Incredible affinity of Kattosh with PPAR‐γ receptors attenuates STZ‐induced pancreas and kidney lesions evidenced in chemicobiological interactions

Abstract Since ancient times, plants have been used as green bioresources to ensure a healthier life by recovering from different diseases. Kattosh (Lasia spinosa L. Thwaites) is a local plant with various traditional uses, especially for arthritis, constipation and coughs. This research investigated the effect of Kattosh stem extract (LSES) on streptozotocin‐induced damage to the pancreas, kidney, and liver using in vitro, in vivo and in silico methods. In vitro phytochemical, antioxidative and anti‐inflammatory effects of LSES were accomplished by established methods followed by antidiabetic actions in in vivo randomized controlled intervention in STZ‐induced animal models for four weeks. In an in silico study, LSES phytocompounds interacted with antidiabetic receptors of peroxisome proliferator‐activated receptor‐gamma (PPAR, PDB ID: 3G9E), AMP‐activated protein kinase (AMPK, PDB ID: 4CFH) and α‐amylase enzyme (PDB ID: 1PPI) to verify the in vivo results. In addition, LSES showed promising in vitro antioxidative and anti‐inflammatory effects. In contrast, it showed a decrease in weekly blood glucose level, normalized lipid profile, ameliorated liver and cardiac markers, managed serum AST and ALT levels, and increased glucose tolerance ability in the animal model study. Restoration of pancreatic and kidney damage was reflected by improving histopathological images. In ligand–receptor interaction, ethyl α‐d‐glucopyranoside of Kattosh showed the highest affinity for the α‐amylase enzyme, PPAR, and AMPK receptors. Results demonstrate that the affinity of Kattosh phytocompounds potentially attenuates pancreatic and kidney lesions and could be approached as an alternative antidiabetic source with further clarification.


| INTRODUC TI ON
Diabetic mellitus (DM) is a metabolic condition that affects millions of people worldwide. It is caused by a shortage of insulin or inadequate insulin synthesis in the pancreas, leading to severe complications such as diabetic neuropathy, nephropathy, retinopathy and cardiovascular disease. 1 According to a recent study of diabetes prevalence worldwide, the disease now affects 463 million people. 2 Despite the availability of various natural and synthetic antidiabetic drugs, diabetes and its micro-and macroimplications are a severe medical concern globally. Modern diabetic treatments are typically connected with problems such as ineffectiveness, high cost and a wide range of side effects. 3 Side effects of antidiabetic sulfonylureas include heartburn, vomiting and skin rashes. If taken for a long time, biguanides (metformin) might cause gastrointestinal problems, anorexia, vomiting and B12 deficiency. 4 Because of the drawbacks of conventional medicines, medicinal plants with claimed antidiabetic activity may be used as an alternative source for the management of diabetes, especially in developing countries, because of their easy accessibility, cost-effectiveness, cultural acceptability and lower side effects. 5 A list of 21,000 medicinal plants from throughout the world has been compiled by the World Health Organization (WHO). There are about 400 plants that can be used to cure diabetes. 6 Kattosh is one such plant that has been utilized to treat several ailments by traditional healers in Bangladesh and other areas of the world.
Lasia spinosa, also known as Kattosh or Kantakachu belonging to the Araceae family, is a perennial herbaceous plant that grows 1 to 2 metres tall and spreads via a long, creeping stoloniferous stem.
It is primarily found in Tropical Asia, from India to New Guinea and South-East Asia, including Bangladesh. Since time immemorial, all parts of this plant have been used for treating a wide range of ailments and diseases in several Asian countries. 7 The stem is used as an expectorant and antitussive in baths to relieve itching caused by roseola, measles, rubella, and other skin illnesses. The tubers are used to cure constipation, rheumatoid arthritis, hyperglycaemia and blood purification in Bangladesh's Rajshahi and Natore regions. 8 The Naga tribes of India believe that the leaves of LS have anthelminthic effects, and they have used them to treat parasitic intestinal worms since ancient times. 9 However, no comprehensive work on the antidiabetic effects of Lasia spinosa is yet conducted by any researcher. Therefore, the current research aimed to explore the potential effect of Kattosh (ethanol extract of Lasia spinosa stem, LSES) to recover the lesions of the pancreas, liver and kidney in the streptozotocininduced albino rat model. ethanol at room temperature (23 ± 0.5°C) for 7 days, with 2 to 3 days of intervals, with occasional stirring. A rotary evaporator (Bibby Scientific, RE200, UK) was used to condense the collected supernatant under reduced pressure at a temperature below 50°C. The concentrated crude extract (LSES) was kept in a Petri dish and dried at 37°C for complete evaporation of the solvent. For future use, the concentrated dark brown semisolid ethanolic stem extract (LSES, 8.0 g) was kept at 4°C.

