Herin Setianingsih*
Anatomy Department, Medical Faculty of Hang Tuah University of Surabaya,
*Corresponding author: [email protected]
Abstract
Sedentary style and behavior of people who tend to eat fatty foods can increase the prevalence of obesity that leads to metabolic syndrome. Excessive food intake in this case high-fat diet can lead to greater caloric intake than its expenditure, resulting in excess fat that will be stored in adipose tissue or will be buried in the visceral organs. Obesity, a condition in which excess fat mass accumulated in the body, is the etiology of non-alcoholic fatty liver disease. Extract of seaweed extract (Kappaphycus alvarezii) is known to have many effects that are useful for treating non-alcoholic fatty liver. The aim of this study was to determine the effect of seaweed extract (Kappaphycus alvarezii) against fatty liver that is seen from the description of fatty degeneration of hepatocyte cell (steatosis) in Wistar rats fed a high-fat diet. This study was a true experimental research with post test control group design.The subjects were 24 male Wistar rats (Rattus norvegicus) divided into 3 groups:
a group fed standard diet for 28 days (K1), a group fed high fat diet for 28 days (K2), dan a group who was given a high-fat diet for 28 days dan on the day-15 until the day-28 was given red seaweed extract (Kappaphycus alvarezii) with a dose of 140mg/200grBB/day (K3). After that, surgery of the liver was done and liver histopathology assessment was measured based on the levels or grading of steatosis. Statistical analysis with Mann-Whitney test in two groups (K2 and K3) with a confidence level of 95% showed p value
= 0.025 (p <α) meaning there was significant difference of the description of fatty degeneration on hepatocyte cell (steatosis) between the groups of rats fed a high-fat diet without Seaweed extract (Kappaphycus alvarezii) compared with rats fed a high-fat diet and seaweed extract (Kappaphycus alvarezii). In conclusion, extract of seaweed extract (Kappaphycus alvarezii) can affect the description of fatty degeneration on hepatocyte cell (steatosis) in Wistar rats (Rattus novergicus) fed a high-fat diet.
Keywords : high-fat diet, steatosis, liver. Kappaphycus alvarezii
Background
Nonalcoholic Fatty Liver Disease (NAFLD) varies from simple fatty liver (steatosis), fatty liver with inflammation (steatohepatitis), fibrosis to cirrhosis (Charlton, 2009). With the increasing prevalence of obesity, type 2 diabetes mellitus, hyperlipidemia, NAFLD is one of the public health problems that should not be ignored (Charlton, 2009). A study in obese populations in developed countries has 60%
simple fatty liver (steatosis) and it is also reported that patients with type 2 diabetes mellitus experience fatty liver by 70%, whereas in patients with dyslipidemia the figure is around 60%
(Trihatmowijoyo Bundjali and ; A Nusi, 2012).
There is an increase of 4-11 times the risk
of individuals with metabolic syndrome for suffering from NAFLD compared to individuals without insulin resistance (Dabhi et al., 2008).
The prevalence of NAFLD in the urban population in Indonesia is estimated to reach 30%
with obesity as the most influential risk factor (Trihatmowijoyo Bundjali and ; A Nusi, 2012).
Most NAFLD patients also suffer from hypercholesterolemia (> 200-220 mg / dL serum) even though the cause is unknown (polygenic hypercholesterolemia). However, being overweight and diet plays an important role. In obesity, there is a decrease in adiponectin levels which will cause a decrease in liver protection against fat resulting in insulin resistance. Insulin resistance increases lipolysis of triglycerides and the release of free fatty acids which will eventually cause fatty liver. Because obesity has
a large influence on fatty liver, a method is needed to reduce weight, among others, by regulating nutrition, exercise, supplementation, and medical intervention (Schreuder et al., 2008).
Our country is a maritime country that has a vast sea area. Empowerment of marine wealth well, will bring enormous benefits, considering that marine biota materials are very easy to obtain.
Seaweed is a good source of food fiber because of its high fiber content, especially its soluble fiber, some studies have shown that seaweed contains a component of agar, carrageenan and alginate which has a strong influence in reducing serum cholesterol (Ren, 1994).
Kappaphycus alvarezii extract contains active components such as flavonoids and triterpenoids which contribute to lower LDL levels. The flavonoids contained in the extract are in the form of quercetin which can inhibit the HMG-CoA reductase enzyme so that it can reduce LDL levels. The other mechanism of action of flavonoids is inhibiting the secretion of Apo- B100 in CaCO2 cells, inhibiting the activity of the enzyme Acyl-CoA Cholesterol Acyl Transferase (ACAT) in HepG2 cells, and decreasing the activity of Microsomal Triglyceride Protein Transfer (MTP) that plays a role in lipoprotein formation by catalyzing displacement lipids to Apo-B molecules so as to reduce total cholesterol and LDL cholesterol levels. While the triterpenoids also have the same mechanism as flavonoids, which inhibits the HMG-CoA reductase enzyme. This compound can also provide inhibition to the pancreatic lipase enzyme which plays a role in digesting triglycerides from food in the small intestine. Inhibition of pancreatic lipase will inhibit fat absorption and reduce blood cholesterol and triglyceride levels.
