# Ameliorating Effect ofMalva neglecta Wallr on Obesity and Diabetes in Wistar Rats: A Mechanistic Study

Authors: Muhammad Furqan Akhtar; Umar Farooq; Ammara Saleem; Mohammad Saleem; Md Habibur Rahman; Ghulam Md Ashraf
Journal: BioMed Research International
 (2022)

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## Abstract

A high caloric food causes deposition of fats that may progress to obesity. Obesity is a risk factor for various metabolic and cardiovascular diseases, including but limited to diabetes mellitus. This study is aimed at determining the ameliorating effect ofMalva Neglecta wallr aqueous-methanolic extract (MNME) on obesity and diabetes in Wistar rats. The MNME was chemically characterized by high-performance liquid chromatography (HPLC). The plant extract was evaluated by in vitro α-amylase inhibition and DPPH scavenging activities. Obesity was induced by administering high sugar and fat diet (HSFD) to rats for six weeks, followed by intraperitoneal injection of alloxan monohydrate (150 mg/kg) to induce diabetes. Oral treatments with MNME 250, 500, and 750 mg/kg/day were given to diabetic obese rats for 14 days. The HPLC analysis showed the presence of phenolic acids and flavonoids. The plant extract showed significant antioxidant (P<0.001) and alpha-amylase (P<0.0001) inhibition activities. The administration of MNME displayed a considerable decrease in fasting blood glucose, body weight, liver function tests, urea, cholesterol, leptin, and insulin levels in diabetic obese rats as compared to the disease control group and maximum effect were observed at 750 mg/kg/day of MNME. The MNME significantly increased (P<0.05−0.001) the levels of GSH, SOD, and CAT in the liver, kidney, and pancreas while notably (P<0.05−0.001) reduced the malondialdehyde level in kidney and pancreas of diabetic obese rats in contrast to disease control rats. This experimental study concludes that the MNME had exhibited antiobesity and antidiabetic activities through reduction of oxidative stress, leptin, α-amylase activity, and insulin resistance due to the presence of phenolic acid and flavonoid compounds.


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## Body

## 1. Introduction

The human body cannot compensate for the effects of overnutrition and concomitant caloric imbalance which leads to obesity. Obesity is one of the leading contributors to illnesses among global population besides infectious diseases. More than 300 million people in the world are affected by obesity, and the threat is continuously mounting in the upcoming generations [1]. There is a substantial global rise in obesity-linked comorbidities and mortalities; however, the exact etiology is ambiguous yet. Various factors contribute to the global prevalence of obesity like consistent availability of high energy (fast) foods, genetic susceptibility, and diminished physical activity [2]. Obesity is persuaded because of raising adipose tissue mass that corresponds to the division of fat cells via adipogenesis and augmented stacking of triglycerides in cytoplasm [3].Body mass index (BMI) can be used to determine obesity. In Caucasians, BMI of 18.5-24.9 is the desirable range of healthy individuals. ABM1≥30kg/m2 is indicative of obesity among all age groups. BMI has a strong association with insulin resistance and diabetes. Other methods of obesity estimation are waist-to-hip ratio (WHR), skinfold method, and bioelectrical impedance analysis [4]. Obesity is considered as an independent high-risk factor responsible for various metabolic and cardiovascular diseases such as diabetes mellitus (DM), hypertension, ischemic stroke, and coronary heart diseases. Obesity is clinically identified by dyslipidemia, insulin resistance, and impaired glucose tolerance [5].The DM is metabolic syndrome associated with impaired fat, carbohydrates, and protein metabolism, well-recognized by polyuria, hyperglycemia, dyslipidemia, polyphagia, polydipsia, weight loss, etc. [6]. Moreover, the reduced insulin synthesis from pancreatic β cells and/or insulin resistance occurs in DM. Most probable complications of this multifaceted disease are diabetic neuropathy, nephropathy, retinopathy, ketonuria, and cardiovascular problems like atherosclerosis [7]. Among the deadliest diseases, diabetes is holding 7th position. The incidence of DM is growing at an alarming pace, and it is assumed that a huge population in China, India, and United States is at risk of this syndrome in 2030 [8]. Insulin resistance is an etiopathological feature of type 2 DM (T2DM), a common metabolic disorder associated with obesity. The obesity-induced insulin resistance causes T2DM via stimulating allostatic burden of the pancreas. Due to a high prevalence and associated comorbidities, there is urgency to sort out a suitable drug, preferably from herbal sources to either delay or prevent the diabetes progression [9].Malva neglecta wallr (family: Malvaceae) is also called common mallow or dwarf mallow in the United States. Its local name is Sonchal in Pakistan. It is annually growing herb from Malva genus [10]. In rural areas, it is consumed as food while on the other hand, it is traditionally used for several health-related problems like wound healing, inflammation, cancer, general weakness, stomach ache, muscular pains, and respiratory diseases [11]. It is rich in alkaloids, tannins, anthocyanin, oils, kaempferol, and hydroxycinnamic acid. Previous studies reported the hepatoprotective, anti-inflammatory, glucose lowering, and antiulcerogenic potential of the plant. M. neglecta is mostly devoid of toxicological properties except nitrate toxicity that rarely occurs due to excessive ingestion [12]. The current project was aimed to evaluate antiobesity and antidiabetic potential of M. neglecta in diabetic obese rats. Furthermore, the quantitative estimation of secondary metabolites and antioxidative properties of plant extract was also assessed.


## 2. Materials and Methods

The chemicals such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), methanol, ascorbic acid, sodium bicarbonate, dinitro-salicylic acid (DNS), sodium potassium tartrate, hydrogen peroxide, trichloroacetic acid (TCA),α-amylase were acquired from Uni Chem®, UK while Folin-ciocalteau (FC) reagent, acarbose, diethyl ether, metformin, pyrogallol, di-thiobis-nitro-benzoic acid (DTNB), starch, alloxan monohydrate were purchased from Sigma Aldrich®, USA.


### 2.1. Extraction and Phytochemical Analysis

The whole plantM. neglecta (5 kg) was collected from super highway Okara, Pakistan. The plant was identified (voucher no. 24-01-19) by a botanist at University of Agriculture, Faisalabad, and the plant sample was placed in the herbarium bank for future reference. The whole plant was shade dried, crushed, and coarsely ground. The coarse powder was macerated in aqueous methanol (30 : 70) for 14 days along with regular shaking. It was filtered by muslin cloth and then through a Whatman filter paper (11 μm pore size). The filtrate was concentrated with a rotary evaporator at 40°C under reduced pressure. The concentrated extract was placed in an incubator at 37°C to get a semisolid mass, and percentage yield was calculated. The M. neglecta aqueous-methanol extract (MNME) was stored at 2-8°C in a refrigerator for future use [13].The qualitative phytochemical analysis was performed to detect the presence of alkaloids, flavonoids, tannins, carbohydrates, protein, saponins, fats, phenolic acids, glycosides, gums and mucilage, and steroids by earlier standard procedures [14].


### 2.2. Determination of Total Phenolic (TPC) and Total Flavonoid Contents (TFC)

The TPC estimation of MNME was carried out by FC method. In a test tube, 0.5 ml of 1 mg/ml plant extract solution was mixed with 2 ml of diluted FC reagent (10%v/v), neutralized with Na2CO3 solution (8.5% w/v), and then incubated at room temperature for 30 min with intermittent shakings to produce distinct blue color. Absorbance was taken by UV-Vis spectrophotometer at 765 nm. For plotting calibration curve, gallic acid served as standard. The TPC of plant extract was expressed as mg/g gallic acid equivalent of plant extract [15].For TFC, the colorimetric method was adopted as described earlier [16]. A 1 ml solvent-free extract was mixed in 3 ml of methanol and incubated for 5 min. Then, 200 μl of 10% w/v aluminum chloride (AlCl3) was added and incubated for 6 min at 25°C. Afterward, 200 μl of 10% potassium acetate (CH3COOK) (1 : 10 w/v in water) was added to it, and the mixture was shaken vigorously to produce pink color. Quercetin was used as standard for plotting calibration curve. Absorbance was measured at 420 nm. The TFC of plant extract was expressed as mg/g quercetin equivalent of plant extract [17].


### 2.3. Quantitative Analysis

For the quantitative estimation of phenolic and flavonoids in the plant extract, reverse-phase high-performance chromatography (HPLC) was performed according to the previous method [7]. First, the sample was prepared by dissolving 50 mg extract in 40 ml of 60% aqueous methanol solution. A 10 ml 6 M HCl was added to and mixed with the sample for 5 min. The sample was heated to 90°C for 2 h. About 20 μl of the sample solution was injected to HPLC equipped with Shim Pack CLC-ODS (C18) column (25cm×4.6mm, 5 μm). The mobile phase was comprised of two gradients, A (H2O: acetic acid-94 : 6 at pH 2.27) and B (acetonitrile 100%) which ran from 0−15min=15% B, 15−30min=45% B, and 30−45=100% B at a flow rate of 1 ml/min. Absorbance was taken with UV-Vis detector (SPD-10AV) at 256 nm. Phytochemicals were detected and quantified by comparing with the retention time of respective standards [18].


### 2.4. In Vitro Antioxidant Activity

The DPPH inhibition assay was conducted to evaluate the antioxidant activity of the plant extract. To prepare 0.4 mM solution, DPPH (40 mg) was dissolved in 100 ml methanol. The plant extract (1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.0156 mg/ml) solutions were prepared in methanol while ascorbic acid served as a standard. One milliliter each of methanol and test solution and 2 ml DPPH solution were mixed and placed in the dark at 25°C for 30 min. The absorbance of the solutions was recorded at 517 nm [19]. The test was performed for three times to calculate mean percentage DPPH inhibition.


### 2.5. In Vitro Antidiabetic Activity

In order to elucidate the antidiabetic activity of plant extract, the in vitroα-amylase inhibitory activity was performed by adopting earlier procedure [20]. The DNS (2.21 g) was sonicated in 75 ml 0.5 N NaOH solution at 70°C for 30 min. In that solution, a 30% w/v sodium potassium tartrate was poured. To prepare starch solution, starch (0.02 g) was dissolved in 20 ml of 20 mM sodium phosphate buffer (pH 6.9). A 25.3 mg of α-amylase was added and mixed with 100 ml DW to prepare the starch solution. About 1 ml of plant extract (1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.0156 mg/ml) was added to 1 ml α-amylase solution and was subjected to incubation for 10 min at 25°C followed by addition and thorough mixing of 2 ml starch solution. The resulting solution was incubated for 30 min at 37°C and 1 ml stop solution (DNS coloring reagent) was added. The reaction mixture was incubated for 5 min in boiling water bath. The solution was then cooled to 25°C, and final volume was adjusted to 10 ml with distilled water. The absorbance was determined at 540 nm, and %age inhibition was estimated. The same procedure was followed for the control solution (methanol instead of plant extract) to determine 100% enzymatic activity. Acarbose was adopted as standard α-amylase inhibitor [21].


### 2.6. Experimental Animals

Wistar rats of either sex (two weeks old) were obtained and acclimatized in the animal house of the Riphah Institute of Pharmaceutical Sciences (RIPS). All rats were provided with standard laboratory conditions (25±3°C, humidity 55-70%, and 12 h light and dark cycles). The animal experiment was approved by a Research Ethical Committee of RIPS with an authorized number of REC/RIPS-LHR/011 [22].


### 2.7. Induction of Obesity

The rats were provided with high fat/high sugar diet (HSFD) (fats 43%, carbohydrates 40%, and proteins 17%) for 16 weeks to induced obesity while normal control group rats were given a standard pallet diet (fats 10%, carbohydrates 60%, and proteins 13%)ad libitum [23, 24]. The animals exhibiting 16% or more increase in body weight as compared to normal control rats were designated as obese rats and selected for induction of diabetes [25]. The study was performed by following the guidelines of National Institute of Health (NIH). All the requisite attempts were made to avoid/reduce animal suffering.


