HEPATOPROTECTIVE EFFECT OF BANANA PEEL FLOUR ON HISTOLOGICAL AND LIVER FUNCTION IN DIABETIC RATS

In diabetes mellitus, non-alcoholic fatty liver disease (NAFLD) is closely linked to hyperglycemia metabolism. This study aimed to find out how a banana peel supplemented diet affected histological and liver function changes in streptozotocin-induced diabetic rats. Vitamins, minerals, dietary fiber, antioxidants, and tryptophan are all contained in banana peel flour (BPF). Serotonin is a neurotransmitter that has been linked to depression and anxiety. This post-test-only control group study was conducted on twenty-five male Wistar rats which were separated into five groups with different treatments. Groups II to V were diabetic rats model groups that consumed standard diet mixed with BPF 0%, 5%, 10%, and 20%, respectively, while group I was a healthy control group fed a standard diet. Hepatic enzyme transaminase (Alanine Aminotransferase-ALT and Aspartate Aminotransferase - AST) and Hematoxylin-Eosin (HE) staining were analyzed with the NAFLD score to examine the liver function and hepatocellular morphology. A change in liver function was observed, as well as a substantial change in the levels of ALT and AST. The NAFLD score with HE staining showed substantial improvements in liver morphology, which was better seen at a 20% BPF dose. The current study supported the hypothesis that BPF had a hepatoprotective effect in diabetic rats, which may be due to the mechanism of controlling the hepatic enzyme transaminase and inducing liver regeneration.


INTRODUCTION
Type 2 diabetes mellitus (T2DM) in developing countries has increased dramatically which is often associated with sedentary lifestyle and obesity (Halter et al., 2014). In patients with DMT2, hyperglycemic conditions cause metabolic abnormalities in the body which if prolonged will have complications, one of which is a liver disease (Patel et al., 2011;Tan et al., 2015) such as Nonalcoholic fatty liver disease (NAFLD). NAFLD is a type of liver disease that occurs without the consumption of alcohol, which can be confirmed by laboratory microscopic and ultrastructural anatomy tests (Eren et al., 2003). The pathogenesis of NAFLD is not known with certainty, but already it has been strongly associated with metabolic syndrome and T2DM.
Indonesia is a tropical country that produces abundant bananas. In 2014, banana production in Indonesia reached 7,008,407 tons. The abundant production of bananas raises a classic problem: there is significant banana peel waste. In general, banana peels have not been used for any real purpose, except as disposable waste and rats feed. One way to use banana peels so that they can be used for a long time and are flexible in their use in 530 Volume 32 Issue 4 (2021) food products is by processing them into the form of flour. Remarkably, the nutritional content contained in BPF has benefits if it can be used as the right raw food material (Keszthelyi and Troost, 2009). The content in banana peels is not much different from the banana fruit itself. The mineral content in banana peels reaches 1,910 mg/kg and vitamin B6 content is 0.8 mg. Besides these essential nutrients, banana peels also contain the amino acid tryptophan as much as 2.5 mg/kg (Meliala et al., 2020), also contain other ingredients such as pectin, lignin, cellulose, and hemicellulose which lower the blood sugar levels, as a result, will improve liver function (Lattimer & Haub, 2010) BPF also contains tryptophan and its precursors, wherein tryptophan is a precursor to serotonin in the body (Rådholm et al 2016). Serotonin (serotonin) is a neurotransmitter that plays a key role in the Central Nervous System (CNS) and the autonomic nervous system (ANS), several studies have been conducted to determine the role of serotonin in regulating body homeostasis through the ANS, with blood glucose levels being one of them (Huskisson et al., 2007), serotonin is involved in the mechanism of hepatic glucose uptake and glycogen metabolism, which helps to support liver cell regeneration and maintain glucose and insulin homeostasis (Lesurtel et al., 2012). The ability of serotonin to regulate hepatic glycolysis via the phosphofructokinase (PFK) activity regulatory pathway (Coelho et al., 2012) Due to their natural tendency to be damaged quickly after harvest, banana peels must be processed into more durable food products with low water content, one of which is flour. BPF is more adaptable and can be found in a variety of diabetic-friendly processed foods. Few studies have investigated the link between BPF dietary quality and liver function parameters, particularly when tryptophan, a serotonin precursor, is present. Based on this, the purpose of this study was to analyze whether BPF has hepatoprotective properties supported by the various phytochemical compositions contained in it.

