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Comparative Study of Betulinic Acid Versus Simvastatin on Total Cholesterol and HDL in Hyperlipidemic Model.

Byline: Zainub A., Ayub F., Jehangir A., Inam T., Lodhi S. and Ayub S.

Keywords: Atherosclerotic cardiovascular disease, betulinic acid, simvastatin, high fat diet, high density lipoproteins, total cholesterol.

INTRODUCTION

The global burden of disease in 2013 revealed that about 30% of all deaths in the world result from cardiovascular diseases. During the years of 2012 and 2013 there were about 2.3 million people who suffered from chronic heart diseases in the UK alone.1 In Europe Cardiovascular events are the primary cause of death. Despite recent decrease in the mortality rate in many countries, it still accounts for more than 4 million deaths in a year i.e. about half of all deaths in Europe.2

WHO and the World Bank estimated that in South Asian region during 1990's every 4th death was from CVD. CHD mortality among population of South Asian origin has increased about 1.5 to 4.0 times more than the indigenous populations.3 Clinical studies conducted during last 20 years showed that healthy improvement in blood levels of lipids can lower down the incidence and mortality of CHD significantly. Prevention and treatment of hyperlipidemia has been of great importance in this regard, and is basically achieved by the use of 'lipid lowering therapy' (LLT). Available data from different researches depicts that lowering the levels of lipids in blood can remarkably reduce the incidence of atherosclerosis which in turn decreases the incidence of CVD.4 High levels of 'low-density lipoprotein' cholesterol (LDL-C) and low levels of 'high-density lipoprotein' cholesterol (HDL-C) in the blood are two main factors leading to CVD.

Drug therapies that decrease LDL-C levels (for example, statins) have shown to decrease the incidence of CVD.5 While higher levels of HDL have proven to be preventive of CVD hence named as "good cholesterol". Keeping in view the statistics, in the clinical practice drugs that are used to lower down lipid levels mainly include statins, cholesterol absorption inhibitors, niacin etc. While statins provide the main support for treating hyperlipidemia.4 This group of drugs (Statins)6 act to decrease the de novo synthesis of cholesterol by blocking the rate-limiting enzyme, HMG-CoA reductase of the de novo pathway. They are competitive inhibitors of hydroxymethylglutaryl Co-A reductases.

Statins decrease the levels of cholesterol in the situations of decreased food intake of cholesterol. The cholesterol de novo synthesis is also responsible for provision of isoprenoids which provide lipid anchorage for many proteins making them able to take part in transduction of signals and metabolism. Thus, apart from lowering lipid levels in blood, statins have many other effects.7 There are many pleiotropic effects of statins. They increase nitric oxide (NO) formation in the endothelium and thus inhibit vascular inflammation. They are also known for reducing inflammatory biomarkers7 like CRP in the circulation.6 Irrespective of the mechanical reason, decreasing the inflammatory process is taken as the second choice (after lowering lipid levels) in inhibiting the advancement of atherosclerotic phenomenon. So, 'anti-inflammatory compounds' which do not have lipid lowering activity are currently being developed to treat atherosclerosis.6

Though statins demonstrate agreeable result in treating hyperlipidemia, only 40% - 60% of the patients using statins adhere to it appropriately.8 Although statins usually show good tolerance, but they are accompanied with many side effects such as digestive system disturbances, myalgia, respiratory problems, and headaches. Recently statins are also being linked with raised levels of glucose and glycosylated hemoglobin (A1C) in blood.9 Moreover a rise in liver enzymes specifically transaminases have also been observed rather infrequently, so it is advised not to recommend statins in acute liver diseases, as well as in cases of chronic ailments with unexplained elevation of liver transaminases.

Recently researchers in food sciences are focusing on developing more treatment options for diseases like hyperlipidemia by identifying active ingredients of food extracts that inhibit lipid accumulation in the body.4

Triterpenoid is a group of chemicals containing many compounds, which are recently attracting attention from researchers in the medical field all over the world due to their favorable bioactivity against different disease. Betulinic acid (3-hydroxy-lup-20 (29)-en-28-oic acid)10 structurally speaking is a pentacyclic lupine type compound belonging to a class named triterpenoid which is distributed broadly in the plant kingdom. Betulin is the precursor of betulinic acid shown in figure 1.

The considerable interest in BA is due to a variety of activities including biological and pharmacological effects like the anti-inflammatory,11,12 anti-bacterial, anti-viral, anti-malarial, anti-HIV, and anti-tumor effects.13 It is believed that BA has a prospective potential in anticancer therapeutic regimens because of its cytotoxic property specifically upon tumor cells.14

Other reported biological and pharmacological activities of BA include hepatoprotective effect, anti-AIDS and anti-depression effects.15,16 As hyperlipidemia leads to cardiovascular disease by inducing inflammatory mechanism, Betulinic Acid may have a potential therapeutic role because of its proved anti-inflammatory effect. So, the role of BA in hyperlipidemia is recently under discussion.15

MATERIAL AND METHOD

This was an experimental randomized control trial of 6 months duration (Nov 2017 - Apr 2018) conducted at Biochemistry Department of Islamic International Medical College, Riphah International University in collaboration with National Institute of Health Islamabad. In this study we included 40 healthy adults (6 - 7 weeks old) male Balb/c mice weighing 30 +- 5 gms and randomly divided them into 4 groups and keeping in 12 hour light/dark cycle with a temperature of 22 +- 5AdegC. These standard conditions were provided at National Institute of Health Islamabad with the help of NIH approved animal handlers.

