Moghadamnia D, Mokthari M, Khatamsaz S. Comparison of Protective Effects of Omega3 Fish Oil and Aqueous Extract of Glycyrrhiza glabra Root on Thioacetamide Induced Lipid Toxicity in Male Rats. Biomed Pharmacol J 2016;9(1)

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Manuscript received on :October 09, 2015
Manuscript accepted on :December 01, 2015
Published online on: 07-03-2016

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D. Moghadamnia^1,2*, M. Mokthari³ and S. Khatamsaz³

¹Department of Biology , Fars Science and Research Branch,Islamic Azad  University ,Fars ,Iran. ²Department of Biology,Shiraz Branch,Islamic Azad University, Shiraz,Iran. ³Department of Biology,Kazerun Branch,Islamic Azad University, kazerun,Iran.

DOI : https://dx.doi.org/10.13005/bpj/917

Abstract

Thioacetamide(TAA) is an organic compound and is a potentotoxin.TAA has dyslipidemic effects  because of its rapid elimination and cumulative injury when it is given intermittently,presumably by free radical-mediated lipid.Glycyrrhiza glabra is a well-know medical plant.Glycyrrhiza glabra root reduce the risk of coronary heart disease.Omga3 fatty acids are widely used  to treatment hypertriglyceridema.Omega3 fatty acid are know to act as hypolipidaemics.The current investigation was designed to explore the possible protective effects of omega3 fish oil and glycyrrhiza glabra aqueous extract on TAA-induced dyslipidemia in male rats.63 wistar male rats were divided into 9 group. Control group.Sham group1.each rat  of the group received  0.4ml olive oil as a solvent omega3 fish oil supplements orally per day for 3 months. Sham group2.each rat of the group received 150 mg/kg of TAA intrapertionealy in a single dose for 3months. Experimental  group 1,2,3. each rat of the 100,200,300 mg/kg of omega3 fish oil supplements orally per day for 3 months and 150 mg/kg of TAA intrapertionealy in a single dose  for 3months .Experimental  group 4,5,6.each rat  of the group  received 100,200,300 mg/kg of aqueous extract of glycyrrhiza glabra root orally per day for 3 months and 150 mg/kg of TAA intrapertionealy in a single dose for 3months and 150 mg/kg of TAA intrapertionealy in a single dose for 3months. The provided blood samples were tested for total cholesterol,HDL,LDLcholesterol,triglyceride and FBS serum levels.pretreatment with 100,300mg/kg of aqueous extract of glycyrrhiza glabra or by 300mg/kg omega3 fish oil supplements significantly reduced thioacetamide-induced elevation in plasma levels of  total cholesterol. Pretreatmen with the omega3 fish oil andaqueous extract of  glycyrrhiza glabra rootat alldosesshowed nosignificant  difference inserum levels oftriglyceride,LDL,HDL cholesterol and FBS comparing with thioacetamide group(P<0.05).In conclusions,The study  suggests  that  thioacetamide-induced  lipid  toxicity in male rats can be  ameliorated by  oral administration of aqueous extract  of glycyrrhiza glabra root and omega3 fish oil.

Keywords

Glycyrrhizaglabra; Omega3 fish oil; Thioacetamide; Dyslipidemia

Introduction

Thioacetamide (TAA) is an experimental hepatoxin(Staňková et al,2010)which is a thiono-sulfur containing compound with liver damaging and carcinogenic activity.Shortly after administration,TAA undergoes metabolism  to acetamide and thioacetamide-S-oxide by the mixed function oxidase system(Farkaad et al,2011).TAA is the most potent nephrotoxic substance because of its repid elimination and cumulative injury when it is given intermittently,presumably by free radical-mediated lipid(Begum et al,2011; Salama et al ,2012).Metabolic studies of TAA-induced tissue damage suggest that TAA is metabolized by the mixed function oxidase system to its toxic metabolites sulfine(sulfoxide) and sulfene(sulfone) which then distributed among serveral organs including plasma,liver,kidney,bone marrow,adernals and other tissues(Jules Clement Assob Nguedia et al,2014).

