholine and mild inhibition of Staphylococcus aureus and white effect. The first time, our group found that the water extract and water extraction and alcohol precipitation from the extract with anti-hepatitis virus and prevent liver fibrosis, reducing enzyme, hepatic fibrosis. In vitro studies have shown that certain anti-hepatitis B virus. Anti-duck hepatitis B virus tests show that the inhibition of Hongbeiyegen body has the role of D-HBV replication, the role of dose and duration related. Weaker than the effect of lamivudine, but to maintain a long time, medication safety. Research group studied the Hongbeiyegen of BCG and LPS-induced immunological liver injury, the results show that Hongbeiyegen damage can significantly improve the immune thymus, liver, spleen index, lower serum alanine Aminotransferase, aspartate aminotransferase, the activity of nitric oxide and liver homogenate TNF-α, IL-1βlevels, suggesting Hongbeiyegen on immunological liver injury has a significant protective effect.Objectives: The research group extracted by different methods Hongbeiyegen extracted and compared different extraction methods from the alkaloid content, find alkaloid extracted Hongbeiyegen the best extraction method, and derived from bio-separation Base. To compare the different extraction methods extract on liver injury, the Hongbeiyegen alkaloids on carbon tetrachloride induced acute liver injury in preliminary studies to understand Hongbeiyegen active ingredient, which in turn Its further research and development lay the theoretical and experimental basis.Methods:1. Hongbeiyegen extraction of different components1.1 crush:to remove sediment impurities, the Hongbeiyegen is crushed into meal and set aside.1.2 Water extraction:Take medicine Hongbeiyegen 8 times the original amount of water, heated under reflux for 2 hours, collected extract. Plus 8 times the amount of water residue refluxed 2 hours, filter, merge extract excess water evaporated, concentrated, frozen reserve.1.3 Alcohol:Alcohol:Take medicine Hongbeiyegen 8 times the original amount of 95% ethanol, heated under reflux for 2 hours, collected extract. Plus 8 times the ethanol residue was refluxed 2h, filtered, combined extract, after the recovery of ethanol, frozen reserve.1.4 Alkaloids:use of acidic water-alkaline-lipophilic solvent extraction, and ultimately get the total alkaloids. By the ratio of the original herbs configured a certain concentration of water liquid suspension. Alkaloids medium and low dose group were diluted with distilled water suspension plus 1/2,1/4 dose, and frozen reserve.2. Hongbeiyegen extracts on carbon tetrachloride induced acute liver injury in mice studies. 2.1 Experiment grouping96 Kunming mice, weighing 18～22 g, evenly divided male and female male and female (Purchased from the Laboratory Animal Center of Southern Medical University). A randomized method was divided into normal group, model group, Hongbeiyegen water extract (7.8g crude drug/kg), Hongbeiyegen alcohol extract (7.8g crude drug/kg), Bifendate group (175mg/kg) Hongbeiyegen alkaloids high-dose(7.8g crude drug/kg), Hongbeiyegen alkaloids middle-dose(3.9g crude drug/kg), Hongbeiyegen alkaloids low-dose(1.95g crude drug/kg),(n=12 in each groud),. In addition to normal mice, the remaining mice were gavage and the dose was 0.5ml/20g, Normal mice were given the same dose of saline.2.2 Method0.12% carbon tetrachloride (prepared with olive oil) 0.1ml/lOg was injected via Abdominal into mice, The control group was given an equal volume of saline, fasting can not help but water for 16 hours.2.3 Index Detection2.3.1 The mice were weighed and the blood was collected from mice orbit, the blood was centrifugated to obtain serum, according to kit instructions to study serum ALT, AST, T.Bili activity.2.3.2 Liver histopathologyAnimals were killed, taken out the liver, washed floating blood with cold with saline for drying. The right hepatic leaves were taken one, and immediately fixed with 10% neutral formalin,70%,80%,90%,95%,100% ethanol dehydration, xylene transparent, embedded in paraffin, slicing machine for 4um slices, conventional dewaxing, HE staining, neutral gum mounting. As well as taken pictures with PM-20 type microscope camera system, observed with a microscope.2.4 Statistiealanalysis All experiments quantitative data were expressed as mean±S.D and assessed by the one-way analysis of variance(ANOVA). If the variances between groups were homogenous (Levene's test), groups were subjected to the multiple comparison Least Significant Differences(LSD)test. In case of no homogeneity variances, differences were evaluated by Welch and the groups were subjected to the multiple comparisons Dunnett's T3 test. The statistical significance between group was indicated using superscript signs in the figures and tables. Sighnificance was defined as P<0.05.Results:1. Using different extraction methods extract of Hongbeiyegen, and compared the content of alkaloids from the final result is by water extraction and ethanol extraction of the alkaloids extracted about 0.06%, the use of ethanol extraction, extraction from slightly higher alkaloid content, extracted from many other non-alkaloid constituents, alkaloids isolated compared Trouble, so a better approach for the extraction of alkaloids ethanol extraction.2. The normal group, model group, serum ALT, AST levels(P<0.05), Showed that mice of this experiment successful model of acute liver injury. Hongbeiyegen water extract, ethanol extract and total alkaloids of high, medium and low dose group compared with the model group were significantly(P<0.05). Compared with the control group, model group, serum bilirubin levels.(P<0.05). The treatment group compared with the model group, the activity of the serum bilirubin was significantly(P<0.05).3. Liver histological examinationAll mice were observed liver pathology. Model changes in liver cells of mice significantly, loss of normal structure of hepatic lobule and liver cell necrosis with varying degrees of inflammatory cell infiltration, some visible increase in size, was void flaky lesions, compared with the normal group was significant difference, Suggesting that successful model. Positive drug DDB liver cells of mice were normal, only to see some slight swelling, no necrosis. Hongbeiyegen water extract group, ethanol extract group, high dose group alkaloids mild swelling of liver cells, no necrosis. Dose of total alkaloid in the mild swelling of liver cells, little necrosis. Low-dose group was significantly swollen liver cells, accompanied by scattered spotty necrosis. Hongbeiyegen three extracts compared with the model group had significantly reduced the pathological changesConclusion:1. The best extraction method for alkaloids:8 times with 95% ethanol extract the total alkaloids of Hongbeiyegen, the method with fewer impurities and higher levels.2. The results showed that Hongbeiyegen water extract, ethanol extract, total alkaloids were with the prevention of carbon tetrachloride induced liver injury.3. The total alkaloids are Hongbeiyegen against acute liver injury in one of the active ingredients, It will lay further research and development of theoretical and experimental basis for Hongbeiyegen. |
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