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Antidiabetic Potency of Jeruju (Acanthus ilicifolius L.) Ethanol Extract and Taurine on Histopathological Response of Mice Kidney (Mus musculus L.) Induced by Alloxan

Diabetes arises due to interference with the work of the pancreas in secreting the hormone insulin. The disease can cause a decrease in body immunity and complications of various vital organs such as the kidneys. Jeruju (Acanthus ilicifolius L.) leaf and taurine is reported to have antioxidant activity that is potential as antidiabetic agents. The purpose of this study was to determine the effect of jeruju and taurine on blood glucose levels and the ability to regenerate kidney organs damaged by alloxan induction. This study used a Completely Randomized Design with 5 treatment groups and 5 replications each. K1 group as a negative control (no treatment given), K2 group as a positive control (only induced by alloxan), group P1 induced alloxan and given 100% ethanol extract of jeruju leaf 22.4 mg/bw/day, group P2 induced alloxan and given alloxan 50% ethanol extract of jeruju leaf dose 22.4 mg/bw/day, group P3 was induced by alloxan and given a taurine dose of 15.6 mg/bw/day for 14 days. Data were analyzed by ANOVA followed by LSD at 5% of significant level. The results showed that the administration of jeruju extract and taurine affected the bodyweight of mice, and was significantly able to reduce blood glucose levels by 69.39% (P1), 67.06% (P2), 73.77% (P3), able to repair kidney damage after alloxan induction.


Introduction
Exposure to excessive free radicals cause oxidative stress where an imbalance occurs between oxidants and antioxidants in the body. The inability of the body to ward off free radicals causes the emergence of various chronic diseases including diabetes which is characterized by high levels of glucose in the blood [1]. Diabetes occurs due to metabolic disorders of the pancreas so that it cannot produce enough insulin or the body cannot use the insulin that is produced effectively. When insulin secretion is disrupted, the energy that should be produced from the glucose overhaul is not formed. As a result, high glucose levels due to a build-up of glucose in the blood [2].
Exposure to free radicals that causes diabetes could be prevented by herbal medicines that contain antioxidants. One of the plants that have potential as antidiabetic agents is jeruju (local name) (Acanthus ilicifiloius L.) or holy mangrove which contains various bioactive compounds such as flavonoids, saponins, tannins, alkaloids, and steroids [3]. In addition to jeruju, the organic compound 2 taurine is reported to be able to overcome diabetes through its antioxidant mechanisms and can improve the pancreas in secreting insulin [4,5]. Therefore, in this study we would like to explore any potential used of ethanolic jeruju leaf extraction and taurine.

Preparation of Jeruju Extract
250 grams of dried Jeruju leaf powder was macerated using 100% pro analysis ethanol solvent and 50% technical ethanol as much as 2.5 liters for 24 hours (ratio 1:10). The filtrate was filtered and concentrated using a rotary evaporator at 50 o C until a thick extract was obtained then made it in paste form by putting into the oven at 50 o C for several hours. Prior given to treated animal, the extract was dissolved by using 1% CMC, while taurine in the form of powder was dissolved using warm distilled water slowly. To indicate bioactive compound, phytochemical procedures was applied as follows.

Diabetes Testing and Provision of Test Materials
Some Diabetes-conditioned mice were preceded by fasting for ± 6-8 hours, then measured blood glucose levels and initial body weight before treatment. Furthermore, alloxan-induced mice subcutaneously at the nape of the neck with a dose of 160 mg/kg bw which was previously dissolved using 0.3 ml aqua-pro injection. Mice were categorized as diabetes if fasting blood glucose levels reached > 200 mg/dL [6]. After positively experiencing an increase in blood glucose levels, groups of mice were given the extract of jeruju leaves of jeruju leaves with a dose of 22.4 mg/bw/day and a taurine dose of 15.6 mg /bw/day orally for 14 days.

