Investigating novel

methods to reduce
cholesterol level
SMTP Final Research Paper 2016
Ng De Rong Tony (4S3-07) [Leader]
Chay Hui Xiang (4S1-03)
Lai Tian Lang (4S2-08)

Hwa Chong Institution (High School)

Mrs Goh-Yip Cheng Wai

1

Sunflower seeds and kidney beans were also shown to be effective in inhibiting HMG-CoA reductase. similar to pravastatin. a study was conducted to investigate the ability of lactic acid bacteria in the removal of cholesterol. prompting the need for a more natural solution to increasing cholesterol levels. and examine the effectiveness of kidney beans and sunflower seeds in inhibiting HMG-CoA reductase in the cholesterol biosynthesis pathway. a commercial cholesterol reducing drug. and kidney beans having comparable percentage inhibition of the enzyme compared to pravastatin. explore the mechanism for the removal of cholesterol by lactic acid bacteria. with sunflower seeds having 100% inhibition of the enzyme. the number one cause of death worldwide. However.Abstract An increase in blood cholesterol contributes to cardiovascular diseases. Hence. these pharmaceutical agents have been shown to cause several side effects. Results showed that Lactobacillus plantarum was the most effective in reducing cholesterol levels and that the mechanism for cholesterol removal included both the binding to cell wall and active uptake into cells. 2 . Statins are currently the most effective in reducing cholesterol levels and treating patients with high cholesterol.

cardiovascular diseases will remain as the leading causes of death. they are known to have severe side effects. This is often accompanied by evidence of renal dysfunction and occasionally renal failure and death (Eriksson. 2012). Muscle adverse effects are the best recognized adverse effects of statins. and they include muscle pain. there has been considerable interest in the beneficial effects of fermented milk products containing lactobacilli and/or bifidobacteria on human lipid metabolism. which inhibit the enzyme HMGCoA reductase can effectively reduce cholesterol levels and treat patients with high cholesterol. Dans. with an estimated 17. fatigue and weakness. McQueen. An increased level of blood cholesterol is a well-known major risk factor for cardiovascular diseases. the 3 . cardiovascular diseases (CVDs) are the number one cause of death globally. Mann and Spoerry (1974) observed that Maasai warriors in Africa showed a reduction in serum cholesterol levels after consumption of large amounts of milk fermented with a wild Lactobacillus strain. Subsequently. Pais.Introduction According to a report by the World Health Organisation (WHO). Although pharmacological agents such as statins. Angelin & Sjöberg. Avezum. It has been reported that hypercholesterolemia contributes to 45% of heart attacks in Western Europe and 35% of heart attacks in Central and Eastern Europe (Yusuf.5 million people having died from CVDs in 2012. Varigos and Li. 2005). in excess of 10 times the upper limit of normal). accounting for 31% of all global deaths. Budaj. Rhabdomyolysis is one of the most serious adverse effects of statins.g. Hence. Hawken. taking the lives of almost 23. 2004).6 million people worldwide. The WHO has predicted that by 2030. given the research conducted which showed that probiotics such as Lactobacillus acidophilus are capable of reducing levels of cholesterol (Li. Ounpuu. 2008). leading to a marked elevation of creatinine kinase (CK) (e. It occurs when muscle damage is severe. Some tests have also shown increased myositis in patients receiving statins (Golomb & Evans. Lanas.

2. 2. Procedure Apparatus and Materials Incubator Shaking incubator Biological safety cabinet Autoclave UV-vis spectrophotometer Bunsen burner Mortar and pestle Microplate reader Centrifuge Microcentrifuge Syringe 0. 3. casei. 3.potential of probiotics in lowering the incidence of cardiovascular diseases could be explored.85% saline solution Cholesterol Tween 20 (10%) Kidney beans (Phaseolus vulgaris) Sunflower seeds (Helianthus annuus) MRS medium Microtitre plate Total cholesterol assay kit (Cell Biolabs.45 m microfilter 0. plantarum and L. Objectives The objectives of the study are to: 1. Investigate the mechanism of cholesterol removal by lactic acid bacteria. L. acidophilus. Investigate the effectiveness of kidney beans and sunflower seeds in inhibiting HMGCoA reductase in the cholesterol biosynthesis pathway. lactis) are effective in removing cholesterol. Hypotheses The hypotheses of the study are as follows: 1. Kidney beans and sunflower seeds are effective in inhibiting HMG-CoA reductase. Inc) HMG-CoA reductase assay kit (Sigma-Aldrich) Lactobacillus casei Lactobacillus acidophilus Lactobacillus plantarum Lactobacillus lactis Variables 4 . Lactic acid bacteria (L. Cholesterol is removed by lactic acid bacteria via binding to the cell wall. Investigate the ability of lactic acid bacteria in removing cholesterol. L.