| Phytochemical group test (qualitative)
The fractions were submitted to qualitative screening for phytochemical groups using a well-established method. Alkaloids, glycosides, steroids, flavonoids, tannins, saponins, phlobatannins, carbohydrates and proteins were all measured in a 10% (w/v) extract solution for each test. 9

| Gas chromatography-mass spectroscopy analysis (GC-MS)
The bioactive compounds extracted from the LSES were analysed The total phenolic content of LSES was determined using a technique described by Singleton and Rossi (1965) with minor modifications. 10 In brief, 5 ml LSES (8 mg/10 ml) or standard (gallic acid) was added to test tubes, followed by a 2.5 ml Folin-Ciocalteu reagent.
After that, 2.5 ml of 7.5% Na2CO 3 was added to the test tubes. Then, the test tubes of all tests were incubated at 25°C for 20 min. Finally, the optical density (OD) of all the test tubes was measured at 760 nm using a UV-VIS 1280 spectrophotometer (Shimadzu Corporation, Japan). All concentrations were tested in triplicates, with distilled water serving as a blank.
TPC was calculated using the following equation: where 'C' is the TPC (mg/g plant extract in GAE), 'c' is the sample concentration obtained from the calibration curve (mg/ml), 'V' is the sample volume, and m is the sample weight (g). The total phenolic content of LSES was determined as mean ± SD and expressed as gallic acid equivalents (GAE) per gram of plant extract.

| Determination of total flavonoid content (TFC)
The total flavonoid content of LSES was determined using Kumaran and Karunakaran's conventional technique, with certain modifications. 11 Initially, 1 ml of LSES (8 mg/10 ml) or 1 ml of standard solution was put in test tubes. The test tubes were then filled with 3 ml of ethanol. After that, 200 L of 10% AlCl 3 was added to all the test tubes holding 200 L of 1 M CH 3 COOK. The test tubes were then filled with 5.6 ml of distilled water and incubated at 30 min. Finally, a spectrophotometer was used to detect absorbance at 415 nm. All experiments were done in triplicate, using water as a control: where 'C' is the TFC (mg/g LSES in Rutin), 'c' is the sample concentration obtained from the calibration curve (mg/ml), 'V' is the sample volume, and m is the sample weight (g). The experiment was repeated three times, with the results presented as mean ± SD, and values were expressed in mg of Rutin equivalent (RE)/g of dried extracts.

| Determination of total antioxidant capacity (TAC)
The phospho-molybdenum method was used to assess the total antioxidant capacity of LSES 12 . To begin, 0.5 ml of LSES or standard was added to 3 ml of the reaction mixture, including 1 ml of 1.8 M H 2 SO 4 , 1 ml of.084 M dipotassium hydrogen phosphate and 1 ml of % ammonium molybdate. The test tubes were then heated to 95°C for 10 min before cooling to room temperature for another 10 min. A spectrophotometer was used to test the final solution's absorbance at 695 nm. The experiment was repeated three times.
The following equation was used to compute total antioxidant capacity, where 'C' stands for total antioxidant capacity; 'c', for sample con- fraction of scavenging activity or inhibition was plotted versus concentration, and the graph was used to establish the IC 50 .

| Hydroxyl radical scavenging activity
The hydroxyl radical scavenging activity of LSES was determined by the method described by Ali Reza. 17 The hydroxyl radical scavenging activity in an aqueous media was measured using deoxyribose. The where A 0 is the absorbance of the control, A 1 is the absorbance of the extract samples, and A2 is the absorbance of the reference. The IC 50 value was determined by plotting the percentage of inhibition against the extract concentration.