MATERIAL AND METHODS
All of the chemicals used in the experiments were analytical grade, as follows: Streptozotocin (STZ)and Anti-HTR2B antibody produced in rabbits were purchased from Sigma Chemicals Co, St Louis, MO, USA. A fine test rabbit, DAB detection kit, was purchased from Wuhan Fine Biotech, China. Materials for hematological research, including Hematoxylin, Eosin, and Victoria blue were used to stain the cells. Liver function evaluation was analyzed by measuring AST and ALT levels in the serum by using Diasys reagents via spectrophotometry method.

Preparation of BPF.
Yellow Kepok bananas (Musa balbisiana Colla) in stage 5 of ripening characterized by yellow skin with the green tip (Aurore et al., 2009) were used in this study. The bananas were obtained from traditional markets in Sleman, Yogyakarta, Indonesia. Banana peel washed under running water, sliced into small pieces, soaked in 0.5% citric acid solution, steamed for 5 minutes, and mashed with a blender. The following is the ratio of banana peel: water = 1: 2 for 3 minutes, then dried in a drum dryer at a pressure of 3-4 bar, at a temperature of 140 o C, then mashed in a disk mill to make flour particles that move through an 80 mesh sieve.

Chemical composition analysis of experimental diets
Proximate composition of the samples including moisture (Method 925.40), ash (Method 950.49), protein (Method 955.04), fat (Method 920.39), and crude fiber (Method 935.53) was determined according to the Association of Analytical Chemist (AOAC) methods (AOAC, 2010). The serotonin and its precursor 5-Hydroxytryptophan (serotoninP) of experimental diet was analyzed via the reversed-phase HPLC method (Ly et al., 2008)

Ethical clearance
The present

BPF treatment
Twenty-five healthy adult male Wistar rats (Rattus norvegicus) weighing 150-220 g were used in this study. The rats were kept in laboratory rats housing, each in their polypropylene cage at the Laboratory of Pharmacology and Toxicology, Faculty of Pharmacy, Universitas Gadjah Mada following the recommendation of the Guide for the Care and Use of Laboratory Rats with constant 12 h light and dark cycle (08:00 AM -08:00 PM), controlled temperature (22±2 0 C) and humidity (55±10%) with free access to cubes of standard diet food and tap water during the experiment. Before starting the experimental work, the rats were subjected to one week of acclimation period and various handling procedures to reduce the stress of novelty and handling.
The rats were classified into five groups with different diets for four weeks according to the methods described below: Group I (normal control, non-diabetic, administrated with diet standard AIN 93-M); Group II (diabetic control, diabetic, administrated with diet standard AIN-93 M); Group III (diabetic rats and administrated with standard diet containing 5% BPF), Group IV (diabetic rats and administrated with standard diet containing 10% BPF, and Group V (diabetic rats and administrated with standard diet containing 20% BPF). Diabetic rats model groups were injected intraperitoneally with STZ (60 mg/kg BW); Sigma-Aldrich, St Louis, MO) diluted in citrate-buffered saline (0.1 mol/l, pH 4.5; Sigma-Aldrich). After STZ induction, the rats had free access to 5% of dextrose for 72 h. A total of twenty rats with blood glucose levels of more than 250 mg/dL were included in this study.
The blood samples were collected from the orbital sinus under anesthesia using 50 mg/kg of ketamine, intramuscularly at day 0 (initial database) and 21 after 12h fasting. The blood was then allowed to stand at room temperature for 30 min. The samples were then centrifuged at 3600 rpm for 10 min to provide serum. The blood glucose analysis results were determined based on an enzymatic colorimetric method using glucose GOD-PAP (Ariastuti et al., 2020). The hepatic enzyme transaminase ALT and AST) were examined with micro lab photometry. Hematoxylin-Eosin (HE) staining was analyzed to examine hepatocellular morphology with a NAFLD score.

Preparation of liver preparate
At the end of the experiment period, the rats were sacrificed under anesthesia, using 50 mg/kg of ketamine and after decapitation, liver tissue was collected, excised, and fixed in 10% buffered formalin at room temperature for no longer in 24h.

