Study On The Toxicity And Antioxidant Effect Of Rheum Palmatum Water Extract In Mice

The misuse of chemical,hormonal feed additives and antibiotics has not only made livestock susceptible to oxidative stress when facing extreme climate change and bacterial infection,but also harmed the immune performance and normal metabolites.In addition,the chemical feed prodcuts also reduced the quality of livestock products and polluted the environment.There are many herbal active ingredients which show antioxidant,antibacterial,antiviral,growth promoting and immune boosting properties,such as anthracene quinones,flavonoids,saponins,polysaccharides and so on.The traditional Chinese herbs have been used as feed additives with long history,and have shown good efficacy in growth and metabolic regulation of livestock.In the current study,the antioxidant capacity of extracts form seven widely used Chinese herbs in northwest China was assessed by in vitro free radical scavenging method to screen the herbs with better antioxidant properties.The water extract of Rheum palmatum（RP）showed the best antioxidant capacity.And it was further evaluated for toxicology and in vivo antioxidant oral dosage in mice.The in vivo antioxidant properties of RP against DL-Homocysteine（DL-Hcy）induced oxidative stress in mice were investigated and the results showed as the following:1.In vitro antioxidant capacity of herbal extractsIn this study,the 1,1-diphenyl-2-picrylhydrazyl（DPPH） and 2,2’-bi-azido-bis-（3-ethylbenzothiazoline-6-sulphonic acid）diammonium salt（ABTS）radical scavenging method were used to study the antioxidant activity of seven Chinese herbal extracts（Codonopsis pilosula,Astragalus membranaceus,Angelica sinensis,Gjlycyrrhiza uralensis Fisch,Rheum palmatum,Isatis Radix,Scutellaria baicalensis Georgi）)in different solvents（distilled water,ethyl acetate,n-butanol,5%sodium carbonate）and the antioxidant activities of different potentcomponents（baicalin,baicalin,rhodopsin,rhodolic acid,aloe rhodopsin）.The results showed that the water extract of Rheum palmatum and Scutellaria baicalensis Georgi showed the best antioxidant capacity.The DPPH clearing rate was highest（91%,87%）when the water exract of Rheum palmatum at 0.6 mg·mL^-1 and the water extract of Scutellaria baicalensis Georgi at 2.0 mg·mL^-1,respectively.The ABTS clearing rate was highest（98%）when the two concentration were at 0.6 mg·mL^-1 and 1.0 mg·mL^-1,respectively.The DPPH clearing rate of the 5%sodium carbonate of Rheum palmatum and Scutellaria baicalensis Georgi were not high,dnly 30%as maximum at about 3.0mg·mL^-1.The ABTS clearing rates of the 5%sodium carbonate of Rheum palmatum and Scutellaria baicalensis Georgi reached the highest（99%）at 0.6 mg·mL^-1 and 1.0mg·mL^-1,respectively.The baicalin showed the highest DPPH（97%）at 0.6 mg·mL^-1and ABTS（98%）at 0.2 mg·mL^-1,respectively.The baicalein has the highest DPPH（95%）at 0.2 mg·mL^-1and ABTS（50%）at 0.6 mg·mL^-1,respectively.The DPPH clearing rate of emodin,rhein and aloe emodin were highest at 3.0 mg·mL^-1,reaching about 38%,61%,40%.The ABTS clearing rate of emodin,rhein and aloe emodin were highest at 3 mg·mL^-1,reaching about 88%,40%,71%,respectively.The highest DPPH clearing rate was shown in the extraction of ethyl acetate(4 mg·mL^-1),and n-butanol(6 mg·mL^-1)in Rheum palmatum.In conclusion,Rheum palmatum and Scutellaria baicalensis Georgi showed higher DPPH and ABTS clearing rate compared to the other five herbal species.2.Acute toxicity test of the water extract of Rheum palmatum（RP）in miceTo assess the in vivo toxicity of RP,a mouse acute toxicity test was designed.twenty-four mice（male and female,n=6）were randomly divided into high(DH,1400mg·kg^-1 BW·d^-1),medium(DM,800 mg·kg^-1 BW·d^-1),low(DL,500 mg·kg^-1 BW·d^-1)and control groups(CK,0 mg·kg^-1 BW·d^-1)by equal volume gavage once a day for14 