Experimental Design
Twenty-five male mice (Mus musculus L.) from Veterinary Region III of Lampung Province (BPPV) Indonesia were used in this study. They were in 3-4 months old with body weight ranges of 30-40 grams. They randomized put in five different treatment groups: Control group (K1) with no alloxan induction nor jeruru extract/taurine, Positive control group (K2) were given alloxan induction to experience increase in blood glucose level, other three groups prior alloxan induction were Treatment 1 group (P1) given jeruju 100% Et extraction, Treatment 2 group (P2) given jeruju 50% Et extraction and Treatment 3 group (P3) given taurine. Histopathological preparations consisted of several stages including fixation, dehydration, embedding, cutting, staining (Hematoxylin-Eosin staining), and mounting stages. Renal damage scoring was an accumulation of glomerulus damage scores and tubular scoring in 5 visual fields. Glomerular damage score ie, normal (0); inflammatory cell infiltration (1); edema spatium bowman (2); and necrosis (3). While the score of damage to the renal tubules is, normal (0); inflammatory cell infiltration (1); congestion (2); swelling of the tubular epithelium (3); and necrosis (4) [7,8].

Data Analysis
Glucose levels data, bodyweight of mice, kidney wet weight were analyzed with One Way ANOVA and LSD (Least Significant Difference) test at 5 % significance level. While scoring kidney damage was analyzed by the Kruskal-Wallis Test and Wilcoxon-Mann-Whitney test at a 5% level. Table 1 as follow. Based on the results of phytochemical tests, extracts of jeruju (Acanthus ilicifolius L.) leaf, whether extracted using 100% ethanol solvent or 50% ethanol, contained of flavonoids, alkaloids, saponins and tannins. These compounds were known for their antioxidant effect that counteract free radicals released by cell damage and triggered various diseases such as diabetes [9,10].  Alloxan induction caused weight loss in groups K2, P1, P2, and P3. It is known that alloxan could damage the structure of the pancreas so that insulin secretion and metabolic rate in the body was disrupted. The administration of ethanolic jeruju leaf extraction was able to increase the average body weight of mice indicated in group P1 and P2 on the 20th day observation, although it was not yet fully restored as the initial body weight. Antioxidant compounds such as flavonoids, tannins, alkaloids, saponins presumably could improve the pancreas in secreting insulin so that the process of glucose metabolism in the body could be restored and used to synthesize other biomolecules resulting in weight gain. On the other hand, Taurine in this study was not able to increase the bodyweight after alloxan induction. On the 6th day, all treatment groups induced by alloxan experienced an increase in blood glucose levels above 300 mg/dL. Alloxan caused an increase in blood glucose since it has cytotoxic effect and caused damage to insulin-producing pancreatic β cells through the formation of free radicals and oxidative stress.

Changes in Mean Fasting Blood Glucose of Mice
Treatment with Et extract of jeruju leaf and taurin could reduce blood glucose levels in P1, P2, and P3 until reaching normal blood glucose levels. The decrease of blood glucose level could be due to the high antioxidant content in the extract and taurine. Ethanol extract of Jeruju (Acanthus ilicifolius L.) leaf reduced blood glucose levels presumably through intra-pancreatic and extra-pancreatic mechanisms. The intra-pancreatic mechanism worked with active compounds of alkaloids and flavonoids that were able to regenerate the damaged pancreatic β cells so as to increase insulin secretion through sympathetic nerve stimulation and protect damaged nerves pancreatic cells from free radicals [11,12]. In the extra-pancreatic mechanism, alkaloids and saponins decrease blood glucose by inhibiting glucose absorption in the intestine so that glucose entering the bloodstream decreases, stimulating glycogen synthesis and inhibiting the enzyme α-glucosidase.
The treatment with taurine dose of 15.6 mg/bw/day showed a decrease in the highest glucose level of 73.77%. Taurine was thought to play a role in the tolerance of blood glucose which reacted in metabolic processes. High antioxidant activity of taurine presumably could suppress free radicals from alloxan so as to reduce blood glucose levels. Other studies also indicated that administration of taurine was able to reduce the blood glucose of alloxan-induced mice [3,13,14]. According to Carneiro taurine was able to control glucose homeostasis through the regulation of gene expression that was useful in insulin secretion and increases peripheral insulin sensitivity [15]. Taurine also was able to modify the factors that cause susceptibility to toxic chemicals and could protect the body from toxicity due to the induction of carbon tetrachloride [16].

