Title Indonesian Journal of Environmental Management and Sustainability e-ISSN:2598-6279 p-ISSN:2598-6260 Research Paper Effect Of Pomegranate (Punica granatum L.) Fraction In Reducing Total Blood Cholesterol Levels And Triglyceride In Mice (Mus muscullus L.) Nurlaily Agustini1*, Arum Setiawan2, Salni2 1Department Bioscience Program, Faculty of Science, Sriwijaya University, Jalan Padang Selasa 524, Palembang, Sumatera Selatan 30139, Indonesia. 2Department of Biology, Faculty of Mathematics & Natural Sciences, Sriwijaya University. Jalan Raya Palembang-Prabumulih km 32, Indralaya, Indonesia. *Corresponding author e-mail: laily1708@yahoo.com Abstract Pomegranate (Punica granatum L.) will be examined by looking at the ability between pomegranate extracts or fractions, which are more effective in lowering total blood cholesterol and triglyceride levels in mice. This study aims to determine the effect pomegranate fraction of methanol water from several concentrate that can reduce the total cholesterol levels and triglyceride in mice. The research method used was an experimental method with a completely randomized design (CRD) pattern. The research used 6 treatment groups, and test animals divided into 6 treatment groups namely negative control given aquades, positive control fed a high-fat diet, given simvastatin, given 25 mg/grBB pomegranate fraction of methanol water, given 50 mg/grBB pomegranate fraction of methanol water, given 100 mg/grBB of pomegranate fraction of methanol water, each treatment group will be divided into 4 male mice. Based on the results of this study, the active fraction that has an effect on reducing blood cholesterol and triglyceride levels with a percentage reduction about 48% and the significance value of 0,000 <α = 0.05 is the treatment given 100mg/grBB pomegranate fraction of methanol water. Keywords pomegranate, total cholesterol, and triglyceride Received: 28 February 2020, Accepted: 20 March 2020 https://doi.org/10.26554/ijems.2020.4.1.14-22 1. INTRODUCTION Along with the development of times and changes in un- healthy lifestyles ranging from unbalanced eating patterns, consumption of foods high in fat and carbohydrates, to the lack of doing sports activities, many suffer from hypercholes- terolemia. Increased levels of cholesterol and triglycerides in the blood Increased levels of cholesterol and triglycerides in the blood cause the formation of free radicals. High choles- terol and triglyceride levels are also closely related to the increased incidence of cardiovascular disease (Budiyono and Candra, 2013; Roslizawaty et al., 2016). Hypercholesterolemia is an increase in cholesterol levels in the blood that exceeds the limits needed by the body. Someone said to suffer from hypercholesterolemia when total plasma cholesterol levels exceed normal conditions, ie above 200 mg / dL. Cholesterol is one part of fat as one of the nutrients the body needs and the highest calorie producer (Wirawan, 2018). Hypercholesterolemia is a trigger for coro- nary heart disease because high cholesterol causes blockages in peripheral vessels that reduce blood supply to the heart. High cholesterol can also be a trigger for hypertension and stroke (Soleha, 2012). Hypercholesterolemia is a trigger for coronary heart disease because of high cholesterol causes blockages in peripheral vessels that reduce blood supply to the heart. High cholesterol can also be a trigger for hypertension and stroke (Alwan et al., 2009). Dietary management and diet modification is one way to help reduce blood lipid levels. The recommended dietary arrangement is by limiting the consumption of foods that contain high cholesterol and fat, especially saturated fat. In addition to limiting foods that are high in cholesterol and fat, consuming foods that have benefits for lowering cholesterol levels (Sundari and Dieny, 2013). Medicinal plants have been used a lot by humans since an- cient times. The development of the times is also increasing human knowledge about pharmacology and medical science, many people turn to chemical drugs that have been tested for laboratory efficacy. Over time with the development of public knowledge and awareness of the dangers posed by chemicals contained in medicines, people are encouraged to return to using traditional medicines with technological de- velopments. More and more plants have been proven to have https://doi.org/10.26554/ijems.2020.4.1.14-22 Agustini et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 14-22 laboratory efficacy and are guaranteed safe for consumption and cure diseases without causing side effect (Aseptianova, 2019). Indonesia as a country that has developed modern health services but the number of people who use traditional medicine remains high. According to the 2001 national socioeconomic survey 57.7% of the population of Indone- sia treated themselves