ORIGINAL�ARTICLE ABSTRACT Objective: To compare the effect of Betulinic acid and Simvastatin on Triglycerides and Low Density Lipoprotein Cholesterol (LDL-C) in Balb/C Mice. Study Design: Experimental randomized control trial. Place and Duration of Study: Study was conducted at Biochemistry department Islamic International Medical College Rawalpindi in collaboration with National Institute of Health Islamabad from November 2017 to April 2018. Materials and Methods: A total of 40 mice were randomly divided into 4 groups. Excluding one of the 4 groups as negative control, the remaining three were fed with high fat diet for 21 days to achieve raised levels of Triglycerides and Low density lipoprotein. After that out of the three high fat diet fed groups, one group was left untreated considering the positive control group and of the other 2 groups one was treated with simvastatin nd and 2nd one was given betulinic acid for the next 21 days. Sampling was done by cardiac puncture on 42 day. Triglycerides and LDL-C levels were measured in serum samples. Data thus obtained was analyzed by one way ANOVA through SPSS 21. Results: Study showed that positive control group showed a rise in mean triglycerides levels from 130mg/dL to 220mg/dL and mean LDL-C from 23mg/dL to 46mg/dL. Betulinic acid showed significantly better control than simvastatin as mean serum levels of Triglycerides were 146mg/dL as compared to 178mg/dL and mean serum levels of LDL-C were 28mg/dL as compared to 34mg/dL of simvastatin. Considering the p<0.05, TGs had a (p<0.001) and LDL-C had a (p<0.001). Conclusion: Betulinic acid showed a far better control over the levels of Triglycerides and LDL-C in HFD fed Balb/C mice as compared to simvastatin. Key Words: Betulinic Acid, LDL-C; low density lipoprotein cholesterol, Simvastatin. which results in increased risk of cardiovascular 5 complications. Globally conducted estimation of deaths because of CVD by 'WHO' revealed 17.5 6 million deaths in the year 2012. While according to 2013 Global Burden of Disease Study, CVD is 7 responsible for 30% of deaths worldwide. In the last th half of the 20 century many epidemiological and experimental studies identified high levels of LDL-C as being atherogenic and advocated a linear relationship with rate of onset of CHD; notable 8 among these are The “Framingham Heart Study” , the “Lipid Research Clinics” (LRC) trial by Patsy and Wies in 2014, and the “Multiple Risk Factor Intervention Trial” (MRFIT) in 2013. NLA (National Lipid Association) expert panel advised Lifestyle and drug therapies intended to reduce morbidity and 1 mortality associated with dyslipidemia. Statins are the mainstay of treatment options in management of 1 hyperlipidemia. They block the de-novo synthesis of cholesterol by inhibiting “3-hydroxy-3-methyl- glutaryl- coenzyme A reductase”, simultaneously upregulating the LDL-C receptors and enhancing the clearance of plasma lipoproteins Thus, statins work . in a “self-limiting” manner and manage cholesterol Introduction Atherosclerosis is a “lipid-driven” inflammation of 1 the arterial wall , caused by the accumulation of 2 excess lipids into the arterial wall. It has been observed over the years that atherogenesis phenomenon is caused by triglyceride rich 3 lipoproteins (i.e low density lipoproteins LDL). Triglycerides in the serum are derived from fats in our f o o d o r f r o m o t h e r e n e r g y s o u r c e s . Hypertriglyceridemia is characterized by elevation in 4 triglyceride levels and is independently associated with cardiovascular disease (CVD). High levels of LDL- cholesterol is an indication of extra lipids in blood, Effect of Betulinic Acid Vs Simvastatin on Hypelipidemic Mice Model Abeerah Zainub, Farhana Ayub, Abdul Khaliq Naveed, Saira Jahan, Saddaf Ayub, Aisha Hasan Correspondence: Dr. Abeerah Zainub Assistant Professor Department of Biochemistry Islamic International Medical College Riphah International University, Islamabad E-mail: abeera@live.com Department of Biochemistry Islamic International Medical College Riphah International University, Islamabad Funding Source: NIL; Conflict of Interest: NIL Received: February 01, 2019; Revised: July 18, 2019 Accepted: July 19, 2019 Betulinic Acid Versus SimvastatinJIIMC 