Average Weight of Mice Wet Kidney Organs
Subcutaneous alloxan induction caused a significant reduction in renal wet weight at K2, P1, P2, and P3 as shown in Figure 2. Reduction in kidney wet weight was thought to be due to the toxic effects of alloxan which resulted in changing in the structure of the kidney organs. The kidney becomes one of the target organs of toxic compounds because it acts as a place of excretion through which some blood passes and functions to filter out harmful substances. In line with this, the antioxidants contained in Et jeruju leaf and taurine were thought to be able to fight alloxan toxic compounds. It was showed that antioxidant compounds such as saponins can affect the ratio or index of organs that can act as nephroprotective [17,18]. The alloxan-induced group (K2) had the highest damage score of 26.40 ± 0.18 and was significantly different from the entire treatment group (p <0.05). Histopathological scoring results described the level of kidney organ damage in each treatment. The damage score was an accumulation of glomerular observation score and renal tubular observation score as seen in Figure 3.  In the K2 group had the worst damage compared to the other treatment groups. It was believed that high blood glucose levels caused kidney structure to change so that its function was disrupted. In the K2 group, the damage was seen in the form of proximal tubular epithelial cell narrowing, inflammation cell infiltration, congestion, and necrosis in both the tubules and glomerulus. Swelling of the tubular epithelium was thought to be due to bleeding, injury to the tubules, and the cell nucleus that begins to fade causing the tubules to narrow. Necrosis or death of glomerular and tubular cells was caused due to interference and exposure to toxic compounds that occur continuously. The high blood glucose levels caused disruption of homeostasis which was characterized by the release of calcium ions from the mitochondria and the beginning of cell death (necrosis) [19]. Cells that experience necrosis will release various mediators which become the beginning of the inflammatory process and attract the appearance of inflammatory cells. Inflammation of inflammatory cells arise as a response of blood vessels to damaged glomerular and tubular cells. Congestion (blood weir) that occurs in the kidneys was characterized by the presence of blood cells that meet the lumen in the blood vessels. Congestion arises because of increased blood volume due to the dilation of small blood vessels or capillaries [20]. Congestion that occurs continuously is directly proportional to the increase in extracellular and extravascular fluid volume accompanied by fluid retention in the tissues and serous cavities that are the cause of edema.

Mean Damage Score and Description of Mice Kidney Histopathology
In group P1, the glomerulus looked normal and no swelling occur. Cells in the proximal and distal tubules appear to have less necrosis compared to the K2 group although there was still some damage such as edema, congestion, and narrowing of the tubular lumen. The improvement in group P1 was thought to be due to the antioxidant activity contained in 100% ethanol extracts of jeruju leaf. These antioxidants play an important role in repairing cells due to exposure to free radicals. Beside, flavonoid and alkaloid compounds in jeruju have antioxidant activity as suppressants for free radicals and were able to repair the pancreatic and liver organs due to toxic alloxan compounds [3].
In the P2 group, the glomerulus looked normal and there was no swelling, the congestion was lighter than in the K2 group, although infiltration of inflammatory cells was still seen, and the narrowing of the tubular epithelium was seen. The rate of improvement in the K2 group was lower than in the P1 group although both of them used jeruju leaf extract. The difference in the rate of kidney repair was thought to be due to differences in the solvent used in the maceration process. Antioxidant compounds such as flavonoids are polar compounds and will be attracted to extractions with polar solvents as well as to get maximum results [21]. Polarity which is more suitable and compatible with the bioactive components contained in jeruju leaf so that it can attract more secondary metabolites of jeruju leaf compared to those used 50% ethanol in P2 group.
In the P3 group, the proximal and distal tubule structures looked normal although some cells still had necrosis, congestion, tubular epithelial narrowing, and inflammatory cell infiltration. P3 improvement rates were still below P1 and P2. The taurine compound administered orally was thought to have good enough antioxidant activity so that it reduced free radicals from alloxan which caused damage to the kidneys. Taurine acts as an antioxidant by taking local and systemic Reactive Oxygen Species (ROS) free radicals, preventing lipid peroxide by stabilizing lipids in cell membranes and being effective in binding free radicals before reacting with cell molecular components. The administration of taurine can reduce tissue injury, apoptosis, and necrosis in kidney epithelial cells that are ischemic due to high ROS levels from free radicals [22, 23,24].

Conclusion
Ethanol extraction of Jeruju leaf with dose of 22.4 mg/bw/day and taurine with of dose 15.6 mg/bw/day for 14 days can reduce blood glucose levels, able to improve kidney damage, and affect the weight body of mice (Mus musculus L.) induced by alloxan.