without medical assistance, 31.2% of them used traditional medicinal plants and 9.8% chose other traditional methods of treatment (Novitasiah et al., 2012).WHO recommends the use of traditional medicines in the maintenance of public health, prevention and treatment of diseases, especially for chronic diseases, degenerative dis- eases and cancer. WHO also supports efforts to improve the safety and efficacy of traditional medicines (Bustanussalam, 2016). Pomegranate is a fruit that comes from the Middle East. The potential for antioxidants in pomegranates is very high, which is around 92% (Nge et al., 2015). The function of antioxidants to inhibit the formation of degenerative dis- eases such as hypercholesterolemia (Mutia et al., 2018). Pomegranate has several active compounds namely alkaloids, flavonoids, saponins, tannins, and triterpenoids (Roswiem, 2014). Based on some previous studies plants containing alkaloids and flavonoids have benefits in reducing choles- terol levels (Zulviana et al., 2017). So far there has not been much research on pomegranate fractions in reducing blood cholesterol and triglyceride levels. But there have been several studies on pomegranates, one of which was conducted by Wahyuni et al. (2013). In that study stated that pomegranate meat can reduce blood cholesterol lev- els. That is due to the presence of phytosterols and some other nutritional content found in pomegranates which are anticholesterol (Wahyuni et al., 2013). Based on the de- scription above, this study aims to determine the effect of pomegranate fraction in reducing blood cholesterol levels and triglyceride in mice. 2. EXPERIMENTAL SECTION 2.1 Materials The tools used in this study are blenders, filters, measuring cups, mortars, maceration containers, rotary evaporators, separating funnels, animal cages, wood chips, animal drink- ing bottles, analytical scales, oral needles, sonde, paper labels, blood vessels, surgical scissors, cotton swabs, masks, rubber gloves, blood cholesterol levels and the Nesco Multi- Check brand striptest. The materials used in this study were fresh pomegranate, 48 male strain mice, distilled water, 96% ethanol, n-hexane, ethyl acetate, used cooking oil, quail eggs, 1% CMC Na, simvastatin, 70% alcohol . 2.2 Experimental Animals The experimental animals used were 48 male Swiss Webster strain mice. The mice used are around 2-3 months old and weigh about 25-40 grams originating from livestock farming in the Bogor agriculture department. Rat cages are first sterilized by drying them in the sun, then given wood dust as the base of the cage. After the sterile cage, male rats were immediately placed and fed with pellets and drinking distilled water ad libitum (to taste). The cage was also given a woven wire as a cover and then the rats were acclimatized for 7 days. The cage is cleaned and the sawdust is replaced every 3 days. 2.3 Methods 2.3.1 Sampling Samples to be used are pomegranates taken from pomegranate farmers weighing 10 kg in Situbondo, Central Java and then processed into extracts and fractions at the Genetic Labora- tory of the Faculty of Mathematics and Natural Sciences, Sriwijaya University. 2.3.2 Making Pomegranate Ethanol Extract (Punica granatum L.) This study used pomegranates, sorted and washed, drained and dried for 2-3 days. After drying, simplicia is then made into powder using a blender, and sieved to get the powder. The extraction process uses maceration method which is pomegranate powder weighed then immersed with 96% ethanol for 2x24 hours then filtered and obtained liquid extract. The liquid extract obtained was concentrated by distillation using arotary evaporator at a temperature of 80 C at a speed of 30 rpm so that it is free of ethanol and produces a thick concentrated extract. Then the ethanol that has been separated and then soaked again with pomegranate powder for 2 days then do it the previous way using a rotary evaporator until it gets the second concentrated extract. The second concentrated extract is then dried with a hairdryer until later the extract will be more concentrated like a paste. 2.3.3 Making a pomegranate fraction (Punica grana- tum L.) Concentrated pomegranate extract was fractionated with 1 liter of n-hexane, shaken for 5 minutes. After that it is allowed to form 2 layers namely the n-hexane layer and the water layer. This treatment is repeated several times until the n-hexane layer looks clear so that the n-hexane fraction is obtained. The water layer is then fractionated with 1 liter of ethyl acetate, repeated several times as in the above treatment so that the water fraction and ethyl acetate fraction are obtained. Then the water fraction is mixed with 100 ml of methanol, shaken for 5 minutes and the water methanol fraction is obtained. All n-hexane, ethyl acetate and methanol water fractions were evaporated with a rotary evaporator