SGOT and SGPT level of Wistar rat after the administration of Channa micropeltes extract

Amy Nindia Carabelly*1, Deby Kania Tri Putri2, Nadya Rezki3, Maharani Laillyza Apriasari4 1Departement of Oral Pathology, Faculty of Dentistry, Lambung Mangkurat University, Banjarmasin-Indonesia 2Departement of Oral Biology, Faculty of Dentistry, Lambung Mangkurat University, Banjarmasin-Indonesia 3Faculty of Dentistry, Lambung Mangkurat University, Banjarmasin-Indonesia 4Departement of Oral Medicine, Faculty of Dentistry, Lambung Mangkurat University, Banjarmasin-Indonesia


INTRODUCTION
Channa striata (Haruan ish) is one of the local peatland species (Huwoyon and Gustiano, 2013) . Majority of South Kalimantan society believe that Channa striata consumption may accelerate wound healing process due to the albumin content. Albumin is the highest protein to be found in plasma which reaches 60%, and it may accelerate wound healing process by the presence of antioxidant property (Nicodemus et al., 2014) ; (Alamsjah et al., 2014) ; (Agustin et al., 2016) . Channa striata capsule at a 0.7-gram dosage which may accelerate wound healing process has been widely distributed (Tawali et al., 2012) . However, the high price and the complexity of Channa striata cultivation emerge the necessity for alternative species such as Channa micropeltes (Toman ish) (Audina et al., 2018) .
Channa micropeltes may be produced in the form of a capsule and be used as an alternative herbal drug to accelerate wound healing of the oral mucosa. Yet, a further study of its safety is required to analyze the toxicity prior its consumption. One of the toxicity analysis used is a sub-chronic toxicity test (Hulla et al., 2014).This test should be performed for 28-90 days to identify hepatotoxicity effect by observing the in luence of a compound toward the change in Serum Glutamic Oxaloacetic Transaminase (SGOT) and Serum Glutamic Pyruvic Transaminase (SGPT) level of the liver (Singh et al., 2011); (Wahyuni et al., 2017). The liver is the main organ for drug metabolism. Several drugs may induce the destruction of the liver cell due to hepatotoxic property (Indahsari and Histopatologi, 2017). Hepatotoxicity term refers to liver dysfunction due to over-dosage of drugs or xenobiotic (Singh et al., 2011).
Serum Glutamic Oxaloacetic Transaminase (SGOT) is an enzyme found inside the body which immediately detected in peripheral circulation when necrosis occurred in a tissue. SGOT enzyme is commonly found in cardiac and liver, while Serum Glutamic Pyruvic Transaminase (SGPT) is frequently detected in the liver and effectively diagnosed the presence of hepatocellular destruction. This enzyme will be secreted by the liver when there is a destruction of liver cells which is depicted in the increase of SGPT level in blood plasma (Nasution et al., 2015); (Qodriyati et al., 2016). SGOT and SGPT level test should be performed to identify the presence of liver abnormality or destruction due to drug consumption. Average SGOT level is 6-30 IU/L while normal SGPT level is 6-45 IU/L (Nurminha and Gambaran, 2013); (Reza and Rachmawati, 2017). When SGOT and SGPT level is higher than normal, necro- sis of hepatocytes in the liver can be detected. The increase in both enzymes level will indicate that the compound should not proceed for further production as an alternative drug (Sari et al., 2015). Based on the background above, it is pivotal to conduct this study to analyze the effect of Channa micropeltes extract capsule at 0.7-gram dosage per oral upon the level of SGOT and SGPT of Wistar rat liver.  At the beginning of the study, the experimental animal was adapted at the animal laboratory of Veterinary Centre (BVET) Regional V Banjarbaru for seven days by feeding them with BR2 and aqua dest ad libitum. A total of 12 rats were divided into three treatment groups, with four rats presented in each group. The groups comprised of negative control without given any treatment, positive control with the administration of Channa striata extract at 0.7gram dosage and treatment group with the administration of Channa micropeltes extract capsule at 0.7gram dosage. The administration of drugs was performed for 28 days each morning and noon per oral using a nasogastric tube.