The resulting solution was then filter-sterilised. Cholesterol was added to MRS broth at a final concentration of 0. In the control set-up (without cells). The test set-up contained 9 ml MRS broth with cholesterol and 1 ml of bacterial culture. 5 .1 mg/ml. the bacterial culture was replaced with 1 ml MRS broth. Removal of cholesterol by lactic acid bacteria Cholesterol was dissolved in Tween 20 at a concentration of 10 mg/ml.Independent variables Species of lactic acid Dependent variables Absorbance of mixture in Controlled variables Concentration and volume of bacteria each well at 450 nm cholesterol (residual cholesterol assay and mechanism of Type of extract cholesterol removal test) Absorbance of mixture in Absorbance of bacteria each well at 340 nm (HMG- culture CoA reductase assay) Living or non-living bacteria Concentration and volume of extract Temperature of incubation Growth of bacteria Lactic acid bacteria were grown in MRS broth at 30°C with shaking for 2 days.

for the blank for control. 50 ml MRS broth (without cholesterol) was added to 50 µl of cholesterol reaction mix. 6 . Figure 1: Contents of each well (for test) in residual cholesterol assay 50 µL of each sample was added to separate wells of a 96-well micro titer plate. After incubation. Cholesterol reaction mix consisted of 46 µl cholesterol assay buffer. For test. 1 ml portions were removed and centrifuged at 12 000 50 l supernatant of bacteria (grown with cholesterol) 50 l supernatant of bacteria with Tween 20 (without rpm for 10 min to remove the cells. 50 µl supernatant of lactic acid bacteria (grown without cholesterol) was added to 50 µl of cholesterol reaction mix. For the blank prepared for the test setup. 50 50 l of cholesterol reaction mix 50 l of cholesterol reaction mix Blank (Test) Test µL cholesterol reaction mix was added to each well and mixed thoroughly. 2 µl substrate mix and 2 µl enzyme mix.Five replicates of each set-up were prepared and they were incubated with shaking at 30°C for 3 days. For control. 50 µl MRS broth (with cholesterol) was added to 50 µl of cholesterol reaction mix. Finally. 50 µl supernatant of lactic acid bacteria (grown with cholesterol) was added to 50 µl of cholesterol reaction mix. The supernatants were removed and assayed for residual cholesterol using the Total Cholesterol and Cholesteryl Ester Colorimetric Assay Kit II (BioVision).

It is then read with a spectrophotometric microplate reader at 450 nm.85% saline solution in a mortar and pestle. The supernatants were filter-sterilised through a 0. the residual cholesterol test was performed. The amount of residual cholesterol was calculated by comparing the absorbance values with those of the cholesterol standard curve.The plate was incubated for 30 min at 37ºC.45 μm microfilter. The first test setup contains 8 ml of MRS broth. the boiled Lactobacillus culture was replaced with living Lactobacillus culture. The mixtures were centrifuged at 7000 rpm for 10 min and the supernatants were collected. Preparation of extracts 5 g of kidney beans and sunflower seeds were grounded in 15 ml of 0. For the second test setup. After centrifugation to pellet the cells. Mechanism of cholesterol removal Lactic acid bacteria were boiled in a 100oC water bath. Five replicates of each set-up were prepared and they were incubated with shaking at 30°C for 3 days. 7 . In the control setup (without cells). 9 ml of MRS broth was added to 1 ml of 10 mg/ml cholesterol. 1 ml of 10 mg/ml cholesterol and 1 ml boiled lactic acid bacteria culture.