until a thick fraction was obtained. After getting the concentrated fraction, dry it using a hairdryer until a concentrated fraction of paste is obtained. © 2020 The Authors. Page 15 of 22 Agustini et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 14-22 2.3.4 Classification and Induction of Experimental Animals The animals used in this study were 48 male Swiss Webster strain mice aged around 2-3 months and weighing around 25-40 grams. The experimental animals will be divided into 6 groups for preliminary tests namely negative control treatment (A0), positive control (A1), pomegranate extract (A2), pomegranate n-hexane fraction (A3), pomegranate acetate fraction (A4), water methanol fraction pomegranate (A5) and 6 groups for further tests namely negative control treatment (P0), positive control (P1), pomegranate water methanol fraction (P3), pomegranate water methanol frac- tion (P4), pomegranate water methanol fraction (P5). Each group consists of 4 mice each. In the positive control group quail yolk was induced by 1% body weight of mice and high-fat diet foods were added. 2.3.5 Dosage Planning The dosages used in the preliminary test were 25 mg/grBB each in pomegranate extract, n-hexane fraction, ethyl ac- etate fraction, and pomegranate water methanol fraction. The treatment between the extract and the most effective fraction will be carried out further tests on the fraction that is most effective in reducing blood cholesterol levels and mice triglycerides. The dose of simvastatin for humans is 20 mg/day so the dose must be converted to mice . The dose of simvastatin for mice is 0.0026 x 20mg / x / tail = 0.052 mg / x / tail. Two 10 mg simvastatin tablets were crushed until smooth and parchment paper was given 1% CMC Na, pitch and measured to obtain 1 g CMC Na. Then give CMC Na 1 g of warm water then crushed with crushed simvastatin and add 100 ml of distilled water (Artha et al., 2017). 2.3.6 Research Test Stage In the preliminary test conducted to determine the effective fraction in reducing blood cholesterol levels in mice. Provi- sion of treatment in this test will be carried out for 7 days, then only found the most effective fraction in reducing total blood cholesterol levels and the most effective fraction is what will be used for further testing stages in reducing blood total cholesterol levels and mice triglycerides. Animal mice will be divided into 6 groups. Each group will be divided into 4 mice and given the treatment as in Table 1. The follow-up test used was the active fraction in re- ducing blood cholesterol levels and mice triglycerides. The active fraction used will be increased to 1, 2, and 4 times the initial test dose (25, 50, 100 mg/grBB). The treatment group in this follow-up test is the same as the treatment group in the preliminary test, which is 6 groups each divided by 4 mice. Further testing was carried out for 14 days. On the 14th day the mice measured blood cholesterol levels, and on the 14th day the triglycerides were measured. Further grouping of tests as in Table 2. 2.3.7 Measurement of Total Blood Cholesterol Lev- els and Triglycerides Measurements were made using the Nesco MultiCheck gauge . This tool will automatically turn on when the strip is inserted and will die when the strip is removed. After the blood enters the strip, the reaction from the strip container will automatically absorb blood into the strip through the capillaries. When the striptest is filled with blood, the device will process the blood for 150 seconds to get the results of measurements of the body’s blood cholesterol levels. Triglycerides measurements will be performed at the Cen- ter Laboratory Kesahatan (BBLK) Palembang with sampel blood of mice. The method used is the CHOD-PAP method. Previously, the blood of mice was collected first in a test tube, then let stand for 20 minutes. After that the blood is centrifuged for 20 minutes at 3000 rpm then the serum is taken as much as 10 µl and then put into a test tube, added as much as 1000 triglyceride reaction solution and mix well using vortex. Let stand for 20 minutes at room temperature and measure the absorption at a wavelength of 500 mm against the blank. Standard absorption measure- ments are carried out in the same way as sample absorption measurements. 2.3.8 Data analysis This study used a Completely Randomized Design (CRD) divided into 6 treatment groups with 4 replications. The data obtained will then be tested for normality with the Kolmogorov-Smirnov Test , homogeneity test with Levene’s , and quantitative tests for total blood cholesterol and triglyc- eride levels obtained by the One Way Anova test , if there are significant differences between treatments followed by the Duncan Multiple Area test (WBD). 