### 2.8. Induction of Diabetes

The same obese rats were given a single dose of alloxan monohydrate (150 mg/kg) by intraperitoneal route to compromise beta-cell function alongside reduced insulin sensitivity for successful induction of diabetes [26]. Experimental animals were subjected to fasting for 18 h prior to the administration of alloxan. Test animals were orally administered with 10% w/v glucose solution to prevent alloxan-induced hypoglycemia. Animals with a blood glucose level of more than 200 mg/dl were considered to be diabetic [27].


#### 2.8.1. Study Design

All 30 diabetic animals were randomly divided into five groups (n=6) while the nondiabetic/nonobese animals (n=06) were kept as a normal control group. Treatment was started on the 3rd day of alloxan administration after successful diabetes induction and kept on for 14 days. Drug solutions (2 ml/kg) were given once a day by the oral gavage at 10 a.m. daily. The dose selection of MNME for animals was based on previous investigations [28].Group 1: normal control group was given 2 ml/kg/day of normal saline.Group 2: diseased control group was given 2 ml/kg/day of normal saline.Group 3: standard group was given metformin 500 mg/kg/day.Group 4: treatment group was given MNME 250 mg/kg/day.Group 5: treatment group was given MNME 500 mg/kg/day.Group 6: treatment group was given MNME 750 mg/kg/day.


#### 2.8.2. Estimation of Blood Glucose Level and Weight Variation

Glucose levels in the blood were measured before the administration of alloxan, after 48 h of injection, and 7th and 14th day posttreatment. Blood was taken from the tail tip to record blood glucose level with an Accu-check® glucometer. To find the effect of MNME on body weight, the body weights of rats were observed at basal, zero, 7th, and 14th day of experimental treatments.


#### 2.8.3. Serum Insulin and Other Biochemical Analyses

After 14 days of treatment, the blood was collected by heart puncture from preanesthetized with diethyl ether. Serum was obtained from the blood by centrifugation for 15 min at 2500 rpm at 4°C. Radioimmunoassay method was applied by using commercially available DSL-1600 insulin kit (Diagnostic Systems Laboratories, Inc., USA) to estimate the insulin concentration in the blood serum which was expressed asμlU/ml [29].Liver and kidney function tests such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine levels were analyzed by standard methods in the automated chemistry analyzer. Lipid profiling was done to estimate LDL, HDL, triglycerides, and cholesterol levels in diabetic animals by standard methods [30]. Glycosylated hemoglobin (HbA1c) was estimated from the blood with glycosylated hemoglobin kits by a Nycocard reader (Axis shield®, Norway).


#### 2.8.4. Histopathological Studies

After 14 days, animals anesthetized with diethyl ether were sacrificed by cervical dislocation. The pancreas, liver, and kidney were dissected from the experimental animals, thoroughly washed with ice-cold normal saline, and then preserved in formaldehyde (10%) followed by embedding in paraffin wax [31]. Organs were sliced to 5 μm thickness with a microtome, and then, these slides were stained with hematoxylin and eosin for histopathological investigations.


#### 2.8.5. Assessment of Oxidative Stress

In order to assess the effect of therapy on oxidative stress parameters in diabetic and obese rats, tissue homogenates (10%w/v) were prepared. For this purpose, tissues (1 g) of pancreas, kidney, and liver were taken from sacrificed animals and processed by following earlier procedure [32]. Protein content was measured in tissue homogenates by Lowry’s method. Bovine serum albumin served as standard, and absorbance was checked at 620 nm.(1) Estimation of Catalase (CAT) Activity. The CAT activity was estimated by H2O2 decomposition method [33]. The supernatant (1 ml) was added to a cuvette already containing 1.95 ml of phosphate buffer (50 mM, pH 7.0) and 1 ml of hydrogen peroxide (H2O2; 30 mM). Changes in absorbance were determined every 5 S for 30 S at 240 nm. Indeed, one CAT activity unit was equal to 1 M of H2O2 decomposed per min at 25°C.(2) Estimation of Malondialdehyde (MDA). The MDA was determined by thiobarbituric acid (TBA) according to the previous procedure [33]. In this procedure, 2.5 ml TCA (10% w/v) was taken in a centrifuge tube along with 0.5 ml supernatant of the tissue homogenates and placed at 100°C for 15 min, and then, these tubes were cooled down to 25°C. The mixture was centrifuged at 1000 rpm at 4°C for 10 min. Afterward, 2 ml supernatant was transferred from each centrifuge tube to test tubes which already contained 1 ml of TBA solution (0.67% w/v). These test tubes were then put at 100°C for 15 min, and absorbance was measured at 532 nm after cooling down the test tubes.(3) Superoxide Dismutase (SOD) Activity. The SOD activity was determined according to a previous method based on autooxidation of pyrogallol in the alkaline medium [34]. Every 3 ml mixture contained 2.8 ml potassium phosphate buffer (0.1 M, pH 7.4), 0.1 ml pyrogallol (2.6 mM in 10 mM HCl) solution, and 0.1 ml tissue homogenate. Changes in the absorbance were measured at 325 nm after every 30 S for 5 min. Under the assay conditions, each unit of SOD was equivalent to the amount of enzyme needed to achieve 50% inhibition of pyrogallol autoxidation.(4) Glutathione (GSH) Level. For GSH estimation, 1 ml tissue homogenate, 1 ml 10% TCA, and 4 ml of PBS were mixed followed by addition of 0.5 ml DTNB reagent. The level of GSH was estimated from the absorbance determined at 412 nm [34].


### 2.9. Statistical Analysis

The results were presented in the form ofMean±SEM. One-way and two-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test was applied to the data using Graphpad Prism 5 (CA, San Diego, USA). The results were considered moderately significant at P<0.01, while these were considered highly significant at P<0.001.


## 2.1. Extraction and Phytochemical Analysis

The whole plantM. neglecta (5 kg) was collected from super highway Okara, Pakistan. The plant was identified (voucher no. 24-01-19) by a botanist at University of Agriculture, Faisalabad, and the plant sample was placed in the herbarium bank for future reference. The whole plant was shade dried, crushed, and coarsely ground. The coarse powder was macerated in aqueous methanol (30 : 70) for 14 days along with regular shaking. It was filtered by muslin cloth and then through a Whatman filter paper (11 μm pore size). The filtrate was concentrated with a rotary evaporator at 40°C under reduced pressure. The concentrated extract was placed in an incubator at 37°C to get a semisolid mass, and percentage yield was calculated. The M. neglecta aqueous-methanol extract (MNME) was stored at 2-8°C in a refrigerator for future use [13].The qualitative phytochemical analysis was performed to detect the presence of alkaloids, flavonoids, tannins, carbohydrates, protein, saponins, fats, phenolic acids, glycosides, gums and mucilage, and steroids by earlier standard procedures [14].


## 2.2. Determination of Total Phenolic (TPC) and Total Flavonoid Contents (TFC)

The TPC estimation of MNME was carried out by FC method. In a test tube, 0.5 ml of 1 mg/ml plant extract solution was mixed with 2 ml of diluted FC reagent (10%v/v), neutralized with Na2CO3 solution (8.5% w/v), and then incubated at room temperature for 30 min with intermittent shakings to produce distinct blue color. Absorbance was taken by UV-Vis spectrophotometer at 765 nm. For plotting calibration curve, gallic acid served as standard. The TPC of plant extract was expressed as mg/g gallic acid equivalent of plant extract [15].For TFC, the colorimetric method was adopted as described earlier [16]. A 1 ml solvent-free extract was mixed in 3 ml of methanol and incubated for 5 min. Then, 200 μl of 10% w/v aluminum chloride (AlCl3) was added and incubated for 6 min at 25°C. Afterward, 200 μl of 10% potassium acetate (CH3COOK) (1 : 10 w/v in water) was added to it, and the mixture was shaken vigorously to produce pink color. Quercetin was used as standard for plotting calibration curve. Absorbance was measured at 420 nm. The TFC of plant extract was expressed as mg/g quercetin equivalent of plant extract [17].


## 2.3. Quantitative Analysis

For the quantitative estimation of phenolic and flavonoids in the plant extract, reverse-phase high-performance chromatography (HPLC) was performed according to the previous method [7]. First, the sample was prepared by dissolving 50 mg extract in 40 ml of 60% aqueous methanol solution. A 10 ml 6 M HCl was added to and mixed with the sample for 5 min. The sample was heated to 90°C for 2 h. About 20 μl of the sample solution was injected to HPLC equipped with Shim Pack CLC-ODS (C18) column (25cm×4.6mm, 5 μm). The mobile phase was comprised of two gradients, A (H2O: acetic acid-94 : 6 at pH 2.27) and B (acetonitrile 100%) which ran from 0−15min=15% B, 15−30min=45% B, and 30−45=100% B at a flow rate of 1 ml/min. Absorbance was taken with UV-Vis detector (SPD-10AV) at 256 nm. Phytochemicals were detected and quantified by comparing with the retention time of respective standards [18].


## 2.4. In Vitro Antioxidant Activity

The DPPH inhibition assay was conducted to evaluate the antioxidant activity of the plant extract. To prepare 0.4 mM solution, DPPH (40 mg) was dissolved in 100 ml methanol. The plant extract (1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.0156 mg/ml) solutions were prepared in methanol while ascorbic acid served as a standard. One milliliter each of methanol and test solution and 2 ml DPPH solution were mixed and placed in the dark at 25°C for 30 min. The absorbance of the solutions was recorded at 517 nm [19]. The test was performed for three times to calculate mean percentage DPPH inhibition.


## 2.5. In Vitro Antidiabetic Activity

In order to elucidate the antidiabetic activity of plant extract, the in vitroα-amylase inhibitory activity was performed by adopting earlier procedure [20]. The DNS (2.21 g) was sonicated in 75 ml 0.5 N NaOH solution at 70°C for 30 min. In that solution, a 30% w/v sodium potassium tartrate was poured. To prepare starch solution, starch (0.02 g) was dissolved in 20 ml of 20 mM sodium phosphate buffer (pH 6.9). A 25.3 mg of α-amylase was added and mixed with 100 ml DW to prepare the starch solution. About 1 ml of plant extract (1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.0156 mg/ml) was added to 1 ml α-amylase solution and was subjected to incubation for 10 min at 25°C followed by addition and thorough mixing of 2 ml starch solution. The resulting solution was incubated for 30 min at 37°C and 1 ml stop solution (DNS coloring reagent) was added. The reaction mixture was incubated for 5 min in boiling water bath. The solution was then cooled to 25°C, and final volume was adjusted to 10 ml with distilled water. The absorbance was determined at 540 nm, and %age inhibition was estimated. The same procedure was followed for the control solution (methanol instead of plant extract) to determine 100% enzymatic activity. Acarbose was adopted as standard α-amylase inhibitor [21].


## 2.6. Experimental Animals

Wistar rats of either sex (two weeks old) were obtained and acclimatized in the animal house of the Riphah Institute of Pharmaceutical Sciences (RIPS). All rats were provided with standard laboratory conditions (25±3°C, humidity 55-70%, and 12 h light and dark cycles). The animal experiment was approved by a Research Ethical Committee of RIPS with an authorized number of REC/RIPS-LHR/011 [22].


## 2.7. Induction of Obesity

The rats were provided with high fat/high sugar diet (HSFD) (fats 43%, carbohydrates 40%, and proteins 17%) for 16 weeks to induced obesity while normal control group rats were given a standard pallet diet (fats 10%, carbohydrates 60%, and proteins 13%)ad libitum [23, 24]. The animals exhibiting 16% or more increase in body weight as compared to normal control rats were designated as obese rats and selected for induction of diabetes [25]. The study was performed by following the guidelines of National Institute of Health (NIH). All the requisite attempts were made to avoid/reduce animal suffering.


