Ganoderma applanatum mushroom provides new insights into the management of diabetes mellitus, hyperlipidemia, and hepatic degeneration: A comprehensive analysis

Abstract This study was undertaken to evaluate the antidiabetic, hypolipidemic, and hepatoprotective effects of methanol and aqueous extracts of Ganoderma applanatum (MEGA, AEGA) in alloxan‐induced diabetic rats. The antidiabetic study was implemented by the induction of alloxan to the rats. The analysis of the hypolipidemic and liver‐protective effects of fungus extracts was studied by estimating the lipid profile and the liver marker enzymes. Besides, in silico screening of the compounds of Ganoderma applanatum has been incorporated thus to check the binding affinity of compounds and enzymes affinity. The Discovery Studio 2020, UCSF Chimera, and PyRx AutoDock Vina have been used to implement the docking analysis. Nine days of oral feeding of MEGA and AEGA of Ganoderma applanatum resulted in a significant (p < .001) reduction in blood glucose, lipid profile, and liver marker enzymes. Besides, Myrocin C scored the highest score in the docking study. The biological and computational approaches suggested the MEGA and AEGA could be a potential source for antidiabetic, hypolipidemic, and hepatoprotective effects.

| 4365 HOSSAIN et Al. are used to treat diabetes. However, due to unwanted side effects of these currently used antidiabetic agents, WHO has recommended the investigation of new entities with lesser or no side-effects for the treatment of diabetic patients (Gengiah et al., 2014).
Diabetes mellitus is a progressive metabolic disease marked by glucose, lipid, and lipoprotein defects, which results in hyperglycemia and induces numerous complications, such as hyperlipidemia and atherosclerosis (Ozder, 2014). Cholesterol is the cell membrane building block and a precursor to steroid hormones. It comprises of several different particulate substances, high-density lipoproteins (HDLs), low-density lipoproteins (LDLs), and very low-density lipoprotein (VLDL). The LDL and VLDL cholesterol levels have been well known to be atherogenic, while HDL cholesterol protects the progression of atherosclerosis (Deng & Chow, 2010). Liver disorders that are now a worldwide health problem can be categorized as acute hepatitis, chronic hepatitis, hepatitis, and cirrhosis. Unfortunately, hepatitis therapy is controversial since generic or synthetic medications are not adequate to cure these diseases and often have significant side effects. Natural remedies have long proven effective for liver disorders (Asadi-Samani et al., 2015). As the source of raw material for the extraction of secondary active metabolites and synthesis components, medicinal plants are widely used in modern drugs (Vijayakumar et al., 2020). The detoxification of the poisoned liver can be profoundly achieved through the use of natural ingredients, including herbal extracts. According to credible scientific data originating from medicinal plant research, plants such as Glycyrrhiza glabra, Silybum marianum, Picrorhiza kurroa, and Phyllanthus species have been extensively and regularly used to treat liver disorders (McBride et al., 2012;Tatiya et al., 2012). Mushrooms have a remarkable place in folk medicines not only for nutritional value but also for therapeutic uses (Yang et al., 2008). Mushrooms were used extensively for medicinal purposes and as flavorful foodstuffs. Mushrooms have emerged in recent years as a significant class of bioactive products, that is, (Jeong et al., 2004) immunological and hypoglycemic activities (Hao et al., 2020), and antitumor . Mushrooms contain several bioactive compounds that make sure appropriate metabolic functioning of metabolic organs such as endocrine glands, pancreas, and liver (Calvo et al., 2016). As a result, mushrooms have shown effectiveness in controlling both blood glucose levels and diabetic complications (Zhang et al., 2016). Ganoderma applanatum belongs to the family Ganodermaceae which is distributed throughout the world (Paterson, 2006). The different parts of the G. applanatum contain Applanatumin A, Applanatumol A, Applanatumol E, Applanatumol P, Applanatumol Q, Applanoxidic acid E, Cytosporone C, Nigragillin, Ganoapplanin, Myrocin C, Sphaeropsidin D, 4,7,11,15,pregnane, Applanoxidic acid (C-G), Stemphone B, Condidymic acid, etc; as its chemical compositions (Elkhateeb et al., 2018;Li et al., 2016;Luo et al., 2015Luo et al., , 2016. Boundless traditional medicinal uses of Ganoderma species have attracted the pharmaceutical industry recently (Loyd et al., 2018). Several studies reported that Ganoderma applanatum has many medicinal properties such as antibacterial, antiviral, antitumor, antifibrotic, antiobesity, antioxidative, and immunomodulatory (Gao et al., 2019;Luo et al., 2017). The goal of the present research is therefore to study the effectiveness of Ganoderma applanatum as a possible natural oral hypoglycemic, hypolipidemic, and hepatoprotective agent, or functional food, in reducing hyperglycemia along with other vascular complications in diabetic rats by pharmacological and computational approaches.