Hematoxylin-Eosin (HE) staining
The tissues were first stained for 6 h at 60-70ᵒC with Harris' hematoxylin solution, then rinsed in tap water until the water was colorless. Following that, the tissue was differentiated twice with 10% acetic acid and 85% ethanol in water for 2 h and 10 h, respectively, before being rinsed with tap water. In the bluing step, the tissue was soaked in a saturated lithium carbonate solution for 12 h and then rinsing it with tap water. Finally, eosin Y ethanol solution was used to stain the cells for 48 h.

Paraffin embedding
The tissues were dehydrated twice for 0.5 h in 95 % ethanol, then soaked in xylene for 1 h at 60-70°C, followed by paraffin for 24 h.

Slicing and imaging
The stained tissues were cut into 4 µm in width using a microtome, dewaxed, mounted with DPX Mountant (Merck KgaA, Darmstadt, Germany), and then imaged using Olympus CX21/Optilens Optilab Standard light microscope (Carl Zeiss, Oberkochen, Germany) and photographed with a digital camera

Assessment of NAFLD
After the HE staining was completed, an assessment of the histology of the liver was done using the histological scoring system for NAFLD which was validated by the pathology committee of the NASH Clinical Research Network. The liver was scored for the presence of steatosis, ballooning, and lobular inflammation. Steatosis was graded 0-3 based on the percentage of hepatocytes in the biopsy involved with grades 0, 1, 2, and 3 showing involvement of < 5%, 5-33%, 33-66%, and > 66% hepatocytes, respectively. The severity of hepatocyte ballooning was graded from 0 to 2 with grades 0, 1, and 2 showing none, scattered and marked ballooning, respectively. Lobular inflammation was graded 0-3 based on inflammatory foci with grades 1, 2, and 3 showing < 2, 2-4, and > 4 inflammatory foci, respectively. (Kleiner et al., 2005) Volume 32 Issue 4 (2021)

Statistical analysis
Data are expressed as mean ± S.D. of 5 rats in each group and statistically analyzed using The One-way ANOVA was followed by Least Significant Difference (LSD) post-hoc test (SPSS 23 for Windows, IBM Corp., Armonk, NY). The P-values <0.05 were used as the criterion for determining the level of significance. For assessment of NAFLD, the data were also evaluated using kappa statistics, which measure agreement. The Kappa estimates were interpreted according to Landis and Koch's guidelines for κ≤0, poor agreement; κ>0 but ≤0.20, slight agreement; κ>0.20 but ≤0.40, fair agreement; κ>0.40 but ≤0.60, moderate agreement; κ>0.60 but ≤0.80, substantial agreement; and κ>0.80 to 1.00, almost perfect agreement (Batistatou et al., 2013). In this study, we found moderate agreement (Kappa coefficient 0.789) between expertise in pathological anatomy and observer, so the bias that occurs could be minimized.

Chemical composition of experimental diet
The proximate composition of the standard diet and standard diet containing 5%,10%, and 20% BPF which includes moisture, ash, fat, protein, crude fiber, and carbohydrate ( Table I). All of the data was collected on a wet basis and expressed as a percentage of the total. The experimental diets with 5%, 10%, and 20% of BPF led to increasing the percentage of crude fiber and carbohydrate while the percentage of protein and fat were decreased. Statistical analysis by ANOVA and LSD test showed some significant differences between the adding of BPF. Increased percentage of carbohydrate content with the addition of BPF is related to the increased fibers and moisture content. The results of proximate composition of a standard diet containing 20% BPF have given the best nutritional value (crude fiber, carbohydrate, ash). Statistical analysis showed some significant differences between the diets (p<0.05). An increased percentage of BPF content is related to the increased serotonin content.

