# Protective Effect ofDolichos biflorus Seed Extract on 3T3-L1 Preadipocyte Differentiation and High-Fat Diet-Induced Obesity in Rats

Authors: Kumaraswamy Athesh; Nayagam Agnel Arul John; Gurunagarajan Sridharan; Pemiah Brindha; Amer M. Alanazi; Kannan R. R. Rengasamy; Balasubramanian Balamuralikrishnan; Wen-Chao Liu; Arumugam Vijaya Anand
Journal: Evidence-Based Complementary and Alternative Medicine
 (2023)

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## Abstract

Obesity is known to be one of the severe health issues worldwide, as its prevalence continues to rise as well as its association with other chronic diseases worsens. Even though various approaches have been underway to prevent or treat obesity, alternative approaches are in need to combat this chronic condition because of the unsatisfactory effectiveness and adverse side effects of the existing approaches.Dolichos biflorus L. seeds have been employed as a weight-loss treatment in folk medicine. Considering the necessity to develop a safe alternative remedy to rising obesity, the current investigation has been set up to assess the antiobesity potential and the mode of action of the aqueous seed extract of D. biflorus (ASEDB) in a cell line (3T3-L1) and high-fat diet (HFD)-induced rats. For in-vitro studies, 3T3-L1 cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) augmented with adipogenic-inducing medium and the influence of the extract (10 µg/mL–500 µg/mL) on 3T3-L1 adipocyte viability, adipogenesis, and lipolysis was assessed. An in-vitro study revealed maintenance of cell viability, reduced triglycerides (TG) accumulation, and promoted lipolysis in 3T3-L1 cells by ASEDB. Following in-vitro analysis, the HFD-induced obese rats were treated with ASEDB at different concentrations (100 mg/kg, 200 mg/kg, and 300 mg/kg) for 60 days and the effect was evaluated through various anthropometric and biochemical parameters. The findings revealed a significant decrement in total body weight, organ weights, fat pad weights, and restoration of abnormal levels of glucose, leptin, insulin, lipid markers, and antioxidant system to normal by ASEDB treatment. Also, pancreatic lipase inhibition analysis of ASEDB revealed a modest level of inhibition with an IC50 value of 213.3 µg/mL. All these findings exposed that ASEDB possesses pronounced antiobesity potential and exhibits its protective effect by suppressing food intake, reducing fat digestion and absorption, limiting adipogenesis, enhancing lipolysis, and alleviating oxidative stress.


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## Body

## 1. Introduction

Obesity, the leading public health issue of the 21st century, is a complex and chronic condition of excessive body fat buildup that results from an imbalance between energy and food intake, basal metabolism, and energy outlay. Body mass index (BMI), the metric determined from a person’s weight and height, categorizes individuals those who are having BMI from 25 to 29.9 kg/m2 as overweight and ≥30 kg/m2 as obese [1]. This additional body fat gets deposited and is predisposed to major health issues like dyslipidemia, coronary artery disease, type 2 diabetes mellitus (T2DM), stroke, and certain malignancies like breast, endometrial, and colon, which shortens life expectancy. In addition, the obese have higher risks of back pain, osteoarthritis, infertility, and deprived psychosocial wellbeing [2]. Even though obesity is multifactorial in etiology, which is influenced by social, physiological, and genetic variables, increased calorie intake from energy-dense meals along with a sedentary lifestyle are thought to be the primary causes of obesity in the majority of instances [3]. According to WHO, the worldwide incidence of obesity was raised threefold between 1975 and 2016. In 2016, it was also reported that more than 340 million children and adults and over 1.9 billion adults were either obese or overweight. Most shockingly, it is noted that around 38.2 million kids under the age of 5 have identified as obese or overweight [4]. As the problem is spreading like an epidemic across the globe and among all age groups over the years, it imposes a promising strategy to treat it.There are several approaches currently in use to combat overweight and obesity, including dieting, exercise, and medication and in extreme circumstances bariatric surgery. However, all these current approaches have certain limitations. For example, lifestyle modifications were stated to have a let-down rate of higher than 85%, over 5 to 10 years if the modifications were not continued [5]. In the case of pharmacotherapy, the options are very less and orlistat is the only FDA-approved medication which is presently been in the market but which was also reported to have adverse effects upon its prolonged usage, like steatorrhea, flatus, fecal incontinence, and nervousness [6]. Bariatric surgery is generally been suggested for immensely obese people, keeping in mind the associated postoperative complications. Thus, it is of paramount importance to develop an effective and safe alternative strategy to control the obesity epidemic. The scientific community currently believes that revealing the antiobesity capabilities of conventional food/plant healers could lead the path to developing a novel drug for obesity management with fewer side effects.Dolichos biflorus L., belonging to the Fabaceae family, is a leguminous eatable pulse cultivated in subtropics areas. It is usually recognized as horse gram in English, and kulthi in the Indian System of Medicine. It is a suberect, branched herb, indigenous to India and is found up to altitudes of 1000 m. Traditionally, its seeds have been used for various ailments like anthelmintic, expectorant, astringent, diaphoretic, diuretic, ophthalmic, and tonic. The seeds have been reported to possess chemo modulatory [7], antilithiatic [8], antihepatotoxic [9], and antioxidant activity [10]. It is also being used in Ayurvedic medicines for the treatment of medroga (obesity), constipation, piles, hepatomegaly, splenomegaly, and renal calculi. Despite the hot water extract of D. biflorus seeds having long historical use for local management of obesity, there is a dearth of published scientific records on its antiobesity potential. From the literature search, it was found that the antiobesity potential of organic solvent extract of D. biflorus seeds itself or in combination [11] has been studied extensively. However, no such extensive data are available for hot water (aqueous) extract. Thus, the current investigation has been framed to assess extensively to know the antiobesity efficacy of the plant D. biflorus using aqueous of seed (ASEDB) in a cell line, 3T3-L1 adipocytes, and HFD-induced obese rats thereby scientifically validating the plant as a traditional healer.


## 2. Materials and Methods

### 2.1. Chemicals

DMEM (Dulbecco’s modified Eagles medium), streptomycin, penicillin, dexamethasone, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), DMSO (dimethylsulfoxide), FBS (fetal bovine serum), and 3-isobutyl-1-methylxanthines were purchased from Himedia, Mumbai, India. Oil red O, free glycerol determination kit, PPL (porcine pancreatic lipase), commercial insulin, and leptin ELISA quantification kits were purchased from Sigma-Aldrich, USA. Analytical kits for the determination of blood glucose, serum FFA (free fatty acid), TG (triglycerides), TC (total cholesterol), HDL-C (high-density lipoprotein cholesterol), ALT (alanine transaminase), and AST transaminase (aspartate transaminase) were supplied by Biosystems, Tamil Nadu, India. All other analytical grade reagents used in the current study were obtained from Merck Ltd., India.


### 2.2. Extract Preparation

Fresh dried seeds ofD. biflorus were acquired from the natural drug mart in Trichy, India. The clean, dried seeds were then pulverized into tiny powders by using a household electric blender. One part of the powdered sample was soaked with six parts of water and boiled with continuous stirring till the content was reduced to one-third of its original volume. The resultant extract after being filtered through a muslin cloth was then concentrated using a boiling water bath. The yield of the dried extract was found to be nearly 21.4% (w/w) and it was kept in an airtight vessel and stored at 4°C till further usage.


### 2.3. Inhibition of Pancreatic Lipase Assay

The PPL (porcine pancreatic lipase) inhibition assay was done based on previously described protocols [12–14] with minor modifications. PPL stock (1 mg/mL) was prepared by using potassium phosphate buffer (0.1 mM, pH 6.0) and was kept at −20°C. To assay the inhibitory activity, various dosages (5 µg/mL, 10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL, and 500 µg/mL) of 0.2 mL of the extracts were preincubated with 0.1 mL of PPL in 0.1 mM (pH 7.2) potassium phosphate buffer, which contains Tween 80 (0.1%) for 1 hour, at 30°C. To this, 0.1 mL of 25 mM p-nitrophenyl butyrate in acetonitrile (substrate solution) was added and the chemical reaction was initiated by incubating the reaction mixture at 37°C for 5 min. The positive control, orlistat, also underwent the protocol. After the incubation period, the amount of p-nitrophenol freed from p-nitrophenyl butyrate was noted at 405 nm using a spectrophotometer. The negative control was also measured with and without the addition of an inhibitor. By using the following formula, the percentage of lipase inhibition was calculated.(1)%Inhibition=100−B−bA−a×100,where B-activity with an inhibitor; b-negative control with an inhibitor; A-activity without an inhibitor; and a-negative control without an inhibitor.