| Drugs and chemicals
Analytical grade drugs and solvents were utilized in the study.
Alloxan monohydrate and glibenclamide were purchased from Sigma Chemicals Co., USA and Square Pharmaceuticals Ltd., Bangladesh, respectively. 0.9% NaCl was purchased from Beximco Pharmaceuticals Ltd., Bangladesh. Other chemicals of analytical grade were supplied by the Department of Pharmacy, University of Chittagong.

| Extract preparation
The fruiting bodies were shade dried for 14 days. They were then oven dried for better grinding and finely powdered using an electric grinder.
In an air-tight container, the powder was stored. Weighed (200 g of the dried powder for methanolic extract and 200 g dried powder for aqueous extract) sample was soaked in 3 L of methanol and 3 L of distilled water respectively in clean, sterilized, and flat-bottomed glass container for 14 days accompanying occasional stirring and agitation at room temperature. It was then filtered using filter papers (Whatman size no.1). The extracts were obtained by evaporation using a rotary evaporator. A gummy concentrated black color residue of methanolic extract and aqueous extract was found with a yield of 24.25 g and 21.98 g, respectively. These extracts were kept in tightly closed glass containers and stored in the refrigerator for further use.

| Animals
Wistar albino rats of both sexes weighing 180-200 g were procured from BCSIR Laboratories, Chattogram. The animals were used in pharmacological and toxicological studies. They were housed in cages in well-ventilated, room temperature conditions with a natural 12-hr day-night cycle at the Animal House of Department of Pharmacy, Faculty of Biological Science, University of Chittagong.
They were fed a balanced rodent pellet diet. Tap water ad libitum was also provided throughout the experimental period. Water was changed and the cages were cleaned every day. Rats were allowed to acclimatize upon arrival for 2 weeks to laboratory condi-

tions. Permission was granted by the Departmental Ethical Review
Committee, Department of Pharmacy, University of Chittagong, before embarking on the animal studies.

| Acute toxicity test
Overnight fasted healthy Wistar albino rats of either sex were divided into five groups (n = 5 in each group). 2 ml of typical saline solution in distilled water was administered orally in the animals of the control group. The rats of the remaining four groups orally received both extracts in increasing dose levels of 100, 400, 700, and 4,000 mg/kg body weight. According to guidelines No. 425 of the Organization for Economic Cooperation and Development, any change in rats' behavioral, neurological, and autonomic profile was observed consistently up to 72 hr. In-depth care of animals is also taken for 7 days (OECD, O., 2001).

| Induction of diabetes in rats
During 24 hr of fasting duration, animals could freely access the water. Overnight fasted albino rats were made diabetic by injecting with the freshly prepared solution of alloxan (120 mg/kg in normal saline 0.9% NaCl) intraperitoneally (Nabeel et al., 2010). Blood glucose level measured after 3 days. Animals with blood glucose levels equal to 200 mg/dl or above were considered diabetic and used in the experiment (Hassan et al., 2019).

| Experimental design
A total of 42 animals (6 standard and 36 diabetic rats) were used in the experiment. The rats were divided into seven groups (I-VII) of six rats (n = 6). Group I (standard control) and Group II (alloxan-induced diabetic control) received 1% tween 80 in normal saline (0.9% NaCl) i.p. for 9 days. Groups III and IV consisted of alloxan-induced diabetic rats which received methanolic extract at a dose of 250 and 500 mg/kg i.p. respectively in the vehicle for 9 days. Group V and Group VI consisted of alloxan-induced diabetic rats treated with aqueous extract at a dose of 250 and 500 mg/kg i.p. respectively in the vehicle for 9 days. Group VII consisted of alloxan-induced diabetic rats which received glibenclamide 1 mg/kg i.p. for 9 days.

| Determination of blood glucose levels
Fasting blood samples were collected from the tail vein of the rats on 1st, 4th, 7th, and 9th day. Blood glucose level was determined by using a one-touch glucometer (Contour Net EZ). The food and water intake was monitored daily for each rat. The periodical body weight difference of the individual animals was also measured during nine experimental periods.

