Protective Effect Of Solanum Nigrum Granules On Experimental Liver Injury And Preliminary Studies On Reducing The Toxicity Of Solanum Nigrum By Using Compatibility

Complex Granula of Solanum nigrum was a new compound Chinese medicine which was researched and developed based on the clinical experiences of traditional Chinese medicine "Long chai Tang"by professor Shi Jin who is a provincial old TCM and doctoral tutor of Nanjing University of Traditional Chinese Medicine. Complex Granula of Solanum nigrum was composed by the herbs of Solanum nigrum, Hedyotis diffusa, Sedum sarmentosum Bunge, Bupleurum chinense, Cape jasmine fruit, Glycyrrhiza uralensis Fisch. It is used in the clinical treatment of chronic hepatitis B. The effect is clear and rapid.The therapeutical effects and safety evaluation about Complex Granula of Solanum nigrum were studied to provide reliable experimental reference for further clinical experiment, The authenticating test would be taken in the Complex Granula of Solanum nigrum combined with medicine couple, in order to ensurce the effect of protecting the liver and to reduce its toxicity. This study would satisfy the requirements in clinical that the toxic TCM would be used in patients as safety and effective medicines. Meanwhile the new technologies and new ideal would be provided to research the toxic TCM with the method of compatibility attenuation. The main contents are as follows:The main contents:1. Effect of complex granula of Solanum nigrum on acute liver injury by CCl4 in rats60 mice cleaned by KM, were randomly divided into 6 groups:normal group, model group, Bifendate contrast group (100mg/kg) and Solanum nigrum granule high (10 g/kg), medium(5 g/kg), low(2.5 g/kg) dose group. According to the dose, intragastrically infused with complex granula of Solanum nigrum and Bifendate contrast group were given once everyday, normal and model group intragastrically infused with normal saline. After 7 days latter, all groups except normal of acute hepatic injury were set up by giving intraperitoneal injeetion of 0.2%CCl4(10 ml/kg). And 12 hours later, all mice were put to death. Blood was taken ftom the back eye pit, and then centrifugation with 3000r/min by 10 minutes to separate the serum, measured the activities of AST and ALT in the serum. The organ index of liver, spleen and thoracic gland were detected.2. Effect of complex granula of Solanum nigrum on chronic liver injury by CCl4 in rats120 Sprague-Dawley rats were divided into 6 groups,including normal control group,model group, Bifendate contrast group and Solanum nigrum granule high(10 g/kg), medium(5 g/kg), low(2.5g/kg) dose group.All rats except normal control group were given 25% carbon tetrachloride (3mL·kg-1bodyweight) twice a week for 10 weeks,once subcutaneously injected once administered. After 3 weeks of the beginning,the rats of Solanum nigrum granule high, medium, low dose group were administered with Solanum nigrum granule at a dose of 10 g/kg body weight,5 g/kg and 2.5g/kg for 7 weeks. The rats of Bifendate contrast group were administered with bifendate at a dose of 50 mg/kg body weight. Administered for 4weeks and 8weeks, The liver function (ALT、AST、ALP、T-bil、TP、ALB in serum) was detected, and A/G was calculated. The changes of serum levels of TNF-α、IL-6、IL-10 and IFN-y were measured with ELISA.The activity of SOD, GSH-Px, and the level of MDA in serum and levels of protein and Hyp in liver tissue were measured. The organ index of liver, spleen and thoracic gland were detected. The histo-pathology of liver was examined.3. Protective effect of complex granula of Solanum nigrum on hepatocyte injury induced by CCl4Human hepatocyte line LO2 was cultured in vitro treated with complex granula of Solanum nigrum. Then the survival of hepatocytes was detected by MTT assay. Models of hepatocyte damage induced by CCl4 was established, it is divided into normal control group, model control group,Bifendate contrast group(5μg/ml),four groups of different concentrations of complex granula of Solanum nigrum group and the survival of hepatocytes were detected by general methods.the same time, after induced hepatocyte injury model was made,induced hepatocyte were divided into model group, normal serum control group,complex granula of Solanum nigrum serum group(20%,10%,5%) and Bifendate serum group5(μg/ml),MTT test cell viability of each group.4. The toxicity test of complex granula of Solanum nigrumThrough the test of acute toxicity, to observe the status of the mice died, and record the number of deaths, and then calculate the median lethal dose (LD50) by Method of Bliss.The long-term toxicity experiment:After long-term gavaging to rats with complex granula of Solanum nigrum, observed any impact in terms of Body weight, general physical signs, behavior, hematology, blood biochemistry and histopathology, and symptoms of poisoning and target organs.5. Attenuated compatibility of Solanum nigrum and LicoriceThrough the test of acute toxicity, to observe the status of the mice died, and record the number of deaths, and then calculate the median lethal dose (LD50) of Solanum nigrum decoction and the decoction with Solanum nigrum and Licorice by Bliss.The content of Solasonine in two diffent decoction were determinated by high performance liquid chromatography (HPLC).Results:1. Effect of complex granula of Solanum nigrum on acute liver injury by CCl4 in ratsResults showed that compared the model group, liver index is not significantly different (P>0.05), activities of serum ALT and AST decreased but not significantly (P>0.05). Experimental results show that complex granula of Solanum nigrum had little effect on acute liver injury by CCI4 in rats.2. Effect of complex granula of Solanum nigrum on chronic liver injury by CCl4 in rats4 weeks later, Compared with nomal