### 2.4. Anti-Adipogenic Effects of ASEDB in 3T3-L1 Adipocytes

#### 2.4.1. 3T3-L1 Preadipocytes Culture and Differentiation

Mouse 3T3-L1 preadipocytes were purchased from National Centre for Cell Science, India, and were cultured in DMEM adding with FBS (10%), 100 IU/mL of penicillin and 100μg/mL of streptomycin were maintained at 37°C with 5% CO2. For adipocyte differentiation, two days old postconfluent preadipocytes were incubated in an inducer (consisting of 0.25 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 10 μg/mL insulin) supplemented DMEM for 48 hours (day 2) and then maintained in FBS (10%) and insulin (10 μg/mL) supplemented DMEM for another 6 days (day 8). The culture medium was replaced with a fresh medium every two days.


#### 2.4.2. Cell Viability Test

The influence of the aqueous seed extracts ofD. biflorus (ASEDB) on 3T3-L1 preadipocyte viability was analyzed through an MTT assay [15]. 3T3-L1 preadipocytes (1 × 105 cells/mL) were taken in a 96-well microtiter plate (in triplicate) and treated with various concentrations of ASEDB (10 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL, and 500 μg/mL) and incubated in FBS-free culture medium at 37°C for 2 days under 5% CO2. Instead of extracts, 0.05% of DMSO was added to the control group. After two days, fresh media (FBS-free) with MTT (0.5 mg/mL) was added and incubated at 37°C for an hour. The purple formazan crystals developed were then solubilized with100 µL of DMSO and measured at 560 nm spectrophotometrically. The percentage of the viable cells were determined by (At/Ac) × 100, where At and Ac represent the absorbance of the test group and the control group, respectively.


#### 2.4.3. Oil-Red-O Staining

Efficacy of ASEDB on anti-adipogenic activity was evaluated by measuring the lipid pile-up in adipocytes after the neutral lipids were stained with Oil Red O [14, 16]. For anti-adipogenic studies, 3T3-L1 cells undergoing the first 2 days of adipogenic differentiation were treated with nontoxic doses of ASEDB (10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL, and 500 µg/mL). Control 3T3-L1 cells were maintained without extracts. All these cells were subsequently moved to a maintenance medium for the following four days. Following 6 days of incubation, the cells were subjected to PBS (phosphate-buffered saline) wash two times and fixed with formaldehyde (10%) in PBS for 30 minutes at 37°C. The cells were then washed twice with distilled water and finally stained by using Oil Red O (3 mg/mL in 60% isopropyl alcohol) for a period of 1 hour at the temperature of 25°C. After 1 hour, the lipid buildup in the cells was assessed microscopically. In addition, the dye that remained in the cells after being eluted with isopropanol (100%) was quantified spectrophotometrically at 510 nm.


#### 2.4.4. Glycerol Release Assay

Glycerol release was quantified based on the method previously reported [17]. Briefly, the differentiated 3T3-L1 cells were treated with varying dosages of ASEDB (10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL, and 500 µg/mL) and incubated for 24 hours. Then, the free glycerol reagent was mixed to the tubes and the reaction was allowed to stand for 5 mins, based on the manufacturer’s instruction. Distilled water (blank) and glycerol standard were also treated similarly and the absorbance was measured at 540 nm.


### 2.5. Effect of ASEDB on Antiobesity Properties

#### 2.5.1. Experimental Rats

Wistar male albino rats weighing between 100 and 125 g were procured from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India, for use in this investigation. All of the animals were housed in polypropylene cages under appropriate environmental controls (65 ± 5% humidity, 23 ± 2°C temperature, and 12/12 hours of light-dark cycle) and were given standard commercially available rat chow pellets (manufactured by Sai Durga Feeds and Foods, Bengaluru, India) used as a standard feed along with unlimited access to water. The rats spent roughly a week like this becoming accustomed to the lab environment before being used for the investigation.All the experiments and protocols carried out in this study complied with OECD guidelines of CPCSEA and were approved by the Institutional Animal Ethics Committee of Srimad Andavan Arts and Science College, Tiruchirappalli, India (Approval No: 790/03/ac/CPCSEA).


#### 2.5.2. Induction of Obesity and Treatment

A total of 40 rats were induced with preobesity before the start of the original experimental period by feeding a calorie-richhigh-fat diet (HFD) for 45 days. The HFD as represented in earlier studies [14, 18] comprised 5% corn oil (45 kcal), 15% corn starch (60 kcal), 20% sucrose (80 kcal), 20% casein (80 kcal), and 35% beef tallow (315 kcal) in addition to 4% mineral and 1% vitamin mixture. After feeding HFD for 45 days, 30 rats which were considered obese-prone (weighing close to 250 g) were chosen for antiobesity studies. In the same period, another 6 rats were maintained by feeding standard rat chow pellets. The experimental rats were divided randomly into 6 groups (n = 6). The rats fed by standard rat chow pellets served to continue as normal control (Group 1), while the HFD-fed preobese rats served as group 2 to group 6. Group 2 was maintained as the obese/HFD control, and kept eating HFD without ASEDB treatment. Groups 3 to 5 continued to feed on HFD and treated with 100 mg/kg, 200 mg/kg, and 300 mg/kg of ASEDB, respectively. Group 6 animals were given standard medication orlistat (30 mg/kg) in addition to HFD. The drugs were injected orally via a feeding tube once a day for 60 days.


#### 2.5.3. Assessment of Antiobesity Activity

Daily food consumption [19] and body weight (at an interval of every 10 days) of experimental animals were documented regularly. The rats were sacrificed by cervical dislocation under diethyl ether anesthesia at the end of study period (60th day) and blood was collected from the heart for various biochemical assays. Necessary organs (kidney, liver, spleen, and heart) as well as fat fads (mesenteric, epididymal, and perirenal) were separated, washed in buffered saline, blotted dry, and weighed immediately to obtain their absolute weights.The adipocyte area of the visceral fat fad was calculated with the help of a Nikon trinocular light microscope (Eclipse, Ci) furnished with network information system (NIS) digital imaging software. The BMI of the rats, i.e., body weight in gram divided by body length in square cm, was determined and recorded on the initial and final days of the experimentation [20]. Body length (from nose to anus) and waist circumference (on the greatest portion of the stomach) of the experimental rats were measured with a tape by possessing the animals in a ventral posture under mild sodium barbiturate (0.1 ml i.p. of 1%) anesthesia.Serum levels of TC, TG, HDL-C, FFA, AST, ALT, and blood glucose were determined using commercial kits. The LDL-C and VLDL-C were determined using the formulas: LDL-C = TC−(HDL-C−(TG/5)); VLDL-C = total serum TG/5 [21]. The insulin and leptin were quantified using commercially available ELISA kits. Lipid peroxides (LPO) [22], glutathione reduced (GSH) [23], superoxide dismutase (SOD) [24], and catalase [25] were analyzed using standard protocols.


### 2.6. Statistical Analysis

All the obtained data were presented as the mean ± SEM (standard error of the mean), and were statistically analyzed by one-way ANOVA, which is followed by Duncan’s multiple range test; <0.05 was taken as statistically significant differences among groups.


## 2.1. Chemicals

DMEM (Dulbecco’s modified Eagles medium), streptomycin, penicillin, dexamethasone, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), DMSO (dimethylsulfoxide), FBS (fetal bovine serum), and 3-isobutyl-1-methylxanthines were purchased from Himedia, Mumbai, India. Oil red O, free glycerol determination kit, PPL (porcine pancreatic lipase), commercial insulin, and leptin ELISA quantification kits were purchased from Sigma-Aldrich, USA. Analytical kits for the determination of blood glucose, serum FFA (free fatty acid), TG (triglycerides), TC (total cholesterol), HDL-C (high-density lipoprotein cholesterol), ALT (alanine transaminase), and AST transaminase (aspartate transaminase) were supplied by Biosystems, Tamil Nadu, India. All other analytical grade reagents used in the current study were obtained from Merck Ltd., India.


## 2.2. Extract Preparation

Fresh dried seeds ofD. biflorus were acquired from the natural drug mart in Trichy, India. The clean, dried seeds were then pulverized into tiny powders by using a household electric blender. One part of the powdered sample was soaked with six parts of water and boiled with continuous stirring till the content was reduced to one-third of its original volume. The resultant extract after being filtered through a muslin cloth was then concentrated using a boiling water bath. The yield of the dried extract was found to be nearly 21.4% (w/w) and it was kept in an airtight vessel and stored at 4°C till further usage.