Alpha-linolenic acid(ALA), ecosapentaenoic acid(EPA) and docosahexaenoic acid (DHA) are grouped together as the omega3 PUFA,there is substantial evidence suggesting that the individual fatty acids may have selective and potentially independent effects on cardiovascular health(Yashodhara et al,2009; Lee et al,2009).Omega3 fatty acids led to management of diverse disease such as psychiatric(Giuseppe Grosso et al,2014),inflammatory bowel disease and cystic fibrosis(Maria Tabbaa et al,2013).The omega3 Fatty acid,present in fish oil,interfere with the arachidonic acid pathway of inflammation(Philip et al,2010)and can also modulate the response of macrophage to endotoxin by inhibition of  TNF-alpha production in vitro(Zheng et al,2010).

Glycyrrhiza glabra , papilionaceae/fabaceae family grows in various parts of the world. Glycyrrhiza glabra,also know as licorice,is a herbaceous perennial,with pinnate leaves and purpule to whitish blue flowers.It roots posses some nutritive value and medical properties(Masoud Sabouri Ghannad et al,2014).phytochemical analysis of glycyrrhetinic acid and liquirtic acid ,flavonoids and other constituents such as coumarins,simple  sugar  and polysaccharide like starch,pectin,amino acids, choline,  phytosterol,mineral salts and various other substance(Drgham Hamza Yousuf Al-Zwean  et al,2014).Many biological activities such as anti-genic activity,anti-ucler effects,protective action against hepatotoxicity,anti tumor promoting activity,antimicrobial effects(Khattak et al,2010; Kaur et al,2009).

Due to the high incidence of cardiovascular diseases and the numerous side effects of chemical drugs, it is needed to look for safer drugs with lower side effects and higher effectiveness.In the present study ,with respect to the low side effects of omega 3 polyunsaturated fatty acids and glycyrrhiza glabra root,the  protective effects of omega-3 fish oil supplement  andaqueous extract  of  glycyrrhiza glabra root against lipid toxicity in thioacetamide induced male rats  were investigated.

Materials And Methods

63male white wistar rats with a weight of 210±10 gr provided from laboratory animal center shiraz azad university were divided 9 equall groups.Control group.each rat of the group underwent no stress such as injection ,oral gavage and etc. Sham group1.each rat of the group received 0.4ml olive oil as a solvent omega3 fish oil orally per day for 3 months.Sham group2.each rat of the group received 150 mg/kg of TAA intrapertionealy in a single dose for 3months. Experimental  group 1. each rat  of the group  received 100 mg/kg of omega3 fish oil supplements orally per day for 3 months and 150 mg/kg of TAA intrapertionealy in a single dose for 3months.Experimental group 2. each rat of the group received 200 mg/kg of omega 3 fish oil supplements  orally  per day for 3 months and 150 mg/kg of TAA intrapertionealy in a single dose  for 3months. Experimental group 3 .each rat of the group received 300 mg/kg of omega 3 fish oil supplements  orally per day for 3 months and 150 mg/kg of TAA intrapertionealy in a single dose for 3months.Experimental  group 4. each rat of the group  received  100 mg/kg of aqueous extract of glycyrrhiza glabra root orally per day for 3 months and 150 mg/kg of TAA intrapertionealy in a single dose for 3months.Experimental group 5. each rat of the group received 200 mg/kg of  aqueous extract of glycyrrhiza glabra root orally  per day for 3 months and 150 mg/kg of TAA intrapertionealy in a single dose  for 3months. Experimental group 6 .each rat of the group received 300 mg/kg of aqueous extract of glycyrrhiza glabra root orally per day for 3 months and150 mg/kg of TAA intrapertionealy in a single dose for 3months(Hai Zhong Huo et al,2011; Hanna M.Sirag et al,2007; M.Meganathan et al,2011).