| Preparation of serum for the test
After 9 days, the foods (except waters) were removed from the rats. The intraperitoneal injections of ketamine (50 mg/kg body weight, i.p.) have been administered to the rats and made them anesthetized. The blood samples were extracted and transferred to tubes directly from the rats via a cardiac puncture. At 3,000 rpm, blood was centrifuged for 10 min to extract and remove red blood cells (Jemai et al., 2009). Serum samples were extracted and collected by a micropipette and placed for analysis in the refrigerator.

| Molecular analysis: molecular docking
For the protein-ligand binding operation of the selected proteinligand complexes, PyRx Autodock Vina was used . A semiflexible docking system has been applied to perform the docking research. A semiflexible docking system has been applied to perform the docking research. The phytochemicals were translated into PDBQT formats with PyRx AutoDock tools.
The rigidity of proteins and ligands was retained for this analysis.
Ligand molecules had given the freedom for 10 degrees. AutoDock determines the molecules to format pdbqt, box style, grid box formation, etc. The grid box with an active position was built in the middle of the box. BIOVIA Discovery Studio Visualizer 2020  was eventually accelerated to evaluate the docking sites for the possible linking approaches.

| Statistical analysis
The data are presented as mean ±standard deviation (SD).
Significance among different groups was determined using the one-way analysis of variance (ANOVA) test, followed by Dunnet's t test (2-sided). Values of p < .05, p < .01, p < .001 were considered as significant. All the data were analyzed using SPSS (Statistical Package for the Social Sciences) version 16.0 software. Charts were drawn using GraphPad Prism software version 6.01.

| Acute toxicity test
The behavioral, neurological, and autonomic profiles of animals remained unchanged during this test duration. No death of rats was observed at the highest dose of both extracts.

| Effect of MEGA and AEGA on blood glucose level
Intraperitoneal administration of a single dose of alloxan monohydrate (120 mg/kg) significantly (p < .001) increases the blood glucose level in diabetic control rats when compared with normal control rats. During the 9 days of treatment, there was a significant (p < .001) reduction in blood glucose level in Group III, Group IV, Group V, Group VI, and Group VII diabetic rats, which respectively received MEGA 250 mg/kg, MEGA 500 mg/kg, AEGA 250 mg/kg, AEGA 500 mg/kg, and glibenclamide 1 mg/kg body weight intraperitoneally as compared with alloxan-induced untreated diabetic Group II rats (Table 1).

| Effect of MEGA and AEGA on body weight
It was observed that the bodyweight of alloxan-induced Group II diabetic animals gradually decreased when compared (p < .001) to the normal rats indicating the impaired glucose metabolism. After 9 days TA B L E 1 Effect of MEGA and AEGA on blood glucose level in different groups of experimental rats Group I normal animals. The serum lipid profile of the control and the experimental animals has been depicted in Table 3.

| Effect of MEGA and AEGA on serum liver function parameters
The serum levels of liver enzymes such as AST, ALT, and ALP significantly (p < .001) increased in alloxan-induced diabetic control rats as compared with normal control animals indicating hepatic damage. The markers of the AST, ALT, and ALP enzymes of the liver were decreased significantly (p < .001) with the administration of MEGA 250 mg/kg, MEGA 500 mg/kg, AEGA 250 mg/kg, and AEGA 500 mg/kg. The AST, ALT, and ALP were 52.33 ± 4.72, 126.33 ± 5.79, and 248.83 ± 5.46 in diabetic mice but with the administration of the MEGA 500 mg/kg, the level of AST, ALT and ALP was reduced to 25.00 ± 3.74, 98.00 ± 4.38, and 224.17 ± 3.87, respectively. The AST, ALT, and ALP also reduced to 30.00 ± 5.10, 98.83 ± 3.25, and 228.00 ± 5.22, respectively. The results of the extracts have been compared with the results of diabetes and glibenclamide 1 mg/kg induced mice. These findings proved the hepatoprotective effect of Ganoderma applanatum mushroom (Table 4).

| D ISCUSS I ON
The most potent source of new bioactive molecules for new therapies is medicinal plants . Therefore, natural plantbased treatments are prevalent in developing countries and have essential priorities for their beneficial features for human health.
Almost 80% of patients also use conventional drugs in developing countries (Kim, 2005). Various plant derivatives exert significant pharmacological actions, such as antioxidants, cytotoxicity, anxiolytics, thrombolytics, neuroprotective, antidepressants, hepatoprotective and neuroprotective actions (Okwu & Uchenna, 2009  Note: All values are Mean ± SD and statistically analyzed using one -way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test, n = 6. Group II compared with normal control. Groups III, IV, V, VI, and VII compared with diabetic control. is a diabetogenic agent that destroys the islets of Langerhans, resulting from a massive reduction in insulin release, causing hyperglycemia (2016). In the present investigation, it was observed that these extracts were able to reduce the blood glucose level from the day-1 of its administration, indicating the strong hypoglycemic tendency of the phytoconstituents present in the extracts. Ganoderma applanatum might aid in the recovery of β cells to secrete insulin; therefore, the blood glucose level was decreased after treatment.