## 2.8. Induction of Diabetes

The same obese rats were given a single dose of alloxan monohydrate (150 mg/kg) by intraperitoneal route to compromise beta-cell function alongside reduced insulin sensitivity for successful induction of diabetes [26]. Experimental animals were subjected to fasting for 18 h prior to the administration of alloxan. Test animals were orally administered with 10% w/v glucose solution to prevent alloxan-induced hypoglycemia. Animals with a blood glucose level of more than 200 mg/dl were considered to be diabetic [27].


### 2.8.1. Study Design

All 30 diabetic animals were randomly divided into five groups (n=6) while the nondiabetic/nonobese animals (n=06) were kept as a normal control group. Treatment was started on the 3rd day of alloxan administration after successful diabetes induction and kept on for 14 days. Drug solutions (2 ml/kg) were given once a day by the oral gavage at 10 a.m. daily. The dose selection of MNME for animals was based on previous investigations [28].Group 1: normal control group was given 2 ml/kg/day of normal saline.Group 2: diseased control group was given 2 ml/kg/day of normal saline.Group 3: standard group was given metformin 500 mg/kg/day.Group 4: treatment group was given MNME 250 mg/kg/day.Group 5: treatment group was given MNME 500 mg/kg/day.Group 6: treatment group was given MNME 750 mg/kg/day.


### 2.8.2. Estimation of Blood Glucose Level and Weight Variation

Glucose levels in the blood were measured before the administration of alloxan, after 48 h of injection, and 7th and 14th day posttreatment. Blood was taken from the tail tip to record blood glucose level with an Accu-check® glucometer. To find the effect of MNME on body weight, the body weights of rats were observed at basal, zero, 7th, and 14th day of experimental treatments.


### 2.8.3. Serum Insulin and Other Biochemical Analyses

After 14 days of treatment, the blood was collected by heart puncture from preanesthetized with diethyl ether. Serum was obtained from the blood by centrifugation for 15 min at 2500 rpm at 4°C. Radioimmunoassay method was applied by using commercially available DSL-1600 insulin kit (Diagnostic Systems Laboratories, Inc., USA) to estimate the insulin concentration in the blood serum which was expressed asμlU/ml [29].Liver and kidney function tests such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine levels were analyzed by standard methods in the automated chemistry analyzer. Lipid profiling was done to estimate LDL, HDL, triglycerides, and cholesterol levels in diabetic animals by standard methods [30]. Glycosylated hemoglobin (HbA1c) was estimated from the blood with glycosylated hemoglobin kits by a Nycocard reader (Axis shield®, Norway).


### 2.8.4. Histopathological Studies

After 14 days, animals anesthetized with diethyl ether were sacrificed by cervical dislocation. The pancreas, liver, and kidney were dissected from the experimental animals, thoroughly washed with ice-cold normal saline, and then preserved in formaldehyde (10%) followed by embedding in paraffin wax [31]. Organs were sliced to 5 μm thickness with a microtome, and then, these slides were stained with hematoxylin and eosin for histopathological investigations.


### 2.8.5. Assessment of Oxidative Stress

In order to assess the effect of therapy on oxidative stress parameters in diabetic and obese rats, tissue homogenates (10%w/v) were prepared. For this purpose, tissues (1 g) of pancreas, kidney, and liver were taken from sacrificed animals and processed by following earlier procedure [32]. Protein content was measured in tissue homogenates by Lowry’s method. Bovine serum albumin served as standard, and absorbance was checked at 620 nm.(1) Estimation of Catalase (CAT) Activity. The CAT activity was estimated by H2O2 decomposition method [33]. The supernatant (1 ml) was added to a cuvette already containing 1.95 ml of phosphate buffer (50 mM, pH 7.0) and 1 ml of hydrogen peroxide (H2O2; 30 mM). Changes in absorbance were determined every 5 S for 30 S at 240 nm. Indeed, one CAT activity unit was equal to 1 M of H2O2 decomposed per min at 25°C.(2) Estimation of Malondialdehyde (MDA). The MDA was determined by thiobarbituric acid (TBA) according to the previous procedure [33]. In this procedure, 2.5 ml TCA (10% w/v) was taken in a centrifuge tube along with 0.5 ml supernatant of the tissue homogenates and placed at 100°C for 15 min, and then, these tubes were cooled down to 25°C. The mixture was centrifuged at 1000 rpm at 4°C for 10 min. Afterward, 2 ml supernatant was transferred from each centrifuge tube to test tubes which already contained 1 ml of TBA solution (0.67% w/v). These test tubes were then put at 100°C for 15 min, and absorbance was measured at 532 nm after cooling down the test tubes.(3) Superoxide Dismutase (SOD) Activity. The SOD activity was determined according to a previous method based on autooxidation of pyrogallol in the alkaline medium [34]. Every 3 ml mixture contained 2.8 ml potassium phosphate buffer (0.1 M, pH 7.4), 0.1 ml pyrogallol (2.6 mM in 10 mM HCl) solution, and 0.1 ml tissue homogenate. Changes in the absorbance were measured at 325 nm after every 30 S for 5 min. Under the assay conditions, each unit of SOD was equivalent to the amount of enzyme needed to achieve 50% inhibition of pyrogallol autoxidation.(4) Glutathione (GSH) Level. For GSH estimation, 1 ml tissue homogenate, 1 ml 10% TCA, and 4 ml of PBS were mixed followed by addition of 0.5 ml DTNB reagent. The level of GSH was estimated from the absorbance determined at 412 nm [34].


## 2.8.1. Study Design

All 30 diabetic animals were randomly divided into five groups (n=6) while the nondiabetic/nonobese animals (n=06) were kept as a normal control group. Treatment was started on the 3rd day of alloxan administration after successful diabetes induction and kept on for 14 days. Drug solutions (2 ml/kg) were given once a day by the oral gavage at 10 a.m. daily. The dose selection of MNME for animals was based on previous investigations [28].Group 1: normal control group was given 2 ml/kg/day of normal saline.Group 2: diseased control group was given 2 ml/kg/day of normal saline.Group 3: standard group was given metformin 500 mg/kg/day.Group 4: treatment group was given MNME 250 mg/kg/day.Group 5: treatment group was given MNME 500 mg/kg/day.Group 6: treatment group was given MNME 750 mg/kg/day.


## 2.8.2. Estimation of Blood Glucose Level and Weight Variation

Glucose levels in the blood were measured before the administration of alloxan, after 48 h of injection, and 7th and 14th day posttreatment. Blood was taken from the tail tip to record blood glucose level with an Accu-check® glucometer. To find the effect of MNME on body weight, the body weights of rats were observed at basal, zero, 7th, and 14th day of experimental treatments.


## 2.8.3. Serum Insulin and Other Biochemical Analyses

After 14 days of treatment, the blood was collected by heart puncture from preanesthetized with diethyl ether. Serum was obtained from the blood by centrifugation for 15 min at 2500 rpm at 4°C. Radioimmunoassay method was applied by using commercially available DSL-1600 insulin kit (Diagnostic Systems Laboratories, Inc., USA) to estimate the insulin concentration in the blood serum which was expressed asμlU/ml [29].Liver and kidney function tests such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine levels were analyzed by standard methods in the automated chemistry analyzer. Lipid profiling was done to estimate LDL, HDL, triglycerides, and cholesterol levels in diabetic animals by standard methods [30]. Glycosylated hemoglobin (HbA1c) was estimated from the blood with glycosylated hemoglobin kits by a Nycocard reader (Axis shield®, Norway).


## 2.8.4. Histopathological Studies

After 14 days, animals anesthetized with diethyl ether were sacrificed by cervical dislocation. The pancreas, liver, and kidney were dissected from the experimental animals, thoroughly washed with ice-cold normal saline, and then preserved in formaldehyde (10%) followed by embedding in paraffin wax [31]. Organs were sliced to 5 μm thickness with a microtome, and then, these slides were stained with hematoxylin and eosin for histopathological investigations.


## 2.8.5. Assessment of Oxidative Stress

In order to assess the effect of therapy on oxidative stress parameters in diabetic and obese rats, tissue homogenates (10%w/v) were prepared. For this purpose, tissues (1 g) of pancreas, kidney, and liver were taken from sacrificed animals and processed by following earlier procedure [32]. Protein content was measured in tissue homogenates by Lowry’s method. Bovine serum albumin served as standard, and absorbance was checked at 620 nm.(1) Estimation of Catalase (CAT) Activity. The CAT activity was estimated by H2O2 decomposition method [33]. The supernatant (1 ml) was added to a cuvette already containing 1.95 ml of phosphate buffer (50 mM, pH 7.0) and 1 ml of hydrogen peroxide (H2O2; 30 mM). Changes in absorbance were determined every 5 S for 30 S at 240 nm. Indeed, one CAT activity unit was equal to 1 M of H2O2 decomposed per min at 25°C.(2) Estimation of Malondialdehyde (MDA). The MDA was determined by thiobarbituric acid (TBA) according to the previous procedure [33]. In this procedure, 2.5 ml TCA (10% w/v) was taken in a centrifuge tube along with 0.5 ml supernatant of the tissue homogenates and placed at 100°C for 15 min, and then, these tubes were cooled down to 25°C. The mixture was centrifuged at 1000 rpm at 4°C for 10 min. Afterward, 2 ml supernatant was transferred from each centrifuge tube to test tubes which already contained 1 ml of TBA solution (0.67% w/v). These test tubes were then put at 100°C for 15 min, and absorbance was measured at 532 nm after cooling down the test tubes.(3) Superoxide Dismutase (SOD) Activity. The SOD activity was determined according to a previous method based on autooxidation of pyrogallol in the alkaline medium [34]. Every 3 ml mixture contained 2.8 ml potassium phosphate buffer (0.1 M, pH 7.4), 0.1 ml pyrogallol (2.6 mM in 10 mM HCl) solution, and 0.1 ml tissue homogenate. Changes in the absorbance were measured at 325 nm after every 30 S for 5 min. Under the assay conditions, each unit of SOD was equivalent to the amount of enzyme needed to achieve 50% inhibition of pyrogallol autoxidation.(4) Glutathione (GSH) Level. For GSH estimation, 1 ml tissue homogenate, 1 ml 10% TCA, and 4 ml of PBS were mixed followed by addition of 0.5 ml DTNB reagent. The level of GSH was estimated from the absorbance determined at 412 nm [34].


## 2.9. Statistical Analysis

The results were presented in the form ofMean±SEM. One-way and two-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test was applied to the data using Graphpad Prism 5 (CA, San Diego, USA). The results were considered moderately significant at P<0.01, while these were considered highly significant at P<0.001.


## 3. Results

The %age yield of the plant extract was 5.67%. The qualitative phytochemical analysis of MNME exhibited the presence of alkaloids, saponins, flavonoids, phenols, tannins, and steroids. The MNME contained TPC138.7±4.3mg/g gallic acid equivalent and TFC 48.8±5.2mg/g quercetin equivalent.