By the end of experimental periods,rats were scarified under diethyl ether anesthesia at fasting state.The portion of blood samples were collected and allowed to coagulate at room temperature;other portion of blood added to it,EDTA(ethylene diamine tetracetic acid) and centrifuged at 5000r.p.m for 15 minutes.The clear,none-haemolysed supernatant sera and plasma were quickly removed divided into four portions for each individual,and stored at -20c for subsequent analysis(Liang Renjie et al,2010; Mebratu Alebachew et al,2014).

Levels of plasma lowdensity lipo-protein cholesterol(LDL), high density lipo protein cholesterol (HDL),total cholesterol,triglyceride(TG) and fasting blood sugar(FBS) were assayed using clinical test kits and anzymatic methods.

All of the valves are reported as mean±SEM.The statistical significance of variations between groups was analysed employing one-way ANOVA pursued by Tukey test analysis using SPSS version 18 with a value of P<0.05 was regarded significant when compared to the control and sham groups.

Results

Administration of thioacetamide let to significant increase  the serum level of total cholesterolcompared to the control group.Pretreatmen with 300 mg/kg of omega3 fish oil supplementssignificantincreased  the serum levels of total cholesterol comparing with thioacetamide group.Pretreatmen with 100,300 mg/kg of  aqueous extract of  Glycyrrhiza glabra root significant increased  the serum levels of total cholesterol comparing with thioacetamide group (P<0.05).

Administration of thioacetamide let to significant reduced  the serum level of  FBScompared to the control group .Pretreatmen with the omega3 fish oil supplements atall doses increased  the serum levels of FBS comparing with thioacetamide group but there was no significant difference.Pretreatmen with the aqueous extract of  Glycyrrhiza glabra root at all doses increased  the serum levels of FBScomparing with thioacetamide group but there was no significant difference(P<0.05).

Administration of thioacetamide  showed  no  significant difference in  serum levels of LDL, HDLCholesteol,and triglyceride in compared to the control group . Pretreatmen with the omega3 fish oil supplements at all doses showed  no  significant  difference inserum levels of LDL, HDLcholesterol and triglyceride comparing with thioacetamide group.Pretreatmen with the aqueous extract of  Glycyrrhiza glabra root at all doses showed  no  significant  difference inserum levels of LDL, HDLcholesterol and triglyceride comparing with thioacetamide group.

Figure1: Effects of omega3fish oil and  aqueous extract  of glycyrrhiza glabra root on serum levels of FBS. Click here to View figure

Values significantly different between control and sham groupswith TAA group.^aP<0.05 .

Values significantly different between experimental groupswith TAA group.^bP<0.05.

Values significantly different between experimental groupswith control and sham groups.^cP<0.05.

Figure 2: Effects of omega3fish oil and  aqueous extract  of glycyrrhiza glabra root on serum levels of total cholesterol. Click here to View figure

Values significantly different between control and sham groups with TAA group.^aP<0.05

Values significantly different between experimental groups with TAA group.^bP<0.05

Values significantly different between experimental groups with control and sham groups.^cP<0.05.

Figure 3: Effects of omega3 fish oil and  aqueous extract  of glycyrrhiza glabra root on serum levels of  triglyceride. Click here to View figure

Values significantly different between control and sham groups with TAA group.^aP<0.05

Values significantly different between experimental groups with TAA group.^bP<0.05

Values significantly different between experimental groups with control and sham groups.^cP<0.05.

Figure4: Effects of omega3fish oil and  aqueous extract  of glycyrrhiza glabra root on serum levels of LDL cholesterol. Click here to View figure

Values significantly different between control and sham groups with TAA group.^aP<0.05

Values significantly different between experimental groups with TAA group.^bP<0.05

Values significantly different between experimental groups with control and sham groups.^cP<0.05.

Figure5: Effects of omega3fish oil and  aqueous extract  of glycyrrhiza glabra root on serum levels of HDL cholesterol. Click here to View figure

Values significantly different between control and sham groups with TAA group.^aP<0.05.

Values significantly different between experimental groups with TAA group.^bP<0.05.

Values significantly different between experimental groups with control and sham groups.^cP<0.05.

Discussion

Various useful drugs like acetaminophen,gentamicin and some environmental and industrial toxin can cause severe renal damage through activation of these drugs to highly reactive free radicals(Olagunju et al,2009).