Groups TB (mg/dl) Albumin (g/dl) AST (U/L) ALT (U/L) ALP (U/L)
It was an indication that the drug treatment ameliorated the impaired carbohydrate metabolism. Induction of diabetes by alloxan leads to loss of body weight due to increased muscle wasting and loss of tissue proteins (Obia et al., 2016). But, the body weights of   These results suggest that Ganoderma applanatum can be used for the management of hyperlipidemia and atherosclerosis. The bioactive components for hypolipidemic activity in Ganoderma applana- tum are yet to be identified. In this evaluation, MEGA and AEGA have shown that they can bind bile acids with cholesterol metabolites and lower the solubility of cholesterol. After administering MEGA and AEGA to the rats, the fecal excretion of cholesterol and bile acid has increased significantly. The suggestion is that reductions in intestinal cholesterol and bile acid absorption after the feeding of MEGA and AEGA may represent a mechanism for MEGA and AEGA's hypolipidemic mechanisms (Nagaoka et al., 2005). Injury to the structural integrity of the liver is seen by an elevated serum transaminase as the cytoplasms are found and discharged into circulation after cellular damage (Pari & Kumar, 2002). Enhancement of bilirubin indicates abnormal liver function, resulting from the higher synthetic function of the liver (Nasrin et al., 2011). In this study, Table 4 shows a significant (p < .001) rise in the bilirubin level of diabetic animals compared with normal control animals. But Ganoderma applanatum mushroom did not affect albumin production and bilirubin level in diabetic rats.
Diabetic complications such as increased gluconeogenesis and ketogenesis may be due to the elevated enzymes (Kumar et al., 2011).
The liver mainly detoxifies xenobiotics. Elevated liver enzymes act as a mediator for liver damage (Hasan et al., 2018). Table 5 shows kidney function analysis including the study of creatinine, urea, and blood urea nitrogen (BUN). Elevation of renal function markers such as plasma levels of urea and creatinine is considered as renal dysfunction or kidney disease (Gowda et al., 2010). There was no significant effect on the kidney function parameters in diabetic control groups during the treatment than the standard control group.
Again, when the diabetic animals treated with the test drugs, there was no significant effect on serum creatinine, serum urea, and BUN level observed in comparison with the diabetic control group. So, the use of Ganoderma applanatum did not damage the kidney. Molecular docking analysis is a systemic way to predict the ligand-protein relationship that is most suitable for gaining knowledge about the biological activity of the bioactive components. Yet to know more details on the basis of possible modes of action and binding interaction between the ligands and different binding sites of protein molecules (Emon, Jahan, et al., 2020). To forecast the conceivable biological (antidiabetic, hypolipidemic and hepatoprotective) mode of G. applanatum have auspicious binging affinity toward dual ppara/g agonist protein, farnesoid X receptor (FXR) agonist protein, hepatitis C virus NS3/4A protease inhibitors, and human IgG Fc domain receptor. The pharmacological and computational profiles of the G. applanatum are found good with very mild toxicity, which is essential to be a possible drug.

| CON CLUS ION
The pharmacological results of the present study exhibited that Ganoderma applanatum mushroom exerts antidiabetic, hypolipidemic, and hepatoprotective effects that are also validated and showed by computational investigations. This could be due to the presence of different types of active constituents in this mushroom.
So, Ganoderma applanatum mushroom may serve as an antidiabetic, hypolipidemic, and hepatoprotective agent to diabetic, hyperlipidemia, and hepatic patients with high glucose level and other diabetic complications.

ACK N OWLED G M ENTS
We are thankful to the Research Cell, University of Chittagong and Department of Pharmacy, University of Chittagong for technical and Laboratory support.

CO N FLI C T O F I NTE R E S T
The authors declare no known conflict of interest to any organization or individuals.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data have been incorporated into the manuscript.