This decrease will cause LDL synthesis to be inhibited (Astawan et al., 2006).
This study was conducted to determine and correlate the effect of red seaweed extract (Kappaphycus alvarezii) on wistar strain male rats (Rattus norvegicus) given a high-fat diet to reduce fatty liver by looking at liver histopathology.
Methods
This research is experimental with Post Test Control Group Design method conducted at
Widya Mandala Catholic University.The population in this study were Wistar strain male rats (Rattus norvegicus) and were treated at the Animal Laboratory of the Faculty of Pharmacy Widya Mandala Catholic University. Ethical feasibility test in this study was obtained from the Hang Tuah University Research Ethics Commission (No. 29 / HC / EC / KEPUHT / 2017).
Research Sample
The subjects in this study were Wistar strain male rats (Rattus norvegicus) obtained from animal husbandry Drh Rachmad Priyadi and treated at the Animal Laboratory of the Faculty of Pharmacy, Widya Mandala Catholic University.
Rat body weight is 150-200 grams on average and 2-3 months old. Healthy male rats were selected by the researchers, based on the agile movement, bright eyes, smooth fur, and good appetite.
The number of groups used in this study were three groups, with a sample size of 8 experimental animals in each group so that the total number of samples needed were 24 experimental animals.
The sample is divided into 3 groups, namely 1. Negative control group (Group 1, K1) After
being adapted for 7 days, this group of white rats were given a standard diet for 28 days.
2. Positive control group (Group 2, K2) After being adapted for 7 days, this group of white rats were given a high fat diet for 28 days.
3. Treatment group (Group 3, K3) After being adapted for 7 days, this group of white rats were given a high-fat diet for 28 days. On the 15th day, this group was also given red seaweed extract (Kappaphycus alvarezii) for 14 days making a high fat diet
A high-fat diet is made on composition that has been shown to increase total cholesterol in the Octavia (2013) study. The composition of high- fat diet in this study is 2 kg of goat fat, 4 kg of lard, 10 kg of pur 521 (standard feed), 2 kg of catfish pellets, 2 kg of flour, 20 chicken egg yolks, and 20 tablespoons of butter. The procedure for making a high-fat diet is to pulverize goat fat and lard in a blender until smooth, then mix all ingredients and pour as much as 2 L. of distilled water. Then the mixture is allowed to stand for 3- 5 hours. After the feed is sufficiently hardened,
then the feed is ground with a meat grinder and cut to 1cm in size.
Statistic Method
From the experimental results, the data were analyzed using statistical package for social science version 20.0 software, with ANOVA.
Normally distributed and homogeneous data were analyzed further using Post-Hoc Multiple Comparison items Tukey HSD, the value of p<0,05.
Results
This research has been conducted at the Widya Mandala Christian University Surabaya Animal Laboratory for 28 days. The study used 24 wistar male rats (Rattus Norvegicus) which were divided into 3 groups: experimental animals given standard feeds, experimental animal groups fed high fat diets and experimental animal groups fed high fat diets and red seaweed extracts (Kappaphycus alvarezii).
Data from measurement of average grading or steatosis level in each group can be seen in Table 1.1
Table 1.1 Average Grading or Steatosis Level in Each Group
Groups Sum of average grading group
Mean
K1 0 0
K2 24 3
K3 20 2,5
Based on Table 1.1 the average grading of steatosis in all three groups showed different results. The highest average steatosis grading was in the K2 group, followed by the K3 group and the lowest in the K1 group.
Data Analysis.
The results of the research data of the dependent variable of the three groups are ordinal scale, therefore the statistical test used is the Kruskal- Wallis non-parametric test.The Kruskal-Wallis test was used to determine whether there were
significant differences in of hepatocyte cell degeneration (steatosis) in 3 groups of rats.
Test Statisticsa,b
Grading_Steatosis Chi-square 19.933
Df 2
Asymp. Sig. .000 a. Kruskal Wallis Test
b. Grouping Variable: treatment group After significant results were obtained or there were significant differences between the three groups of rats in the Kruskal-Wallis test, the data analysis was continued with Post Hoc analysis using the Mann-Whitney test.
The Mann-Whitney test was used to determine meaningful differences in of hepatocyte cell fat degeneration (steatosis) between 2 groups of rats of all the groups.
Table 1.2 Combined Results of the Significance of the Mann-Whitney Non-Parametric Test in the Rat Group
Groups Sig. Conclusion
K1 K1 .000 P < α, reject H0
K2 K2 .000 P < α, reject H0
K3 K3 .025 P < α, reject H0
From the results of the Mann-Whitney non- parametric test above, conclusions can be taken as follows:
1. There is a significant difference in the picture of hepatocyte cell degeneration (steatosis) in the K1 group (negative control) and the K2 group (high fat diet).
2. There was a significant difference in the picture of hepatocyte cell degeneration (steatosis) in the K1 group (negative control) and K3 group, the high fat diet group + red seaweed extract (Kappaphycus alvarezii) on the 15th day with a dose of 140 mg / 200 grBB during 14 days.