Contents were mixed well. 8 .Inhibition of HMG CoA Reductase The method used is according to that in the HMG. HMG-CoA + 2 NADPH + 2 H+ -> mevalonate + 2 NADP+ + CoA-SH The following mixtures were prepared in a 96-well microtitre plate: Volume/μl 1x assay buffer Inhibitor NADPH HMG CoA HMGR Control 182 - 4 12 2 Blank 184 - 4 12 - Test 82-132 50-100 4 12 2 Blank 84-134 50-100 4 12 - Pravastatin 181 1 4 12 2 Blank 183 1 4 12 - Five replicates were prepared for each set-up.CoA Reductase Assay Kit (SigmaAldrich). it should remain constant or decrease at a lower rate than without inhibitor. If not. The absorbance was taken at 340 nm every 5 min for up to 20 min. The absorbance should decrease due to a decrease in NADPH concentration if no inhibitor is present.

casei. Figure 2: Graph of removal of cholesterol by lactic acid bacteria showed that L. casei. casei. plantarum was the most effective in reducing cholesterol levels. L.Results Removal of cholesterol by lactic acid bacteria In the preliminary screening test. followed by L. lactis. lactis were effective in reducing cholesterol levels 9 . lactis and L. the amount of residual cholesterol was higher for control setups without bacteria as compared to L. L. plantarum and L. This is shown in Figure 2. L. plantarum and L.

the amount of residual cholesterol was higher for control setups without bacteria as compared to that of test setups with L. 10 . acidophilus were effective in reducing cholesterol levels to a different extent. This is shown in Figure 3. plantarum and L. casei and L. The Kruskal-Wallis test showed that the amount of residual cholesterol at the end of the experiment for all three setups were significant. plantarum was the most effective in reducing cholesterol levels. acidophilus. with a p-value of 0. Figure 3: Graph of removal of cholesterol by lactic acid bacteria showed that L. followed by L. Once again. plantarum. acidophilus. L. acidophilus and L.039.In another repeat conducted with both L.

plantarum was more effective than non-living L. plantarum. This is shown in Figure 4. plantarum was not as effective as living L. 11 . plantarum in reducing cholesterol levels. The Kruskal-Wallis test showed that there was a significant difference in the amount of residual cholesterol formed in the three groups. with a p-value of 0. it should be noted that non-living L. However.Mechanism of cholesterol removal For the mechanism of cholesterol removal test. as compared to that in the presence of living L. plantarum in reducing cholesterol levels.004. there was a lower amount of residual cholesterol in control setups without bacteria. Figure 4: The mechanism of cholesterol removal test showed that living L. plantarum and non-living L.

the activity of the enzyme HMG-CoA reductase was determined. From the graph.Inhibition of HMG CoA Reductase For the inhibition of HMG-CoA reductase experiment.003. Figure 5: The inhibition of HMG-CoA reductase test showed that sunflower seed extract was strongly inhibitory towards HMG-CoA reductase. The Kruskal-Wallis test showed that the difference in HMG-CoA reductase activity amongst the three setups was significant. with a p-value of 0. sunflower seed extract showed great effectiveness in inhibiting HMG-CoA reductase. This is shown in Figure 5. 12 .

In a repeat conducted with varying amounts of kidney bean extract. This is shown in Figure 6. HMG-CoA reductase was inhibited to a greater extent in the presence of 0. 13 .10 ml kidney bean extract. as compared to that of 0. Figure 6: The inhibition of HMG-CoA reductase test showed that kidney extract was inhibitory towards HMG-CoA reductase.05 ml kidney bean extract and control without inhibitor.

as shown by the Kruskal-Wallis test. HMG-CoA reductase activity was higher for control without inhibitor as compared to that of kidney bean extract and pravastatin. 14 .In a final repeat conducted with 80 μl of kidney bean extract. with a p-value of 0. Figure 7: The inhibition of HMG-CoA reductase test showed that kidney extract was inhibitory towards HMG CoA reductase.012. The difference in HMG CoA reductase activity was significant. This is shown in Figure 7.

