THE ANTI-OXIDANT ACTIVITIES OF ETHANOL EXTRACT OF MAHOGANY ( Sweitenia mahagoni Jacq . ) SEEDS IN THE LIVER TISSUES OF DIABETIC EXPERIMENTAL RATS

The aim of this research was to analyze the anti-oxidant activities of ethanol extract of mahogany seeds on the liver tissues of diabetic experimental rats to overcome oxidative stress condition. This study used male Rattus norvegicus strain Sprague Dawley which were divided into 5 groups: (i) negative control group (K-), (ii) positive control group/diabetes mellitus (DM) (K+), (iii) DM group treated with 500 mg/kg BW ethanol extract of mahogany seeds (EM), (iv) DM group treated with acarbose (KO), and (v) non-DM group treated with 500 mg/kg BW ethanol extract of mahogany seed (KE). Alloxan with dose of 110 mg/kg BW was used to induce diabetes in rats. The treatments were conducted for 28 days. At the end of treatment, the liver tissues were collected and analyzed for malondialdehyde (MDA) content using thiobarbituric acid reactive substance (TBARS) method and anti-oxidant cooper zinc superoxide dismutase (Cu,Zn-SOD) content using immunohistochemical technique. The result showed that level of MDA were significantly different (P<0.05) in K-; K+; EM; KO; and KE with values were 0.81±0.17; 1.19±0.26; 1.10±0.04; 0.95±0.13; and 0.92±0.0 μmol/gram, respectively. The anti-oxidant content (Cu, Zn-SOD) of rat liver tissue in K+ lower than in K-, while anti-oxidant content in EM and KE were higher compared to K+. The study concluded that ethanol mahogany seeds extract decreased blood glucose levels and increased the anti-oxidant status in the liver tissues of diabetic rats. ____________________________________________________________________________________________________________________


INTRODUCTION
International Diabetes Federation (IDF, 2013) reported that the number of people with diabetes mellitus (DM) in 2013 reached 382 million people globally and was estimated to increase to 592 million people by 2035. Nowadays, the number of people with DM in Indonesia reached 8.5 million people, demanding a serious consideration and handling in order to decrease the number which rising in each year. Diabetes Mellitus is a metabolic disorder marked with high glucose levels in blood. The decreasing metabolism of glucose, fat, and protein in diabetics occurred because of insulin deficiency and resistance (Gardner and Shoback, 2007). In diabetic sufferers, the production of reactive oxygen and free radicals increased, leading to an oxidative stress condition (Giacco and Brownlee, 2010), a condition which the amount of anti-oxidant is lower than the number of free radicals.
As an alternative handling of oxidative stress condition, diabetics may consume high anti-oxidant plants in order to prevent complication caused by diabetes and help lower glucose level in blood.
Several studies showed that the ethanol extract of mahogany stem (Mahonia oiwakensis Hayata) that commonly grow in Taiwan have an anti-oxidant, analgesic, anti-inflammation, and hepatoprotector properties (Chao et al., 2013). Debasis et al. (2011) andSuryani (2013) reported that methanol extract of Mahogany seed (Sweitenia mahagoni Jacq.) are hypoglycemic, able to increase insulin levels and decrease TNF-α expression on diabetic rats that was induced with streptozotocin (STZ). Another study reported that ethanol extract of mahogany seed is able to inhibit the activity of α-glucosidase enzyme in alloxan-induced diabetic rats (Hasan et al., 2011) and in sucrose-induced hyperglycemia rats (Wresdiyati et al., 2015). Mahogany seed extract has phytochemical compounds such as flavonoids, saponins, and triterpenoids (Wresdiyati et al., 2015). It is known that flavonoid acts as a natural anti-oxidant compounds, but the effect of ethanol extract of mahogany seed in the liver of diabetic rats has never been reported. Therefore, this study aimed to analyze anti-oxidant activity of ethanol extract of mahogany seed in liver tissue of diabetic rats model in order to prevent oxidative stress condition.

MATERIALS AND METHODS
This study used 25 rats (Rattus norvegicus) strain Sprague Dawley obtained from Laboratory Animal Management Unit of Faculty of Veterinary Medicine, Bogor Agricultural University. The rats were divided into 5 groups: negative control group (K-), positive control group/diabetes mellitus (DM) (K+), DM group treated with 500 mg/kg BW of the ethanol extract of mahogany seeds (EM), DM group treated with 10 mg/kg of acarbose (KO), and non-DM group treated with 500 mg/kg BW of the ethanol extract of mahogany seed (KE). The alloxan (110 mg/kg BW) was injected intraperitoneally to induce diabetes in all rats except rat in group K-and KE. The experiment was conducted for 28 days. Feed ration was weighed every day, while blood glucose levels and rat body weight were measured once in four days. On day 29 rats were anesthetized using a combination of ketamine (75 mg/kg BW) and silacin (8 mg/kg BW). Rat liver was taken in order to analyze the level of malondialdehyde (MDA) and concentration of anti-oxidant Cu, Zn-SOD through immunohistochemistry technique.

