{"id":42934,"date":"2022-03-31T10:48:53","date_gmt":"2022-03-31T10:48:53","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=42934"},"modified":"2022-04-08T06:46:14","modified_gmt":"2022-04-08T06:46:14","slug":"potency-of-balinese-kecombrang-etlingeraelatior-extract-as-antioxidant-against-the-activity-of-superoxide-dismutase-sod-glutathione-gsh-and-fatty-liver-in-obese-rats","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol15no1\/potency-of-balinese-kecombrang-etlingeraelatior-extract-as-antioxidant-against-the-activity-of-superoxide-dismutase-sod-glutathione-gsh-and-fatty-liver-in-obese-rats\/","title":{"rendered":"Potency of Balinese Kecombrang (Etlingeraelatior) Extract As Antioxidant Against The Activity of Superoxide Dismutase (SOD), Glutathione (GSH) And Fatty liver in Obese rats"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Obesity is condition of being overweight due to the amount of fat in the body that is stored in adipose tissue. The cause of obesity is excessive intake of nutrients compared to body\u2019s needs so that excess intake will be stored as energy reserves in the form of fat which in the long term will make more fat accumulative in the body.1<\/sup>,<\/sup>2<\/sup><\/p>\n

In obesity there is an increase in adipose mass in the body. The increase in adipose mass in obesity causes changes in adipokines which will also cause a decrease in insulin sensitivity. Adipokines are specific structural proteins that are secreted by adipose into the circulation.1<\/sup>,<\/sup>3<\/sup>,<\/sup>4<\/sup><\/p>\n

Decreased insulin sensitivity will increase triglyceride lipolysis in adipose tissue. This leads to excessive production of free fatty acids in obesity which in turn will increase the production of small dance LDL which is easily oxidized. LDL oxidation will produce Reactive\u00a0Oxygen Species (ROS), so that in hyperlipidemic conditions excessive ROS are formed due to the amount of oxidized LDL.1\u20133\u00a0<\/sup>ROS are highly reactive and capable of oxidizing surrounding molecules. ROS include superoxide radicals (O2<\/sub>-), hydrogen peroxide (H2<\/sub>O2<\/sub>), hydroxyl radicals\u00a0(OH–<\/sup>) and various other compounds. ROS\u00a0 and free radicals will always be fought by the body\u2019s defense system known as antioxidants.3<\/sup>,<\/sup>5<\/sup><\/p>\n

Antioxidants are able to slow down, delay and prevent damage to cellular components due to free radicals. Antioxidants consist of endogenous and exogenous antioxidants. Endogenous antioxidants are the main defense system in the body including superoxide\u00a0(GSH), and glutathione peroxide (GPX). SOD enzyme will convert O2<\/sub>–<\/sup> into H2<\/sub>O2<\/sub> will be catalyzed by catalase and gluthione peroxide enzymes and together with other enzymes. Catalase and other antioxidant enzymes will work together to neutralize ROS that are formed in normal\u00a0 amounts so that the balance between free radicals and antioxidants is maintained, but in obesity there is an increase in ROS which causes the balance of oxidation-reduction (redox) reactions to disturbed, resulting in a decrease in enzyme activity both SOD and GSH in the body which is\u00a0called oxidative stress. High oxidative stress can be indicated by low cellular antioxidant status. Decrease endogenous enzyme activity and increased production of ROS that occur in obese patients will be the beginning of the development of various degenerative diseases.6\u20138<\/sup><\/p>\n

The occurrence of oxidative stress due to excess Reactive Oxygen Species (ROS) means that these endogenous antioxidants must receive additional exogenous antioxidants from food and beverage intake that is consumed every day. Antioxidants have a very important role for the\u00a0health of the human body, because they function to inhibit and neutralize oxidation reactions that involve free radicals. Antioxidants in food and drinks can be natural and synthetic antioxidants.5,6<\/sup>\u00a0Amarowiczet al<\/em>. (2000) reported that the use of synthetic antioxidants for a long\u00a0time can cause side effects in the form of inflammation to liver damage and increase the risk of carcinogenesis in experimental animals.7<\/sup>\u00a0Therefore it is necessary to consume natural antioxidants which are found in fruits, vegetables, flowers and other parts of plants that contain\u00a0vitamins A, C, E, folic acid, carotenoids, anthocyanins, phenolic compounds, flavonoids, saponins and tannins to prevent obesity so that \u00a0body fitness is maintained. 8\u201312<\/sup><\/p>\n