3. RESULTS AND DISCUSSION 3.1 Normality Test and Homogeneity Test Based on the results of the normality test using the Kolmogorov- Smirnov test and homogeneity test using the Leven’s test that the results of the study reduced blood cholesterol levels in mice in the preliminary test that is normally distributed and homogeneous data (P> 0.05). in further tests the de- crease in blood cholesterol and triglyceride levels in mice also had normal distribution and homogeneous data (P> 0.05). The results of the normality test and homogeneity test are presented in Table 3 and Table 4. 3.2 Effectiveness of active fractions on Pomegranate (Punica granatum L.) in reducing Total Blood Cholesterol levels in mice (Mus muscullus L.) Based on the preliminary test results, it can be seen that the effect of treatment on extracts and fractions with the avarage in the decrease in total blood cholesterol levels of mice is A0 = 165.65 mg / dl, A1 = 208.30 mg / dl, A2 = 170.91 mg / dl A3 = 177.58 mg / dl, A4 = 189.75 mg / dl, A5 = 169.65 mg / dl. Then he analyzed the significance of © 2020 The Authors. Page 16 of 22 Agustini et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 14-22 Table 1. Dose of Treatment in the Preliminary Test Group Treatment Treatment Days 1-5 Days 6-10 A0 Distilled water Distilled water 0.1ml/10grBB 0.1ml/10grBB A1 MDLT Distilled water 0.1ml/10grBB 0.1ml/10grBB A2 MDLT 25 mg/grBB of 0.1ml/10grBB pomegranate extract A3 MDLT 25 mg/grBB of n-hexane 0.1ml/10grBB pomegranate fraction A4 MDLT 25 mg/kg body weight of 0.1ml/10grBB pomegranate ethyl acetate fraction A5 MDLT 25 mg/kgBB of pomegranate 0.1ml/10grBB water methanol fraction Table 2. Dose of Treatment in Advanced Tests Group Treatment Treatment Days 1-7 Days 8-14 P0 Distilled water Distilled water 0.1ml / 10grBB 0.1ml / 10grBB P1 MDLT Distilled water 0.1ml / 10grBB 0.1ml / 10grBB P2 MDLT Simvastastin drug 0.1ml / 10grBB P3 MDLT 25 mg/kgBB of pomegranate 0.1ml / 10grBB water methanol fraction P4 MDLT 50 mg/kgBB of pomegranate 0.1ml / 10grBB water methanol fraction P5 MDLT 100 mg / kgBB of pomegranate 0.1ml / 10grBB water methanol fraction Table 3. Normality test result of total blood cholesterol levels and triglyceride in the preliminary and advanced test Research variable n p Distribution Total cholesterol Preliminary Test 24 0.18 Normal Advanced Test 24 0.363 Normal Triglycerides Advanced Test 24 0.209 Normal the data with the One Way Anova test which showed that the value of F = 26.316 and the value of p = 0,000. This means that the mean total cholesterol in the blood group after treatment was highly significant (p <0.05). Data on the results of the study of the differences in the average reduction in total blood cholesterol levels of mice and their graphs in the preliminary test are shown in Table 5 and Table 4. Homogenity test result of total blood cholesterol levels and triglyceride in the preliminary and advanced test Research variable F p Information Total cholesterol Preliminary Test 1,508 0.237 Homogeneous Advanced Test 0.521 0.757 Homogeneous Triglycerides Advanced Test 0.284 0.916 Homogeneous Figure 1. Preliminary test results showed that the treatment given 25 mg/grBB of pomegranate fraction of methanol water was more effective in reducing total blood cholesterol levels in mice compared to other treatments. Therefore, the study continued with increasing the dose of active fraction that is the pomegranate fraction of water methanol to 2 5, 50 and © 2020 The Authors. Page 17 of 22 Agustini et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 14-22 Table 5. Preliminary test results of total blood cholesterol levels in mice Treatment group N Average ± SD A0 ( negative control ) 4 165.65 ± 5.70 A1 ( positive control ) 4 208.30 ± 7.70 A2 ( Pomegranate 4 170.91 ± 4.95 Extract 25 mg / grBB ) A3 ( fraction of n-hexane 4 177.58 ± 4.02 25 mg / grBB ) A4 ( 25 mg / grBB 4 189.75 ± 9.41 ethyl acetate fraction ) A5 ( 25 mg / grBB 4 169.65 ± 3.72 water methanol fraction ) Figure 1. Graph of average differences blood total cholesterol levels of mice after being treated in a preliminary test Note: A0; negative control (not treated), A1; positive control (MDLT-induced), A2; MDLT + pomegranate extract 25 mg / grBB, A3; MDLT + n-hexane fraction 25 mg / grBB, A4; MDLT + ethyl acetate fraction 25 mg / grBB, A5; MDLT + methanol water fraction 25 mg / grBB. 100 mg / grBB then compared with the antihypercholesterol drugs namely simvastatin. 