Liver Tissue Processing
Prior to the process of analyzing the content of antioxidant Cu, Zn-SOD with immunohistochemistry techniques, the liver tissue has been sampled fixed in Bouin fixation solution for 24 hours. After that the next process was dehydrating the tissue using alcohol with stratified concentration (70%, 80%, 90%, 95% until absolute alcohol I, II, III) and continued with tissue clearing stage with xylole solution (xylole I, II, III). Next, the tissue was infiltrated with paraffin (paraffin I, II, III) carried out in the oven and followed by embedding the tissue in paraffin mold. Then, the tissue block was cut (sectioning) thickness 4 µm using microtome. The pieces of tissue in the form of ribbons were attached to a glass object that had been given glue (0.2% neofren ® in toluene). Furthermore, the immunohistochemistry staining stage was performed.

The Analysis of Cu, Zn-SOD Concentration with Immunohistochemistry Technique
Anti-oxidant Cu, Zn-SOD profile was detected by using Immunohistochemistry technique with the method previously used by Wresdiyati et al. (2014). Liver tissue that had been deparafinized was soaked in H 2 O 2 in order to block endogen peroxidase for 15 minutes before washed with PBS. Then, the tissue was incubated in a 10% normal serum to block non-specific protein for 30-60 minutes, before washed with PBS. After that, the tissues was re-incubated in background sniper for 15 minutes, incubated in antibody Cu, Zn-SOD for 48 hours and washed with PBS. After that, the tissue incubated in Trekkie Universal Link for 20 minutes, washed with PBS, re-incubated with Trekk Avidin-HRP for 10 minutes, and washed with PBS. The reaction result between antigen and antibody then visualized using diamino benzidine (DAB) and counterstain with hematoxylin. The tissue then dehydrated with alcohol and cleansed with xylole. After the process, the tissue was mounted with entellan ® . The existence of Cu, Zn-SOD marked with the appearance of brown color on the nucleus and cell cytoplasm. Qualitative observation were based on the intensity of a brown color in nucleus and cell cytoplasm, in which the more equal color distribution refer to the more content of Cu, Zn-SOD, while cell that did not contain Cu, Zn-SOD will not reacted marked with the appearance of blue color (hematoxylin) from counterstain. Quantitative observation were performed by counting the amount of hepatocytes that both positively and negatively reacted with Cu, Zn-SOD in the observed tissue. The number of hepatocytes was measured using a McMaster Biophotonics Image J. software program.

Body Weight and Total Ration of Rat Experiment Consumption
The change of rat body weight after 28 days trial can be seen in Figure 1. With the exception of group K+ (had a tendency with stable body weight), the rats body weight from all groups were increased. The lower body weight of K+ rats as compared to other groups possibly caused by low production of insulin, which caused the glucose unable to enter into cell. This condition made glucose unable transformed to be energy, so it could not be restored in glycogen form which in turn caused weight loss in diabetic rats. Total consumption of experimental rat can be seen at Table 1. Table 1 showed that total ration consumed by K+ groups significantly higher (P<0.05) than K-and KE groups, but the amount of ration consumed in K+ did not followed by weight gain (Figure 1). Probably a low insulin production in K+ group caused by DM was responsible for inability of the glucose in blood to enter to cell. This condition leads to the decrease in energy of the body that influence hunger and increase rat total ration consumption.
In the group of diabetic rats that given ethanol extract of mahogany seed (EM) and acarbose (KO) had no significant difference in total ration consumption (P>0.05) with K-group. This result also showed that rat in EM and KO groups (Figure 1) had similar body weight with rat in of K-group.
Similarly, the difference of ration intake of KE and K-groups was not significant (P>0.05), but the KE group had less weight gain, as seen in the K-group ( Figure 1). This is related to the presence of flavonoid compounds in mahogany seed extracts that have the potential to inhibit the activity of α-glucosidase (Wresdiyati et al.,201), in the process of carbohydrates breakdown and glucose absorption in the intestine (Wu et al., 2012). This will inhibit the breakdown of carbohydrate compounds into glucose and delay the absorption of glucose by the small intestine (Adisakwattana and Chanathong, 2011), thus will affect the weight loss. Therefore, the use of ethanol mahogany seed extract in non-DM conditions has the  potential to be utilized as a natural medicine for weight loss.