Kecombrang (Etlingera elatior<\/em>) is one of the local plants that is a\u00a0 typical Balinese food which contains a lot of antioxidant compounds, namely vitamin C, flavonoids, phenol groups,\u00a0steroids and essential oils. Antioxidant compounds can increase enzyme activity of superoxide dismutase (SOD), glutathione (GSH), catalase (CAT) and reduce oxidative stress (MDA).13,14<\/sup><\/p>\n

Based on the above background, the researcher intends to observe the potency of kecombrang extract on SOD and GSH activity and fatty liver\u00a0 in obese wistar rats.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Materials<\/strong><\/p>\n

The samples used were kecombrang flowers and stems (Etlingera elatior<\/em>) obtained in Sukawati, Gianyar, Bali, Indonesia.<\/p>\n

Lard<\/p>\n

Duck egg yolk<\/p>\n

Female wistar rats<\/p>\n

Superoksida Dismutase (SOD)Assay kit (Elabscience, E-BC-K020)<\/p>\n

Glutathione (GSH) Elisa Kit (Elabscience, E-EL-0026)<\/p>\n

Collection and determination of plants<\/strong><\/p>\n

Kecombrang flowers and stems were collected from the Sukawati area, Gianyar, Bali, in February 2020, determined by the Head ofthe Plant Conservation Center of the Botanical Garden \u2019Eka Karya\u2019 Bali-LIPI. The flowers and stems \u00a0obtained were thencleaned with running water,\u00a0 and cut into small pieces then dried at room temperature in an open room to amoisture content of \u00b1 8%, then dried flowers and stems \u00a0were ground in a blender flask and filtered to 100 mesh fineness.Powder material was extracted by maceration using ethanol solvent 12<\/sup>.<\/p>\n

Kecombrang flowers and stems extraction<\/strong><\/p>\n

Five hundred\u00a0 grams of powder\u00a0 from the flowers and stems of kecombrang\u00a0 were put in a 4.5liter beaker separately then extracted with 3 liters of ethanol solvent\u00a0 for\u00a0 24 hours at room temperature. \u00a0Maseration can be repeated five times. The filtrate collected, combined and evaporated.15\u00a0<\/sup>This Kecombrang flower and stem extracts were used for phytochemical\u00a0 test, and \u00a0the invivo assay.<\/p>\n

Phytochemical Test<\/strong><\/p>\n

The phytochemical tests were carried out qualitatively \u00a0according to Bogoriani et al<\/em>.(2021).12<\/sup><\/p>\n

High Fat Composition<\/strong><\/p>\n

A high fat diet (HFD) composition was done\u00a0 by mixing 60% standard\u00a0 diets\/CP 550 \u00a0and 20% lard and 20% duck egg yolks. Diet is provided in pellet form and given for 60 days.11<\/sup><\/p>\n

Animals of Experiment<\/strong><\/p>\n

White wistar rats as a research protocol were taken from the laboratory of the Center for Study of Animal Diseases (CSAD) of Veterinary Medicine Faculty of Udayana University (No : 26\/UN14.2.9\/PT.01.04\/2020). Twenty-four female wistar rats, 11-12 weeks old and 100-150 g of\u00a0weight were divided into 4 groups, one control group (6 normal rats with standard diet Cp 550), 3 treatment groups (18 rats were obese to the Lee obesity index > 0.3) by a calculation\u00a0 of Bogorianiet al<\/em>., 2020 with a high-fat diet for 8 weeks.10<\/sup>\u00a0After the rats became obese and then\u00a0divided into 3 groups. The treatment group I was rats fed only high_ fat, the treatment group II was rats fed high_ fat and \u00a0added kecombrang flowers extract 100 mg\/kg bw\/day, the treatment III is equal to the treatment group I, and added kecombrang stems extract with dose 100\u00a0mg\/kgbw\/day, each group of 6 rats. Rats\u00a0 from each group were individually caged at room temperature, with a bright cycle: dark 12:12 hours. Rats were treated for 30 days and free to drink ad libitum. After 30 days of treatment, the rats were fasted by withdrawing all food and\u00a0drink for 14 hours. All drugs given to rats through oral once daily. At the end of the study the rats were anesthetized with Chloroform. Blood\u00a0 was taken through the orbital sinus, collected in a blood tube and centrifuged at 5000 g for 15 minutes at 4 \u00b0 C to obtain serum and then frozen\u00a0until analysis. After the blood collection, the rats were dissected and parameters were measured: SOD, GSH and fatty liver by histopathology.<\/p>\n