3.3 Effects of Pomegranate ( Punica granatum .L) Active Fraction in Lowering Total Blood Choles- terol Levels and Triglycerides in Mice ( Mus muscullus L.) 3.3.1 Total Blood Cholesterol Levels The results of the study after the administration of the treat- ment with various concentrations in the treatment groups P0, P1, P2, P3, P4, and P5 showed a difference in the aver- age amount of total cholesterol in the blood of mice after treatment. The average total cholesterol level of men cit blood is P0 = 167.69 mg / dl, P1 = 215.48 mg / dl, P2 = 166.12 mg / dl, P3 = 182.15 mg / dl, P4 = 175, 82 mg / dl, P5 = 166.71 mg / dl. The avaragetotal cholesterol level in blood of mice can be seen in Table 6 and the graph in Figure 2. After he analyzed the significance with the One Way Anova test showed that the value of F = 26.825 and the value of p = 0.000. This means that the total cholesterol in the blood of the six groups after treatment was significantly Table 6. Result of total blood cholesterol levels of mice after being given treatment Treatment group N Average ± SD P0 (negative control) 4 167.69 ± 5.87 P1 (positive control) 4 215.48 ± 10.98 P2 (simvastatin) 4 166.12 ± 5.14 P3 ( 25 mg / grBB 4 182.15 ± 6.85 water methanol fraction ) P4 ( 50 mg / grBB 4 175.82 ± 5.11 water methanol fraction ) P5 ( 100 mg / grBB 4 166.71 ± 8.14 water methanol fraction ) Figure 2. Graph of differences in mean levels total cholesterol of the blood of mice after treatment Note: P0; negative control, P1; positive control (MDLT-induced), P2; MDLT + simvastatin, P3; MDLT + methanol water fraction 25 mg / grBB, P4; MDLT + mehanol water fraction 50 mg / grBB, P5; MDLT + methanol water fraction 100 mg / grBB different (p<0.05). In the Post Hoc Duncan test, it was shown that the P2 treatment given simvastatin when com- pared with P5, P0, and P4 was not significantly different in reducing total blood cholesterol levels in mice. Whereas P2, P5, PO, and P4 were significantly different from the decrease in total cholesterol levels in mice. In the treatment of P5 given 100 mg/grBB of methanol waterfraction, almost comparable to the treatment of P2 given antihypercholes- terolemia drugs simvastatin in giving the effect of reducing total cholesterol levels in blood of mice. Pos Hoc Duncan’s test can be seen in Table 7. Based on the results above shows that the mice given 100 mg/grBB of pomegranate water methanol fraction has the effect of reducing total blood cholesterol levels which is almost comparable to the blood given simvastatin. This is because 92% of pomegranates have antioxidant content (Nge et al., 2015). The presence of antioxidant compounds such as flavonoids, alkaloids, and saponins. In other treat- ment groups such as at doses of 25 and 50 mg / grBB the pomegranate water methanol fraction also had a decrease but this was not significant. The treatment group gave a dose of 100 mg / grBB of pomegranate water methanol fraction was able to reduce the maximum and is a dose which is appropriate compared to other doses, this reduc- tion is almost comparable to the decrease in the treatment © 2020 The Authors. Page 18 of 22 Agustini et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 14-22 Table 7. Post Hoc Duncan Test effectiveness of pomegranate fraction (Punica granatum L. ) againts decreased total blood cholesterol levels in mice (Mus muscullus L.) Treatment group n Subset for alpha = 0.05 1 2 3 P2(simvastatin) 4 166 12a P5(100mg/grBB water 4 166 71a methanol fraction) P0(positive control) 4 167 69a P4(50mg/grBB water 4 175 82ab 175 82b methanol fraction) P3(25mg/grBB water 4 182 12b methanol fraction) P1 (negative control) 4 215 48c Sig. 0.101 0.237 1,000 group given simvastatin which if consumed continuously has adverse side effects for the body. Mechanism of flavonoid compounds can reduce total cholesterol levels by inhibiting 3- Hydroxy -3- Methyl-Glutaryl-CoenzymeA (HMG-CoA) reductase that causes a decrease in cholesterol synthesis and increases the number of LDL receptors contained in the liver cell membranes and extrahepatic tissue so that levels total cholesterol will decrease, with a decrease in total cholesterol levels, LDL which functions as a means of transporting lipids in the blood will decrease in levels (Nge et al., 2015). Saponins form a complex that is insoluble with cholesterol, thereby preventing the absorption of cholesterol in the small intestine. In addition, saponins reduce the absorption of bile sap by forming micelle complexes that cannot be ab- sorbed because the molecular weight is too large (Matsui et al., 2009). Tannins will bind to the body’s protein and will coat the intestinal wall, so that fat absorption is inhib- ited. Tannins in plants inhibit the absorption of cholesterol in the surface of the small intestine by reacting with mu- cosal protein and intestinal epithelial cells and increase the formation of gallbladder acid from cholesterol and then ex- creted through feces so that it will reduce cholesterol levels (Mu’nisa et al., 2014). Pomegranates also contain fiber and vitamin C (Wahyuni et al., 2013). Fiber can inhibit the ab- sorption of cholesterol in the small intestine and ultimately will reduce the concentration of cholesterol in plasma and increase cholesterol synthesis by the liver, bile synthesis, and excretion of cholesterol through feces. Therefore, food fiber has been widely used and recommended