days.The behavioural activity,food intake and body weight of the mice were observed and recorded.The histopathological changes of liver,kidney and intestine were observed.The results showed that the mice in all dose groups showed diarrhoea and weight loss compared with the CK group,and the severity of diarrhoea increased and the weight loss increased with increasing RP dosage（P<0.05）,but the difference in daily food intake between the dose groups was not significant（P>0.05）.The antioxidant index of liver showed that the activity of glutathione peroxidase（GSH-Px）was significantly lower in the other three dose groups compared with the CK group（P<0.05）,while the contents of superoxide dismutase（SOD）and malondialdehyde（MDA）were not significantly different（P>0.05）.The results of histopathological sections showed that the severity of the lesions in the liver,kidney and small intestine was more severe as the dose of RP increased.In DH group,the hepatic sinus shrink,disappearing of hepatic cord,steatosis and mononuclear cell hepatic tissue infiltration appeared;mice had dilated renal tubular lumens,swollen epithelial cells,vacuolar degeneration and disintegrated and shed epithelial cells;There was shortening of the villi,inflammatory cell infiltration in the intestinal mucosa,lymphatic expansion and intestinal gland enlargement in the DH mice compared to CK group（P<0.05）.3.Antioxidative stress test of RPTo investigate the antioxidant effect of RP on oxidative stressed mice,forty（n=10）male mice were randomly divided into DL-Hcy,DL-Hcy+50RP,DL-Hcy+150RP and control group（CK group）.The oxidative stress model mice was induced by homocysteine（DL-Hcy）.Except for the CK group,DL-Hcy was injected subcutaneously twice a day（8 h interval）.DL-Hcy+50RP and DL-Hcy+150RP groups were gavaged with 50 and 150 mg·kg^-1 BW·d^-1 of RP for 21 d.The intake,body weight and behavioural activities of the mice were observed and recorded.Serum and organs were also collected from the mice,and blood physiological and biochemical parameters,tissue reactive oxygen content and histopathological changes were measured.The results showed that DL-Hcy and RP had no significant effect on daily weight gain and feed intake of mice（P>0.05）.Compared with CK group,the DL-Hcy group showed significantly higher liver SOD and GSH-Px concentrations,significantly lower MDA concentration（P<0.05）,significantly higher reactive oxygen species（ROS）levels in kidney and spleen as well as blood glucose concentrations（P<0.05）,and severe pathological damage in liver,kidney and small intestine tissues.Compared with the DL-Hcy group,the RP groups(50,150 mg·kg^-1BW·d^-1)significantly reduced ROS level in kidney and spleen tissues,and glucose concentrations and the activities of SOD and GSH-Px,and inhibited the release of pro-inflammatory factors,and improved hepatic steatosis,chronic nephritis,and enterocolitis.In conclusion,RP showed the best antioxidant properties among the seven common Chinese herbs with different organic solvents,followed by Scutellaria baica-lensis Georgi.In addition,baicalin and baicalein also showed good DPPH and ABTS scavenging effects.The acute toxicity of feeding rhubarb water extract to mice showed that the gavage doses of 500,800 and 1400 mg·kg^-1 BW·d^-1 caused lesion of liver,kidney and intestine,triggered inflammatory reactions and reduced the activity of liver antioxidant enzymes.The results of gavage of RP in DL-Hcy oxidative stress model mice showed that RP had curing effect on the effective damaged internal organs（liver and kidney）in oxidative stressed mice at the dose of 50 and 150 mg·kg^-1BW·d^-1.The RP also reduced blood glucose and increased antioxidant properties of oxidative stressed mice.In addition,the higher dose(150 mg·kg^-1 BW·d^-1)showed better antioxidant effect than the lower dose(50 mg·kg^-1 BW·d^-1).