### 3.1. HPLC Analysis

The HPLC analysis of MNME showed the presence of various flavonoids and phenolic acids such as quercetin, kaempferol, gallic acid, chlorogenic acid, syringic acid, and cinnamic acid in the chromatogram. Kaempferol was present in the highest amount (128.14 ppm) followed by cinnamic acid (7.93 ppm) as shown in Table1.Table 1
Phytochemicals detected by HPLC inMalva neglecta methanolic extract.
S#Compound nameRetention timeArea (mV.s)Amount (ppm)1Quercetin3.0531.80.6892Gallic acid4.7801.31.133Chlorogenic acid15.07324.5671.914Syringic acid16.44084.0830.615Cinnamic acid24.793226.6687.936Kaempferol2.200298.000128.14


### 3.2. Free Radical Scavenging Potential

The free radical scavenging potential of MNME increased concentration dependently. The highest inhibition of DPPH was shown at 1 mg/ml (70.0±3.07%). The %age inhibition of DPPH by the MNME was noticeably (P<0.001) different from the ascorbic acid at all respective concentrations as mentioned in Table 2.Table 2
The percent inhibition of DPPH andα-amylase exhibited by aqueous methanol extract of Malva neglecta.
Concentrations (mg/ml)%age inhibition of DPPH%age inhibition ofα-amylaseMalva neglecta extractAscorbic acidMalva neglecta extractAcarbose170.0±3.07∗∗∗85.4±1.2576.4±5.2∗∗∗∗91.2±7.160.559.8±3.11∗∗∗71.3±2.3769.3±4.1∗∗∗∗83.6±6.310.02551.0±1.94∗∗∗66.8±1.2658.4±4.3∗∗∗∗79.2±4.140.12544.1±1.27∗∗∗56.8±1.2252.4±5.6∗∗∗∗72.5±9.130.062540.0±2.27∗∗∗48.9±1.2737.8±6.3∗∗∗∗55.3±4.300.0312535.7±1.17∗∗∗43.4±1.0924.5±7.1∗∗∗∗47.9±6.280.015629.2±1.22∗∗∗36.1±1.1519.4±4.4∗∗∗∗38.2±3.32Data were presented asmean±S.D. (N=03). ∗∗∗P<0.001 significant values were observed in comparison with ascorbic acid. ∗∗∗∗P<0.0001 showed significant values as compared with the standard acarbose.


### 3.3.α-Amylase Inhibitory Activity

To estimate the in vitro hypoglycemic activity of MNME,α-amylase inhibitory assay was performed. The α-amylase activity was concentration dependently inhibited by MNME all tested levels with the highest activity observed at 1 mg/ml (76.4±5.2%) as revealed in Table 2. The %age inhibition of α-amylase activity by MNME was substantially (P<0.001) lower than the acarbose in respective concentration.


### 3.4. Effect of Plant Extract on Hyperglycemia

The fasting blood glucose level (FBG) did not vary in all the experimental groups at basal stage. The FBG was recorded after alloxan administration on the 0, 7, and 14th day. There was a profound rise in blood glucose level in all groups as compared to the normal control group. The FBG in the disease control group was successively raised on the 7 and 14th day in contrast to the normal control rats.Treatment with standard therapy (metformin) and MNME significantly ameliorated the alloxan-induced hyperglycemia on the 7 and 14th day in contrast to the disease control group. There was an insignificant difference between normal control (105.40±7.48mg/dl) and standard group (124.76±5.34mg/dl) on the 14th day. Moreover, all treatment groups were notably different (P<0.001−0.0001) from the normal, diseased, and standard groups on the 7 and 14th day of treatment.The FBG was significantly (P<0.0001) decreased by MNME on the 7 and 14th day of treatment in contrast to the disease control group. The FBG was reduced dose dependently with MNME at 250 (281.30±10.01mg/dl), 500 (248.68±9.02mg/dl), and 750 mg/kg (184.59±7.21mg/dl) on the 14th day. The FBG with MNME at 250 and 500 mg/kg was noticeably (P<0.001) higher than that of MNME 750 mg/kg treated group on the 14th day of treatment. The effect of MNME at 750 mg/kg on FBG at 7 and 14th days was insignificantly different from the standard treatment on respective days as depicted in Figure 1.Figure 1
The effect of Malvaneglecta extract on glucose levels of diabetic and obese rats. Data were shown as mean±S.D. (n=6) and evaluated by two-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗∗∗P<0.00001 showed significant difference in comparison to the disease control group while disease control significantly different &&&&P<0.00001 in comparison to the normal control group. MNME: methanolic extract of Malva neglecta.


#### 3.4.1. Effect on Body Weight

The animal body weights were insignificantly different at preliminary stages of the experiment. Body weights of diseased animals were significantly greater than initial body weight after HSFD for 16 weeks. After alloxan administration, the weight of all rats reduced significantly (P<0.01−0.0001) on the 7th day in contrasts to normal control group. The body weight of treated rats reduced significantly (P<0.05−0.01) at MNME 500 and 750 mg/kg on the 7 and 14th day of treatment in contrast to the disease control group. Likewise, the body weight reduced substantially (P<0.05−0.001) in the standard treatment group on the 7 and 14th day in contrast to disease control group. The effect of MNME at 500 and 700 mg/kg MNME on body weight on the 7 and 14th day was insignificantly different while MNME at 250 mg/kg insignificantly reduced the body weight in contrast to the disease control group on the 7 and 14th day of treatment as shown in Figure 2.Figure 2
The effect ofMalva neglecta extract on body weight of diabetic obese rats. Data were shown as mean±S.D. (n=6) and evaluated by two-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significantly different in comparison with the disease control group while &&P<0.01 and &&&&P<0.0001 in comparison to normal control group. MNME: methanolic extract of Malva neglecta.


#### 3.4.2. Leptin and Lipid Profile

In the current study, leptin level profoundly increased (P<0.05) in the disease control as compared to the normal control group that was significantly restored upon treatment with metformin and MNME at 500 and 750 mg/kg as mentioned in Table 3. The serum levels of triglycerides (TG), total cholesterol (TC), and low-density lipoproteins (LDL) were significantly (P<0.05−0.01) raised in the diseased control group as compared to normal control and were notably (P<0.01−0.001) reduced by metformin and MNME at 500 and 750 mg/kg treatment in diseased rats. In contrast, the high-density lipoprotein (HDL) level was insignificantly different among different treatments (Table 3).Table 3
The effect ofMalva neglecta extract on leptin, lipid profile, HbA1c, and insulin level in diabetic obese rats.
Treatment groupsLeptin (ug/ml)Triglycerides (mmol/L)Cholesterol (mmol/L)LDL (mmol/L)HDL (mmol/L)HbA1c (%)Insulin (μU/ml)Normal control0.042±0.001∗1.67±0.29∗5.88±0.22∗2.69±0.43∗∗0.98±0.065.83±0.61∗∗∗0.36±0.05∗∗∗Disease control0.053±0.0011.78±0.346.87±0.333.61±0.231.02±0.0412.16±0.110.08±0.01Metformin treatment0.038±0.01∗∗∗∗1.63±0.31∗∗∗5.47±0.32∗∗2.53±0.44∗0.95±0.036.46±0.26∗∗∗0.25±0.03∗∗∗MNME 250 mg/kg0.0508±0.0031.70±0.526.11±0.442.95±0.260.98±0.0610.13±0.43∗∗∗0.11±0.05MNME 500 mg/kg0.048±0.002∗∗1.69±0.245.81±0.562.59±0.27∗0.97±0.068.61±0.38∗∗∗0.19±0.02∗∗MNME 750 mg/kg0.045±0.001∗∗∗∗1.66±0.34∗∗5.74±0.41∗2.43±0.33∗0.94±0.046.93±0.73∗∗∗0.19±0.01∗∗Data were shown asmean±S.D. (n=6). ∗∗∗∗P<0.0001, ∗∗∗P<0.001, ∗∗∗P<0.01, and ∗P<0.05 showed significantly different in comparison to the disease control group. MNME: methanolic extract of Malva neglecta; LDL: low density; HDL: high-density lipoprotein; HbA1c: glycosylated hemoglobin.


#### 3.4.3. Effect on HbA1c and Insulin

The percentage of HbA1c was elevated in diabetic rats (12.16±0.11%) as compared to the normal control group. Standard therapy of metformin and all doses of the plant extract significantly decreased (P<0.01) HbA1c percentage. The serum insulin level declined substantially in the diseased rats in contrast to normal control rats. However, the insulin level was notably (P<0.05−0.01) restored with the plant extract at 500 and 750 mg/kg and metformin treatment as compared to the disease control group as demonstrated in Table 3.


#### 3.4.4. Effect on Oxidative Stress Biomarkers

(1) CAT Activity. The activity of CAT was significantly reduced in the liver, kidney, and pancreas of the disease control group than the normal control group. The CAT activity was remarkable (P<0.05−0.001) and dose dependently restored in the liver, kidney, and pancreas of rats treated with MNME at 250, 500, and 750 mg/kg in contrast to disease control rats as shown in Table 4. This showed that MNME had produced the antioxidant effect by increasing the production CAT in the pancreas especially at MNME 500 and 750 mg/kg (Table 4).Table 4
The effect ofMalva neglecta extract on catalase (U/mg of protein) activity in different organs of diabetic obese rats.
GroupsLiverKidneyPancreasNormal control70±2.5∗55±0.5∗30±0.5∗Disease control59±2.639±0.519±0.5Standard treatment65±1.0∗∗50±1.0∗26±1.5∗∗∗MNME 250 mg/kg58±1.5∗∗∗42±0.520±0.5MNME 500 mg/kg61±0.5∗∗47±0.523±0.5∗MNME 750 mg/kg63±0.5∗∗∗50±0.5∗25±1.0∗∗Data were presented asmean±S.D. (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significantly different as compared to the disease control group. MNME: methanolic extract of Malva neglecta.(2) MDA Level. The MDA level was raised noticeably in the liver, kidney, and pancreas of untreated diabetic rats as compared to normal rats. The MDA level was significantly (P<0.01−0.001) reinstated with the plant extract and metformin treated groups in kidney and pancreas as compared to respective tissues of disease control group. The MDA level was insignificantly restored in the liver of metformin and plant extract treated groups as equated with disease control group as shown in Table 5.Table 5
Effect ofMalva neglecta extract on MDA (μg/mg) levels in different organs of diabetic obese rats.
GroupsLiverKidneyPancreasNormal control0.835±0.06∗0.936±0.12∗∗∗0.165±0.11∗∗∗Diseased control1.3±0.221.751±0.130.391±0.02Standard therapy0.942±0.171.311±0.05∗∗∗0.216±0.04∗∗∗MNME 250 mg/kg1.1±0.151.631±0.12∗0.311±0.06∗∗∗MNME 500 mg/kg0.991±0.031.561±0.01∗∗0.291±0.02∗∗∗MNME 750 mg/kg0.983±0.051.393±0.03∗∗∗0.263±0.04∗∗∗Data were presented asmean±standarddeviation (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significant differences in comparison to the disease control group. MNME: methanolic extract of Malva neglecta.(3) SOD Activity. The disease control rats exhibited a decrease in SOD activity in the liver, kidney, and pancreas tissue homogenate as compared to normal control rats which indicated a decline in antioxidant defense. The SOD activity (μg/mg of protein) in the pancreas was significantly (P<0.05−0.001) and dose dependently restored by MNME at 250, 500, and 750 mg/kg in the liver, kidney, and pancreas tissues. A rise in SOD activity was also notable in all tested tissues of metformin-treated group, in contrast to the disease control group, as shown in Table 6.Table 6
The effect ofMalva neglecta extract on superoxide dismutase (U/mg of protein) activity in different organs of diabetic obese rats.
TreatmentsLiverKidneyPancreasNormal control8.1±0.5∗∗14.2±0.5∗∗∗13±0.01∗∗∗Disease control5.8±0.57.81±0.58.13±0.5Standard treatment7.1±0.5∗12.1±0.5∗∗∗12.3±0.5∗∗∗MNME 250 mg/kg6.31±0.59.8±0.5∗∗∗8.91±0.5MNME 500 mg/kg6.6±0.510.1±0.5∗∗∗9.2±0.5∗MNME 750 mg/kg7.1±0.5∗11.6±1.0∗∗∗9.6±0.5∗∗Data were presented asmean±S.D. (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significantly different in comparison to the disease control group. MNME: methanolic extract of Malva neglecta.