to maintain blood cholesterol concentrations to remain normal while vitamin C is mentioned that plants that have vitamin C have the potential to reduce cholesterol (Hernawati, 2015; Wahyuni et al., 2013). Besides flavonoids, saponins, and tannins, in some literature pomegranates also contain alkaloids, and triterpenoids (Roswiem, 2014). Giving 100 mg/grBB of pomegranate water methanol fraction in mice is almost comparable to hypercholesterol mice given antihypercholesterol drugs in the form of sim- vastatin. Simvastatin is a statin antihypercholesterol drug which causes side effects such as diarrhea, nausea, vomiting, headaches and body aches. While pomegranates are an alter- native in reducing total blood cholesterol levels because the nutritional content of pomegranates is quite adequate as a source of vitamins. So that white pomegranate meat is safer to be used as a blood cholesterol-lowering drug compared to simvastatin drug (Wahyuni et al., 2013). 3.4 Triglycerides The results of the study after the administration of treat- ments with various concentrations in the treatment groups P0, P1, P2, P3, P4, and P5 showed that there were dif- ferences in the average amount of mice triglycerides after treatment. The average triglyceride men cit are P0 = 101.63 mg/dl, P1 = 153.02 mg/dl, P2 = 90 mg/dl, P3 = 82.48 mg/dl, P4 = 77.08 mg/dl, P5 = 74.61 mg/dl. The average of mice triglycerides can be seen in Table 8 and the graph in Figure 3. Table 8. Result of triglycerides in mice after being treated Treatment group N Average ± SD P0 ( negative control ) 4 101.63 ± 5.32 P1 ( positive control ) 4 153.02 ± 6.36 P2 (simvastatin) 4 90.00 ± 8.82 P3 ( 25 mg / grBB 4 82.48 ± 10.29 water methanol fraction ) P4 ( 50 mg / grBB 4 77.08 ± 8.77 water methanol fraction ) P5 ( 100 mg / grBB 4 74.61 ± 8.95 water methanol fraction ) Figure 3. Graph of differences in mean triglyceride of mice after treatment Note: P0; negative control, P1; positive control (MDLT-induced), P2; MDLT + simvastatin, P3; MDLT + methanol water fraction 25 mg / grBB, P4; MDLT + mehanol water fraction 50 mg / grBB, P5; MDLT + methanol water fraction 100 mg / grBB. After he analyzed the significance with the One Way Anova test, it showed that the value of F = 50.522 and p = 0.000. This means that the mean total cholesterol in the © 2020 The Authors. Page 19 of 22 Agustini et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 14-22 Table 9. Post Hoc Duncan Test effectiveness of pomegranate fraction (Punica granatum L. ) againts decreased triglyceride in mice (Mus muscullus L.) Treatment group n Subset for alpha = 0.05 1 2 3 4 P5 (100mg / grBB water methanol fraction) 4 74 6175a P4 (50mg / grBB water methanol fraction) 4 77 0825a P3 (25mg / grBB water methanol fraction) 4 82 4875ab 82 4875b P2 (simvastatin) 4 90 0000bc 90 0000c P0 (positive control) 4 101 63c P1 (negative control) 4 153 02d Sig. 0.218 0.215 0.62 1,000 blood of the six groups after treatment was significantly dif- ferent (p<0.05). In the Post Hoc Duncan test, it was shown that the P2 treatment given simvastatin when compared with P5, P0, and P4 was not significantly different in reduc- ing total blood cholesterol levels in mice. Whereas P2, P5, PO, and P4 were significantly different from the decrease in total cholesterol levels in mice. Post Hoc Duncan test results showed that the treatment of P5 given a pomegranate frac- tion of methanol water 100 mg/grBB when compared with the treatment of P4, and P3 were not significantly different to the decrease in triglyceride in mice. Whereas P5, P4, and P3 were significantly different compared to treatments P2 and P0. In the treatment given pomegranate water methanol fraction from several concentrations is comparable to the decrease in the treatment given simvastatinn can be seen in Table 9. Based on the results of the above study, it is known that 92% of pomegranates contain powerful antioxidants (Nge et al., 2015). The benefits of pomegranates in the fight against free radicals that can cause degenerative diseases have been widely studied by scientists (Muflihunna et al., 2014). An increase in cholesterol levels in the blood can cause formation. Free radicals are compounds that are very reactive and have unpaired electrons in their outer skin. Free radicals can come from pollution, dust and can be produced from metabolic processes that can be bad (Yuliani and Di- enina, 2015). Free radicals will complement their unpaired electrons by attracting nearby macromolecular electrons such as proteins, nucleic acids and DNA. Macromolecules in cells if oxidized and degraded can cause damage to cells (Astuti, 2012). Therefore, the formation of free radicals due to oxidation of triglycerides and cholesterol can damage endothelial cells causing atherosclerosis (Jufri et al., 2015). Chemical content of pomegranates, namely flavonoids, pal- ifenol and