| Lipid peroxidation inhibitory effect
The lipid peroxidation inhibition assay of LSES was carried out using Ruberto's method. 18 To begin, a test tube was filled with 100 µl of LSES or standard, 500 µl of bovine brain homogenate solution and 200 l of 0.2 mM ferric chloride. The reaction mixture was then incubated for 30 min at 37°C before adding 2 ml of 1% ice-cooled TBA-TCA-BHT solution. The reaction mixture was then placed on ice after 60 min of incubation at 90°C. The reaction mixtures were then centrifuged for 10 min at 56 g, and the supernatants were collected to determine the absorbance at 532 nm with a spectrophotometer. The following formula was used to calculate lipid peroxidation activity: The IC50 value (the sample concentration necessary to scavenge 50% of lipid peroxidation) was determined by plotting the percentage of inhibition against the extract concentration, and the findings were expressed as mean ± SD.

| Membrane stabilization activity
The membrane-stabilizing activity assay was carried out using a well-established standard method. 19 First, four millilitres of fresh blood was drawn from subjects. The Alsever solution (4 ml) was then added. The solutions were centrifuged for 10 min at 3000 g. The packed cells were then washed with isosaline. After making a 10% v/v suspension solution, it was stored in the freezer at 4°C. Then, 0.5 mg/ml plant extract or standard was mixed in 10 ml distilled water to make a stock solution with a 500 µg/ml final concentration.

| Experimental animals and ethical statements
For the investigation, male adult albino rats of the Wistar strain (Avg. 150-200 g body weight) were employed. They were taken from Jahangirnagar University's animal breeding unit in Dhaka. In a 12-h light-dark cycle, all the animals were separately housed in polycarbonate cages filled with wood husk at a temperature of (25 ± 2°C) and humidity of 55%-60%. All the rats were fed a commercial rat pellet diet throughout the trial. All targeted animal studies adhered to the Animal Ethics Review Board's rules and regulations at the University of Chittagong (approval no. EACUBS2018-5).

| Acute toxicity evaluation
The acute toxicity test was conducted in a traditional laboratory set-

| Test for oral glucose tolerance (OGTT)
At the end of the third week, each animal's glucose tolerance capacity was assessed using the oral glucose tolerance test (OGTT).
Blood samples were taken from the tail tip incision at 0 (immediately before glucose ingestion), 30 to 120 min after glucose delivery. The glucose levels were taken on a regular basis. 24

| Collection of animal organs and blood
At the end of the intervention, the animals were sacrificed using halothane anaesthesia, and blood and organs were collected. A heparinized tube was used to collect whole blood from a heart puncture. The obtained blood was centrifuged for 15 min at 3000 rpm at 25-37°C, and the serum was kept at −20°C for

| Qualitative screening for phytochemicals
The qualitative phytochemical analysis of secondary metabolites in the LSES was carried out. Where alkaloids, flavonoids, steroids, tannins, carbohydrates and protein were found, no saponins, phlobatannins or cardiac glycosides were present in the LSES (Table S1).

| Gas chromatography-mass spectroscopy (GC-MS) data for LSES
The GC-MS analysis of LSES yielded 34 compounds with retention times ranging from 10.117 to 30.261, as indicated in Table 1, and the chromatogram is shown in Figure S1.

| Effect of LSES on lipid peroxidation inhibition, membrane stabilization and protein denaturation inhibition
The effect of LSES on the inhibition of lipid peroxidation, membrane stabilization and inhibition of protein denaturation is summarized in   Note: Here, total flavonoid content (TFC), total phenolic content (TPC), total antioxidant capacity (TAC) and total proanthocyanidin content (TPACC) of LSES were expressed as mg/g of dry weight. All the IC 50 values of LSES are expressed as (µg/ml). Values are presented as mean ± SD.

| Acute toxicity evaluation
All the treated rats showed neither any toxic effect, nor any lethal effect. The present study shows administration of dose up to 2000 mg/kg did not reveal any signs of toxicity or mortality in rats during the entire observation period. Therefore, LD 50 of LSES extract may be greater than 2000 mg/kg (2 g/kg).