Channa micropeltes extraction
Channa micropeltes was obtained from traditional market Martapura, Kalimantan Selatan, and used in this study had a total weight of 11 kg. The part utilized for the study was the lesh of Channa micropeltes. The extract was made at Pharmaceutical Laboratory, Faculty of Mathematics and Science ULM. Fish was cleaned from scale, blood, head and guts and the lesh were later weighed at 9.84 kg. The lesh was steamed inside a pan for 30 minutes under 70-80 o C temperature. Light yellow liquid was secreted from the lesh to be collected and separated in a total of 750 ml. Channa micropeltes lesh was later covered with lannel fabric and Whatman paper no 1 to be inserted into a hydraulic press for pressing. Channa micropeltes extract was then put into a reaction tube as much as 7.5 ml and centrifuge for 15 minutes on 6000 rpm speed. The supernatant liquid was collected from the centrifuged extract. A total of 700 ml of liquid was obtained to be separated from 50 mL sedimentation. Further, Channa micropeltes extract was evaporated in a rotary evaporator for 8 hours until thickened. The extract was evaporated a second time in a water bath until dried in the form of granules.

Channa striata extract capsule
In this study, the capsulate of 0.7 gram Channa striata extract distributed in the market was employed.
Below presents the formulation of 0.7 gram Channa striata extract capsule is shown in Table 1 .

Formulation of Channa micropeltes extract capsule
Dried Channa micropeltes extract was inserted into a mortar and mixed with aerosol, talk, Mg stearate and amylum. All compounds were crushed using stamper until homogenous. Granules were then weighed using an analytical scale and put on a parchment to be inserted inside a gelatinous capsule shell. The capsule was then stored inside dark bottle glass. Formulas of Channa micropeltes extract capsule are presented in Table 2 (Nurani et al., 2017).

Animal treatment
Experimental rats were randomly selected and were administered with standard dosage given orally for 28 days every morning and noon. Calculation of the dosage was obtained from human dose, which is converted by multiplying it with 0.018 (Togubu et al., 2013). The capsule of Channa micropeltes extract was divided into two in which one capsule contained 500 mg granules. Thus, a dosage conversion for rat obtained: 500 mg x 0.018 = 9 mg/g BW.
One capsule of 0.7 gram Channa striata extract in a weight of 750 mg might present a conversion of dosage as 750 mg x 0.018 = 13.5 mg/g BW.
These were the treatment given for each group: Group A (negative control) in which four rats were given BR2 feed for 28 days each morning and noon; Group B (positive control) in which four rats given Channa striata extract capsule at 13.5 mg/g BW dosage dissolved in aqua dest for 28 days each morning and noon using nasogastric tube; Group C (treatment group) in which four rats given Channa micropellets extract capsule at 9 mg/g BW dosage dissolved in aqua dest for 28 days every morning and noon using nasogastric tube.

Collection of blood plasma
On day 29, rats were sacri iced to collect their blood. Each rat was sacri iced by putting it in a container of cotton fumed with 5 ml diethyl ether. The container was covered tightly so that the diethyl ether would not evaporate. After waiting for several minutes until the rat was unconscious, blood was then obtained by an intracardial technique using a syringe. Blood was centrifuged until blood plasma was secreted. Plasma was then separated into a microtube.

Identi ication of SGOT and SGPT level
SGOT and SGPT analysis was conducted at Toxicology Laboratory of Veterinary Centre (BVET) Regional V Banjarbaru with IFCC methods and interpreted using Genesis 20 spectrophotometry with 365 nm wavelength. Blood plasma mixed with reagent kit under 37 o C room temperature. Blood plasma was mixed in a total of 100 µL with a reagent kit in a total of 1000uL. After mixed homogenously, absorbency was observed in minute 1, 2 and 3. Data were presented in the result of absorbance (A). Thus, the result of SGOT and SGPT level activity (IU/L) should be obtained by multiplying the average subtraction from absorbance (A) minute 1, 2 and 3 with 3235 factors. The measurement of activity employed this formula: { ((△A minute 1 and 2)+(△A minute 2 and 3)) 2

} x 3235
The result then would be input to computer software SPSS 23.0 for Windows.21

Statistical analysis
Data were analyzed using Saphiro-Wilk test and then proceeded into variance homogeneity test of Levene's. It was revealed that the data were normally distributed and homogenous (p>0.05) thus Oneway ANOVA parametric test with a 95% con idence level (α=0.05) was performed. Data analysis was then followed by Post-Hoc Bonferroni test.