catechins as well as vitamin A and vitamin C. Where compounds -This compound is a powerful antioxidant that is useful to prevent the development of free radicals in the body and repair damaged body cells (Muflihunna et al., 2014). From the results of research that has been done shows that mice given 100 mg / grBB are more effective in re- ducing triglycerides. This is caused by the presence of antioxidant compounds such as flavonoids, alkaloids, and saponins. In other treatment groups such as at doses of 25 and 50 mg / grBB the pomegranate water methanol fraction also had a decrease but this was not significant because the treatment group gave a dose of 100 mg / grBB of pomegranate water methanol fraction was able to reduce the maximum and is a dose which is appropriate compared to other doses, this reduction is almost comparable to the decrease in the treatment group given simvastatin which if consumed continuously has adverse side effects for the body. The mechanism of flavonoid compounds can reduce triglyceride levels through increased activity of the LPL enzyme, with the increase in the enzyme VLDL that carries triglycerides will hydrolyse into fatty acids and glycerol. The released fatty acids will be absorbed by muscles and other tissues, then oxidized to produce energy and adipose tissue will store them as energy reserves (Mu’nisa et al., 2014). The flavonoids can inhibit Fatty Acid Synthase (FAS), which is a very important enzyme in the metabolism of fat. Barriers to FAS can directly reduce the formation of fatty acids, thereby reducing the formation of triglycerides (Zhang et al., 2009). Saponin mechanism can reduce triglyceride levels, namely by inhibiting the absorption of cholesterol and triglycerides in the intestine and increasing the reaction of bile acid forma- tion from cholesterol then excreted through feces (Arauna and Aulanni’am, 2016). Saponins bind with bile acids and cholesterol (from food), then form micelles that cannot be absorbed by the intestine and also inhibit the work of the LPL enzyme (Rahmawati et al., 2016). Saponins can in- hibit the absorption of cholesterol and triglycerides in the intestine by forming insoluble complex bonds in cholesterol, binds to bile acids to form micelles and increases binding of cholesterol and triglycerides by fiber (Ekananda, 2015). While tannin compounds can reduce triglyceride levels by reducing the absorption of cholesterol and triglycerides in the small intestine and increasing the excretion of bile acids (Cahaya and Ayu, 2017). © 2020 The Authors. Page 20 of 22 Agustini et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 14-22 4. CONCLUSIONS The conclusion from the results of this study is that mice given pomegranate fractions are effective in reducing total cholesterol and triglyceride levels due to the presence of antioxidant compounds as antihypercholesterol. The active dose that can reduce blood cholesterol and triglyceride levels in mice is in the treatment group given 100 mg / grBB and that was very significantly different to other treatment groups given doses of 25 and 50 mg / grBB of pomegranate water methanol fraction. REFERENCES Alwan, R. L., T. A, Armstrong, and C. M (2009). Noncom- municable disease mortality and life expectancy in immi- grants to Israel from the former Soviet Union: county of origin compared with host country. Bulletin of the World Health Organization, 87(1); 20–29 Arauna, Y. and O. D. Aulanni’am (2016). Studi kadar triglis- erida dan gambaran histopatologi hepar hewan model tikus (Rattus novergicus) huperkolesterolemia yang dit- erapi dengan ekstrak air benalu mangga (Dendrophthoe Petandra)[skripsi]. Malang: Fakultas Kedokteran Hewan Universitas Brawijaya Artha, C., A. Mustika, and S. W. Sulistyawati (2017). Pen- garuh Ekstrak Daun Singawalang Terhadap Kadar LDL Tikus Putih Jantan Hiperkolesterolemia. e-Journal Ke- dokteran Indonesia, 5(2); 105–109 Aseptianova, A. (2019). PEMANFAATAN TANAMAN OBAT KELUARGA UNTUK PENGOBATAN KELU- ARGA DI KELURAHAN KEBUN BUNGA KECA- MATAN SUKARAMI-KOTA PALEMBANG. Batoboh, 3(1); 1 Astuti, S. (2012). Isoflavon kedelai dan potensinya sebagai penangkap radikal bebas. Jurnal Teknologi & Industri Hasil Pertanian, 13(2); 126–136 Budiyono, W. and A. Candra (2013). Perbedaan Kadar Kolesterol Total dan Trigliserida Sebelum dan Setelah Pemberian Sari Daun Cincau Hijau (Premna oblongifolia Merr) pada Tikus Dislipidemia. Ph.D. thesis, Diponegoro University Bustanussalam, B. (2016). Pemanfaataan Obat Tradisional (Herbal) Sebagai Obat Alternatif. Biotrends, 7(1); 20–25 Cahaya, G. and P. R. Ayu (2017). Pengaruh Jus Biji Pepaya (Carica Papaya L.) terhadap Kadar Kolesterol Darah pada Dislipidemia. Jurnal Majority, 7(1); 77–82 Ekananda, N. (2015). Bay leaf in dyslipidemia therapy. Jurnal Majority, 4(4) Hernawati, S. (2015). Ekstrak Buah Delima sebagai Al- ternatif Terapi Recurrent