group, model group was found to significantly increase the levels of ALT、AST、ALP and T-bil in serum (P<0.01). And the level of TP were decreasd (P<0.05). Compared with model group,Complex granula of Solanum nigrum low(2.5g/kg) dose group decreasd ALT(P<0.05), all Solanum nigrum groups increase the levels of TNF-a and IL-6(P<0.05～0.01), decrease IFN-y(P<0.01), high(10 g/kg), medium(5 g/kg)dose group increase IL-10 (P<0.05) and GSH-PX (P<0.05～0.01), MDA obviously inereased in Solanum nigrum groups.The rest showed no significant difference(P>0.05).The variance of ALT、AST. ALP between groups missed. Possible cause was that modeling time was not long, chronic liver injury model was not stable enough, resulting in individual differences between groups. And we should extend modeling time, so that a stable model of chronic liver injury would be made.8 weeks later, compared with model group, Complex granula of high(10 g/kg), medium(5 g/kg) dose group was found to significantly decrease the levels of ALT、AST(P<0.05～0.01),and decrease ALP and T-bil in high(10 g/kg) dose group (P<0.05～0.01), TP. ALB increase in all Solanum nigrum groups(P<0.05～0.01). TNF-α、IL-6 increased (P<0.05～0.01) and IFN-y decrease significantly (P<0.01).Meanwhile it was also decreased MDA content (P<0.01) and prevented the reduction of SOD, GSH-PX activities in serum (P<0.05～0.01). The level of Hyp in liver homogenate was decreased (P<0.05～0.01). And it could also improve the pathological changes in liver tissue. Experimental results show that Complex granula of Solanum nigrum has significantly effect on chronic liver injury induced by CCl4 in rats.The protective effect may be related to its antioxidant activity. It could rid of excess oxygen free radicals and improve immunity.3. Protective effect of complex granula of Solanum nigrum on hepatocyte injury induced by CCl4Complex granula of Solanum nigrum showed no cytotoxicity for proliferation and growth of cell LO2 within a certain dose range (1～10mg/ml). Compared with model group, Solanum nigrum liquid has no significant difference (P>0.05).Complex granula of Solanum nigrum serum groups (10%) OD value were significantly higher than CCl4 model group(P<0.05).Experimental results show that high concentrations of Complex granula of Solanum nigrumhave certain cytotoxic on LO2.10% of the drug-containing serum can significantly reduce injury of CCl4 on LO2 hepatocytes.4. The toxicity test of complex granula of Solanum nigrumExperiment on the acute toxicity of complex granula of Solanum nigrum by calculating the value of LD50 were 79.83±5.19.It is about 44 times of the human clinical dosage.The result showed that large doses have some toxicity, the symptoms of dead mice are mainly deep breathing, covered shaking, jump channeling and so on, consistent with solanine poisoning symptoms.In chronic toxicity test,compared with normal group,male rats growth slowly (P<0.05-0.01). The organ index of liver, spleen and kidney increased (P<0.05～0.01).After administered 3 months, the hematologic index results indicated that the percent of HGB and Reticulocyte had a statistical significance (P<0.05～0.01). The biochemical index results indicated that T-BIL、GLU、Cl-on rats in high dose group of complex granula of Solanum nigrum,T-BIL、 CREA、Cl-in middle dose group and ALT、Na+、Cl-in low dose group had a statistical significance(P<0.05～0.01).After administered 6 months,the hematologic index results indicated that Reticulocyte in all dose groups,WBC、GRN in high dose group,LYM、GRN in middle dose group had a statistical significance(P<0.05～0.01).The biochemical index results indicated that ALB、GLU in high dose group,GLU in middle dose group and AST、Cl- in low dose group had a statistical significance(P<0.05～0.01).When convalescent in 1 month, the hematologic index results indicated that Reticulocyte in all dose groups,RBC、WBC in high dose group,MID in low dose group had a statistical significance (P<0.05～0.01).The biochemical index results indicated that T-cho、K+、Na+ in high and middle dose group,TG、GLU、Na+、Cl- in low dose group had a statistical significance(P<0.05～ 0.01).The histological examinations indicated that one rat in normal group had A large number of inflammatory cells in the prostate stroma. One rat in normal group and one rat in high group had hepatic cell necrosis and infiltration of lymphocytes,and there were no apparent change in internal organs among other rats and not yet pathology change caused by toxicity.Toxicology experiments suggested that Complex granula of Solanum nigrum probably produce toxic reaction to the liver, spleen and kidney. It should be pay attention to the designed of dosage and medication cycle in clinical application. We should avoid large doses, taking a long time, while enhance the function and morphological examination of liver, spleen, and kidney (eg B ultrasonic) to ensure the safety of medication.5. Attenuated compatibility of Solanum nigrum and LicoriceExperiment on the acute toxicity of Solanum nigrum decoction by calculating the value of LD50 were 1.52(g/ml).Decoction of Solanum nigrum and Licorice were 1.87(g/ml).The content of Solasonine in Solanum nigrum decoction were 1.31mg/g, and the content of Solasonine in Solanum nigrum and Licorice decoction were 1.70mg/g.Conclusion:These results suggested that Complex granula of Solanum nigrum has significantly effect on chronic liver injury induced by CCl4 in rats.The protective effect may be related to its antioxidant activity. It could rid of excess oxygen free radicals.Toxicology experiments suggested that Complex granula of Solanum nigrum probably produce toxic reaction to the liver, spleen and kidney. Compatibility of Solanum nigrum and licorice could reduce the toxicity of Solanum nigrum.The protective effect was not related to the reduction of Solasonine.