RESULTS AND DISCUSSION
Average SGOT level is 6-30 IU/L, while normal SGOT level is 6-45 IU/L. Graphic of SGOT dan SGPT level mean value in Wistar rat is presented in Figure 1. Based on Figure 1, it can be concluded that the average value for SGOT and SGPT level in group A, B and C are at a normal level. Data were then examined using Saphiro-Wilk and Levene's Test, which resulted in normal distribution and homogeneity among three groups (p>0.05). Data were further analyzed using one-way ANOVA test and a signi icant value obtained for SGOT level was 0.006 (p<0.05) while SGPT level was 0.308 (p>0.05) thus presenting a signi icant difference between each treatment. Data were then continued with Posthoc Bonferroni analysis which can be observed in Table 3.
Serum Glutamic Oxaloacetic Transaminase (SGOT) and Serum Glutamic Pyruvic Transaminase (SGPT) enzyme are two enzymes which may detect the destruction of liver cell (Nasution et al., 2015). In this research, there is no signi icant difference between the SGOT level of negative control and Channa micropeltes extract treatment group. Hence, as between positive control of Channa striata extract group and Channa micropeltes extract treatment group. This reveals that Channa striata extract may increase the SGOT level but still in the normal range; thus, no toxicity effect resulted in the liver. No signi icant difference was observed on the impact of Channa striata, and Channa micropeltes extract toward SGPT level among all groups, which describes that Channa striata and Channa micropeltes extract do not induce hepatocellular destruction.
Channa micropeltes (Channa micropeltes) contains omega-3 fatty acids, omega-6 fatty acid, zinc, vitamin C and albumin (Nicodemus et al., 2014); (Firlianty, 2016); (Irwanda et al., 2015). Albumin content in Channa micropeltes reaches 5.35% (Fajriani et al., 2018).This albumin content will undergo distribution and metabolism (Throop et al., 2004). At a metabolic stage, albumin is synthesized at the liver cell, speci ically hepatocytes, and it is converted into preproalbumin (Arroyo et al., 2014). Preproalbumin will then be imported into the endoplasmic reticulum, and ission of N-terminal prepropeptide will present as it is assisted by serine protease to be released into interstitial of the liver, sinusoid and liver vein (Arroyo et al., 2014); (Kebamo and Tesema, 2015). The aerobe route of albumin metabolism in the liver cell will form a by product of oxygen molecules which is classi ied as Reactive Oxygen Species (ROS) (Lee and Wu, 2015); (Li et al., 2015). Albumin possesses the antioxidant property to bind free radical produced by ROS and stimulate antioxidant enzyme such as superoxide dismutase (SOD) through the activation of nuclear factor-erythroid-2 related factor 2 (NRf2) (Widayati et al., 2012); (Ma, 2013); (Cahyani and Rustanti, 2015). NRf2 functions as the irst defence against oxidative stress in the cytoplasm. Unless induced by the presence of oxidant and electrophile, NRf2 will be presented in the inactive form (Vriend and Reiter, 2015); (Layal, 2016). It will bind with receptor molecule such as kelch like ECH association protein 1 (Keap-1) and later formulate NRf2-Keap1 complex. In the presence of an oxidant, Nrf2 will be translocated to the nucleus and will form Antioxidants Response Element (ARE) and will be able to stimulate antioxidant enzyme activity such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) which can neutralize ROS component (Widayati et al., 2012); (Vriend and Reiter, 2015).
The increase of superoxide dismutase (SOD) level has a role against free radical inside mitochondria by inducing or changing anion peroxide (O 2− ) into hydrogen peroxide (H 2 O 2 ), a form of free radical (Widayati et al., 2012); (Fukai and Ushio-Fukai, 2011). Hydrogen peroxide (H2O2) will then be transformed into water (H 2 O) and oxygen (O 2 ) by GPx and CAT (Tsutsui et al., 2011);Werdhasari, 2014;(Qu et al., 2016). The decrease of intracellular or extracellular ROS level will affect the biochemical process, including the protection against microorganisms and the function of the liver cell. When ROS decrease, the occurrence of oxidative stress can be prevented. Thus liver cell remained living and freed from radical (Hardiningtyas et al., 2014). Liver cells which are free from radical will halt cell destruction. Thus, SGOT and SGPT enzyme as identifying marker for cytoplasm and mitochondria destruction in the liver cell will be presented in the normal level (Rachmawati and Ulfa, 2018); (Giannini, 2005).
Channa micropeltes contains several hepatoprotective compounds other than albumins, such as zinc, omega-3 fatty acid and vitamin C. Omega-3 fatty acid is proven to heal liver injury, stabilize and also decrease SGOT and SGPT level (Sukarsa and Studi, 2004); (Chavan et al., 2013). Zinc is shown to reduce SGOT and SGPT level, thus de late liver cell destruction effect (Unsal et al., 2008). Vitamin C possesses an antioxidant property which depicts a hepatoprotective effect by binding free radical, which decrease oxidative stress in the liver cell (Sabiu et al., 2015).

CONCLUSIONS
It can be concluded from this study that there is no effect of Channa micropeltes extract capsule at 0.7-gram dosage per oral upon SGOT and SGPT level changes in Wistar rat liver. This result should be deployed as the foundation of Channa micropeltes extract capsule development as an alternative herbal drug to accelerate wound healing of the oral mucosa with no destructive effect upon the liver.