Apthous Stomatitis (RAS). STOMATOGNATIC-Jurnal Kedokteran Gigi, 12(1); 20– 25 Jufri, N. N., B. Wirjatmadi, and M. Adriani (2015). Com- bined food (Bekatul dan Lemak) menurunkan kadar koles- terol total, trigliserida, dan LDL pada tikus galur wistar. Jurnal Kedokteran Brawijaya, 28(3); 208–212 Matsui, Y., K. Kobayashi, H. Masuda, H. Kigoshi, M. Akao, H. Sakurai, and H. Kumagai (2009). Quantitative analy- sis of saponins in a tea-leaf extract and their antihyper- cholesterolemic activity. Bioscience, biotechnology, and biochemistry; 0906031484–0906031484 Muflihunna, A., S. Syarif, and D. Rahmawati (2014). Uji Aktivitas Antioksidan Varian Jus Delima (Punicagrana- tum L.) Dengan Metode Frap. As-Syifaa Jurnal Farmasi, 6(2); 145–153 Mutia, S., F. Fauziah, and Z. Thomy (2018). Pengaruh Pemberian Ekstrak Etanol Daun Andong (Cordyline fru- ticosa (L.) A. Chev) Terhadap Kadar Kolesterol Total dan Trigliserida Darah Tikus Putih (Rattus norvegicus) Hiperkolesterolemia. Journal of BioLeuser, 2(2) Mu’nisa, A., A. F. Arsal, et al. (2014). Pengaruh Pembe- rian Minyak Mandar yang Ditambahkan Bubuk Daun Sukun (Arthocarpus altilis) terhadap Kadar Kolesterol pada Mencit (Mus musculus). bionature, 15(2) Nge, S. T., M. Martosupono, and F. F. Karwur (2015). The Polyphenolics and Health Effects of Pomegranate. Sains Medika, 6(1); 30 Novitasiah, H. R., E. Yuniati, and R. Ramadhanil (2012). Studi Etnobotani Komparatif Tumbuhan Rempah yang Bernilai Sebagai Obat di Desa Tombi Kecamatan Ampibabo Kabupaten Parigi Moutong Sulawesi Tengah. Biocelebes, 6(2) Rahmawati, Y. W., E. U. Ulfa, and E. Rachmawati (2016). Pengaruh Ekstrak Metanol Daun Kayu Kuning (Arcange- lisia flava (L.) Merr) terhadap Histopatologi Aorta Tikus Wistar Hiperlipidemia (The Influence of Methanol Ex- tract of Yellow Root (Arcangelisia flava (L.) Merr) Leaves on Aortic Histopathology in Hyperli. Pustaka Kesehatan, 4(2); 241–248 Roslizawaty, R., R. Rusli, N. Nazaruddin, S. Syafruddin, I. S. Bangun, and J. Jumaidar (2016). Pengaruh Pem- berian Ekstrak Etanol Sarang Semut (Myrmecodia sp.) terhadap Peningkatan Aktivitas Enzim Lipoprotein Li- pase (LPL) dan Perbaikan Histopatologis Hati Tikus (Rat- tus norvegicus) Hiperkolesterolemia. Jurnal Kedokteran Hewan-Indonesian Journal of Veterinary Sciences, 10(1); 77–81 Roswiem, A. P. (2014). Aktivitas Jus Buah Delima (Punica granatum L.) terhadap Peroksidasi Lipid Darah Tikus yang Diinduksi Parasetamol. YARSI Medical Journal, 22(2); 114–124 Soleha, M. (2012). Kadar Kolesterol Tinggi Dan Faktor- Faktor Yang Berpengaruh Terhadap Kadar Kolesterol Darah. Jurnal Biotek Medisiana Indonesia, 1(2); 85–92 Sundari, S. and F. F. Dieny (2013). Pengaruh Pemberian Yoghurt Kedelai Hitam (Black Soyghurt) terhadap Kadar Kolesterol Total dan Trigliserida pada Laki-laki Penderita Dislipidemia Usia 40-55 Tahun. Ph.D. thesis, Diponegoro University © 2020 The Authors. Page 21 of 22 Agustini et. al. Indonesian Journal of Environmental Management and Sustainability, 4 (2020) 14-22 Wahyuni, F. D., I. N. Asyiah, and S. Hariyadi (2013). Pen- garuh Ekstrak n-Heksana Daging Buah Delima Putih (Punica granatum) terhadap Penurunan Kadar Koles- terol Darah pada Tikus Putih (Rattus norvegicus L.) dan Pemanfaatannya sebagai Buku Suplemen. Pancaran Pendidikan, 2(4); 89–99 Wirawan, W. (2018). Uji Efektivitas Fraksi Daun Salam Terhadap Kadar Kolesterol Total Tikus. Jurnal Mandala Pharmacon Indonesia, 4(1) Yuliani, N. N. and D. P. Dienina (2015). Uji Aktivitas Antioksidan Infusa Daun Kelor (Moringa oleifera, Lamk) dengan Metode 1, 1-diphenyl-2-picrylhydrazyl (DPPH). Jurnal info kesehatan, 13(2); 1060–1082 Zhang, S.-Y., X.-F. Ma, C.-G. Zheng, Y. Wang, X.-L. Cao, and W.-X. Tian (2009). Novel and potent inhibitors of fatty acid synthase derived from catechins and their inhibition on MCF-7 cells. Journal of enzyme inhibition and medicinal chemistry, 24(3); 623–631 Zulviana, E., N. Rahman, and S. Supriadi (2017). Pengaruh Pemberian Ekstrak Buah Kelor (Moringa oleifera) Ter- hadap Penurunan Kadar Kolestrol Pada Darah Hewan Mencit (Mus musculus). Jurnal Akademika Kimia, 6(1); 15–20 © 2020 The Authors. Page 22 of 22 INTRODUCTION EXPERIMENTAL SECTION Materials Experimental Animals Methods Sampling Making Pomegranate Ethanol Extract (Punica granatum L.) Making a pomegranate fraction (Punica granatum L.) Classification and Induction of Experimental Animals Dosage Planning Research Test Stage Measurement of Total Blood Cholesterol Levels and Triglycerides Data analysis RESULTS AND DISCUSSION Normality Test and Homogeneity Test Effectiveness of active fractions on Pomegranate (Punica granatum L.) in reducing Total Blood Cholesterol levels in mice (Mus muscullus L.) Effects of Pomegranate ( Punica granatum .L) Active Fraction in Lowering Total Blood Cholesterol Levels and Triglycerides in Mice ( Mus muscullus L.) Total Blood Cholesterol Levels Triglycerides CONCLUSIONS