#### 3.4.5. GSH Level

The level of GSH in the liver, kidney, and pancreas was noticeably (P<0.05) decreased in diseased rats as compared to the normal control group; however, it was significantly (P<0.01−0.001) restored in treatment groups. The GSH level was notably raised in the liver of treated animals with metformin and MNME 750 mg/kg treatment. The GSH level was notably restored in kidney and pancreas of treated rats with metformin and MNME 750 mg/kg as depicted in Table 7.Table 7
Effect ofMalva neglecta on reduced glutathione (μM/mg) level in different organs of diabetic obese rats.
GroupsLiverKidneyPancreasNormal control22.3±1.5∗∗∗15.2±0.5∗∗∗8.6±0.3∗∗∗Diseased control15.6±0.59.1±0.43.2±0.4Standard therapy19.5±0.7∗∗14.3±0.4∗∗6.1±0.5∗∗∗MNME 250 mg/kg16.3±0.310.4±0.63.0±0.2MNME 500 mg/kg18.2±0.412.2±0.3∗4.2±0.4∗MNME 750 mg/kg19.4±0.5∗∗13.4±0.6∗∗6.2±0.5∗∗Data were presented asmean±standarddeviation (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significant differences in comparison to the disease control group. MNME: methanolic extract of Malva neglecta.


#### 3.4.6. Renal and Hepatic Function

The urea and creatinine levels were noticeably elevated in disease control in comparison to the normal control group. The urea and creatinine levels were evidently (P<0.001−0.01) alleviated in the treatment groups, as shown in Table 8.Table 8
The effect ofMalva neglecta extract on the liver and renal function tests of diabetic obese rats.
Treatment groupsUrea (mg/dl)Creatinine (mg/dl)ALT (U/L)AST (U/L)Bilirubin (mg/dl)Alkaline phosphatase (U/L)Normal control31±3.4∗∗∗0.35±0.06∗∗∗418±23∗∗∗140±8∗∗∗0.41±0.01∗∗∗723±22∗∗∗Diseased control67±2.31.05±0.101075±43170±50.73±0.022483±45Standard therapy35±2.6∗∗∗0.38±0.07∗∗∗515±31∗∗∗150±7∗∗∗0.52±0.04∗∗∗1487±63∗∗∗MNME 250 mg/kg51±4.4∗∗∗0.81±0.02∗∗836±21∗∗∗166±80.71±0.03∗1891±34∗∗∗MNME 500 mg/kg45±2.5∗∗∗0.62±0.07∗∗∗721±33∗∗∗161±8∗∗0.67±0.01∗∗∗1780±43∗∗∗MNME 750 mg/kg39±3.4∗∗∗0.47±0.03∗∗∗611±27∗∗∗154±6∗∗∗0.61±0.02∗∗∗1561±41∗∗∗Data were presented asmean±standarddeviation (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, significant values were observed in comparison of treatment groups with the disease control. ALT: alanine aminotransferase; AST: aspartate aminotransferase; MNME: methanolic extract of Malva neglecta.In the disease control group, the level of ALT, AST, bilirubin, and ALP was substantially raised (P<0.001) in contrast to normal rats. The liver function was significantly restored in metformin and MNME 750 mg/kg treated groups (Table 8).


#### 3.4.7. Histopathological Studies

Pancreas of normal control rats showed normal structure in contrast to disease control rats which showed severe inflammation and necrosis of islets of Langerhans. Metformin treatment resulted in reduced inflammation. Animals treated with MNME at 250 mg/kg showed inflammation and necrosis. Pancreas of rats treated with MNME 500 mg/kg demonstrated a reduced inflammation. Treatment with MNME 750 mg/kg resulted in the least inflammation and necrosis of islets of Langerhans (Figure3).Figure 3
Histopathological photomicrographs of pancreas of diabetic obese rat treated withMalva neglecta extract at 40× magnification. Pancreas of (a) normal control rat. (b) Disease control rat showing severe inflammation and necrosis. (c) Metformin treated rats showing little inflammation. (d) MNME 250 mg/kg treated rat showing inflammation and necrosis. (e) MNME 500 mg/kg treated rats showing less inflammation. (f) MNME 750 mg/kg showing the least inflammation and necrosis. Here, the box showed inflammation, and the arrow showed necrosis.
(a)(b)(c)(d)(e)(f)The liver of normal control rats presented normal structure while the diseased control group displayed congestion of portal vein and hemorrhage. Animals treated with metformin exhibited dilated portal vein. Photomicrographs of the liver of MNME 250 mg/kg treated group displayed normal lobular structure as well as partial congestion. Rats treated with MNME 500 mg/kg revealed the normal lobular structure and partial congestion while MNME 750 mg/kg treatment resulted in the normal lobular structure and dilated portal vein as displayed in Figure4.Figure 4
Histopathological photomicrographs of the liver of diabetic obese rat treated withMalva neglecta extract at 40× magnification. Liver of (a) normal control rat. (b) Disease control rat displaying congestion of portal vein, and hemorrhage. (c) Metformin-treated animal exhibiting dilated portal vein. (d) MNME 250 mg/kg treated rats exhibited lobular structure and partial congestion. (e) MNME 500 mg/kg treated rats revealed partial congestion. (f) MNME 750 mg/kg showing normal lobular structure and dilated portal vein. Here, the star showed congestion, and the triangle showed necrosis.
(a)(b)(c)(d)(e)(f)Histological evaluation of the kidney of normal rat displayed the normal structure of glomeruli and intact epithelial cells. The disease control group revealed the necrosis of tubular cells. Treatment with metformin showed the recovery of epithelial and tubular cells in diabetic obese animals. Animals treated with MNME 250 mg/kg still displayed the damaged epithelial cells. The treatment of diabetic obese rats with MNME 500 and 750 mg/kg improved the tubular structure and epithelial cells as demonstrated in Figure5.Figure 5
Histopathological photomicrographs of the kidney of diabetic obese rat treated withMalva neglecta extract at 40× magnification. Kidney of (a) normal rats. (b) Diseased control rats showing the necrosis of tubular cells. (c) Metformin treated animals showing the recovered epithelial and tubular cells. (d) MNME 250 mg/kg treated rats displaying the destroyed epithelial structures. (e) MNME 500 mg/kg treated rats showing the improved tubular and epithelial cell structure. (e) Rat treated with MNME 750 mg/kg exhibiting improved tubular and epithelial cell. Here, the arrow showed intact glomeruli, and the triangle showed slugged off epithelial cells and deranged tubular structure.
(a)(b)(c)(d)(e)(f)


## 3.1. HPLC Analysis

The HPLC analysis of MNME showed the presence of various flavonoids and phenolic acids such as quercetin, kaempferol, gallic acid, chlorogenic acid, syringic acid, and cinnamic acid in the chromatogram. Kaempferol was present in the highest amount (128.14 ppm) followed by cinnamic acid (7.93 ppm) as shown in Table1.Table 1
Phytochemicals detected by HPLC inMalva neglecta methanolic extract.
S#Compound nameRetention timeArea (mV.s)Amount (ppm)1Quercetin3.0531.80.6892Gallic acid4.7801.31.133Chlorogenic acid15.07324.5671.914Syringic acid16.44084.0830.615Cinnamic acid24.793226.6687.936Kaempferol2.200298.000128.14


## 3.2. Free Radical Scavenging Potential

The free radical scavenging potential of MNME increased concentration dependently. The highest inhibition of DPPH was shown at 1 mg/ml (70.0±3.07%). The %age inhibition of DPPH by the MNME was noticeably (P<0.001) different from the ascorbic acid at all respective concentrations as mentioned in Table 2.Table 2
The percent inhibition of DPPH andα-amylase exhibited by aqueous methanol extract of Malva neglecta.
Concentrations (mg/ml)%age inhibition of DPPH%age inhibition ofα-amylaseMalva neglecta extractAscorbic acidMalva neglecta extractAcarbose170.0±3.07∗∗∗85.4±1.2576.4±5.2∗∗∗∗91.2±7.160.559.8±3.11∗∗∗71.3±2.3769.3±4.1∗∗∗∗83.6±6.310.02551.0±1.94∗∗∗66.8±1.2658.4±4.3∗∗∗∗79.2±4.140.12544.1±1.27∗∗∗56.8±1.2252.4±5.6∗∗∗∗72.5±9.130.062540.0±2.27∗∗∗48.9±1.2737.8±6.3∗∗∗∗55.3±4.300.0312535.7±1.17∗∗∗43.4±1.0924.5±7.1∗∗∗∗47.9±6.280.015629.2±1.22∗∗∗36.1±1.1519.4±4.4∗∗∗∗38.2±3.32Data were presented asmean±S.D. (N=03). ∗∗∗P<0.001 significant values were observed in comparison with ascorbic acid. ∗∗∗∗P<0.0001 showed significant values as compared with the standard acarbose.


## 3.3.α-Amylase Inhibitory Activity

To estimate the in vitro hypoglycemic activity of MNME,α-amylase inhibitory assay was performed. The α-amylase activity was concentration dependently inhibited by MNME all tested levels with the highest activity observed at 1 mg/ml (76.4±5.2%) as revealed in Table 2. The %age inhibition of α-amylase activity by MNME was substantially (P<0.001) lower than the acarbose in respective concentration.


## 3.4. Effect of Plant Extract on Hyperglycemia

The fasting blood glucose level (FBG) did not vary in all the experimental groups at basal stage. The FBG was recorded after alloxan administration on the 0, 7, and 14th day. There was a profound rise in blood glucose level in all groups as compared to the normal control group. The FBG in the disease control group was successively raised on the 7 and 14th day in contrast to the normal control rats.Treatment with standard therapy (metformin) and MNME significantly ameliorated the alloxan-induced hyperglycemia on the 7 and 14th day in contrast to the disease control group. There was an insignificant difference between normal control (105.40±7.48mg/dl) and standard group (124.76±5.34mg/dl) on the 14th day. Moreover, all treatment groups were notably different (P<0.001−0.0001) from the normal, diseased, and standard groups on the 7 and 14th day of treatment.The FBG was significantly (P<0.0001) decreased by MNME on the 7 and 14th day of treatment in contrast to the disease control group. The FBG was reduced dose dependently with MNME at 250 (281.30±10.01mg/dl), 500 (248.68±9.02mg/dl), and 750 mg/kg (184.59±7.21mg/dl) on the 14th day. The FBG with MNME at 250 and 500 mg/kg was noticeably (P<0.001) higher than that of MNME 750 mg/kg treated group on the 14th day of treatment. The effect of MNME at 750 mg/kg on FBG at 7 and 14th days was insignificantly different from the standard treatment on respective days as depicted in Figure 1.Figure 1
The effect of Malvaneglecta extract on glucose levels of diabetic and obese rats. Data were shown as mean±S.D. (n=6) and evaluated by two-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗∗∗P<0.00001 showed significant difference in comparison to the disease control group while disease control significantly different &&&&P<0.00001 in comparison to the normal control group. MNME: methanolic extract of Malva neglecta.


### 3.4.1. Effect on Body Weight

The animal body weights were insignificantly different at preliminary stages of the experiment. Body weights of diseased animals were significantly greater than initial body weight after HSFD for 16 weeks. After alloxan administration, the weight of all rats reduced significantly (P<0.01−0.0001) on the 7th day in contrasts to normal control group. The body weight of treated rats reduced significantly (P<0.05−0.01) at MNME 500 and 750 mg/kg on the 7 and 14th day of treatment in contrast to the disease control group. Likewise, the body weight reduced substantially (P<0.05−0.001) in the standard treatment group on the 7 and 14th day in contrast to disease control group. The effect of MNME at 500 and 700 mg/kg MNME on body weight on the 7 and 14th day was insignificantly different while MNME at 250 mg/kg insignificantly reduced the body weight in contrast to the disease control group on the 7 and 14th day of treatment as shown in Figure 2.Figure 2
The effect ofMalva neglecta extract on body weight of diabetic obese rats. Data were shown as mean±S.D. (n=6) and evaluated by two-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significantly different in comparison with the disease control group while &&P<0.01 and &&&&P<0.0001 in comparison to normal control group. MNME: methanolic extract of Malva neglecta.