Design of Experiment<\/strong><\/p>\n

After acclimatization to the laboratory conditions, rats of experiment were randomly divided in to four groups (6 rats each) placed in individual cages and classified as follow:<\/p>\n

Group I ( the group of normal control): rats fed standard diet.<\/p>\n

Group II (the group of obesity induced) : rats fed high-fat diet (HFD)<\/p>\n

Group III (the group of obesity induced + Kecombrang flowers extract 100 mg\/kg bw\/day)<\/p>\n

Group IV (the group of obesity induced + Kecombrang stems extract 100 mg\/kg bw\/day)<\/p>\n

Biochemical analysis<\/strong><\/p>\n

SOD and GSH activity test<\/strong><\/p>\n

Determination of serum SOD activity using the WST-1 method of the Superoxide Dismutase (SOD) Assay kit (Elabscience, E-BC-K020). \u00a0Observe the serum samples, certrifuge for 10 min at 2000 g if it\u2019s muddy. Collect the supernatant and carry out the assay immediately.\u00a0The supernatant is diluted into difference concentration with normal saline, then take the pre-experiment.<\/p>\n

Operation steps<\/strong><\/p>\n

Control well: add 20 \u03bcL of double distilled water and 20 \u03bcL of enzyme working solution. Blankcontrol<\/sub> well: add 20 \u03bcL of double distilled water and 20 \u03bcL of enzyme diluents. Sample well: add 20 \u03bcL of sample and 20 \u03bcL of enzyme working solution. Blanksample<\/sub> well: add 20 \u03bcL of enzyme diluents<\/p>\n

Add \u03bcL of substrate application solution with a multi-channel pipettor into each well and mix fully.<\/p>\n

Incubate at 37o<\/sup>C for 20 min. Measure OD values of each well with Microplate Reader.<\/p>\n

Determination of serum GSH activity using GSH (Glutathione) ELISA Kit. Each well which already contains \u00a050\u03bcL sample\/standard is then added \u00a050\u03bcL Biotinylated Detection and Incubate for 45 minutes at 37o<\/sup>C. Then aspirated and washed 3 times. 3. Each wall Added\u00a0100\u03bcL Horseradish Peroxidase (HRP) conjugated and \u00a0incubated for 30 minutes at 37o<\/sup>C, then aspirated and washed 5 times. \u00a0Add 90\u03bcL substrate reagent and incubated 15 minute at 37o<\/sup>C, add\u00a050\u03bcL of \u00a0stop solution to each well. Read at 450nm immediately and calculation of results.<\/p>\n

Histopathological Observation<\/strong><\/p>\n

A portion of liver tissue of normal rats group, High-Fat Diet group, and the group of rats with kecombrang flower and stem extracts with a dose of 100 mg\/kg\/day each were stored in\u00a0containers in15% formalin solution and subjected to histopathological study Observed microscopically for histopathological changes that is normal liver, HFD liver, and recovered liver was studied and compared.<\/p>\n

Statistical Calculation<\/strong><\/p>\n

All the values were expressed as mean \u00b1 standard deviation . The data of results were analyzed by one-way ANOVA, and the difference among the treatments \u00a0groups was \u00a0determined with LSD. Values p <0.05 was used to consider to be significant.10<\/sup><\/p>\n

Results and Discussion<\/strong><\/p>\n

Kecombrang Flowers and Stems Ethanol Extract with Maceration Method\u00a0<\/strong><\/p>\n