One of the most extensively studied chemical and industrial toxicants is TAA.                                                                                                                                                      TAA is know to induce centrilobular hepatic necrosis,liver cirrhosis,hepatocellular carcinoma and bile duct proliferation(Kadir et al,2013; Atef et al,2011)with injury to the terminal portion of the proximal renal tubule and dyslipidemia.

Pretreatmen with 100,300 mg/kg of  aqueous extract of  Glycyrrhiza glabra root significant increased  the serum levels of total cholesterol comparing with thioacetamide group.Pretreatmen with the aqueous extract of  Glycyrrhiza glabra rootat all doses decreased  the serum levels of triglyceride comparing with thioacetamide group but there was no significant difference(P<0.05).

Glycyrrhizic acid a constituent of glycyrrhiza glabra,reduces plasma cholesterol by down-regulating hepatic HMG COA reductase(HMGR) mRNA expression in hamster fed a high fructose-fat diet. Glycyrrhizic acid treatment significantly decrease apolipoprotein B(APO B),lipase a(LP a)  and cholesteroliester-transport protein(CETP) concentrations but increased APO A-1 levels and  APO A-1 / APO A-2 ratio.The contents of cholesterol and triglyceride in hepatic tissue were significantly lower in the glycyrrhizic acid group than in the control group(Maurya et al,2011).Glycyrrhizic acid improved insulin sensitivity and lipid profiles and induced up-regulation of total PPAR gamma and lipoprptein lipase(LPL) expression level in rats and decrease in triacylglycerol , total cholesterol and a elevation in HDL cholesterol(Yoke Yin et al,2010).Licochalcone A(LA) a constituent of glycyrrhiza glabra,suppressed hepatic triglyceride accumulation through modulation of AMP-SREBP pathway.LA inhibited lipogenesis via supperession of sterol  regulatory element-binding protein1(SREP1C) and target  enzymes     (stearoyl-COA desaturase1,fatty acid synthase and glycerol-3-phosphate acyltransferase)      transcription.LA up-regulated gen expression of proteins such as PPAR alpha and fatty acid transport(FAT/CD36) which are responsible for lipolysis and fatty acid transport(Quan et al,2013).Chalcones of glycyrrhiza glabra roots decrease that levels of plasma total cholesterol and triglyceride.Chalcones showed strong inhibition against pancreatic lipase(Rahul et al,2011).Glabrol from glycyrrhiza glabra roots act as antihypercholesterolemic agent.Glabrol inhibited acyl-coenzyme A cholesterol acyltransferase(ACAT) and decreased cholesterol ester formation(Jung et al,2007).Beta-sitosterol  from glycyrrhiza glabra roots reduced intracellular levels of triglyceride and cholesterol in L6 cells.Benefical effects of beta-sitosterol on lipid metabolismIn L6 myotube cells are mediated by AM-activated protein kinase(Seung-Lark Hwang et al,2008).Glabirdin  from glycyrrhiza glabra roots showed inhibitory effect on adipogenesis in a dose dependent manner.The inhibitory effects of glabirdin resulted from inhibiting the induction of transcription factor CAAAT enhancer binding protein alpha and PPAR gamma(Jiyun Ahn et al,2013).

Pretreatmen with 300 mg/kg of omega3 fish oil supplementssignificanincreased  the serum levels of total cholesterol comparing with thioacetamide group. Pretreatmen with omega3 fish oil supplements at all doses decreased  the serum levels of triglyceride comparing with thioacetamide group but there was no significant difference(P<0.05).