3. There is a significant difference in the picture of hepatocyte fat degeneration (steatosis) in the K2 group (high fat diet) and K3 (high fat diet + red seaweed extract (Kappaphycus alvarezii) on
the 15th day with a dose of 140mg / 200 grBB for 14 day).
Discussion
The results obtained in the Mann-Whitney Test are in accordance with Table 5.5 between the K1 control group) and the group with a high-fat diet (K2) with a value of α = 0.05, obtained p = 0.000, because the P value <0.05, it can be concluded that there are significant differences between the two groups. This clearly proves that administering a high-fat diet can improve the picture of hepatocyte fat degeneration (steatosis).
The discovery of the picture of hepatocyte cell degeneration (steatosis) in the group of rats refers to non-alcoholic fatty liver disease or commonly known as NAFLD abbreviation.
The term NAFLD is used to describe a broad spectrum of fatty liver changes starting from mild ones, namely simple fatty liver (steatosis), fatty liver with inflammation (NASH), fibrosis, to cirrhosis (Dabhi et al., 2008). Non- alcoholic fatty liver disease (NAFLD) is a group of liver disorders similar to fatty liver in alcoholic drinkers, where liver fat content exceeds 5 percent or from liver biopsy results found that at least 5 to 10 percent of hepatocyte cells contain fat, but this happens in individuals who do not drink alcohol or only consume in very small amounts, namely up to 20 grams of ethanol per day (Trihatmowijoyo Bundjali and ; A Nusi, 2012).
There are many factors that influence non- alcoholic fatty liver and are divided into external and internal factors. One external factor that affects non-alcoholic fatty liver (NAFLD) is eating behavior or diet (Hu, 2006).
Giving a high-fat diet can increase fat in the intestine and its absorption which then causes deposition not only in adipocytes, but also in the muscles and liver. Increased fat mass has an important role, because in addition to being a storage organ of the energy substrate, adipocytes are also thought to be endocrine organs. Increased fat mass and adipocyte differentiation lead to the production of various cytokines and a decrease in adiponectin which causes insulin resistance syndrome which ultimately increases the production of free fatty acids in the liver. These hepatic fatty acids can undergo oxidation or esterification. However, a very high increase in
free fatty acids can exceed the oxidation ability so that there is an increase in esterification of fatty acids to triacylglycerol which is then stored in the hepatocyte cytoplasm and becomes steatosis. The oxidation process that also occurs results in ROS that triggers oxidative stress which can cause damage to hepatocyte cells which are characterized by changes in the cell nucleus to picnotics, namely cell nucleus shrinking, irregular boundaries, and dark color and varying hepatocyte cell size, as well as progression from fatty heart (Hu, 2006).
The above statement is also supported by previous studies, such as those carried out by Hartono (2011) in wistar strain white rats and the results showed that administration of a high-fat diet for 4 weeks had shown a picture of hepatocyte cell degeneration (steatosis). In addition to obtaining a picture of steatosis, another impression that is seen is marked hepatocyte cell damage Similar to this study, the administration of a high-fat diet for 20 weeks has shown a significant improvement in the picture of hepatocyte fat degeneration (steatosis) which is accompanied by damage to the cells into various picnics and cell sizes.
According to the research conducted, the content of fucoxanthin from Sargassum sp. has an anti-inflammatory effect. The content acts as a competitive inhibitor of cyclooxygenase and / or lipoxygenase in inflammatory reactions which results in decreased production of prostaglandins and leukotrienes and suppresses nitric oxide induced by lipopolysaccharide (Yang and Ming, 2006).
Other studies supporting the effectiveness of Sargassum sp, among others in a study conducted by (Awang et al., 2015) using rats with type 2 diabetes due to high-calorie diets and low- dose streptozotocin injections which were then observed for histopathological features of several organs such as pancreas, liver, and ren from these rats. The results obtained were groups of rats given extracts from Sargassum sp. with a dose of 150mg / kgBW for 22 days, it gave a picture of histopathological improvement of pancreatic, hepatic, and ren organs compared to the control group .
The other research in their study used rats with hepatitis due to the induction of carbon tetrachloride and then observed histopathological
features of their hepatic organs. The results obtained were significant liver damage from these rats. Provision of red seaweed extract (Kappaphycus alvarezii) is said to be able to restore liver histopathology to normal levels (Madkour, Khalil and Dessouki, 2015).
Aulanni'am et al (2012) study also proved the effectiveness of Sargassum duplicatum, which was an improvement in the picture of damage to jejunal rats induced by indomethacin. The extract was given orally for 7 days at a dose of 100 mg / kg of rat .
Conclusion
1. Provision of red seaweed extract (Kappaphycus alvarezii) at a dose of 140 mg / 200 grBB has a significant effect on the hepatocyte cell fat degeneration (steatosis) in white rats (Rattus novergicus) of the Wistar strain given a high-fat diet.
2. Provision of red seaweed extract (Kappaphycus alvarezii) at a dose of 140 mg / 200 grBB can slow down hepatocyte cell damage in white rats (Rattus novergicus) of the Wistar strain given a high-fat diet.
Acknowledgement
The authors wish to thank Hang Tuah University of Medical Science for its financial support.
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