Effect on rat's liver function
The changes in AST and ALT levels by BPF intervention in the diet. There was a significant decrease (p = 0.022 and (Figure 1) p = 0.005) in both a standard diet containing 5% and 20% BPF groups compared to the diabetic control group and intervention with BPF 20% appeared to significantly (p = 0.026) reduce the serum level of AST compared to the normal control group. Our study also showed that the effect of the 5% BPF intervention was not significantly different when compared to the 10% BPF (p = 0.501) and 20% BPF (P=0.177), and the 10% BPF did not show a significant difference (p = 0.070) when compared with the 20% BPF diet intervention group.
The levels of ALT in the serum of the standard diet containing 5%, 10%, and 20% groups showed a significant decrease (P=0.000) compared to the diabetic control group (Figure 2). The interesting thing that we found in this study was that the decrease in serum ALT levels in the 5% BPF intervention was not significantly different (p = 0.594) compare to the group given the 10% BPF and when compared to the 5% BPF dietary intervention, the decrease in the 20% BPF dietary intervention was more significant (p = 0.003).  This result confirmed that AST serum was alleviated by hepatoprotective activities from BPF, but it was not dose-dependent. The results were following previous reports (Famii & Ebuka, 2019), but different from those of other studies, which found that groups given a 50 percent ethanolic whole plant extract of Musa paradisiaca in doses of (200 and 400 mg/kg) once daily for 14 days were able to avoid hepatotoxicity in a dose-dependent manner (Sultana et al., 2012). In this study, the simple hepatic function can be seen from the levels of the transaminase enzyme. AST is an enzyme produced in mitochondria found in the liver, heart, kidneys, and brain. This enzyme plays a role in converting aspartate and α-ketoglutarate to oxaloacetate and glutamate (Wurtman et al., 2003).
These results indicate that the effect of BPF intervention on serum ALT and AST levels are not dose-dependent but the effects are comparable. All the groups BPF intervention produced a significant decrease in serum of ALT when compared to the diabetic control group. Therefore, the results of the present study indicated that BPF can significantly reduce the levels of several key markers of hepatic injury, and these effects were comparable to the normal control rats group. ALT is the main enzyme found in liver cells that serves to catalyze the transfer of amino acids from alanine to αketoglutarate. The leakage of transaminase enzyme from the cells causes an increase in transaminase enzyme in serum (Wurtman et al., 2003). The level of enzyme ALT exhibited a significant decrease in the experimental groups with BPF-fed diet compared to the diabetic control group (p <0.05) (Figure 2). The results were following previous reports and the hepatoprotective potential of BPF is possible as a result of its antioxidant properties Mitochondrial damage can also occur due to oxidative stress and reactive oxygen species (ROS) that are produced in large quantities in a hyperglycemic state, resulting in an increase in AST levels due to the intracellular release of enzymes into the blood, however, ALT is the main enzyme found in liver cells. This enzyme is also found in small amounts in muscles, the heart, kidneys, and skeletal muscles. The increase in the ALT is more prominent when there is partial damage to liver cells mainly in the membranes of the liver cells. The hyperglycemic state is related to insulin resistance in every cell in the body, one of which is the liver. Insulin resistance affects the permeability of the cell membrane so that the change in permeability and accumulation of ROS causes the ALT enzyme in the cytoplasm of the liver cells to leak and there will be an increase in AST levels in the bloodstream. In this condition, the liver is adversely affected by fat deposition, inflammation, with or without fibrosis (Salvatore et al., 2008). Banana peel with various nutritional compounds caused a decrease of the AST enzyme which was seen from a significant difference in the AST parameter. This is because Kepok BPF contains vitamins, minerals, proteins, and antioxidants that can improve enzyme regulation in the liver (Meliala et al., 2020;Mosa & Khalil, 2015). Antioxidants are molecules that can prevent other molecules from oxidizing. Free radicals are produced during oxidation reactions, which can harm cells. Antioxidants can neutralize free radicals. The antioxidants contained in Kepok BPF are vitamin C, pectin, saponins, and tannins, where tannins are the largest content, reaching 1,236 mg per 100 grams of BPF (Meliala et al., 2020).
In addition, BPF also has a high content of tryptophan dan serotonin, where serotonin has been known to have various biological functions in liver cell metabolism. One of them is the effect of serotonin in the Hepatic Stellate Cell (HSC) which has an important role in the response to liver damage (Ruddell et al., 2008). Serotonin has a role in HSC proliferation and inhibition of apoptosis of hepatocytes, but other studies suggest that serotonin can also increase the apoptotic process of hepatocytes and prevent the proliferation of HSCs (Antonio et al., 2007). The group of diabetic rats fed with variation of Kepok BPF had the impact reducing AST and ALT levels.