## 2.3. Inhibition of Pancreatic Lipase Assay

The PPL (porcine pancreatic lipase) inhibition assay was done based on previously described protocols [12–14] with minor modifications. PPL stock (1 mg/mL) was prepared by using potassium phosphate buffer (0.1 mM, pH 6.0) and was kept at −20°C. To assay the inhibitory activity, various dosages (5 µg/mL, 10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL, and 500 µg/mL) of 0.2 mL of the extracts were preincubated with 0.1 mL of PPL in 0.1 mM (pH 7.2) potassium phosphate buffer, which contains Tween 80 (0.1%) for 1 hour, at 30°C. To this, 0.1 mL of 25 mM p-nitrophenyl butyrate in acetonitrile (substrate solution) was added and the chemical reaction was initiated by incubating the reaction mixture at 37°C for 5 min. The positive control, orlistat, also underwent the protocol. After the incubation period, the amount of p-nitrophenol freed from p-nitrophenyl butyrate was noted at 405 nm using a spectrophotometer. The negative control was also measured with and without the addition of an inhibitor. By using the following formula, the percentage of lipase inhibition was calculated.(1)%Inhibition=100−B−bA−a×100,where B-activity with an inhibitor; b-negative control with an inhibitor; A-activity without an inhibitor; and a-negative control without an inhibitor.


## 2.4. Anti-Adipogenic Effects of ASEDB in 3T3-L1 Adipocytes

### 2.4.1. 3T3-L1 Preadipocytes Culture and Differentiation

Mouse 3T3-L1 preadipocytes were purchased from National Centre for Cell Science, India, and were cultured in DMEM adding with FBS (10%), 100 IU/mL of penicillin and 100μg/mL of streptomycin were maintained at 37°C with 5% CO2. For adipocyte differentiation, two days old postconfluent preadipocytes were incubated in an inducer (consisting of 0.25 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 10 μg/mL insulin) supplemented DMEM for 48 hours (day 2) and then maintained in FBS (10%) and insulin (10 μg/mL) supplemented DMEM for another 6 days (day 8). The culture medium was replaced with a fresh medium every two days.


### 2.4.2. Cell Viability Test

The influence of the aqueous seed extracts ofD. biflorus (ASEDB) on 3T3-L1 preadipocyte viability was analyzed through an MTT assay [15]. 3T3-L1 preadipocytes (1 × 105 cells/mL) were taken in a 96-well microtiter plate (in triplicate) and treated with various concentrations of ASEDB (10 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL, and 500 μg/mL) and incubated in FBS-free culture medium at 37°C for 2 days under 5% CO2. Instead of extracts, 0.05% of DMSO was added to the control group. After two days, fresh media (FBS-free) with MTT (0.5 mg/mL) was added and incubated at 37°C for an hour. The purple formazan crystals developed were then solubilized with100 µL of DMSO and measured at 560 nm spectrophotometrically. The percentage of the viable cells were determined by (At/Ac) × 100, where At and Ac represent the absorbance of the test group and the control group, respectively.


### 2.4.3. Oil-Red-O Staining

Efficacy of ASEDB on anti-adipogenic activity was evaluated by measuring the lipid pile-up in adipocytes after the neutral lipids were stained with Oil Red O [14, 16]. For anti-adipogenic studies, 3T3-L1 cells undergoing the first 2 days of adipogenic differentiation were treated with nontoxic doses of ASEDB (10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL, and 500 µg/mL). Control 3T3-L1 cells were maintained without extracts. All these cells were subsequently moved to a maintenance medium for the following four days. Following 6 days of incubation, the cells were subjected to PBS (phosphate-buffered saline) wash two times and fixed with formaldehyde (10%) in PBS for 30 minutes at 37°C. The cells were then washed twice with distilled water and finally stained by using Oil Red O (3 mg/mL in 60% isopropyl alcohol) for a period of 1 hour at the temperature of 25°C. After 1 hour, the lipid buildup in the cells was assessed microscopically. In addition, the dye that remained in the cells after being eluted with isopropanol (100%) was quantified spectrophotometrically at 510 nm.


### 2.4.4. Glycerol Release Assay

Glycerol release was quantified based on the method previously reported [17]. Briefly, the differentiated 3T3-L1 cells were treated with varying dosages of ASEDB (10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL, and 500 µg/mL) and incubated for 24 hours. Then, the free glycerol reagent was mixed to the tubes and the reaction was allowed to stand for 5 mins, based on the manufacturer’s instruction. Distilled water (blank) and glycerol standard were also treated similarly and the absorbance was measured at 540 nm.


## 2.4.1. 3T3-L1 Preadipocytes Culture and Differentiation

Mouse 3T3-L1 preadipocytes were purchased from National Centre for Cell Science, India, and were cultured in DMEM adding with FBS (10%), 100 IU/mL of penicillin and 100μg/mL of streptomycin were maintained at 37°C with 5% CO2. For adipocyte differentiation, two days old postconfluent preadipocytes were incubated in an inducer (consisting of 0.25 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 10 μg/mL insulin) supplemented DMEM for 48 hours (day 2) and then maintained in FBS (10%) and insulin (10 μg/mL) supplemented DMEM for another 6 days (day 8). The culture medium was replaced with a fresh medium every two days.


## 2.4.2. Cell Viability Test

The influence of the aqueous seed extracts ofD. biflorus (ASEDB) on 3T3-L1 preadipocyte viability was analyzed through an MTT assay [15]. 3T3-L1 preadipocytes (1 × 105 cells/mL) were taken in a 96-well microtiter plate (in triplicate) and treated with various concentrations of ASEDB (10 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL, and 500 μg/mL) and incubated in FBS-free culture medium at 37°C for 2 days under 5% CO2. Instead of extracts, 0.05% of DMSO was added to the control group. After two days, fresh media (FBS-free) with MTT (0.5 mg/mL) was added and incubated at 37°C for an hour. The purple formazan crystals developed were then solubilized with100 µL of DMSO and measured at 560 nm spectrophotometrically. The percentage of the viable cells were determined by (At/Ac) × 100, where At and Ac represent the absorbance of the test group and the control group, respectively.


## 2.4.3. Oil-Red-O Staining

Efficacy of ASEDB on anti-adipogenic activity was evaluated by measuring the lipid pile-up in adipocytes after the neutral lipids were stained with Oil Red O [14, 16]. For anti-adipogenic studies, 3T3-L1 cells undergoing the first 2 days of adipogenic differentiation were treated with nontoxic doses of ASEDB (10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL, and 500 µg/mL). Control 3T3-L1 cells were maintained without extracts. All these cells were subsequently moved to a maintenance medium for the following four days. Following 6 days of incubation, the cells were subjected to PBS (phosphate-buffered saline) wash two times and fixed with formaldehyde (10%) in PBS for 30 minutes at 37°C. The cells were then washed twice with distilled water and finally stained by using Oil Red O (3 mg/mL in 60% isopropyl alcohol) for a period of 1 hour at the temperature of 25°C. After 1 hour, the lipid buildup in the cells was assessed microscopically. In addition, the dye that remained in the cells after being eluted with isopropanol (100%) was quantified spectrophotometrically at 510 nm.


## 2.4.4. Glycerol Release Assay

Glycerol release was quantified based on the method previously reported [17]. Briefly, the differentiated 3T3-L1 cells were treated with varying dosages of ASEDB (10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, 250 µg/mL, and 500 µg/mL) and incubated for 24 hours. Then, the free glycerol reagent was mixed to the tubes and the reaction was allowed to stand for 5 mins, based on the manufacturer’s instruction. Distilled water (blank) and glycerol standard were also treated similarly and the absorbance was measured at 540 nm.


## 2.5. Effect of ASEDB on Antiobesity Properties

### 2.5.1. Experimental Rats

Wistar male albino rats weighing between 100 and 125 g were procured from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India, for use in this investigation. All of the animals were housed in polypropylene cages under appropriate environmental controls (65 ± 5% humidity, 23 ± 2°C temperature, and 12/12 hours of light-dark cycle) and were given standard commercially available rat chow pellets (manufactured by Sai Durga Feeds and Foods, Bengaluru, India) used as a standard feed along with unlimited access to water. The rats spent roughly a week like this becoming accustomed to the lab environment before being used for the investigation.All the experiments and protocols carried out in this study complied with OECD guidelines of CPCSEA and were approved by the Institutional Animal Ethics Committee of Srimad Andavan Arts and Science College, Tiruchirappalli, India (Approval No: 790/03/ac/CPCSEA).


### 2.5.2. Induction of Obesity and Treatment

A total of 40 rats were induced with preobesity before the start of the original experimental period by feeding a calorie-richhigh-fat diet (HFD) for 45 days. The HFD as represented in earlier studies [14, 18] comprised 5% corn oil (45 kcal), 15% corn starch (60 kcal), 20% sucrose (80 kcal), 20% casein (80 kcal), and 35% beef tallow (315 kcal) in addition to 4% mineral and 1% vitamin mixture. After feeding HFD for 45 days, 30 rats which were considered obese-prone (weighing close to 250 g) were chosen for antiobesity studies. In the same period, another 6 rats were maintained by feeding standard rat chow pellets. The experimental rats were divided randomly into 6 groups (n = 6). The rats fed by standard rat chow pellets served to continue as normal control (Group 1), while the HFD-fed preobese rats served as group 2 to group 6. Group 2 was maintained as the obese/HFD control, and kept eating HFD without ASEDB treatment. Groups 3 to 5 continued to feed on HFD and treated with 100 mg/kg, 200 mg/kg, and 300 mg/kg of ASEDB, respectively. Group 6 animals were given standard medication orlistat (30 mg/kg) in addition to HFD. The drugs were injected orally via a feeding tube once a day for 60 days.