### 3.4.2. Leptin and Lipid Profile

In the current study, leptin level profoundly increased (P<0.05) in the disease control as compared to the normal control group that was significantly restored upon treatment with metformin and MNME at 500 and 750 mg/kg as mentioned in Table 3. The serum levels of triglycerides (TG), total cholesterol (TC), and low-density lipoproteins (LDL) were significantly (P<0.05−0.01) raised in the diseased control group as compared to normal control and were notably (P<0.01−0.001) reduced by metformin and MNME at 500 and 750 mg/kg treatment in diseased rats. In contrast, the high-density lipoprotein (HDL) level was insignificantly different among different treatments (Table 3).Table 3
The effect ofMalva neglecta extract on leptin, lipid profile, HbA1c, and insulin level in diabetic obese rats.
Treatment groupsLeptin (ug/ml)Triglycerides (mmol/L)Cholesterol (mmol/L)LDL (mmol/L)HDL (mmol/L)HbA1c (%)Insulin (μU/ml)Normal control0.042±0.001∗1.67±0.29∗5.88±0.22∗2.69±0.43∗∗0.98±0.065.83±0.61∗∗∗0.36±0.05∗∗∗Disease control0.053±0.0011.78±0.346.87±0.333.61±0.231.02±0.0412.16±0.110.08±0.01Metformin treatment0.038±0.01∗∗∗∗1.63±0.31∗∗∗5.47±0.32∗∗2.53±0.44∗0.95±0.036.46±0.26∗∗∗0.25±0.03∗∗∗MNME 250 mg/kg0.0508±0.0031.70±0.526.11±0.442.95±0.260.98±0.0610.13±0.43∗∗∗0.11±0.05MNME 500 mg/kg0.048±0.002∗∗1.69±0.245.81±0.562.59±0.27∗0.97±0.068.61±0.38∗∗∗0.19±0.02∗∗MNME 750 mg/kg0.045±0.001∗∗∗∗1.66±0.34∗∗5.74±0.41∗2.43±0.33∗0.94±0.046.93±0.73∗∗∗0.19±0.01∗∗Data were shown asmean±S.D. (n=6). ∗∗∗∗P<0.0001, ∗∗∗P<0.001, ∗∗∗P<0.01, and ∗P<0.05 showed significantly different in comparison to the disease control group. MNME: methanolic extract of Malva neglecta; LDL: low density; HDL: high-density lipoprotein; HbA1c: glycosylated hemoglobin.


### 3.4.3. Effect on HbA1c and Insulin

The percentage of HbA1c was elevated in diabetic rats (12.16±0.11%) as compared to the normal control group. Standard therapy of metformin and all doses of the plant extract significantly decreased (P<0.01) HbA1c percentage. The serum insulin level declined substantially in the diseased rats in contrast to normal control rats. However, the insulin level was notably (P<0.05−0.01) restored with the plant extract at 500 and 750 mg/kg and metformin treatment as compared to the disease control group as demonstrated in Table 3.


### 3.4.4. Effect on Oxidative Stress Biomarkers

(1) CAT Activity. The activity of CAT was significantly reduced in the liver, kidney, and pancreas of the disease control group than the normal control group. The CAT activity was remarkable (P<0.05−0.001) and dose dependently restored in the liver, kidney, and pancreas of rats treated with MNME at 250, 500, and 750 mg/kg in contrast to disease control rats as shown in Table 4. This showed that MNME had produced the antioxidant effect by increasing the production CAT in the pancreas especially at MNME 500 and 750 mg/kg (Table 4).Table 4
The effect ofMalva neglecta extract on catalase (U/mg of protein) activity in different organs of diabetic obese rats.
GroupsLiverKidneyPancreasNormal control70±2.5∗55±0.5∗30±0.5∗Disease control59±2.639±0.519±0.5Standard treatment65±1.0∗∗50±1.0∗26±1.5∗∗∗MNME 250 mg/kg58±1.5∗∗∗42±0.520±0.5MNME 500 mg/kg61±0.5∗∗47±0.523±0.5∗MNME 750 mg/kg63±0.5∗∗∗50±0.5∗25±1.0∗∗Data were presented asmean±S.D. (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significantly different as compared to the disease control group. MNME: methanolic extract of Malva neglecta.(2) MDA Level. The MDA level was raised noticeably in the liver, kidney, and pancreas of untreated diabetic rats as compared to normal rats. The MDA level was significantly (P<0.01−0.001) reinstated with the plant extract and metformin treated groups in kidney and pancreas as compared to respective tissues of disease control group. The MDA level was insignificantly restored in the liver of metformin and plant extract treated groups as equated with disease control group as shown in Table 5.Table 5
Effect ofMalva neglecta extract on MDA (μg/mg) levels in different organs of diabetic obese rats.
GroupsLiverKidneyPancreasNormal control0.835±0.06∗0.936±0.12∗∗∗0.165±0.11∗∗∗Diseased control1.3±0.221.751±0.130.391±0.02Standard therapy0.942±0.171.311±0.05∗∗∗0.216±0.04∗∗∗MNME 250 mg/kg1.1±0.151.631±0.12∗0.311±0.06∗∗∗MNME 500 mg/kg0.991±0.031.561±0.01∗∗0.291±0.02∗∗∗MNME 750 mg/kg0.983±0.051.393±0.03∗∗∗0.263±0.04∗∗∗Data were presented asmean±standarddeviation (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significant differences in comparison to the disease control group. MNME: methanolic extract of Malva neglecta.(3) SOD Activity. The disease control rats exhibited a decrease in SOD activity in the liver, kidney, and pancreas tissue homogenate as compared to normal control rats which indicated a decline in antioxidant defense. The SOD activity (μg/mg of protein) in the pancreas was significantly (P<0.05−0.001) and dose dependently restored by MNME at 250, 500, and 750 mg/kg in the liver, kidney, and pancreas tissues. A rise in SOD activity was also notable in all tested tissues of metformin-treated group, in contrast to the disease control group, as shown in Table 6.Table 6
The effect ofMalva neglecta extract on superoxide dismutase (U/mg of protein) activity in different organs of diabetic obese rats.
TreatmentsLiverKidneyPancreasNormal control8.1±0.5∗∗14.2±0.5∗∗∗13±0.01∗∗∗Disease control5.8±0.57.81±0.58.13±0.5Standard treatment7.1±0.5∗12.1±0.5∗∗∗12.3±0.5∗∗∗MNME 250 mg/kg6.31±0.59.8±0.5∗∗∗8.91±0.5MNME 500 mg/kg6.6±0.510.1±0.5∗∗∗9.2±0.5∗MNME 750 mg/kg7.1±0.5∗11.6±1.0∗∗∗9.6±0.5∗∗Data were presented asmean±S.D. (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significantly different in comparison to the disease control group. MNME: methanolic extract of Malva neglecta.


### 3.4.5. GSH Level

The level of GSH in the liver, kidney, and pancreas was noticeably (P<0.05) decreased in diseased rats as compared to the normal control group; however, it was significantly (P<0.01−0.001) restored in treatment groups. The GSH level was notably raised in the liver of treated animals with metformin and MNME 750 mg/kg treatment. The GSH level was notably restored in kidney and pancreas of treated rats with metformin and MNME 750 mg/kg as depicted in Table 7.Table 7
Effect ofMalva neglecta on reduced glutathione (μM/mg) level in different organs of diabetic obese rats.
GroupsLiverKidneyPancreasNormal control22.3±1.5∗∗∗15.2±0.5∗∗∗8.6±0.3∗∗∗Diseased control15.6±0.59.1±0.43.2±0.4Standard therapy19.5±0.7∗∗14.3±0.4∗∗6.1±0.5∗∗∗MNME 250 mg/kg16.3±0.310.4±0.63.0±0.2MNME 500 mg/kg18.2±0.412.2±0.3∗4.2±0.4∗MNME 750 mg/kg19.4±0.5∗∗13.4±0.6∗∗6.2±0.5∗∗Data were presented asmean±standarddeviation (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significant differences in comparison to the disease control group. MNME: methanolic extract of Malva neglecta.


### 3.4.6. Renal and Hepatic Function

The urea and creatinine levels were noticeably elevated in disease control in comparison to the normal control group. The urea and creatinine levels were evidently (P<0.001−0.01) alleviated in the treatment groups, as shown in Table 8.Table 8
The effect ofMalva neglecta extract on the liver and renal function tests of diabetic obese rats.
Treatment groupsUrea (mg/dl)Creatinine (mg/dl)ALT (U/L)AST (U/L)Bilirubin (mg/dl)Alkaline phosphatase (U/L)Normal control31±3.4∗∗∗0.35±0.06∗∗∗418±23∗∗∗140±8∗∗∗0.41±0.01∗∗∗723±22∗∗∗Diseased control67±2.31.05±0.101075±43170±50.73±0.022483±45Standard therapy35±2.6∗∗∗0.38±0.07∗∗∗515±31∗∗∗150±7∗∗∗0.52±0.04∗∗∗1487±63∗∗∗MNME 250 mg/kg51±4.4∗∗∗0.81±0.02∗∗836±21∗∗∗166±80.71±0.03∗1891±34∗∗∗MNME 500 mg/kg45±2.5∗∗∗0.62±0.07∗∗∗721±33∗∗∗161±8∗∗0.67±0.01∗∗∗1780±43∗∗∗MNME 750 mg/kg39±3.4∗∗∗0.47±0.03∗∗∗611±27∗∗∗154±6∗∗∗0.61±0.02∗∗∗1561±41∗∗∗Data were presented asmean±standarddeviation (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, significant values were observed in comparison of treatment groups with the disease control. ALT: alanine aminotransferase; AST: aspartate aminotransferase; MNME: methanolic extract of Malva neglecta.In the disease control group, the level of ALT, AST, bilirubin, and ALP was substantially raised (P<0.001) in contrast to normal rats. The liver function was significantly restored in metformin and MNME 750 mg/kg treated groups (Table 8).