2019 Vol. 14, No.3 121 9 levels in blood. Although generally statins are 10 endured well but poor compliance with them may be caused by many of the side effects, including ga st ro i n te st i n a l d i st u r b a n c e s , b o d y - a c h e s , respiratory problems, and headaches. Of all these adverse effects Liver and Muscle related problems 11,12,13,14 are remarkably higher in numbers. Recently, in blood, higher levels of glucose and glycosylated hemoglobin Hb-A1C have also been reported in 13 association with administration of Statins. As evidence gap is still there, the American College of Cardiology/American Heart Association (ACC/AHA) Blood Cholesterol Guidelines 2013 included dyslipidemia in the recommendations for high 15 priority research areas. Keeping the recommendation in view we chose a noble compound named Betulinic acid (BA), a triterpene with a pentacyclic structure. This is found 16 in the in leaves of Ziziphus spina-christi as well as 17 from stem of white bark birch tree , and in various o t h e r p l a n t s i n t ro p i c a l re g i o n s s u c h a s Tryphyllumpeltaum, Ancistrocladusheyneaus, Diospyorosleucomelas, Tetraceraboliviana, and Syzygiumformosanum. BA and its derivatives have been the subject of intense study which is primarily focused on their anti-cancer effects, anti-HIV, anti- bacterial, anti-inflammatory, antimalarial, anti- 18,19 helminthic, and other pharmaceutical properties. The direct effect of Betulinic Acid on lipid metabolism is recently being evaluated for the better treatment options available to treat dyslipidemia. As it proved to be beneficial in the treatment of 20 nonalcoholic fatty liver disease (NAFLD). In this study we compared the effect of Betulinic acid and Simvastatin on Triglycerides and Low Density Lipoprotein Cholesterol in Balb/C Mice. Materials and Methods This experimental randomized control trial was conducted in department of Biochemistry at Islamic I n t e r n a t i o n a l M e d i c a l C o l l e g e o f R i p h a h International University in collaboration with National Institute of Health Islamabad in 6 months duration from November 2017 to April 2018. After getting approval from the Ethical Review Committee (ERC) of Islamic International Medical College, and Regulatory Authority of National Institute of Health Islamabad a total of 40 subjects were randomly selected from a population of male mice bred in the animal house of NIH according to international standards for experimental purposes. For this our inclusion criteria was Balb/c adult mice (6-7 weeks old). Only healthy male mice weighing 30±5gms were selected under the guidance of a veterinary doctor and animal house supervisor appointed by NIH. These 40 subjects were randomly divided into 4 groups where every member of the population had the same chance of being in any of the four groups and all four groups were exposed to 12 hour light/dark cycle while being provided with a temperature of 22±5°C, kept and maintained at NIH with the help of NIH approved animal handlers. Subjects were equally divided into 4 groups. Group I was named NC for negative control and was given normal rodent chow throughout the experiment (42 days). Group II named PC (positive control) was provided with High Fat Diet (HFD consisted of 25% fats, 25% sucrose and 50% standardized Rodent 20,21 chow purchased from NIH) throughout the experiment (42 days). Group III named BA (betulinic acid) was provided with HFD for 21 days. Afterwards they were treated with betulinic acid while being fed with standardized rodent chow from day 22 to day 42 i.e the last day of experiment. Group IV named SIM (simvastatin) was also provided with HFD for 21 days. And from day 22 onwards they were treated with simvastatin while being fed with standardized rodent chow from day 22 to day 42 i.e last day of experiment. On day 21 after overnight fast serum samples of 2 subjects from HFD groups (i.e PC, BA and SIM) were collected on random selection method to ensure established hyperlipidemia. After the confirmation of established hyperlipidemia, two groups i.e BA and SIM were administered with treatment drugs Betulinic Acid and Simvastatin respectively, at the dosage searched from literature (i.e; Betulinic acid: 