| Effect of LSES on weekly body weight, blood glucose levels and oral glucose tolerance
The body weight of distinct experimental animal groups during the intervention period is displayed in the graph in Figure 2A. During the intervention period, the body weights of the animals fluctuated. Except for the NC group, all groups' body weight decreased significantly (p < 0.05) after the first week. However, the body weights of all groups increased significantly (p < 0.05) after the fourth week, except for the DC group, which gradually declined significantly (p < 0. 05).
The animals' blood glucose levels during the intervention period are depicted in the graph below ( Figure 2B). The DC group had significantly higher blood glucose levels than the other groups (p < 0.05). The blood glucose levels of distinct groups exhibited a substantial (p < 0.001) decrease in the first week of intervention.
Some of them were almost the same as the NC group.   group kidney weights were significantly (p < 0.05) lower than the DC group. The kidney weights of the LSES50 and LSES200 groups were essentially identical to those of the NC and RC groups. However, the pancreas weights of the LSES200 group were significantly (p < 0.001) higher than all groups. All other treatment groups also showed higher pancreatic weights than the DC group.

| Effect of LSES on the weights of liver, kidney, and pancreas and oral glucose tolerance
After the 4 th week of intervention, blood glucose levels of all groups were less than 16.66 mmol/L except for the DC group. The oral glucose tolerance test (OGTT) data were collected during the third week of the experiment ( Table 5). The DC group's glucose tolerance was considerably (p < 0.001) lower than the other groups.
The glucose tolerance ability of the LSES50 group was significantly (p < 0.001) higher than that of the other groups, and it was comparable to that of the RC group. Table 6 summarizes the effects of LSES on animal liver glycogen, serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), lipid profiles, lactate dehydrogenase (LDH), creatinine kinase-MB (CK-MB), urea, creatinine and uric acid levels.

| Effect LSES on liver glycogen and other biochemical parameters
The concentrations of liver glycogen in the treatment groups, RC and NC, were considerably (p < 0.001) lower than in the DC group.
The treatment groups' serum ALT levels were considerably (p < 0.001) lower than the DC group. LSES200 was found to be the most effective dose for lowering ALT levels compared with other doses. The treatment groups' serum AST levels were considerably (p < 0.001) lower than the DC. LSES200 showed the highest efficacy in managing the AST levels of STZ-induced animals. Table 6 demonstrates that the cholesterol levels of the treatment groups were considerably (p < 0.001) lower than the DC group in terms of total cholesterol, triglyceride (TG), low-density lipoprotein (LDL) and Note: Liver, kidney and pancreas weight for the intervention of LSES in Albino rats over four weeks at certain temperature and pressure (n = 6). Data are expressed as mean ± SD. All data were analysed by the statistical software SPSS (Statistical Package for the Social Sciences, Version 22.0; IBM Corporation, NY) followed by Tukey's post hoc test for significance. p < 0.05 was considered as significant when compared to the DC group. The star mark on the data represents the values are significant compared with each other for the intervention period. Here, ***p < 0.001; **p < 0.01; and *p < 0.05.
The superscript letters (a-e) denote the level of significance of the experimental groups at least in the experimental environment.

| Histopathological analyses of pancreas and kidneys
The experimental parameters' histopathological findings were  Table 7. Compared with NC, the size of the islet of Langerhans in the pancreas was shown to be smaller in the DC group and degeneration. On the contrary, other treatment groups showed less degeneration, with the width of the islet of Langerhans and the area occupied by β-cell/islet of Langerhans being much higher than the DC group. In addition, the kidney tissues of the DC and RC groups show more tubular epithelial cell degeneration, tubular epithelial cell necrosis and hyperaemic interstitial arteries than the LSES groups, whereas LSES100 was shown to be more promising than the other two doses.

| Pharmacokinetic properties (LSES)
Using Lipinski's rule of five and Veber's rules (absorption, distribution, metabolism and excretion/transport), the researchers discovered the drug-like features of selected substances. Only two compounds defied one of Lipinski's five principles, while the others followed both Lipinski's and Veber's standards ( Table 8). As a result, all compounds revealed drug-like properties and could be taken orally.