Group I was named as NC for negative control and throughout the trial of 42 days it was provided with normal standardized rodent chow (approved and purchased from National Institute of Health Islamabad). Group II labeled PC (positive control) was given high fat diet (HFD)17,18 all along the experiment. HFD, constituted of 25% fats, 25% sucrose and 50% standardized Rodent chow,19,20 was prepared at NIH.

Group III named as BA (betulinic acid) was given HFD only for 21 days to generate the HFD model and then treatment with betulinic acid was started while their feed consisted of standardized rodent chow from day 22 to day 42 i.e. the last day of experiment. Group IV was given the name as SIM (after simvastatin). This was also given HFD for 21 days like group III (BA). And from day 22 onwards their treatment of 21 days with simvastatin was started while they were fed with standardized rodent chow from day 22 to day 42 i.e. last day of experiment.

On day 21 when HFD was discontinued a serum sample of two randomly selected subjects from HFD fed groups were taken after an overnight fast. Through this we ensured that hyperlipidemia has been established. After that the two drugs at the same dosage were administered to both of the treatment groups i.e. BA and SIM. Calculated dosage for 30 gm weight subject was 0.3mg per mouse considering Simvastatin: 10 mg/ kg/day21 Betulinic acid: 10 mg/kg/day17 which was given in 1 ml of distal water through oral route.

On the last day of the experiment, fasting serum samples were taken after keeping all standardized aseptic measures into account and anaesthetizing the subjects. Samples were collected through cardiac puncture and 1.5 +- 0.5 ml of blood was taken from each subject and carefully stored in SST (serum separating tube) which were labeled and placed upright in a stand in a cold storage box. After separating serum by centrifugation at 2500 rpm for 10 min, samples were tested for total cholesterol and HDL levels using reagents purchased from Merck by semi-automated biochemical analyzer Merck 300.

SPSS (Statistical Package for Social Sciences software) version 21 was used to analyze the data collected from results of total cholesterol and HDL levels of the subjects' samples. Data analysis showed that it was normally distributed and as the quantitative variables were expressed using Means +- S.E.M. Analysis of Variance (one-way ANOVA) was applied to compare 4 groups and p-value of < 0.05 was determined as significant.

Table 1: Results of total Cholesterol levels in Serum.

Group###Mean +- SEM of TC###Value of Significance

NC###127.5 +- 5.457

PC###229.4+-12.01###p < 0.001

SIM###127.2 +- 4.895

BA###150.7 +- 6.975###p < 0.001

Table 2: Results of HDL levels in Serum.

Group###Mean +- SEM of HDL###Value of significance

NC###33.00 +- 3.235

PC###22.17 +- 0.9458###p < 0.01

SIM###31.00 +- 1.483

BA###48.67 +- 1.116###p < a0.001

Significantly lower values of Mean +- SEM of serum HDL 22.17 +- 0.9458 (p < 0.01) was seen in positive control group when matched with negative control 33.00 +- 3.235. Mean +- SEM of BA 48.67 +- 1.116 and SIM 31.00 +- 1.483 showed significant rise (p < 0.001) and (p < 0.05) in serum HDL individually.

DISCUSSION

Dyslipidemia has been accountable for an estimated 4.4 million deaths every year. Although it is responsible for a considerable portion of atherosclerotic cardiovascular disease, it is a manageable problem.22 For the management of atherosclerosis CVD a four step approach including life style modification, use of drugs to lower down blood cholesterol levels (for example statins) and excess body weight23 Despite the promising results, shown by statins in lowering blood cholesterol multiple reports recently have shown that they are associated with many side effects.24,9,25 Therefore as per the guidelines issued by ACC/AHA further research to evaluate better treatment options for management of hyperlipidemia is needed.23

So keeping these recommendations in view in our study we focused on a noble compound, a triterpenoid named betulinic acid and observed its effect on Balb/c mice model of hyperlipidemia.

In our study HFD model we gave a trial of BA in comparison with simvastatin, it was observed that TC levels in serum were found to be lowered in hyper-lipidemic mice treated with BA at a dose of 10 mg/kg/ day as compared with SIM (as shown in the Fig. 2). This is abridged by the claims of Ahangpour et al.31 and Peng et al.32

Conversely a decrease in the levels of highly dense lipoproteins is observed in hyperlipidemic state.30 Current study showed BA raised serum HDL levels in a remarkably significant manner (p < 0.001) as compared with simvastatin (p<0.05) when compared with PC group. This is depicted in Fig. 3 and is in conformity with the discoveries of Ahangpour et a[31 and Peng et al.32

It is concluded that, BA efficiently decreased total cholesterol levels like simvastatin but it has improved the HDL levels more efficiently than simvastatin. Considering the cardio-protective effect of HDL BA will be a better choice of treatment than simvastatin. More research to evaluate this nobel compound's side effect are recommended.

ACKNOWLEDGEMENTS

We pay our sincere thanks to Director, Institute of BioMedical and Genetic Engineering, Islamabad and Dean Riphah Academy of Research and Education, Rawalpindi.

Author's Contribution

All authors have contributed equally in the Multidisciplinary Laboratory of this respective research project conducted at Islamic International Medical College, Rawalpindi.

Funding Source

Research was partially granted by Riphah Academy of Research and Education Rawalpindi and partially managed by the authors.

Conflict of Interest

All authors declare no conflict of interest.

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Publication:Biomedica
Date:Dec 31, 2018
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