20 22 10mg/kg/day and Simvastatin: 10mg/kg/day ). Considering 30gm weight, their doses were 0.3mg per mouse. Drugs obtained were in powder forms and readily soluble in water so they were administered in dissolved form through oral route. During this time HFD was discontinued for the treatment groups BA and SIM. On the final day after an overnight fast samples were taken after anaesthetizing the mice in a closed lid glass jar containing cotton wool soaked in JIIMC 2019 Vol. 14, No.3 122 Betulinic Acid Versus Simvastatin chloroform. Sampling was done through cardiac puncture 1.5±0.5ml of blood was collected from each subject and was stored in previously labeled SST (serum separating tube with clot activating gel) and were placed upright in a stand in a cold storage box. Samples were analyzed within 24 hours of collection. After separating serum by centrifugation at 2500rpm for 10 min, samples were analyzed with reagents purchased from Merck. Protocol of technique used for Triglycerides levels was endpoint direct method and for LDL-C levels was enzymatic direct endpoint method. Semiautomated biochemical analyzer Merck 300 was used for the analysis of Triglycerides and LDL-C. Collected data was analyzed using SPSS (Statistical Package for Social Sciences) software version 21, used for the analysis of data. Normally distributed q u a n t i ta t i ve va r i a b l e s w e re ex p re s s e d a s Means±S.E.M. as it was an analysis between 4 groups so One-way ANOVA (analysis of variance) was applied and the differences among group means was observed while a p-value of <0.05 was set to be significant. Results Levels of serum triglycerides were compared in the form of Mean±S.E.M. serum triglyceride in positive control group was 220 ±7.727 mg/dL as compared to negative control group 130.7 ±4.883 mg/dL with a p<0.001. While treatment groups BA with 146.2 ±16.03 mg/dL and SIM with 178.8 ±6.959 mg/dL showed significant reduction with p<0.001 and p<0.05 respectively. This is evident in Fig 1. PC. Steric (*) denotes comparison of BA and SIM with PC, our results showed *** P<0.001 when compared with PC. Mean±S.E.M of serum LDL-C levels of positive control group was 46.86±1.844 mg/dL with a p<0.001 as compared to negative control group 23.17±2.088 mg/dL. While treatment group BA had 28.00±3.033 mg/dL with p<0.001 and SIM had 34.83±1.537 mg/dL with p<0.01. The comparative analysis is depicted in Fig 2. Fig 1: Graphical Presenta�on of the Results of Serum Triglycerides (Mg/Dl) In All 4 Groups. Hash (#) denotes comparison between NC and PC, our results showed P<0.001 comparison of NC and Fig 2: Graphical Presenta�on of the Results of Serum LDL-C (Mg/Dl) In All 4 Groups. Hash (#) denotes comparison between NC and PC, our results showed P<0.001 comparison of NC and PC. Steric (*) denotes comparison of BA and SIM with PC, our results showed *** P<0.001 when compared with PC. Discussion Dyslipidemia has been responsible for a considerable 23 proportion of Atherosclerotic CVD in the world. For its management a four step approach has been recommended to lower ASCVD which include modification of life style, lowering blood cholesterol levels by the use of drugs (statins). However over the course of decades only approach that has shown overwhelming body of evidence is the drug (statins) 15 therapy approach. Therefore in the guidelines for management of Hyperlipidemia issued by American College of Cardiology / American Heart Association (ACC/AHA), research in this field to the develop better treatment options for management of 15 hyperlipidemia has been recommended. So, we observed effects of betulinic acid on Triglycerides and LDL levels in serum of hyperlipidemic Balb/c mice. JIIMC 2019 Vol. 14, No.3 123 Betulinic Acid Versus Simvastatin Our study demonstrated that BA treated group kept lipid levels in blood near normal which is in accordance with Quan HY et al. who studied that betulinic acid alleviates non alcoholic fatty liver 20 disease (2013) and Ahangarpour et al. who studied the effect of betulinic acid on leptin, adiponectin, hepatic enzyme levels and lipid profiles in streptozotocin–nicotinamide-induced diabetic mice 24 (2018). 