### 3.4.7. Histopathological Studies

Pancreas of normal control rats showed normal structure in contrast to disease control rats which showed severe inflammation and necrosis of islets of Langerhans. Metformin treatment resulted in reduced inflammation. Animals treated with MNME at 250 mg/kg showed inflammation and necrosis. Pancreas of rats treated with MNME 500 mg/kg demonstrated a reduced inflammation. Treatment with MNME 750 mg/kg resulted in the least inflammation and necrosis of islets of Langerhans (Figure3).Figure 3
Histopathological photomicrographs of pancreas of diabetic obese rat treated withMalva neglecta extract at 40× magnification. Pancreas of (a) normal control rat. (b) Disease control rat showing severe inflammation and necrosis. (c) Metformin treated rats showing little inflammation. (d) MNME 250 mg/kg treated rat showing inflammation and necrosis. (e) MNME 500 mg/kg treated rats showing less inflammation. (f) MNME 750 mg/kg showing the least inflammation and necrosis. Here, the box showed inflammation, and the arrow showed necrosis.
(a)(b)(c)(d)(e)(f)The liver of normal control rats presented normal structure while the diseased control group displayed congestion of portal vein and hemorrhage. Animals treated with metformin exhibited dilated portal vein. Photomicrographs of the liver of MNME 250 mg/kg treated group displayed normal lobular structure as well as partial congestion. Rats treated with MNME 500 mg/kg revealed the normal lobular structure and partial congestion while MNME 750 mg/kg treatment resulted in the normal lobular structure and dilated portal vein as displayed in Figure4.Figure 4
Histopathological photomicrographs of the liver of diabetic obese rat treated withMalva neglecta extract at 40× magnification. Liver of (a) normal control rat. (b) Disease control rat displaying congestion of portal vein, and hemorrhage. (c) Metformin-treated animal exhibiting dilated portal vein. (d) MNME 250 mg/kg treated rats exhibited lobular structure and partial congestion. (e) MNME 500 mg/kg treated rats revealed partial congestion. (f) MNME 750 mg/kg showing normal lobular structure and dilated portal vein. Here, the star showed congestion, and the triangle showed necrosis.
(a)(b)(c)(d)(e)(f)Histological evaluation of the kidney of normal rat displayed the normal structure of glomeruli and intact epithelial cells. The disease control group revealed the necrosis of tubular cells. Treatment with metformin showed the recovery of epithelial and tubular cells in diabetic obese animals. Animals treated with MNME 250 mg/kg still displayed the damaged epithelial cells. The treatment of diabetic obese rats with MNME 500 and 750 mg/kg improved the tubular structure and epithelial cells as demonstrated in Figure5.Figure 5
Histopathological photomicrographs of the kidney of diabetic obese rat treated withMalva neglecta extract at 40× magnification. Kidney of (a) normal rats. (b) Diseased control rats showing the necrosis of tubular cells. (c) Metformin treated animals showing the recovered epithelial and tubular cells. (d) MNME 250 mg/kg treated rats displaying the destroyed epithelial structures. (e) MNME 500 mg/kg treated rats showing the improved tubular and epithelial cell structure. (e) Rat treated with MNME 750 mg/kg exhibiting improved tubular and epithelial cell. Here, the arrow showed intact glomeruli, and the triangle showed slugged off epithelial cells and deranged tubular structure.
(a)(b)(c)(d)(e)(f)


## 3.4.1. Effect on Body Weight

The animal body weights were insignificantly different at preliminary stages of the experiment. Body weights of diseased animals were significantly greater than initial body weight after HSFD for 16 weeks. After alloxan administration, the weight of all rats reduced significantly (P<0.01−0.0001) on the 7th day in contrasts to normal control group. The body weight of treated rats reduced significantly (P<0.05−0.01) at MNME 500 and 750 mg/kg on the 7 and 14th day of treatment in contrast to the disease control group. Likewise, the body weight reduced substantially (P<0.05−0.001) in the standard treatment group on the 7 and 14th day in contrast to disease control group. The effect of MNME at 500 and 700 mg/kg MNME on body weight on the 7 and 14th day was insignificantly different while MNME at 250 mg/kg insignificantly reduced the body weight in contrast to the disease control group on the 7 and 14th day of treatment as shown in Figure 2.Figure 2
The effect ofMalva neglecta extract on body weight of diabetic obese rats. Data were shown as mean±S.D. (n=6) and evaluated by two-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significantly different in comparison with the disease control group while &&P<0.01 and &&&&P<0.0001 in comparison to normal control group. MNME: methanolic extract of Malva neglecta.


## 3.4.2. Leptin and Lipid Profile

In the current study, leptin level profoundly increased (P<0.05) in the disease control as compared to the normal control group that was significantly restored upon treatment with metformin and MNME at 500 and 750 mg/kg as mentioned in Table 3. The serum levels of triglycerides (TG), total cholesterol (TC), and low-density lipoproteins (LDL) were significantly (P<0.05−0.01) raised in the diseased control group as compared to normal control and were notably (P<0.01−0.001) reduced by metformin and MNME at 500 and 750 mg/kg treatment in diseased rats. In contrast, the high-density lipoprotein (HDL) level was insignificantly different among different treatments (Table 3).Table 3
The effect ofMalva neglecta extract on leptin, lipid profile, HbA1c, and insulin level in diabetic obese rats.
Treatment groupsLeptin (ug/ml)Triglycerides (mmol/L)Cholesterol (mmol/L)LDL (mmol/L)HDL (mmol/L)HbA1c (%)Insulin (μU/ml)Normal control0.042±0.001∗1.67±0.29∗5.88±0.22∗2.69±0.43∗∗0.98±0.065.83±0.61∗∗∗0.36±0.05∗∗∗Disease control0.053±0.0011.78±0.346.87±0.333.61±0.231.02±0.0412.16±0.110.08±0.01Metformin treatment0.038±0.01∗∗∗∗1.63±0.31∗∗∗5.47±0.32∗∗2.53±0.44∗0.95±0.036.46±0.26∗∗∗0.25±0.03∗∗∗MNME 250 mg/kg0.0508±0.0031.70±0.526.11±0.442.95±0.260.98±0.0610.13±0.43∗∗∗0.11±0.05MNME 500 mg/kg0.048±0.002∗∗1.69±0.245.81±0.562.59±0.27∗0.97±0.068.61±0.38∗∗∗0.19±0.02∗∗MNME 750 mg/kg0.045±0.001∗∗∗∗1.66±0.34∗∗5.74±0.41∗2.43±0.33∗0.94±0.046.93±0.73∗∗∗0.19±0.01∗∗Data were shown asmean±S.D. (n=6). ∗∗∗∗P<0.0001, ∗∗∗P<0.001, ∗∗∗P<0.01, and ∗P<0.05 showed significantly different in comparison to the disease control group. MNME: methanolic extract of Malva neglecta; LDL: low density; HDL: high-density lipoprotein; HbA1c: glycosylated hemoglobin.


## 3.4.3. Effect on HbA1c and Insulin

The percentage of HbA1c was elevated in diabetic rats (12.16±0.11%) as compared to the normal control group. Standard therapy of metformin and all doses of the plant extract significantly decreased (P<0.01) HbA1c percentage. The serum insulin level declined substantially in the diseased rats in contrast to normal control rats. However, the insulin level was notably (P<0.05−0.01) restored with the plant extract at 500 and 750 mg/kg and metformin treatment as compared to the disease control group as demonstrated in Table 3.


## 3.4.4. Effect on Oxidative Stress Biomarkers

(1) CAT Activity. The activity of CAT was significantly reduced in the liver, kidney, and pancreas of the disease control group than the normal control group. The CAT activity was remarkable (P<0.05−0.001) and dose dependently restored in the liver, kidney, and pancreas of rats treated with MNME at 250, 500, and 750 mg/kg in contrast to disease control rats as shown in Table 4. This showed that MNME had produced the antioxidant effect by increasing the production CAT in the pancreas especially at MNME 500 and 750 mg/kg (Table 4).Table 4
The effect ofMalva neglecta extract on catalase (U/mg of protein) activity in different organs of diabetic obese rats.
GroupsLiverKidneyPancreasNormal control70±2.5∗55±0.5∗30±0.5∗Disease control59±2.639±0.519±0.5Standard treatment65±1.0∗∗50±1.0∗26±1.5∗∗∗MNME 250 mg/kg58±1.5∗∗∗42±0.520±0.5MNME 500 mg/kg61±0.5∗∗47±0.523±0.5∗MNME 750 mg/kg63±0.5∗∗∗50±0.5∗25±1.0∗∗Data were presented asmean±S.D. (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significantly different as compared to the disease control group. MNME: methanolic extract of Malva neglecta.(2) MDA Level. The MDA level was raised noticeably in the liver, kidney, and pancreas of untreated diabetic rats as compared to normal rats. The MDA level was significantly (P<0.01−0.001) reinstated with the plant extract and metformin treated groups in kidney and pancreas as compared to respective tissues of disease control group. The MDA level was insignificantly restored in the liver of metformin and plant extract treated groups as equated with disease control group as shown in Table 5.Table 5
Effect ofMalva neglecta extract on MDA (μg/mg) levels in different organs of diabetic obese rats.
GroupsLiverKidneyPancreasNormal control0.835±0.06∗0.936±0.12∗∗∗0.165±0.11∗∗∗Diseased control1.3±0.221.751±0.130.391±0.02Standard therapy0.942±0.171.311±0.05∗∗∗0.216±0.04∗∗∗MNME 250 mg/kg1.1±0.151.631±0.12∗0.311±0.06∗∗∗MNME 500 mg/kg0.991±0.031.561±0.01∗∗0.291±0.02∗∗∗MNME 750 mg/kg0.983±0.051.393±0.03∗∗∗0.263±0.04∗∗∗Data were presented asmean±standarddeviation (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significant differences in comparison to the disease control group. MNME: methanolic extract of Malva neglecta.(3) SOD Activity. The disease control rats exhibited a decrease in SOD activity in the liver, kidney, and pancreas tissue homogenate as compared to normal control rats which indicated a decline in antioxidant defense. The SOD activity (μg/mg of protein) in the pancreas was significantly (P<0.05−0.001) and dose dependently restored by MNME at 250, 500, and 750 mg/kg in the liver, kidney, and pancreas tissues. A rise in SOD activity was also notable in all tested tissues of metformin-treated group, in contrast to the disease control group, as shown in Table 6.Table 6
The effect ofMalva neglecta extract on superoxide dismutase (U/mg of protein) activity in different organs of diabetic obese rats.
TreatmentsLiverKidneyPancreasNormal control8.1±0.5∗∗14.2±0.5∗∗∗13±0.01∗∗∗Disease control5.8±0.57.81±0.58.13±0.5Standard treatment7.1±0.5∗12.1±0.5∗∗∗12.3±0.5∗∗∗MNME 250 mg/kg6.31±0.59.8±0.5∗∗∗8.91±0.5MNME 500 mg/kg6.6±0.510.1±0.5∗∗∗9.2±0.5∗MNME 750 mg/kg7.1±0.5∗11.6±1.0∗∗∗9.6±0.5∗∗Data were presented asmean±S.D. (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significantly different in comparison to the disease control group. MNME: methanolic extract of Malva neglecta.


## 3.4.5. GSH Level

The level of GSH in the liver, kidney, and pancreas was noticeably (P<0.05) decreased in diseased rats as compared to the normal control group; however, it was significantly (P<0.01−0.001) restored in treatment groups. The GSH level was notably raised in the liver of treated animals with metformin and MNME 750 mg/kg treatment. The GSH level was notably restored in kidney and pancreas of treated rats with metformin and MNME 750 mg/kg as depicted in Table 7.Table 7
Effect ofMalva neglecta on reduced glutathione (μM/mg) level in different organs of diabetic obese rats.
GroupsLiverKidneyPancreasNormal control22.3±1.5∗∗∗15.2±0.5∗∗∗8.6±0.3∗∗∗Diseased control15.6±0.59.1±0.43.2±0.4Standard therapy19.5±0.7∗∗14.3±0.4∗∗6.1±0.5∗∗∗MNME 250 mg/kg16.3±0.310.4±0.63.0±0.2MNME 500 mg/kg18.2±0.412.2±0.3∗4.2±0.4∗MNME 750 mg/kg19.4±0.5∗∗13.4±0.6∗∗6.2±0.5∗∗Data were presented asmean±standarddeviation (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, ∗∗P<0.01, and ∗P<0.05 showed significant differences in comparison to the disease control group. MNME: methanolic extract of Malva neglecta.


## 3.4.6. Renal and Hepatic Function

The urea and creatinine levels were noticeably elevated in disease control in comparison to the normal control group. The urea and creatinine levels were evidently (P<0.001−0.01) alleviated in the treatment groups, as shown in Table 8.Table 8
The effect ofMalva neglecta extract on the liver and renal function tests of diabetic obese rats.
Treatment groupsUrea (mg/dl)Creatinine (mg/dl)ALT (U/L)AST (U/L)Bilirubin (mg/dl)Alkaline phosphatase (U/L)Normal control31±3.4∗∗∗0.35±0.06∗∗∗418±23∗∗∗140±8∗∗∗0.41±0.01∗∗∗723±22∗∗∗Diseased control67±2.31.05±0.101075±43170±50.73±0.022483±45Standard therapy35±2.6∗∗∗0.38±0.07∗∗∗515±31∗∗∗150±7∗∗∗0.52±0.04∗∗∗1487±63∗∗∗MNME 250 mg/kg51±4.4∗∗∗0.81±0.02∗∗836±21∗∗∗166±80.71±0.03∗1891±34∗∗∗MNME 500 mg/kg45±2.5∗∗∗0.62±0.07∗∗∗721±33∗∗∗161±8∗∗0.67±0.01∗∗∗1780±43∗∗∗MNME 750 mg/kg39±3.4∗∗∗0.47±0.03∗∗∗611±27∗∗∗154±6∗∗∗0.61±0.02∗∗∗1561±41∗∗∗Data were presented asmean±standarddeviation (n=6) and evaluated by one-way ANOVA followed by Tukey’s test. ∗∗∗P<0.001, significant values were observed in comparison of treatment groups with the disease control. ALT: alanine aminotransferase; AST: aspartate aminotransferase; MNME: methanolic extract of Malva neglecta.In the disease control group, the level of ALT, AST, bilirubin, and ALP was substantially raised (P<0.001) in contrast to normal rats. The liver function was significantly restored in metformin and MNME 750 mg/kg treated groups (Table 8).