### 2.5.3. Assessment of Antiobesity Activity

Daily food consumption [19] and body weight (at an interval of every 10 days) of experimental animals were documented regularly. The rats were sacrificed by cervical dislocation under diethyl ether anesthesia at the end of study period (60th day) and blood was collected from the heart for various biochemical assays. Necessary organs (kidney, liver, spleen, and heart) as well as fat fads (mesenteric, epididymal, and perirenal) were separated, washed in buffered saline, blotted dry, and weighed immediately to obtain their absolute weights.The adipocyte area of the visceral fat fad was calculated with the help of a Nikon trinocular light microscope (Eclipse, Ci) furnished with network information system (NIS) digital imaging software. The BMI of the rats, i.e., body weight in gram divided by body length in square cm, was determined and recorded on the initial and final days of the experimentation [20]. Body length (from nose to anus) and waist circumference (on the greatest portion of the stomach) of the experimental rats were measured with a tape by possessing the animals in a ventral posture under mild sodium barbiturate (0.1 ml i.p. of 1%) anesthesia.Serum levels of TC, TG, HDL-C, FFA, AST, ALT, and blood glucose were determined using commercial kits. The LDL-C and VLDL-C were determined using the formulas: LDL-C = TC−(HDL-C−(TG/5)); VLDL-C = total serum TG/5 [21]. The insulin and leptin were quantified using commercially available ELISA kits. Lipid peroxides (LPO) [22], glutathione reduced (GSH) [23], superoxide dismutase (SOD) [24], and catalase [25] were analyzed using standard protocols.


## 2.5.1. Experimental Rats

Wistar male albino rats weighing between 100 and 125 g were procured from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India, for use in this investigation. All of the animals were housed in polypropylene cages under appropriate environmental controls (65 ± 5% humidity, 23 ± 2°C temperature, and 12/12 hours of light-dark cycle) and were given standard commercially available rat chow pellets (manufactured by Sai Durga Feeds and Foods, Bengaluru, India) used as a standard feed along with unlimited access to water. The rats spent roughly a week like this becoming accustomed to the lab environment before being used for the investigation.All the experiments and protocols carried out in this study complied with OECD guidelines of CPCSEA and were approved by the Institutional Animal Ethics Committee of Srimad Andavan Arts and Science College, Tiruchirappalli, India (Approval No: 790/03/ac/CPCSEA).


## 2.5.2. Induction of Obesity and Treatment

A total of 40 rats were induced with preobesity before the start of the original experimental period by feeding a calorie-richhigh-fat diet (HFD) for 45 days. The HFD as represented in earlier studies [14, 18] comprised 5% corn oil (45 kcal), 15% corn starch (60 kcal), 20% sucrose (80 kcal), 20% casein (80 kcal), and 35% beef tallow (315 kcal) in addition to 4% mineral and 1% vitamin mixture. After feeding HFD for 45 days, 30 rats which were considered obese-prone (weighing close to 250 g) were chosen for antiobesity studies. In the same period, another 6 rats were maintained by feeding standard rat chow pellets. The experimental rats were divided randomly into 6 groups (n = 6). The rats fed by standard rat chow pellets served to continue as normal control (Group 1), while the HFD-fed preobese rats served as group 2 to group 6. Group 2 was maintained as the obese/HFD control, and kept eating HFD without ASEDB treatment. Groups 3 to 5 continued to feed on HFD and treated with 100 mg/kg, 200 mg/kg, and 300 mg/kg of ASEDB, respectively. Group 6 animals were given standard medication orlistat (30 mg/kg) in addition to HFD. The drugs were injected orally via a feeding tube once a day for 60 days.


## 2.5.3. Assessment of Antiobesity Activity

Daily food consumption [19] and body weight (at an interval of every 10 days) of experimental animals were documented regularly. The rats were sacrificed by cervical dislocation under diethyl ether anesthesia at the end of study period (60th day) and blood was collected from the heart for various biochemical assays. Necessary organs (kidney, liver, spleen, and heart) as well as fat fads (mesenteric, epididymal, and perirenal) were separated, washed in buffered saline, blotted dry, and weighed immediately to obtain their absolute weights.The adipocyte area of the visceral fat fad was calculated with the help of a Nikon trinocular light microscope (Eclipse, Ci) furnished with network information system (NIS) digital imaging software. The BMI of the rats, i.e., body weight in gram divided by body length in square cm, was determined and recorded on the initial and final days of the experimentation [20]. Body length (from nose to anus) and waist circumference (on the greatest portion of the stomach) of the experimental rats were measured with a tape by possessing the animals in a ventral posture under mild sodium barbiturate (0.1 ml i.p. of 1%) anesthesia.Serum levels of TC, TG, HDL-C, FFA, AST, ALT, and blood glucose were determined using commercial kits. The LDL-C and VLDL-C were determined using the formulas: LDL-C = TC−(HDL-C−(TG/5)); VLDL-C = total serum TG/5 [21]. The insulin and leptin were quantified using commercially available ELISA kits. Lipid peroxides (LPO) [22], glutathione reduced (GSH) [23], superoxide dismutase (SOD) [24], and catalase [25] were analyzed using standard protocols.


## 2.6. Statistical Analysis

All the obtained data were presented as the mean ± SEM (standard error of the mean), and were statistically analyzed by one-way ANOVA, which is followed by Duncan’s multiple range test; <0.05 was taken as statistically significant differences among groups.


## 3. Results

### 3.1. Inhibitory Effect of Pancreatic Lipase by ASEDB

The porcine pancreatic lipase (PPL) inhibitory action of ASEDB at various concentrations (5µg/mL to 500 µg/mL) was assessed by evaluating the breakdown of p-nitrophenyl butyrate into p-nitrophenol. Figure 1 demonstrates that ASEDB has a substantial level of inhibition potential on the activity of PPL with IC50 of 213.3 µg/mL when matched to the standard drug orlistat, whose IC50 value was found to be 9.2 µg/mL.Figure 1
The pancreatic lipase inhibitory effect of ASEDB. Values are the mean of 3 experiments; ASEDB–aqueous seed extract ofDolichos biflorus L. Orlistat was used as a positive control.


### 3.2. Anti-Adipogenic and Lipolytic Effect

First, to assess the possibility of 3T3-L1 preadipocytes upon ASEDB exposure, the cells were treated with a varied dosage of ASEDB (10μg/mL–500 μg/mL) and the observed findings were expressed as percentage viability, comparing the cell viability of negative control’s (Figure 2(a)). These data made it clear that none of the ASEDB doses administered had an impact on the cells’ viability. Thus, the selected dosages of ASEDB (10 μg/mL−500 μg/mL) were deemed nontoxic in 3T3-L1 cell lines and considered for further investigation.Figure 2
Anti-adipogenic and lipolytic effect of ASEDB on 3T3-L1 adipocytes: (a) cell viability, (b) intracellular lipid content, (c) microscopic photographs of Oil-Red-O staining, and (d) glycerol release. ASEDB–aqueous seed extract ofDolichos biflorus L. Values are the mean ± SEM (n = 3). ∗∗p<0.05, significantly different from the control group.
(a)(b)(c)(d)Oil Red O staining was used to assess the anti-adipogenic potential of ASEDB. The observed findings (Figure2(b)), as well as photomicrographs of lipid staining (Figure 2(c)), clearly revealed that ASEDB dose-reliantly decreased lipid accumulation during 3T3-L1 differentiation. At the higher dose of 500 μg/mL, ASEDB exerted around 35.25 ± 1.37% reductions in fat accumulation than control cells.The lipolytic effect of ASEDB was assessed by estimating the quantity of glycerol liberated from the differentiated 3T3-L1 adipocytes and the obtained data are presented in Figure2(d). ASEDB had shown a reasonable level of lipolytic activity which was evidenced through a notable increase (p<0.05) in the release of glycerol approximately by 21, 31, and 33% at doses of 100, 250, and 500 μg/mL of ASEDB, respectively.


### 3.3. Antiobesity Effect of ASEDB on HFD-Induced Obese Rats

#### 3.3.1. Effect of ASEDB on Food Consumption

The food consumption of the experimental groups was determined by subtracting the quantity of remaining chow from the preweighed chow, every 24 hours per cage basis and the received data are represented in Figure3 as average food intake. The data have indicated that animals fed with HFD consumed significantly more food on average than animals fed with the ND. Administration of ASEDB along with HFD however showed a slight suppression of food intake, without showing any significant dose-dependent variation. However, orlistat-treated group 6 rats showed significantly lower (p<0.05) food consumption than ASEDB-treated rats.Figure 3
Levels of average food intake in experimental rats. Group 1–fed with ND; group 2–fed with HFD; group 3–HFD + ASEDB (100 mg/kg bw); group 4–HFD + ASEDB (200 mg/kg bw); group 5–HFD + ASEDB (300 mg/kg bw); group 6–HFD + orlistat (30 mg/kg bw); ND–normal diet; HFD–high fat diet; ASEDB–aqueous seed extract ofDolichos biflorus L; values are the mean of 6 rats; ∗p<0.05, significantly different from group 1; ∗∗p<0.01, significantly different from group 2; #p<0.05, group 3, 4, and 5 significantly different from group 2; no significant differences between ASEDB-treated groups (group 3, 4, and 5).