Omega3 polyunsaturated ameliorated diet-induced hyperlipidemia and fatty liver through induction of CYP7A1 expression and activation of cholesterol catabolism to bile acid(Kim et al,2012).Omega3 readly incorporated into hepatic phospholipids,reduced stearoyl-COA desaturase16 , stearoyl-COA desaturase,delta 6 desaturase,delta5 desaturase activities and reduced the lipogenesis index(Lamaziere et al,2013).Both caloric restriction and omega3 supplement diets are able to prevent hypercholesterolemia,by regulating HMG-COAR activation state by controlling ROS production and P38 phosphorylation.Moreover also the age-dependent less of LDL membrane exposition in prevented(Chaiara  Martini et al,2008).Combination of fish oil and fish protein hydrolysate decrease the plasma cholesterol level(Wergedahl et al,2009).Omega3  fatty acid negatively regulate triglyceride biosynthesis.Omega3 fatty acid deficiency increase stearoyl-COA desaturase(sd1)and suggest that down regulation of sd1 is a mechanism by which omega3 fatty acid repress constitutive triglyceride biosynthesis(Rylon Hofacer et al,2012).DHA supplementation reducedAPO C III concentrations which inhibits the activity lipoprotein lipase(LPL) that control triglyceride(TG) clearance from blood. Thus,a reduction in the concentrations of APO C III increased activity of LPL and hence increased clearance of plasma TG(Bays et al,2008).Omega3 regulate  APO C III there effects on PPAR alpha which down regulates  APO C III expression and NF-KB which  up regulated APO C III expression(Ooi et al,2008).Docosahexanoic acid(DHA) is an FXRa ligand has been shown to decreases the expression of hepatic lipase and APO C III and increase APO C II and VLDL-receptor gene expression in HPG2 cells(Davidson et al,2009).Omega3 fatty acids reduced activity of key enzymes in TG biosynthesis such as phosphatidic acid phoshohydrolase and diacylglycerol acyltransferase.

Pretreatmen with the aqueous extract of  Glycyrrhiza glabra root at all doses increased  the serum levels of FBS comparing with thioacetamide group but there was no significant difference.

18 beta-glycyrrhetinic acid.a glycine of glycyrrhizin possesses a potential anti hyperglycemia effects that is comparable with glibenclamide(Kalaiarasi et al,2009).Glycyrrhizic acid inhibit11 beta-hydroxysteroid dehydrogenase1 activity(Chia et al,2010).Glycyrrhizin improved significantly the diabetogenic effects of streptozotocin namely enhanced blood glucose level,glucose intolerant behavior,decreased seum insulin levels(Sen et al,2011).Glycyrrhizic acid improved insulin sensitivity and lipid profile and induced up regulation of total PPAR gamma and lipoprotein lipase expression levels in rats(Yoke et al,2010).Glabridin,a polyphenolic flavonoid from licorice significantly decreasing fasting blood glucose.These results demonstrated that glabridin possesses hypoglycemic effects(Wu et al,2013).Glycyrrhizin ameliorates insulin resistance,dyslipidemia and oxidative stress in fructose-induced metabolic syndrome X in rat model.The decrease levels of PPAR gamma and glucose transport(GLUT4) proteins in slekeletal muscle of metabolic syndrome were elevated by glycyrrhizin,indicating improved fatty acid oxidation and glucose homeostasis(Sil et al,2013).

Pretreatmen with the omega3 fish oil supplements atall doses increased  the serum levels of FBScomparing with thioacetamide group but there was no significant difference.

Omega3 fattty acids may increase the plasma glucose level through other mechanism.                                                             Long-chain omega3 can decrease glucose utilization and increase glucagon-stimulated C-peptide(Manas Kaushik et al,2009) or could increase hepatic gluconeogenesisby increase uptake and oxidation of free fatty acids in liver(Shokouh Sarbolouki et al,2013).Thus,omega3 fatty acid and fish consumption may increase the diagnosis of T2DM by increasing circulating concentration of glucose but whithout causing other adverse metabolic abnormalities.

The overall result from this study demonstrated TAA-induced lipid toxicity by biochemical analysis.The concurrent treatment with omega3 fish oil and or by aqueous extract  ofglycyrrhiza glabra root clearly provided a considerable degree of protection in a dose-dependent manner against the deleterious lipid side effects of TAA.

In conclusion,omega3 fish oil and aqueous extract of  glycyrrhiza glabra root could act on the lipid metabolism as potent antioxidant to prevent ongoing TAA-induced lipid toxicity.

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