25 Triglycerides are absorbed from intestines. High levels of Triglyceridess result in increased LDL formation by liver giving rise to ox-LDL and vascular 26 inflammation. Betulinic Acid was determined to 27 effectively lower down Triglycerides. And in this study, at same dosage of 10mg/kg body wt, BA showed a better control of serum Triglycerides (p<0.001) as compared to SIM (p<0.05) in hyperlipidemic mice as evident in Fig. 1. Additional studies which were conducted by Hai Yan Quan et 20,27 24 al and Ahangarpour et al., backed our findings in their animal based researches. With HFD, LDLc level rise because of excess Triglycerides absorption from intestines and their further accumulation in lipoprotein particle giving 25 rise to LDLc. It was observed that reducing LDLc 26 levels decreased endothelial inflammation. Present study determined that Serum LDL-c levels in BA and SIM were found to be lowered when compared with PC (hyperlipidemic) group and BA showed significantly (p<0.001) better results as compared with SIM (p<0.01). This is depicted in Fig.2 and is in agreement with the findings of studies on mice by 24 28 Ahangpour et al. and Juan Peng et al. Conclusion It is concluded that, Betulinic Acid and simvastatin both have comparable effects in the rectification of hyperlipidemia in Balb/C mice. Simvastatin efficiently treats hypertriglyceridemia and lowers down LDL-C but betulinic acid shows an overall better control. Hence it may be a good alternative to statins but further researches in this field are needed because Betulinic Acid is a novel compound whose safety profile is yet to be evaluated by the researchers. REFERENCES 1. Jacobson TA, Ito MK, Maki KC, Orringer CE, Bays HE, Jones PH, et al. National Lipid Association Recommendations for Patient-Centered Management of Dyslipidemia: Part 1—Full Report. J Clin Lipidol. 2015;9(2). 2. Owens AP, Byrnes JR, Mackman N. Hyperlipidemia, tissue factor, coagulation, and simvastatin. Vol. 24, Trends in Cardiovascular Medicine. 2014. p. 95–8. 3. Nordestgaard BG. Triglyceride-Rich Lipoproteins and Atherosclerotic Cardiovascular Disease: New Insights from E p i d e m i o l o g y, G e n et i c s , a n d B i o l o g y. C i rc Re s . 2016;118(4):547–63. 4. Ruchel JB, Braun JBS, Adefegha SA, Guedes Manzoni A, Abdalla FH, de Oliveira JS, et al. Guarana (Paullinia cupana) ameliorates memory impairment and modulates acetylcholinesterase activity in Poloxamer-407-induced hyperlipidemia in rat brain. Physiol Behav [Internet]. 2017;168:11–9. Available from: http://dx.doi.org / 10.1016/j.physbeh.2016.10.003 5. Mann S, Beedie C, Jimenez A. Differential effects of aerobic exercise, resistance training and combined exercise modalities on cholesterol and the lipid profile: review, synthesis and recommendations. Vol. 44, Sports Medicine. 2014. p. 211–21. 6. Rao W, Su Y, Yang G, Ma Y, Liu R, Zhang S, et al. Cross- sectional associations between body mass index and hyperlipidemia among adults in northeastern China. Int J Environ Res Public Health. 2016;13(5). 7. Bhatnagar P, Wickramasinghe K, Williams J, Rayner M, Townsend N. The epidemiology of cardiovascular disease in the UK 2014. Heart. 2015. 8. Mahmood M, Hoque H, Mahmood SA, Quayum MA. Prevention of Ischaemic Heart Disease. Univ Hear J. 2014;10(1). 9. Zimmer M, Bista P, Benson EL, Lee DY, Liu F, Picarella D, et al. CAT-2003: A novel sterol regulatory element-binding protein inhibitor that reduces steatohepatitis, plasma lipids, and atherosclerosis in apolipoprotein E*3-Leiden mice. Hepatol Commun [Internet]. 2017;1(4):311–25. Available from: http://doi.wiley.com/10.1002/hep4.1042 10. De Vera MA, Bhole V, Burns LC, Lacaille D. Impact of statin adherence on cardiovascular disease and mortality outcomes: a systematic review. Br J Clin Pharmacol [ I n t e r n e t ] . 2 0 1 4 ; 7 8 ( 4 ) : 6 8 4 – 9 8 . Av a i l a b l e f r o m : http://doi.wiley.com/10.1111/bcp.12339 11. El-Salem K, Ababneh B, Rudnicki S, Malkawi A, Alrefai A, Khader Y, et al. Prevalence and risk factors of muscle complications secondary to statins. Muscle and Nerve. 2011;44(6):877–81. 12. S c h a c h t e r M . C h e m i c a l , p h a r m a c o k i n e t i c a n d pharmacodynamic properties of statins: An update. Fundam Clin Pharmacol. 2005;19(1):117–25. 