#### 3.3.2. Influence of ASEDB on Body Weight, Fat Pad Weights, Organ Weights, Adipocyte Area, BMI, and Waist Circumference (WC)

An initial body weight of the experimental models were noted on the day one, followed by the changes in those weights brought on by ASEDB supplementation; these changes were documented once every 10 days. Obtained results have shown that there was a significant upsurge in a gain of body weight in HFD control rats (Group 2) when matching to the normal control (Group 1) rats. On the 60th day of the experimental period, group 2 animals have shown approximately 15% more weight gain than group 1 animals. However, treatment with various dosages of ASEDB (100 mg/kg, 200 mg/kg, and 300 mg/kg BW) significant reduction (p<0.05) was noted in the percentage body weight gain of the animals are in proportion to the dose when compared to HFD control rats. Adipocyte area, the weights of the liver, heart, kidney, and spleen, as well as the mesenteric, perirenal, and retroperitoneal fat pad weights, were also observed to be increased in HFD fed, untreated rats but dramatically decreased (p<0.05) in ASEDB-treated rats in a dose-reliable manner (Figure 4).Figure 4
Effect of ASEDB on (a) body weight; (b) fat pad weight; (c) adipocyte area; (d) organ weights; and (e) microscopic images of adipocytes showing variations in adipocyte area among different groups. WAT–white adipose tissue; ASEDB–aqueous seed extract ofDolichos biflorus L.; values are the mean ± S.E.M of 6 rats; #p<0.05, significantly different from group 1; ∗p<0.05, significantly different from group 2; ∗∗p<0.01, significantly different from group 2.
(a)(b)(c)(d)(e)The BMI and WC of the animals were measured on days 1 and 60 and the found data are depicted in Figure5. When compared to obese rats that were administered HFD without receiving any treatment (Group 2), the administration of ASEDB plus HFD significantly decreased the rats’ BMI and WC in proportionate to the concentration.Figure 5
Effect of ASEDB on (a) body mass index; (b) waist circumference of experimental rats. Values are the mean ± S.E.M (n = 6); #significant difference from normal control (group 1) at p<0.01; ∗significant difference from HFD control (group 2) at p<0.05; ∗∗Significant difference from HFD control (group 2) at p < 0.01.
(a)(b)


#### 3.3.3. Effect of ASEDB on Different Biochemical Parameters

The effect of ASEDB on blood glucose, leptin, and insulin levels in the animals is given in Table1. The increased levels of glucose, leptin, and insulin found in HFD-induced rats were efficiently returned to near-normal levels in ASEDB-treated animals. The data on the effect of ASEDB on hepatic triglycerides and serum lipid profiles are also given in Table 1. HFD feeding to the experimental animals raised the levels of hepatic and serum triglycerides, FFA, TC, LDL-C, and VLDL-C while HDL-C was found to be reduced. However, administrations of ASEDB effectively restored serum lipids and hepatic triglycerides to normalcy in proportion to the dosage. The AST and ALT showed elevated levels in the serum of HFD-fed rats than normal rats. However, on ASEDB treatment, these alterations were reversed to normal, dose-dependently (Table 1).Table 1
Effect of ASEDB on different biochemical parameters.
ParametersGroup 1Group 2Group 3Group 4Group 5Group 6Glucose (mg/dL)71.08 ± 1.65149.16 ± 2.58#125.84 ± 1.67106.43 ± 1.8293.28 ± 1.87∗76.52 ± 2.12∗∗Insulin (µIU/mL)3.42 ± 0.207.35 ± 0.22#6.55 ± 0.165.61 ± 0.235.02 ± 0.16∗4.04 ± 0.25∗∗Leptin (ng/mL)3.12 ± 0.247.56 ± 0.32#6.36 ± 0.385.63 ± 0.364.95 ± 0.18∗3.83 ± 0.17∗∗Hepatic TG (mg/g tissue)9.58 ± 0.4818.02 ± 0.49#16.22 ± 0.6115.33 ± 0.5214.54 ± 0.13∗11.26 ± 0.23∗∗Serum TG (mg/dL)73.85 ± 1.92169.24 ± 2.54#146.34 ± 1.77127.92 ± 2.69114.47 ± 1.93∗92.19 ± 1.47∗∗FFA (mg/dL)20.12 ± 2.0846.71 ± 1.89#34.86 ± 1.2032.80 ± 1.7129.56 ± 2.32∗21.90 ± 1.03∗∗TC (mg/dL)98.02 ± 1.41174.16 ± 2.48#158.31 ± 1.96135.71 ± 1.98108.65 ± 1.96∗99.54 ± 1.59∗∗HDL-C (mg/dL)67.28 ± 0.9843.05 ± 0.71#46.93 ± 0.8849.21 ± 1.3253.91 ± 0.91∗∗58.99 ± 1.25∗∗LDL-C (mg/dL)42.15 ± 1.66157.55 ± 1.43#132.43 ± 1.91103.48 ± 1.2473.60 ± 1.58∗57.33 ± 1.34∗∗VLDL-C (mg/dL)15.25 ± 0.5233.16 ± 1.83#31.28 ± 1.0225.58 ± 1.1422.09 ± 0.63∗17.08 ± 0.59∗∗ALT (U/L)39.23 ± 1.0474.19 ± 0.80#57.18 ± 1.2951.03 ± 0.8845.73 ± 0.93∗43.89 ± 1.27∗∗AST (U/L)67.83 ± 1.47150.56 ± 1.32#135.79 ± 0.62121.69 ± 1.13102.06 ± 1.34∗79.63 ± 1.46∗∗Values are expressed in the mean ± S.E.M (n = 6); #significantly differ from normal control at p<0.01; ∗significantly differ from the HFD-treated group at p<0.05; ∗∗significant difference from the HFD-treated group at p<0.01.


#### 3.3.4. Effect of ASEDB on Hepatic Antioxidant Status

The data obtained on antioxidant status in liver tissues of HFD-fed obese rats revealed a significant elevation in peroxidation of lipids and depletion in the level of GSH and activities of enzymatic antioxidants such as SOD and CAT. However, the analyses of data from HFD-rats treated with ASEDB have shown improved levels of these alterations in a dose-reliable manner (Table2).Table 2
Effect of ASEDB on hepatic enzymatic and nonenzymatic antioxidant parameters.
ParametersGroup 1Group 2Group 3Group 4Group 5Group 6LPO (n moles of MDA formed/g tissue)78.25 ± 1.04144.56 ± 1.72#137.42 ± 1.31118.78 ± 1.52112.12 ± 1.18∗97.42 ± 1.42∗∗GSH (mg/g tissue)4.47 ± 0.052.33 ± 0.06#2.51 ± 0.113.56 ± 0.103.98 ± 0.07∗4.25 ± 0.18∗∗SOD (µmoles of epinephrine oxidized/mg protein)5.06 ± 0.191.98 ± 0.07#2.35 ± 0.152.71 ± 0.233.17 ± 0.24∗4.23 ± 0.26∗∗CAT (µmoles of H2O2 utilized/min/mg protein)34.72 ± 1.2914.27 ± 0.84#17.31 ± 0.7120.50 ± 0.9424.41 ± 1.32∗30.07 ± 1.23∗∗Values are expressed in the mean ± S.E.M (n = 6); #significantly differ from normal control at p<0.01; ∗significantly differ from the HFD-treated group at p<0.05; ∗∗significant difference from the HFD-treated group at p<0.01.


## 3.1. Inhibitory Effect of Pancreatic Lipase by ASEDB

The porcine pancreatic lipase (PPL) inhibitory action of ASEDB at various concentrations (5µg/mL to 500 µg/mL) was assessed by evaluating the breakdown of p-nitrophenyl butyrate into p-nitrophenol. Figure 1 demonstrates that ASEDB has a substantial level of inhibition potential on the activity of PPL with IC50 of 213.3 µg/mL when matched to the standard drug orlistat, whose IC50 value was found to be 9.2 µg/mL.Figure 1
The pancreatic lipase inhibitory effect of ASEDB. Values are the mean of 3 experiments; ASEDB–aqueous seed extract ofDolichos biflorus L. Orlistat was used as a positive control.