Maceration results from 500 grams of dry powder of flowers and stems using ethanol solvent obtained thick extracts of 85 grams and 80 grams, respectively. The yield of powder\u00a0extract\u00a0 were 17% and 16%, respectively. The calculation of extraction yield according to the formula from Bogorianiet al<\/em>., 2021.12<\/sup><\/p>\n

Phytochemical Test<\/strong><\/p>\n

The results of phytochemical screening on the extract of kecombrang flowers and stems are presented in Table 1<\/p>\n

Table 1: Result of phytochemical test from \u00a0methanol extract of kecombrang flower and stem<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
No<\/strong><\/td>\nPhytochemical compound<\/strong><\/td>\nTest<\/strong><\/td>\nResult<\/strong><\/td>\n<\/tr>\n
Flower<\/strong><\/td>\nStem<\/strong><\/td>\n<\/tr>\n
1<\/td>\nPolyphenols<\/td>\nFeCl3<\/sub> test<\/td>\n+<\/td>\n+<\/td>\n<\/tr>\n
2<\/td>\nFlavonoids<\/td>\nMg and HCl<\/td>\n+<\/td>\n+<\/td>\n<\/tr>\n
3<\/td>\nSaponins<\/td>\nFoam test<\/td>\n+<\/td>\n+<\/td>\n<\/tr>\n
4<\/td>\nAlkaloids<\/td>\nMayer test<\/td>\n+<\/td>\n+<\/td>\n<\/tr>\n
5<\/td>\nSteroids<\/td>\nLiebermann-burchard test<\/td>\n+<\/td>\n+<\/td>\n<\/tr>\n
6<\/td>\nTannins<\/td>\nFeCl3 test<\/td>\n+<\/td>\n+<\/td>\n<\/tr>\n
7<\/td>\nPhytosterols<\/td>\nLiebermann-burchard test<\/td>\n+<\/td>\n+<\/td>\n<\/tr>\n
8<\/td>\nAmino acids<\/td>\nNinhydrin reagent<\/td>\n+<\/td>\n+<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

+ = presence; – = absence<\/p>\n

Table 1 shows that ethanol extract of kecombrang flowers and stems contained all of the tested metabolites such as polyphenols, flavonoids, saponins, alkaloids, steroids, tannins, phytosteroids and amino acids. All these metabolites have been reported to have activity as drugs\u00a0such anti glycemia, antioxidant, anticancer, anti-lipidemia, anti obesity, anti microba and immunomodulatory activities. 12,16<\/sup>\u00a0<\/sup>Ethanol extracts of kecombrang flowers and stems were\u00a0found to be quite effective solvent in extraction. \u00a0Ethanol extracts \u00a0ofkecombrang flowers and stems proved exhibited the positive reaction in all the assays. The results of phytochemical screening are almost the same as those conducted\u00a0 by Bogoriani et al<\/em>. (2021)12<\/sup>.<\/p>\n

Effect of Kecombrang flower and stem extracts administration on Lee Obesity index, \u00a0SOD and GSH concentrations and Fatty liver in Obesity induced in female rats.<\/strong><\/p>\n

After 30 days of treatment, the blood of the rats was taken for analysis of the SOD and GSH concentrations \u00a0and was dissected for histophatological determination. The concentrations of SOD and GSH\u00a0 \u00a0can be seen in Table 2.<\/p>\n

Table 2 shows that the extracts of kecombrang flowers and stems have an effect on increasing serum SOD and GSH concentrations with a significant difference (p <0.05)when compared to the high-fat diet group. The effect of kecombrang flowers extract on GSH\u00a0concentration was significantly different (p <0.05), while the stems extract was not significantly different (p> 0.05) than the normal control group. The effect of 100 mg \/ kg bw of kecombrang flower and stem extracts intake on \u00a0SOD activity of obesity-induced rats were significantly\u00a0different (p < 0.05) compared to the normal control group. An increase in\u00a0 SOD and GSH concentrations and reduce fatty liver \u00a0because kecombrang flower and stem extracts contain antioxidant compounds such as tannins, flavonoids, vitamin C and phenolic compounds.\u00a0Maimulyanti and Prihadi, 2015 reported that flowers and stems contain tannin compounds, flavonoids, saponins and steroids. Micronutrients contained in plants such as vitamins A, C, E, folic acid, carotenoids, anthocyanins, and polyphenols have the ability to scavenge free radicals\u00a0so that they can be used as a substitute for consumption of synthetic antioxidant. 4,16\u201318<\/sup><\/p>\n