| DISCUSS ION
Diabetes mellitus is a major public health issue that affects people  ions are known to cause ascorbic acid and glucose oxidation. 34 Lipid peroxidation is also increased in diabetes, which could be related to the increased generation or decreased elimination of reactive oxygen species (ROS). 35 LSES extract has DPPH free radical scavenging activity, iron-chelating activity, nitric oxide scavenging activity, hydroxyl radical scavenging activity, lipid peroxidation activity, protein denaturation inhibition activity and membrane stabilization activity.
Inhibitors of α-amylase slow the breakdown of carbohydrates and reduce the postprandial blood glucose spike in people with diabetes. 36 Controlling the catalytic activity of this enzyme lowers glucose production in the postprandial stage, which could be a therapeutic advantage for people with diabetes. The α-amylase inhibitory activity of LSES will be a game-changer for diabetic rats for the management of diabetic complications. During the experiment, it was revealed that the DC group's body weight was severely reduced. The treatment group's body weight dropped as well, although it was restored after a few days; NC has no decrement. This decrement was attributed to muscle and adipose tissue loss caused by an increase in tissue protein and fatty acid breakdown driven by a fall in plasma insulin levels. Protein synthesis is hindered, and breakdown is increased in the absence of insulin, resulting in an increase in amino acid levels in the blood, which can subsequently be used for gluconeogenesis. 37 When diabetic rats were compared to normal control rats, their fasting blood glucose levels were considerably higher. LSES treatment lowered fasting blood glucose levels considerably, and LSES50 has higher activity than the RC group. The extract's antidiabetic properties could be linked to secondary metabolites found in the plant. The glucose tolerance test (OGTT) assesses the body's ability to metabolize and remove sugar (glucose) from the bloodstream. The OGTT can be used to detect diabetes 1, gestational diabetes (diabetes during pregnancy) and prediabetes, among other things (high blood sugar that predicts type 2 diabetes). All treatment groups in this experiment showed better glucose tolerance than the diabetic control group. These results could be owing to the STZ-induced diabetic rats receiving LSES, which caused the pancreas to heal and secrete insulin into the bloodstream.
In the current investigation, STZ-treated DC rats had an in-   ALT is a cytoplasmic enzyme found in abundance in the liver; an increase in ALT in the blood indicates liver disease; however, AST is less specific as a marker of liver injury. Hepatocellular injury may be the reason for the high levels of AST and ALT in the serum of STZinduced diabetic rats. 40 In the present investigation, ALT and AST levels are higher in the DC group when compared to other groups. In the case of diabetic cardiomyopathy, a diagnosis of cardiac enzymes is required. Biomarkers for myocardial infarction include aspartate aminotransferase (AST), creatine kinase-isoenzyme (CK-MB) and lactate dehydrogenase (LDH). These enzymes are closely bound to the contractile apparatus of cardiac muscle tissue, and they are released into the bloodstream when the heart muscle is injured.
In diabetic animals, blood levels of CK-MB and LDH are increased. 43 However, compared with the diabetic control (DC) group, LSES dramatically reduced levels of cardiac enzymes such as CK-MB, showing a preventive role in renal injury. The LSES100 treatment group recovered faster than the other treatment groups.
F I G U R E 7 Best rank poses of (A) 2D and (B) 3D molecular interactions of ethyl alpha-d-glucopyranoside docked with the active-site 1PPI α-amylase for antidiabetic potential In structural molecular biology and computer-assisted drug design (CADD), a molecular docking study is vital for developing a new drug, and a molecular docking tool evaluates the prediction of new compound binding interactions against key proteins. 44 Molecular docking is also utilized to find out what the putative molecular mechanisms of action of specific pharmacological agents are. 45 However, molecular docking was used to understand further the molecular mechanism and its relationship to the study's findings.
A total of eight compounds were chosen by PASS prediction from LSES GC-MS data to understand the biological activity better. These If medicine or chemical breaks all these requirements, it is not regarded to have good oral bioavailability. 48 However, this study found that some compounds in LSES have a high oral bioavailability of the bioactive substances tested. As a result, based on a study depicted as a drug-like quality, these phytocompounds could be viewed as viable medication candidates with good oral bioavailability. It is obvious from the preceding discussions that LSES is very efficient in treating type 2 diabetes because it reduces the diabetes-related parameters in the STZ-induced rat model; histological examination and computational studies have proven its relevance.

| CON CLUS ION
The injection of LSES to STZ-diabetic rats decreased weekly blood