## 3.4.7. Histopathological Studies

Pancreas of normal control rats showed normal structure in contrast to disease control rats which showed severe inflammation and necrosis of islets of Langerhans. Metformin treatment resulted in reduced inflammation. Animals treated with MNME at 250 mg/kg showed inflammation and necrosis. Pancreas of rats treated with MNME 500 mg/kg demonstrated a reduced inflammation. Treatment with MNME 750 mg/kg resulted in the least inflammation and necrosis of islets of Langerhans (Figure3).Figure 3
Histopathological photomicrographs of pancreas of diabetic obese rat treated withMalva neglecta extract at 40× magnification. Pancreas of (a) normal control rat. (b) Disease control rat showing severe inflammation and necrosis. (c) Metformin treated rats showing little inflammation. (d) MNME 250 mg/kg treated rat showing inflammation and necrosis. (e) MNME 500 mg/kg treated rats showing less inflammation. (f) MNME 750 mg/kg showing the least inflammation and necrosis. Here, the box showed inflammation, and the arrow showed necrosis.
(a)(b)(c)(d)(e)(f)The liver of normal control rats presented normal structure while the diseased control group displayed congestion of portal vein and hemorrhage. Animals treated with metformin exhibited dilated portal vein. Photomicrographs of the liver of MNME 250 mg/kg treated group displayed normal lobular structure as well as partial congestion. Rats treated with MNME 500 mg/kg revealed the normal lobular structure and partial congestion while MNME 750 mg/kg treatment resulted in the normal lobular structure and dilated portal vein as displayed in Figure4.Figure 4
Histopathological photomicrographs of the liver of diabetic obese rat treated withMalva neglecta extract at 40× magnification. Liver of (a) normal control rat. (b) Disease control rat displaying congestion of portal vein, and hemorrhage. (c) Metformin-treated animal exhibiting dilated portal vein. (d) MNME 250 mg/kg treated rats exhibited lobular structure and partial congestion. (e) MNME 500 mg/kg treated rats revealed partial congestion. (f) MNME 750 mg/kg showing normal lobular structure and dilated portal vein. Here, the star showed congestion, and the triangle showed necrosis.
(a)(b)(c)(d)(e)(f)Histological evaluation of the kidney of normal rat displayed the normal structure of glomeruli and intact epithelial cells. The disease control group revealed the necrosis of tubular cells. Treatment with metformin showed the recovery of epithelial and tubular cells in diabetic obese animals. Animals treated with MNME 250 mg/kg still displayed the damaged epithelial cells. The treatment of diabetic obese rats with MNME 500 and 750 mg/kg improved the tubular structure and epithelial cells as demonstrated in Figure5.Figure 5
Histopathological photomicrographs of the kidney of diabetic obese rat treated withMalva neglecta extract at 40× magnification. Kidney of (a) normal rats. (b) Diseased control rats showing the necrosis of tubular cells. (c) Metformin treated animals showing the recovered epithelial and tubular cells. (d) MNME 250 mg/kg treated rats displaying the destroyed epithelial structures. (e) MNME 500 mg/kg treated rats showing the improved tubular and epithelial cell structure. (e) Rat treated with MNME 750 mg/kg exhibiting improved tubular and epithelial cell. Here, the arrow showed intact glomeruli, and the triangle showed slugged off epithelial cells and deranged tubular structure.
(a)(b)(c)(d)(e)(f)


## 4. Discussion

The present study was conducted to demonstrate the antiobesity and antidiabetic potential of a wild herbM. neglecta extract through several in vitro and in vivo tests. The effect of the plant extract was observed on FBG, body weight, renal and liver function tests, insulin, leptin, and oxidative stress biomarkers in HFD-fed diabetic rats at various concentrations. It has been reported that diabetes induced by alloxan with HFD is free radical mediated and concomitantly accompanied with a wide range of complications [35].Theα-amylase is involved in carbohydrate digestion contributing to hyperglycemia whereas its inhibition lowers the blood glucose level as observed in the findings of the present study. The MNME eased hyperglycemia via inhibiting α-amylase. The MNME dose dependently exhibited alpha-amylase inhibition due to the presence of phenolic acids that might be conclusively contributing towards its hypoglycemic potential [36].Alloxan causes necrosis in pancreaticβ-cells via generating excessive reactive oxygen species (ROS). The hepatocellular uptake of alloxan is restricted that makes the liver less sensitive to alloxan-induced damage through ROS as compare to β cells [37]. The ROS generated by alloxan gradually causes fragmentation of DNA and alters calcium homeostasis. Alloxan possesses a high affinity to the sulfhydryl (-SH) containing compounds such as reduced glutathione, cysteine, and several proteins; however, these -SH compounds are prerequisites for the production of insulin. Alloxan becomes attached to sugar-binding sides of glucokinase to deactivate it by adversely affecting blood glucose control [38]. Alloxan produces persistent hyperglycemia by causing oxidation at the cellular level through the generation of ROS [39]. In the alloxan-induced diabetic rats, there was a significant increase in FBG levels as compared to the normal control group. There was a significant decrease of FBG in animals treated with standard therapy and MNME 500 and 750 mg/kg for 14 days in comparison to the diseased control group. Furthermore, there was a considerable weight loss in all treatment groups except MNME 250 mg/kg due to muscle wasting and abrupt catabolism of tissue proteins. Weight loss might be due to halt of gluconeogenesis and improved action of insulin [40].The standard biochemical marker for estimation of obesity and diabetes is leptin and HbA1c, respectively. In the standard therapy group, metformin produced a substantial reduction in leptin levels while the groups treated with plant extract at 500 and 750 mg/kg significantly decreased the leptin levels as compared to disease control while MNME 250 mg/kg insignificantly affected leptin level. It can be deduced that the MNME exerted a considerable effect against obesity and caused remarkable glycemic control. The TG, TC, and LDL were restored with metformin and MNME at 500 and 750 mg/kg, while MNME 250 mg/kg marginally affected lipid profile.The plasma insulin level also served as a parameter to estimate diabetes. The MNME dose dependently restored the insulin level in diabetic obese animals, with the most pronounced effect observed at 750 mg/kg dose; however, MNME 250 mg/kg dose insignificantly restored the insulin level. The improved glycemic control in rats treated with MNME and metformin could be related to improved insulin sensitivity on the skeletal muscles. The MNME was also enriched with phenols and flavonoid that might be responsible for its antidiabetic and antiobesity activity. Phenolic-rich plant extract improved the insulin sensitivity in skeletal muscle to maintain overall glucose homeostasis in the blood stream and tissues [41].Alloxan produces its effect in multiple ways, as dialuric acid is produced by successive reduction that generates superoxide radicals. The dismutation of these superoxide radicals results in the generation of highly reactive hydroxyl radicals through Fenton reaction [42]. The concentration of these reactive hydroxyl groups has an inverse relation with that of GSH. In the presence of low concentrations of GSH, the production of these radicals is increased. Therefore, the antioxidants like SOD, CAT, and other nonenzymatic hydroxyl radical scavengers protect against the pathogenesis of diabetes [43].The SOD plays a pivotal role against ROS such as superoxide anion by converting them into unreactive hydrogen peroxide and molecular oxygen to protect against the destruction of pancreatic cells [44]. In the current study, it was found that the MNME showed a dose-dependent action against free radicals as the activity of pancreatic SOD at MNME 500 and 750 mg/kg as compared to the disease control group likewise in the liver and kidney. The CAT causes conversion of hydrogen peroxide into water and molecular oxygen. There was a dose-dependent increase in CAT activity in pancreatic, liver, and kidney tissues with MNME at 500 and 750 mg/kg as compared to the diseased control group.Lipid peroxidation in the liver, pancreas, and kidney was assessed by determining the level of MDA. In the present investigation, there was a marked increase of MDA in diabetic obese rats that was restored by the treatment with MNME at all dose levels (P<0.001) in pancreatic tissue. In the liver, there was an insignificant decrease of MDA caused by MNME treatment as compared to the disease control group. In kidneys, a dose-dependent effect of MNME on MDA was evident at all dose levels. The GSH is an important intra- and extracellular antioxidant that protects from oxidative stress [45]. Hydroxyl radicals (OH-) are converted into water while glutathione thiyl radical (GS-) is converted to GSSG [46]. A change in level of GSH may trigger the development of disease. In the present experimental observations, the GSH levels were restored in pancreas, liver, and kidney by MNME at 500 and 750 mg/kg.Liver function markers were observed to analyze the protective effect of MNME against alloxan-induced hepatic damage. Liver enzymes were increased in all diabetic obese rats as compared to the normal control group possibly due to leakage from the cellular cytosol of damaged hepatocytes [47]. The MNME exhibited hepatoprotective by reversing organ damage as evident from the level of AST, ALT, Bil, and ALP levels mainly at the highest dosage. Moreover, the plasma levels of urea and creatinine also gradually increased during DM. In the present study, the urea and creatinine levels raised in diabetic rats were restored by MNME treatment [48].High levels of cholesterol and triglycerides contribute towards the pathogenesis and complications linked to obesity and DM. These high levels in the serum enhance the development of macro- and microvascular complications such as coronary heart disease, atherosclerosis, and other cardiovascular diseases [9]. Insulin has an inhibitory action against hormone-sensitive lipase, thus, a deficiency of insulin or insulin resistance leads towards dyslipidemia, as observed in the disease control rats [49]. The dyslipidemia was notably restored by MNME 750 mg/kg and metformin-treated groups which suggest the preventive effect against the cardiovascular disorders associated with DM and obesity.Chemical evaluation of MNME displayed the presence of various phytochemicals including gallic acid, quercetin, kaempferol, p-coumaric acid, and sinapic acid. Antidiabetic activity of gallic acid and quercetin has been proven through a number of studies [40]. The phytochemicals such as gallic acid increased the secretion of insulin by reversing the damage to β-cells of the pancreas, increased the glucose uptake through skeletal muscles, and decreased oxidative stress [50]. It is also found that insulin played a vital role in decreasing intracellular lipase that had hydrolyzed triglycerides into fatty acids and increased cholesterol [51]. In the current study, MNME produced significant effects on lipid profile and decreased the levels of triglycerides and cholesterol due to the presence of phenolic acids.


## 5. Conclusion

It is concluded from the present study that the MNME had exhibited antidiabetic and antiobesity effect in rats. The hypoglycemic and antiobesity potentials of MNME were the most pronounced at 750 mg/kg/day due to the presence of phenolic acids and flavonoids. The MNME exhibited its action via reducing oxidative stress, insulin resistance, leptin, Hb1Ac, andα-amylase activity. Moreover, it restored the lipid profile, renal, and liver function tests. This study provides the pharmacological basis for use of MNME as antidiabetic and antiobesity agent. The bioassay-based isolation of active principles responsible for these pharmacological actions should be carried out.



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*Source: 2614599-2022-05-28.xml*