The Hypoglycemic Effect of Ethanol Mahogany Seeds Extract
The changes in blood glucose levels of experimental rat for 28 days are presented in Figure 2. It showed that the blood glucose levels in the group of DM rats given ethanol extract of mahogany seeds (EM) and acarbose (KO) was decreased, while in the K+ group was increased. The similar finding has been reported by Hasan et al. (2011) that the ethanol extract of mahogany seed (Swietenia mahagoni) has hypoglycemic effect, so it can lower blood glucose levels in DM rat. The decrease of blood glucose level is possibly caused by the active compounds contained in the ethanol mahogany seed extract such as flavonoids and saponin that have hypoglycemic activity (Wresdiyati et al., 2015). Flavonoid compounds also have potential as anti-oxidant by scavenging free radical compounds (Lukacinova et al., 2008) and repairing damage to pancreatic tissue caused by free radical activity (Wresdiyati et al., 2008;Suryani et al., 2013). Because of this healing process, the β cells in Islets of Langerhans of pancreatic tissue will increase its insulin secretion. This causes glucose to be absorbed into cells and indirectly decreases the blood glucose levels in the body's circulation.
In the KO group also showed a decrease in blood glucose levels. Acarbose has been widely used as a drug for the treatment of diabetes because it acts as an αglucosidase inhibitor (Sivakumar et al., 2012). Inhibition of this enzyme causes inhibition of carbohydrate breakdown into glucose and delayed the absorption of glucose by the small intestine (Adisakwattana and Chanathong 2011;Wu et al., 2012), so blood glucose levels of rats with DM will be restored in normal value.

MDA and Anti-oxidant Cu, Zn-SOD Levels in Liver tissue
The results of MDA level and anti-oxidant content of Cu, Zn-SOD analysis on rat liver tissue with DM were presented in Table 2. This table shows that MDA levels in the K+ group were higher than in the K-group (P<0.05). This indicates that the condition of DM can cause the occurrence of oxidative stress conditions, because in a state of hyperglycemia in DM condition may stimulate increased production of free radicals (Tiwari et al., 2013). This increased production can occur through various mechanisms, such as through the mechanism of glycation-oxidation of proteins and fats, increase production of advanced glycation end product (AGEs), activation of protein kinase C (PKC), polyolsorbitol pathways, and glucose auto-oxydation (Giacco and Brownlee, 2010).
The EM, KO, and KE groups had MDA levels not significantly difference than in the K+ group (P>0.05). In all these three groups (EM, KO, and KE), there was also damage to lipid peroxidation through free radical activity that is naturally generated by the body through the body's metabolic processes. Nevertheless, through the administration of ethanol mahogany seed extract in the EM and KE groups, MDA levels in these group can be suppressed so as not to differ significantly compared with K-group (P>0.05). These results are suspected to be related to the compounds contained in the ethanol mahogany seed extract, namely flavonoids (Wresdiyati et al., 2015). Flavonoids are potential anti-oxidant by eliminating excess free radical compounds (Matos et al., 2015), so that MDA production as lipid peroxidation products can be suppressed. MDA levels have been widely used as an indicator of the occurrence of oxidative damage, because MDA is the final product of lipid peroxidation produced by oxidation by free radicals (Tiwari et al., 2013).
The results of qualitative analysis on the antioxidant content of Cu, Zn-SOD on liver tissue showed that the anti-oxidant content of Cu, Zn-SOD in liver tissue of K+ group was lower than in K-group. Antioxidant content in EM and KE group rats also appeared to be higher than in the K+ group ( Figure 3).
Quantitative analysis of the anti-oxidant content of Cu, Zn-SOD on liver tissue was performed by counting the number of liver cell nuclei at various levels of Cu, Zn-SOD with the results are presented in Table 2. The results showed that the anti-oxidant content of Cu, Zn-SOD in the K + group was lower than that of K-, EM, and KE. This is indicated by the number of liver cells which positively reacted (+++) was lower in the K+ group than in the K-, EM, and KE group (P<0.05), and the number of liver cells that negatively reacted (-) was higher than that of K-, EM, KO, and KE groups (P<0.05).
The anti-oxidant content of Cu, Zn-SOD in the rats group given the ethanol extract of mahogany seeds (EM and KE) was higher than in the K+ group. This was demonstrated by the number of liver cell reacted positivey (+++) in the mahogany seeds extract groups (EM and KE) was higher than in the K+ group (P<0.05), while the number of cells that negatively reacted (-) was significantly (P<0.05) lower in the EM and KE groups than in the K + groups.
Anti-oxidant Cu, Zn-SOD is one of the endogenous anti-oxidant present in cells that act as a defense mechanism by inhibiting oxidation reactions of free radical activity by converting them into more stable compounds (Valko et al., 2006). The high content of anti-oxidant Cu, Zn-SOD in groups of rats given ethanol extract of mahogany seed (EM and KE) probably due to by flavonoid compound contained in mahogany seed (Wresdiyati et al., 2015). Flavonoids can act as anti-oxidant through various mechanisms, such as through scavenging or quenching free radical compounds by donating hydrogen atoms from hydroxyl groups when reacting with free radicals, metal ionizing, or by enzymatically inhibiting the formation of new free radical compounds (Lukacinova et al., 2008;Han et al., 2012). Flavonoids in mahogany seed extract will work synergistically with anti-oxidant Cu, endogenous Zn-SOD in neutralizing the activity of free radical compounds, so that the anti-oxidant content of Cu, Zn-SOD in rat liver tissue can be improved in DM condition.

CONCLUSION
This study concluded that the ethanol extract of mahogany seed has effect on lowering blood glucose levels and improve anti-oxidant status in liver tissue of diabetic rats.