Effect of BPF on the histopathology of the liver
The NASH Clinical Research Network's pathology committee has approved a histological scoring system for NAFLD, which looked for steatosis, lobular inflammation, and ballooning degeneration to see if there was any hepatocellular damage in liver cells stained with HE. The mean results of observations of rat liver cells with HE staining on all parameters, namely steatosis, lobular inflammation, ballooning degeneration, and the total score (Table II) and the representative images of the histopathological observation of the liver of rats at the different groups Figure 3). Rats in the normal control group showed normal architecture of the liver with healthy hepatocytes and central vein. Liver from the rats in the BPF fed diet groups showed severe fatty deposition, ballooning, and lobular inflammation.
As a result, the One Way Anova test was conducted, followed by LSD's post-hoc analysis. There were significant (p <0.05) differences between groups of BPF intervention in steatosis, lobular inflammation, ballooning degeneration, and total parameters. In the steatosis parameter, diabetic rats given BPF 5%, 10%, and 20% showed significant (p = 0.004, p = 0.018, and p = 0.000) reduction in liver steatosis compared to diabetic rats with a standard diet.
The liver of rats with BPF 10% and 20% intervention showed significant (p =0.016 and p =0.038) lobular inflammation compared to the diabetic control, but an insignificant result (p =0.083) was seen in the liver of rats treated with BPF 5%. Table 2 also shows that the presence of stratified doses of BPF had no significant impact on the appearance of lobular inflammation, where the liver group with 5% and 20% BPF intervention was not significantly different (p = 0.428 and p = 0.172)  compared to the liver condition in the group with 10% BPF intervention. This shows the effect of BPF on the occurrence of lobular inflammation, not dose-dependent. There were significant differences between groups in terms of ballooning degeneration parameters. The presence of diabetes conditions caused the degeneration view to balloon, indicating a significant difference from normal conditions and the group that consumed BPF at doses of 5% (p = 0.002), 10% (p = 0.000), and 20% (p = 0.016). Mutidose of BPF administration did not have a significant impact on the appearance of ballooning degeneration, where the liver group with 10% and 20% BPF intervention did not show a significant difference (p =0.205 and p =0.393) compared to the liver condition in the group with BPF 5% intervention. This shows that the effect of BPF on the microscopic feature of ballooning degeneration is not dose-dependent.
The total score parameter showed a significant difference between groups, with the normal rat group having the lowest score, followed by the treatment groups, which consisted of diabetic rats treated with a standard diet containing 5%, 10%, and 20% of BPF, and then the diabetic rats' group with standard feed which had the highest overall score, but the effect caused by the 5% BPF intervention was not significantly different from the 10% and 20% BPF intervention with p-value = 0.212 and 0.104. Hyperglycemia causes organs in the body, including the liver, to lose their balance in the diabetic condition. The liver will increase glucose uptake, resulting in biochemical and morphological changes in the organ.
Observations in the liver cell with HE staining were assessed using a histological scoring system for NAFLD, which was observed for steatosis, lobular inflammation, and ballooning degeneration to determine whether there was any hepatocellular damage. The following is a sample of the HE staining from each group (Figure 1). Changes in the liver had begun to occur in the hyperglycemic state, as evidenced by an increase in the ALT and AST enzymes, as well as structural changes in the liver. The mitochondria, rough endoplasmic reticulum, and nucleus of hepatocytes showed the most ultrastructural variation. However, whether these structural changes are caused by pure hyperglycemia or by STZ and alloxan, which are used to cause experimental liver damage in rats, is still unknown. (Luchchesi et al., 2013). Damage to the hepatic cell structure is closely related to duration, with damage beginning to appear at week 2 and progressing to week 26 in experimental rats with uncontrolled hyperglycemia conditions (Bilal et al., 2017).
Due to the presence of its phenolic hydroxyl group, serotonin in foods acts as an antioxidant compound in the mechanism of radical scavenging activity, according to various research findings (Gulcin, 2008;Sarikaya & Gulcin, 2013). The consumption of foods containing serotonin as an antioxidant can boost their physiologic concentrations in the blood, boosting antioxidant defenses, improving mood, and treating sleep disorders, depression, and anxiety (Gonçalves et al., 2021), serotonin is also the precursor of melatonin, this one is capable to reduce fibrosis, inflammation, and liver tissue injury in rats (Colares et al., 2016).

CONCLUSIONS
The intervention of BPF with several variations in diabetic rats caused a significant improvement in the morphology of cells in the liver, according to this study. This could be due to the presence of its phenolic hydroxyl group from serotonin acts as an antioxidant compound in the mechanism of radical scavenging activity high content of antioxidant active substances in BPF, as well as from serotonin, allowing for a good regeneration process in the liver cells. Based on these results, BPF provided a hepatoprotective effect in the diabetic rat model, which might occur through the mechanism of controlling gluconeogenesis and liver regeneration.