## 3.2. Anti-Adipogenic and Lipolytic Effect

First, to assess the possibility of 3T3-L1 preadipocytes upon ASEDB exposure, the cells were treated with a varied dosage of ASEDB (10μg/mL–500 μg/mL) and the observed findings were expressed as percentage viability, comparing the cell viability of negative control’s (Figure 2(a)). These data made it clear that none of the ASEDB doses administered had an impact on the cells’ viability. Thus, the selected dosages of ASEDB (10 μg/mL−500 μg/mL) were deemed nontoxic in 3T3-L1 cell lines and considered for further investigation.Figure 2
Anti-adipogenic and lipolytic effect of ASEDB on 3T3-L1 adipocytes: (a) cell viability, (b) intracellular lipid content, (c) microscopic photographs of Oil-Red-O staining, and (d) glycerol release. ASEDB–aqueous seed extract ofDolichos biflorus L. Values are the mean ± SEM (n = 3). ∗∗p<0.05, significantly different from the control group.
(a)(b)(c)(d)Oil Red O staining was used to assess the anti-adipogenic potential of ASEDB. The observed findings (Figure2(b)), as well as photomicrographs of lipid staining (Figure 2(c)), clearly revealed that ASEDB dose-reliantly decreased lipid accumulation during 3T3-L1 differentiation. At the higher dose of 500 μg/mL, ASEDB exerted around 35.25 ± 1.37% reductions in fat accumulation than control cells.The lipolytic effect of ASEDB was assessed by estimating the quantity of glycerol liberated from the differentiated 3T3-L1 adipocytes and the obtained data are presented in Figure2(d). ASEDB had shown a reasonable level of lipolytic activity which was evidenced through a notable increase (p<0.05) in the release of glycerol approximately by 21, 31, and 33% at doses of 100, 250, and 500 μg/mL of ASEDB, respectively.


## 3.3. Antiobesity Effect of ASEDB on HFD-Induced Obese Rats

### 3.3.1. Effect of ASEDB on Food Consumption

The food consumption of the experimental groups was determined by subtracting the quantity of remaining chow from the preweighed chow, every 24 hours per cage basis and the received data are represented in Figure3 as average food intake. The data have indicated that animals fed with HFD consumed significantly more food on average than animals fed with the ND. Administration of ASEDB along with HFD however showed a slight suppression of food intake, without showing any significant dose-dependent variation. However, orlistat-treated group 6 rats showed significantly lower (p<0.05) food consumption than ASEDB-treated rats.Figure 3
Levels of average food intake in experimental rats. Group 1–fed with ND; group 2–fed with HFD; group 3–HFD + ASEDB (100 mg/kg bw); group 4–HFD + ASEDB (200 mg/kg bw); group 5–HFD + ASEDB (300 mg/kg bw); group 6–HFD + orlistat (30 mg/kg bw); ND–normal diet; HFD–high fat diet; ASEDB–aqueous seed extract ofDolichos biflorus L; values are the mean of 6 rats; ∗p<0.05, significantly different from group 1; ∗∗p<0.01, significantly different from group 2; #p<0.05, group 3, 4, and 5 significantly different from group 2; no significant differences between ASEDB-treated groups (group 3, 4, and 5).


### 3.3.2. Influence of ASEDB on Body Weight, Fat Pad Weights, Organ Weights, Adipocyte Area, BMI, and Waist Circumference (WC)

An initial body weight of the experimental models were noted on the day one, followed by the changes in those weights brought on by ASEDB supplementation; these changes were documented once every 10 days. Obtained results have shown that there was a significant upsurge in a gain of body weight in HFD control rats (Group 2) when matching to the normal control (Group 1) rats. On the 60th day of the experimental period, group 2 animals have shown approximately 15% more weight gain than group 1 animals. However, treatment with various dosages of ASEDB (100 mg/kg, 200 mg/kg, and 300 mg/kg BW) significant reduction (p<0.05) was noted in the percentage body weight gain of the animals are in proportion to the dose when compared to HFD control rats. Adipocyte area, the weights of the liver, heart, kidney, and spleen, as well as the mesenteric, perirenal, and retroperitoneal fat pad weights, were also observed to be increased in HFD fed, untreated rats but dramatically decreased (p<0.05) in ASEDB-treated rats in a dose-reliable manner (Figure 4).Figure 4
Effect of ASEDB on (a) body weight; (b) fat pad weight; (c) adipocyte area; (d) organ weights; and (e) microscopic images of adipocytes showing variations in adipocyte area among different groups. WAT–white adipose tissue; ASEDB–aqueous seed extract ofDolichos biflorus L.; values are the mean ± S.E.M of 6 rats; #p<0.05, significantly different from group 1; ∗p<0.05, significantly different from group 2; ∗∗p<0.01, significantly different from group 2.
(a)(b)(c)(d)(e)The BMI and WC of the animals were measured on days 1 and 60 and the found data are depicted in Figure5. When compared to obese rats that were administered HFD without receiving any treatment (Group 2), the administration of ASEDB plus HFD significantly decreased the rats’ BMI and WC in proportionate to the concentration.Figure 5
Effect of ASEDB on (a) body mass index; (b) waist circumference of experimental rats. Values are the mean ± S.E.M (n = 6); #significant difference from normal control (group 1) at p<0.01; ∗significant difference from HFD control (group 2) at p<0.05; ∗∗Significant difference from HFD control (group 2) at p < 0.01.
(a)(b)


### 3.3.3. Effect of ASEDB on Different Biochemical Parameters

The effect of ASEDB on blood glucose, leptin, and insulin levels in the animals is given in Table1. The increased levels of glucose, leptin, and insulin found in HFD-induced rats were efficiently returned to near-normal levels in ASEDB-treated animals. The data on the effect of ASEDB on hepatic triglycerides and serum lipid profiles are also given in Table 1. HFD feeding to the experimental animals raised the levels of hepatic and serum triglycerides, FFA, TC, LDL-C, and VLDL-C while HDL-C was found to be reduced. However, administrations of ASEDB effectively restored serum lipids and hepatic triglycerides to normalcy in proportion to the dosage. The AST and ALT showed elevated levels in the serum of HFD-fed rats than normal rats. However, on ASEDB treatment, these alterations were reversed to normal, dose-dependently (Table 1).Table 1
Effect of ASEDB on different biochemical parameters.
ParametersGroup 1Group 2Group 3Group 4Group 5Group 6Glucose (mg/dL)71.08 ± 1.65149.16 ± 2.58#125.84 ± 1.67106.43 ± 1.8293.28 ± 1.87∗76.52 ± 2.12∗∗Insulin (µIU/mL)3.42 ± 0.207.35 ± 0.22#6.55 ± 0.165.61 ± 0.235.02 ± 0.16∗4.04 ± 0.25∗∗Leptin (ng/mL)3.12 ± 0.247.56 ± 0.32#6.36 ± 0.385.63 ± 0.364.95 ± 0.18∗3.83 ± 0.17∗∗Hepatic TG (mg/g tissue)9.58 ± 0.4818.02 ± 0.49#16.22 ± 0.6115.33 ± 0.5214.54 ± 0.13∗11.26 ± 0.23∗∗Serum TG (mg/dL)73.85 ± 1.92169.24 ± 2.54#146.34 ± 1.77127.92 ± 2.69114.47 ± 1.93∗92.19 ± 1.47∗∗FFA (mg/dL)20.12 ± 2.0846.71 ± 1.89#34.86 ± 1.2032.80 ± 1.7129.56 ± 2.32∗21.90 ± 1.03∗∗TC (mg/dL)98.02 ± 1.41174.16 ± 2.48#158.31 ± 1.96135.71 ± 1.98108.65 ± 1.96∗99.54 ± 1.59∗∗HDL-C (mg/dL)67.28 ± 0.9843.05 ± 0.71#46.93 ± 0.8849.21 ± 1.3253.91 ± 0.91∗∗58.99 ± 1.25∗∗LDL-C (mg/dL)42.15 ± 1.66157.55 ± 1.43#132.43 ± 1.91103.48 ± 1.2473.60 ± 1.58∗57.33 ± 1.34∗∗VLDL-C (mg/dL)15.25 ± 0.5233.16 ± 1.83#31.28 ± 1.0225.58 ± 1.1422.09 ± 0.63∗17.08 ± 0.59∗∗ALT (U/L)39.23 ± 1.0474.19 ± 0.80#57.18 ± 1.2951.03 ± 0.8845.73 ± 0.93∗43.89 ± 1.27∗∗AST (U/L)67.83 ± 1.47150.56 ± 1.32#135.79 ± 0.62121.69 ± 1.13102.06 ± 1.34∗79.63 ± 1.46∗∗Values are expressed in the mean ± S.E.M (n = 6); #significantly differ from normal control at p<0.01; ∗significantly differ from the HFD-treated group at p<0.05; ∗∗significant difference from the HFD-treated group at p<0.01.