Table 2: The effect of kecombrang extracts intake on mean Lee Obesity Index, concentration of SOD and GSH and Fatty liver in obesity-induced female rats<\/strong><\/p>\n\n\n\n\n\n\n\n
Parameter<\/strong><\/td>\nControl<\/strong><\/td>\nHFD<\/strong><\/td>\nFlower<\/strong><\/td>\nStem<\/strong><\/td>\n<\/tr>\n
Lee Obesity Index <\/strong><\/td>\n0.290\u00b10.006<\/td>\n0.355\u00b10.003<\/td>\n0.216\u00b10.008a,b,<\/sup><\/td>\n0.231\u00b10.016a,b<\/sup><\/td>\n<\/tr>\n
GSH (\u03bcg\/mL)<\/strong><\/td>\n37.82 \u00b1 0.03<\/td>\n13.64 \u00b10.17<\/td>\n39.78 \u00b1 0.14a,b<\/sup><\/td>\n37.76 \u00b1 0.17b<\/sup><\/td>\n<\/tr>\n
SOD\u00a0 (U\/mL)\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/strong><\/p>\n

\u00a0<\/strong><\/td>\n

0.49\u00b1 0.01<\/td>\n0.14 \u00b10.01<\/td>\n0.43 \u00b10.02a,b<\/sup><\/td>\n0.41 \u00b1 0.51a,b<\/sup><\/td>\n<\/tr>\n
Fatty liver (cells in five points of view)<\/strong><\/td>\n0.00 \u00b10.00<\/td>\n68 \u00b1 0.00<\/td>\n4.33 \u00b1 0.51a,b<\/sup><\/td>\n20 \u00b1 0.00a,b<\/sup><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Results are expressed in Mean \u00b1 SD. SD = standard deviation;\u00a0 a<\/sup> exhibits a significant difference from normal group ( p < 0.05); b<\/sup> exhibits a significant difference from High -Fat Diet (HFD) (p < 0.05)<\/p>\n\n\n\n
\"Vol15No1_Pot_Way_fig1\"<\/a><\/td>\nFigure 1: (A). <\/strong>Effect of kecombrang \u00a0flower and stem extracts intake on mean concentration of SOD\u00a0 in obese rats showed a significant difference\u00a0 (p < 0.05) from HFD <\/strong>(B). <\/strong>Effect of kecombrang flower and stem extracts intake on mean concentration of GSH in obese rats showed a significant difference (p < 0.05) from HFD.<\/strong><\/p>\n

Click here to view figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Obese rats with a high-fat diet showed a significant reduction in SOD and GSH concentrations and there was an increase Lee obesity index and fatty liver (p <0.05). This is because of obesity is one of the metabolic syndromes which is a risk factor for cardiovascular disease and is the\u00a0main cause of morbidity and mortality in the world. 2,18<\/sup>\u00a0<\/sup>Noronha et al<\/em>., 2001 reported that Sindrum metabolic (SM) \u00a0is affected by high levels of oxidative stress, which gradually develops into a disease vascular.19\u00a0<\/sup>Volpato et al<\/em>. 2004 added that the risk of SM is higher in women than\u00a0in men.20<\/sup><\/p>\n