L. (Golomb & Evans. plantarum removes cholesterol by allowing cholesterol to bind onto its cell wall and assimilation by L. This study utilises natural substances such as lactic acid bacteria. Hence the consumption of increased amounts of lactic acid bacteria. acidophilus and L. commonly available and with few known side effects. L. The results for the mechanism of cholesterol removal in lactic acid bacteria also complemented the study conducted by Li (2012). Kidney beans and sunflower seeds have also been shown to be effective in reducing cholesterol levels via the inhibition of the enzyme HMG-CoA reductase. who showed that probiotics such as Lactobacillus acidophilus were capable of reducing levels of cholesterol via coprecipitation and assimilation. plantarum was effective in removing cholesterol. plantarum cells. Li also recognised two other cholesterol removing mechanisms of probiotics. hence is virtually zero cost. This also reduces the usage of statins. lactis. casei in reducing cholesterol levels. plantarum are more effective than L. namely reducing the host absorption of cholesterol and the deconjugation of bile salts. the intake of which can be increased by a change in diet. Furthermore. Yang and Huo (2011). 15 . sunflower seeds and kidney beans to remove cholesterol. it can be seen that L. who showed that L. fatigue and weakness.Conclusion From the study. as well as the adsorption and incorporation of cholesterol to membrane. but were able to allow it to bind to its cell wall. as non-living cells could not assimilate cholesterol. 2008) Our results were comparable to that of Guo. based on results showing that both living and non-living cells exhibited cholesterol reducing properties and that living cells removed more cholesterol than non-living cells. which causes muscle pain. kidney beans and sunflower seeds to overcome the problem of high cholesterol levels in humans shows great potential.

reducing cholesterol levels. We can also encourage the intake of lactic acid bacteria with cholesterol-rich meals to remove cholesterol before absorption. such as through cultured milk. hence the reproducibility of results in practice cannot be determined. Some future work for consideration include investigating the effect of bile salts on cholesterol removal as it is one of the methods described by Pereira and Gibson (2002). due to complications with experimentation on humans.One limitation of the study is the inability to conduct the tests in vivo. such that the body produces less cholesterol. fatigue and rhabdomyolysis. This study has potential applications such as recommending a change in diet for patients facing high cholesterol problems to include more kidney beans and sunflower seeds. 16 . We can also repeat the experiment with other species of lactic acid bacteria to see which species is the most effective in terms of cholesterol removal. It also provides a cheaper alternative for patients with high blood cholesterol as a change in diet is low cost compared to purchasing commercial drugs. hence allowing less cholesterol to be absorbed into the bloodstream. lowering cholesterol levels. This reduces the use of statins which poses adverse effects to human health such as causing pain. to further analyse the mechanism of removal of cholesterol by lactic acid bacteria. allowing patients to choose from the most effective species of bacteria and encouraging producers of cultured milk to incorporate such bacteria into their products. and is present naturally in the alimentary canal.

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and Li.net/publication/8350865_Yusuf_S_et_al_INTERHEART_St udy_Investigators_Effect_of_potentially_modifiable_risk_factors_associated_with_my ocardial_infarction_in_52_countries_the_INTERHEART_study_casecontrol_study_Lancet_364_937- Acknowledgements We are grateful to Hwa Chong Institution for providing the facilities to carry out this work.. (2002). 2016 from: http://www. 37(4). L. McQueen. Lancet.. S. 937–952. Retrieved March 19.int/mediacentre/factsheets/fs317/en/ [8] Yusuf. P. G. Ounpuu. Varigos. 317. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. A. Effects of Consumption of Probiotics and Prebiotics on Serum Lipid Levels in Human. (2004). Fact sheet no. 2016 from: http://www. 364 (9438).I. J.researchgate. A.nlm.R. Cardiovascular Disease.who.gov/pubmed/12236466 [7] World Health Organisation (WHO) (2015).. S. 2016 from: https://www. Retrieved March 20.. Mdm Lim Cheng Fui and Mr Xie Shun Quan of the SRC Biology lab for their assistance while conducting experiments.. Hawken.ncbi. 259-81. Retrieved March 19.. T. Pais. F.. Mrs Goh-Yip Cheng Wai. for her valuable guidance throughout this project. Critical Reviews in Biochemistry and Molecular Biology.. Lanas.S. 18 . M. Dans.[6] Pereira. Avezum. our mentor. and Gibson.. D. S. Budaj.nih.