13. Park Y, Rha S-W, Choi BG, Choi SY, Goud A, Lee S, et al. Impact of Simvastatin on Development of New-Onset Diabetes Mellitus in Asian Population: Three-Year Clinical Follow Up Results. J Am Coll Cardiol [Internet]. 2014;63(12):A2128. Av a i l a b l e f r o m : h t t p : / / l i n k i n g h u b . e l s e v i e r. c o m / retrieve/pii/S0735109714621318 14. Aghasadeghi K, Nabavi S, Amirmoezi F, Attar A. Vitamin E supplementation for treatment of statin induced hepatocellular damage: A randomized, double-blind, p l a c e b o - c o n t ro l l e d t r i a l . I n t C a rd i o va s c Re s J . JIIMC 2019 Vol. 14, No.3 124 Betulinic Acid Versus Simvastatin 2018;12(1):29–33. 15. Stone NJ, Robinson JG, Lichtenstein AH, Bairey Merz CN, Blum CB, Eckel RH, et al. 2013 ACC/AHA guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults: A report of the American college of cardiology/American heart association task force on practice guidelines. Vol. 63, Journal of the American College of Cardiology. 2014. p. 2889–934. 16. Nader MA, Baraka HN. Effect of betulinic acid on neutrophil recruitment and inflammatory mediator expression in lipopolysaccharide-induced lung inflammation in rats. Eur J Pharm Sci. 2012; 17. Lee SY, Kim HH, Un S, 3� P. Letter to the editor: RECENT STUDIES ON BETULINIC ACID AND ITS BIOLOGICAL AND PHARMACOLOGICAL ACTIVITY. EXCLI J [Internet]. 2015;14:199–203. Available from: http://dx.doi.org/ 10.17179/excli2015-150 18. Kobayashi RK, Gaziri LC, Vidotto MC. Functional activities of the Tsh protein from avian pathogenic Escherichia coli (APEC) strains. J Vet Sci. 2010;11(4):315–9. 19. Wu J, Niu Y, Bakur A, Li H, Chen Q. Cell-free production of pentacyclic triterpenoid compound betulinic acid from betulin by the engineered Saccharomyces cerevisiae. Molecules. 2017;22(7). 20. Quan HY, Kim DY, Kim SJ, Jo HK, Kim GW, Chung SH. Betulinic acid alleviates non-alcoholic fatty liver by inhibiting SREBP1 activity via the AMPK-mTOR-SREBP signaling pathway. Biochem Pharmacol [Internet]. 2013;85(9):1330–40. Available from: http://dx.doi.org /10.1016/ j.bcp. 2013.02.007 21. Del Pozo R, Mardones L, Villagrán M, Muñoz K, Roa S, Rozas F, et al. Efecto de una dieta alta en grasas en el proceso de formación de cálculos biliares de colesterol. Rev Med Chil. 2017;145(9):1099–105. 22. Song X, Wang J, Wang P, Tian N, Yang M, Kong L. 1H NMR- based metabolomics approach to evaluate the effect of Xue-Fu-Zhu-Yu decoction on hyperlipidemia rats induced by high-fat diet. J Pharm Biomed Anal [Internet]. 2013;78–79:202–10. Available from: http://dx.doi.org/ 10.1016/j.jpba.2013.02.014 23. Venkitachalam L, Wang K, Porath A, Corbalan R, Hirsch AT, Cohen DJ, et al. Global variation in the prevalence of elevated cholesterol in outpatients with established vascular disease or 3 cardiovascular risk factors according to national indices of economic development and health system performance. Circulation. 2012;125(15):1858–69. 24. Ahangarpour A, Shabani R, Farbood Y. The effect of betulinic acid on leptin, adiponectin, hepatic enzyme levels and lipid profiles in streptozotocin-nicotinamide-induced diabetic mice. Res Pharm Sci. 2018;13(2):142–8. 25. Ta s k i n e n M R , B o ré n J . N e w i n s i g h t s i n t o t h e pathophysiology of dyslipidemia in type 2 diabetes. Atherosclerosis. 2015;239(2):483–95. 26. Caliceti C, Rizzo P, Ferrari R, Fortini F, Aquila G, Leoncini E, et al. Novel role of the nutraceutical bioactive compound berberine in lectin-like OxLDL receptor 1-mediated endothelial dysfunction in comparison to lovastatin. Nutr Metab Cardiovasc Dis [Internet]. 2017;27(6):552–63. A v a i l a b l e f r o m : h t t p : / / d x . d o i . o r g / 1 0 . 1 0 1 6 / j.numecd.2017.04.002 27. Quan HY, Kim DY, Kim SJ, Jo HK, Kim GW, Chung SH. Betulinic acid alleviates non-alcoholic fatty liver by inhibiting SREBP1 activity via the AMPK-mTOR-SREBP signaling pathway. Biochem Pharmacol. 2013;85(9):1330–40. 28. Peng J, Lv YC, He PP, Tang YY, Xie W, Liu XY, et al. Betulinic acid downregulates expression of oxidative stress-induced lipoprotein lipase via the PKC/ERK/c-Fos pathway in R AW 2 6 4 . 7 m a c r o p h a g e s . B i o c h i m i e [ I n t e r n e t ] . 2015;119:192–203. Available from: http://dx.doi.org/ 10.1016/j.biochi.2015.10.020 JIIMC 2019 Vol. 14, No.3 125 Betulinic Acid Versus Simvastatin