### 3.3.4. Effect of ASEDB on Hepatic Antioxidant Status

The data obtained on antioxidant status in liver tissues of HFD-fed obese rats revealed a significant elevation in peroxidation of lipids and depletion in the level of GSH and activities of enzymatic antioxidants such as SOD and CAT. However, the analyses of data from HFD-rats treated with ASEDB have shown improved levels of these alterations in a dose-reliable manner (Table2).Table 2
Effect of ASEDB on hepatic enzymatic and nonenzymatic antioxidant parameters.
ParametersGroup 1Group 2Group 3Group 4Group 5Group 6LPO (n moles of MDA formed/g tissue)78.25 ± 1.04144.56 ± 1.72#137.42 ± 1.31118.78 ± 1.52112.12 ± 1.18∗97.42 ± 1.42∗∗GSH (mg/g tissue)4.47 ± 0.052.33 ± 0.06#2.51 ± 0.113.56 ± 0.103.98 ± 0.07∗4.25 ± 0.18∗∗SOD (µmoles of epinephrine oxidized/mg protein)5.06 ± 0.191.98 ± 0.07#2.35 ± 0.152.71 ± 0.233.17 ± 0.24∗4.23 ± 0.26∗∗CAT (µmoles of H2O2 utilized/min/mg protein)34.72 ± 1.2914.27 ± 0.84#17.31 ± 0.7120.50 ± 0.9424.41 ± 1.32∗30.07 ± 1.23∗∗Values are expressed in the mean ± S.E.M (n = 6); #significantly differ from normal control at p<0.01; ∗significantly differ from the HFD-treated group at p<0.05; ∗∗significant difference from the HFD-treated group at p<0.01.


## 3.3.1. Effect of ASEDB on Food Consumption

The food consumption of the experimental groups was determined by subtracting the quantity of remaining chow from the preweighed chow, every 24 hours per cage basis and the received data are represented in Figure3 as average food intake. The data have indicated that animals fed with HFD consumed significantly more food on average than animals fed with the ND. Administration of ASEDB along with HFD however showed a slight suppression of food intake, without showing any significant dose-dependent variation. However, orlistat-treated group 6 rats showed significantly lower (p<0.05) food consumption than ASEDB-treated rats.Figure 3
Levels of average food intake in experimental rats. Group 1–fed with ND; group 2–fed with HFD; group 3–HFD + ASEDB (100 mg/kg bw); group 4–HFD + ASEDB (200 mg/kg bw); group 5–HFD + ASEDB (300 mg/kg bw); group 6–HFD + orlistat (30 mg/kg bw); ND–normal diet; HFD–high fat diet; ASEDB–aqueous seed extract ofDolichos biflorus L; values are the mean of 6 rats; ∗p<0.05, significantly different from group 1; ∗∗p<0.01, significantly different from group 2; #p<0.05, group 3, 4, and 5 significantly different from group 2; no significant differences between ASEDB-treated groups (group 3, 4, and 5).


## 3.3.2. Influence of ASEDB on Body Weight, Fat Pad Weights, Organ Weights, Adipocyte Area, BMI, and Waist Circumference (WC)

An initial body weight of the experimental models were noted on the day one, followed by the changes in those weights brought on by ASEDB supplementation; these changes were documented once every 10 days. Obtained results have shown that there was a significant upsurge in a gain of body weight in HFD control rats (Group 2) when matching to the normal control (Group 1) rats. On the 60th day of the experimental period, group 2 animals have shown approximately 15% more weight gain than group 1 animals. However, treatment with various dosages of ASEDB (100 mg/kg, 200 mg/kg, and 300 mg/kg BW) significant reduction (p<0.05) was noted in the percentage body weight gain of the animals are in proportion to the dose when compared to HFD control rats. Adipocyte area, the weights of the liver, heart, kidney, and spleen, as well as the mesenteric, perirenal, and retroperitoneal fat pad weights, were also observed to be increased in HFD fed, untreated rats but dramatically decreased (p<0.05) in ASEDB-treated rats in a dose-reliable manner (Figure 4).Figure 4
Effect of ASEDB on (a) body weight; (b) fat pad weight; (c) adipocyte area; (d) organ weights; and (e) microscopic images of adipocytes showing variations in adipocyte area among different groups. WAT–white adipose tissue; ASEDB–aqueous seed extract ofDolichos biflorus L.; values are the mean ± S.E.M of 6 rats; #p<0.05, significantly different from group 1; ∗p<0.05, significantly different from group 2; ∗∗p<0.01, significantly different from group 2.
(a)(b)(c)(d)(e)The BMI and WC of the animals were measured on days 1 and 60 and the found data are depicted in Figure5. When compared to obese rats that were administered HFD without receiving any treatment (Group 2), the administration of ASEDB plus HFD significantly decreased the rats’ BMI and WC in proportionate to the concentration.Figure 5
Effect of ASEDB on (a) body mass index; (b) waist circumference of experimental rats. Values are the mean ± S.E.M (n = 6); #significant difference from normal control (group 1) at p<0.01; ∗significant difference from HFD control (group 2) at p<0.05; ∗∗Significant difference from HFD control (group 2) at p < 0.01.
(a)(b)


## 3.3.3. Effect of ASEDB on Different Biochemical Parameters

The effect of ASEDB on blood glucose, leptin, and insulin levels in the animals is given in Table1. The increased levels of glucose, leptin, and insulin found in HFD-induced rats were efficiently returned to near-normal levels in ASEDB-treated animals. The data on the effect of ASEDB on hepatic triglycerides and serum lipid profiles are also given in Table 1. HFD feeding to the experimental animals raised the levels of hepatic and serum triglycerides, FFA, TC, LDL-C, and VLDL-C while HDL-C was found to be reduced. However, administrations of ASEDB effectively restored serum lipids and hepatic triglycerides to normalcy in proportion to the dosage. The AST and ALT showed elevated levels in the serum of HFD-fed rats than normal rats. However, on ASEDB treatment, these alterations were reversed to normal, dose-dependently (Table 1).Table 1
Effect of ASEDB on different biochemical parameters.
ParametersGroup 1Group 2Group 3Group 4Group 5Group 6Glucose (mg/dL)71.08 ± 1.65149.16 ± 2.58#125.84 ± 1.67106.43 ± 1.8293.28 ± 1.87∗76.52 ± 2.12∗∗Insulin (µIU/mL)3.42 ± 0.207.35 ± 0.22#6.55 ± 0.165.61 ± 0.235.02 ± 0.16∗4.04 ± 0.25∗∗Leptin (ng/mL)3.12 ± 0.247.56 ± 0.32#6.36 ± 0.385.63 ± 0.364.95 ± 0.18∗3.83 ± 0.17∗∗Hepatic TG (mg/g tissue)9.58 ± 0.4818.02 ± 0.49#16.22 ± 0.6115.33 ± 0.5214.54 ± 0.13∗11.26 ± 0.23∗∗Serum TG (mg/dL)73.85 ± 1.92169.24 ± 2.54#146.34 ± 1.77127.92 ± 2.69114.47 ± 1.93∗92.19 ± 1.47∗∗FFA (mg/dL)20.12 ± 2.0846.71 ± 1.89#34.86 ± 1.2032.80 ± 1.7129.56 ± 2.32∗21.90 ± 1.03∗∗TC (mg/dL)98.02 ± 1.41174.16 ± 2.48#158.31 ± 1.96135.71 ± 1.98108.65 ± 1.96∗99.54 ± 1.59∗∗HDL-C (mg/dL)67.28 ± 0.9843.05 ± 0.71#46.93 ± 0.8849.21 ± 1.3253.91 ± 0.91∗∗58.99 ± 1.25∗∗LDL-C (mg/dL)42.15 ± 1.66157.55 ± 1.43#132.43 ± 1.91103.48 ± 1.2473.60 ± 1.58∗57.33 ± 1.34∗∗VLDL-C (mg/dL)15.25 ± 0.5233.16 ± 1.83#31.28 ± 1.0225.58 ± 1.1422.09 ± 0.63∗17.08 ± 0.59∗∗ALT (U/L)39.23 ± 1.0474.19 ± 0.80#57.18 ± 1.2951.03 ± 0.8845.73 ± 0.93∗43.89 ± 1.27∗∗AST (U/L)67.83 ± 1.47150.56 ± 1.32#135.79 ± 0.62121.69 ± 1.13102.06 ± 1.34∗79.63 ± 1.46∗∗Values are expressed in the mean ± S.E.M (n = 6); #significantly differ from normal control at p<0.01; ∗significantly differ from the HFD-treated group at p<0.05; ∗∗significant difference from the HFD-treated group at p<0.01.