In fact, under SM conditions, there is an increase in glucose delivery to the adipose tissue. Endothelial cells in adipose tissue stimulate an increase in glucose uptake via glucose transporter, so that it increases the activity of nicotinamide adenine dinucleotide phosphate\u00a0(NADPH) oxidase, and the production of mitochondrial reactive oxygen compounds. An increase in reactive oxygen species (ROS) causes oxidative stress and activates inflammatory signals, so that the activated endothelium attracts proinflammatory macrophages. 3<\/sup>\u00a0Weisberg et\u00a0al. <\/em>(2003) reported that macrophages infiltrating the adipose tissue of obese people are the main source of inflammatory cytokines. Since then macrophages have been known to produce ROS, because macrophages infiltrated into adipose also contribute to increasing NADPH oxidase\u00a0 activity and ROS production in these tissues. 21<\/sup>\u00a0Oxidative stress is defined as an imbalance in ROS \u00a0production and endogenous antioxidant status. This condition is a comorbidity of obesity,\u00a0diabetes mellitus, and impaired kidney function. \u00a0The low activity of SOD proves the high level of oxidative stress in the body, so it is unable to eliminate the amount of oxidants (free radical) 1,3,22<\/sup><\/p>\n

High oxidative stress is also associated with the condition of obese sufferers. In the condition of obesity, the wider adipose tissue can lead to hypoxic conditions (lack of O2). Debevecet al<\/em>. 2017 explained that chronic hypoxia increases oxidative stress by producing excessive ROS without\u00a0compensating for antioxidant enzyme activity. Several studies have shown that during hypoxia, the production of ROS increases so that it suppresses the action of the SOD enzyme. 23<\/sup>\u00a0This occurs because hypoxia is a partial inhibition of the activity of the electron transport chain due to the leakage of electrons\u00a0from complex I, resulting in the formation of ROS. Yuan et al<\/em>. (2008) added that during the hypoxia \/ reoxygenation cycle, ROS is formed enzymatically through the xanthine oxidase pathway. On the other hand, ROS is also formed during ischemia \/ reperfusion, therefore the level of oxidative stress in obese\u00a0patients is higher which results in lower activity of the SOD and GSH enzymes. 24<\/sup><\/p>\n

Liver Histopathology<\/strong><\/p>\n

The results of the study of liver histopathology are shown in Fig. 2 and Table 2. Intracellular fat examination was carried out in five fields of view with Hematoxylin Eosin (HE) staining using 400 x magnification can be seen in each image.\u00a0 It shows that there is a decrease\u00a0fatty liver in the group treated with 100 mg \/ kg bw flower and stem extracts with a significant difference (p< 0.05) compared to the\u00a0 a high-fat diet group. Obese rats show an increase in fatty liver. Characteristics of fatty liver is characterized by the accumulation of fat in liver cells which\u00a0is almost equal to 5% of hepatocytes.25<\/sup>\u00a0<\/sup>An increase in fat in the liver of rats with a high-fat diet triggers liver cell damage.26<\/sup>\u00a0Fatty liver is often associated with a high-fat diet, obesity, and insulin resistance which will lead to impaired lipolysis in the periphery, thus increasing fat\u00a0uptake to the liver. One of the causes of fatty liver is the high consumption of food sources of\u00a0 fat (atherogenic) so that it accumulates free fatty acids in the liver which are then esterified into triglycerides. The pathophysiology of fatty liver is affected by an imbalance between the\u00a0 synthesis and \u03b2-oxidation of triglycerides from fatty acids in the liver due to the accumulation of free fatty acids in the blood circulation of a high-fat diet.25\u00a0<\/sup><\/p>\n\n\n\n
\"Vol15No1_Pot_Way_fig2\"<\/a><\/td>\nFigure 2: Histopathology of female wistar rat liver using \u00a0HE staining (400 x magnification) a. control, b. A high-fat diet, c. Kecombrang stem samples, d. Kecombrang flower samples. Information: a. normal hepatocytes, b. degeneration of fat<\/strong><\/p>\n

Click here to view figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Conclusion<\/strong><\/p>\n

The results of this study concluded that the extract of kecombrang flowers and stems have activity as an antioxidant in vivo so that it has the potential to increase body immunity.<\/p>\n

Acknowledgment<\/strong><\/p>\n

This research was supported by PNBP funds from Udayana University. We also thank students and all research members and PUIPAR \u00a0who have helped a lot.<\/p>\n

Conflict of Interest<\/strong><\/p>\n

There are no conflict of interest.<\/p>\n

Funding Sources<\/strong><\/p>\n

There are no funding source.<\/p>\n

References<\/strong><\/p>\n

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