## 3.3.4. Effect of ASEDB on Hepatic Antioxidant Status

The data obtained on antioxidant status in liver tissues of HFD-fed obese rats revealed a significant elevation in peroxidation of lipids and depletion in the level of GSH and activities of enzymatic antioxidants such as SOD and CAT. However, the analyses of data from HFD-rats treated with ASEDB have shown improved levels of these alterations in a dose-reliable manner (Table2).Table 2
Effect of ASEDB on hepatic enzymatic and nonenzymatic antioxidant parameters.
ParametersGroup 1Group 2Group 3Group 4Group 5Group 6LPO (n moles of MDA formed/g tissue)78.25 ± 1.04144.56 ± 1.72#137.42 ± 1.31118.78 ± 1.52112.12 ± 1.18∗97.42 ± 1.42∗∗GSH (mg/g tissue)4.47 ± 0.052.33 ± 0.06#2.51 ± 0.113.56 ± 0.103.98 ± 0.07∗4.25 ± 0.18∗∗SOD (µmoles of epinephrine oxidized/mg protein)5.06 ± 0.191.98 ± 0.07#2.35 ± 0.152.71 ± 0.233.17 ± 0.24∗4.23 ± 0.26∗∗CAT (µmoles of H2O2 utilized/min/mg protein)34.72 ± 1.2914.27 ± 0.84#17.31 ± 0.7120.50 ± 0.9424.41 ± 1.32∗30.07 ± 1.23∗∗Values are expressed in the mean ± S.E.M (n = 6); #significantly differ from normal control at p<0.01; ∗significantly differ from the HFD-treated group at p<0.05; ∗∗significant difference from the HFD-treated group at p<0.01.


## 4. Discussion

Obesity is reaching epidemic proportions worldwide and has led to a rise in the burden of health care, a decline in life expectancy, and many other socioeconomic problems. This epidemic of obesity alerts the public and mainstream researchers to the need to increase the efficacy of the therapies available. In the present study, we have studied the antiobesity properties of ASEDB in the differentiation of 3T3-L1 preadipocytes and HFD-induced obesity in the experimental rats.Although numerous factors may be responsible for the pathogenesis of obesity, the availability of energy-rich foods and excessive calorie consumption are the prominent contributors to obesity in maximum cases [26]. Dietary fats are mostly comprised of mixed TGs which get broken down by pancreatic lipase (PL) and serve as the major source of extra calories relative to other nutrients. Therefore, the reduction of the activity of PL and also reduction of the digestion and absorption of dietary fat from the intestine are the cornerstone strategies in the therapeutic attempt to reduce the excessive intake of energy [27]. So far, orlistat, which is derived from Streptomyces toxytricini, is the lone drug available in the market to inhibit PL activity. This drug can reduce fat absorption by about 30%. However, its use is limited because its prolonged usage can result in side effects including oily spotting, steatorrhea, fecal urgency, and bloating [28, 29]. Hence, the quest for a safe PL inhibitor is still in pipeline and many researchers in recent days screened hundreds of natural products for possible lipase inhibitory effects. In the current investigation, we analyzed the pancreatic lipase inhibition ability of ASEDB and the found data exposed that ASEDB inhibited PL moderately in different concentrations when compared with the standard drug orlistat. Even though ASEDB was not more effective in inhibiting lipase activity than orlistat, its effect was superior to that of many other dietary plants which were reported previously [30]. Given that the secondary metabolites of ASEDB such as tannins, saponins, polyphenols, or triterpenes have a well-established ability to inhibit pancreatic lipases [31–33], this inhibitory effect of ASEDB might be mediated by these secondary metabolites.Obesity is highlighted by the accumulation of additional calories in the adipose tissue, which is mostly composed of fat cells. Because this expansion of adipose mass is associated with both hyperplasia and hypertrophy, the efficiency of ASEDB was studied on the progressions of the proliferation of the adipocyte and differentiation with 3T3-L1 preadipocytes. 3T3-L1 cell line, developed from murine Swiss 3T3 cells, is a well-known model for studying adipogenesis and obesity [14]. These cells display a fibroblast-like morphology and upon suitable conditions undergo differentiation (the process also known as adipogenesis) and develop into mature adipocytes [34, 35]. We first examined the effect of various concentrations of ASEDB on 3T3-L1 preadipocyte viability. As revealed in Figure 2(a), an insignificant reduction in cell viability was noticed as the concentration increased, but still, the viability of the cell was retained at over 95% for all the administered concentrations. Therefore, these concentrations were taken as noncytotoxic doses and were used for further study.The 3T3-L1 preadipocytes have been exposed to an adipogenic-inducing medium and the same has been treated with various doses of ASEDB. Then, the concentrations of intracellular lipid build-up in maturing 3T3-L1 adipocytes were measured. 3T3-L1 preadipocytes possess the triglyceride accumulation ability upon differentiation. Oil Red O staining results showed that ASEDB decreased the size and number of lipid droplets in a dose-reliant manner and indicates the inhibitory activity on the accumulation of lipids in differentiated 3T3-L1 adipocytes using ASEDB. As the mechanism of antiobesity potential mainly depends upon the balance between lipolysis and lipogenesis [17, 36], we then studied the lipolytic effect of ASEDB on matured 3T3-L1 adipocytes. Adipocytes release glycerol into the medium in proportionate to triglyceride hydrolysis, and this release was found to be significantly elevated after the treatment of matured 3T3-L1 adipocytes with ASEDB, expressing the antiadipogenic effects. The findings of the present study are in agreement with the earlier reported study [11]. Adipocyte differentiation is promoted by a cascade of transcription factors acting sequentially. Likewise, lipolysis was also identified to be regulated by the expression of genes associated with hormone-sensitive lipase and the oxidation of fatty acid. It is presumed that ASEDB inhibits the development as well as the build-up of fat in the cells by obstructing the proliferation and differentiation of adipocytes by promoting lipolysis by regulating the expression of lipolysis-associated genes. Furthermore, in-depth gene expression studies are needed to confirm this. Many natural compounds, such as epigallocatechin gallate (EGCG), ginkgolide C, capsaicin, resveratrol, and curcumin have also been reported to inhibit adipogenesis and induce the process of lipolysis through regulated gene expression [34].If a person consumes additional calories, then they use them up or expend; such additional calories get deposited as adipose tissues and result in weight gain. Thus, body weight gain is assessed as one of the early signs of the progression of obesity [37]. In this study, HFD feeding resulted in 15.3% more weight gain in obese control rats (group 2) than in normal control rats (group 1) over 60 days. Increased body weight in group 2 animals might be associated with energy balance and accompanying fat deposition in visceral regions caused by HFD feeding. On the contrary, ASEDB supplementation in addition to HFD significantly (p<0.05) reduced the percent weight gain following the dose to HFD-fed group 2 rats. The decreases in body weight may partially be associated with suppressed food intake as evidenced in this study in ASEDB-treated rats. Also, food intake was not drastically suppressed in these ASEDB-treated rats, and their food intake was still higher than that of normal control rats. This discards the possibility of provocation of ASEDB-induced toxicity. Earlier studies also reported its nontoxicity nature and its safe usage as a food source [38].Obesity features an upsurge in the number and size of adipocytes at the cellular level because of unnecessary fat deposition [39]. Thus, measurement of adipocyte fat pad weights would help to understand the magnitude of obesity. In this study, we observed the weights were increased in the retroperitoneal, mesenteric, and perirenal fat pads in the obese (HFD-fed) control rats when compared to the normal control rats. However, ASEDB administration, significantly reduced (p<0.05) the weight of such fat pads. Furthermore, the measurement of the size of adipocytes in terms of their area after 9 weeks of treatment with ASEDB had shown a substantial decrease in the area of adipocytes following the dose. Determination of organ weight could be another important index of the severity of obesity, as excess calories slowly deposit in organs like the liver, heart, kidney, and spleen in addition to adipose tissues [40]. It was found that HFD feeding resulted in around 46%, 39%, 41%, and 44% more weight gain in the liver, kidney, heart, and spleen of obese control rats (group 2), respectively, than normal diet-fed rats (group 1). However, ASEDB treatment had shown a significant (p<0.05) decrease in the weight of the organ in a dose-dependent manner. It was reported previously that continuous exposures to HFD decline the resting metabolic rate as well as thermogenesis, thereby promoting the accumulation of triglycerides in abdominal regions [41].The anthropometric methods such as BMI and WC are standard practical approaches, which are recommended for diagnosis, risk evaluation, and exact tracking of overall body fat distribution. Assessing central obesity (visceral adiposity) through BMI and WC helps to predict metabolic troubles like CVDs, atherosclerosis, hypertension, and dyslipidemia [42]. It was reported that a 1 cm rise in WC raises the threat of the arousal of hypertension by 2.49 times [43]. The rise in WC is also a useful predictor for the subsequent progression of T2DM [44]. In this study, it has been noted that HFD administered for 60 days results in a considerable rise in the BMI and WC; however, ASEDB treatment brought them back significantly (p<0.05) in proportionate to dose. This decline in BMI and WC is well correlated with reduced fat accumulation in fat pads and visceral organs.The adipose tissue acts as a dynamic endocrine organ, which secretes several biologically active adipokines including leptin, theob gene cytokine product. In this study, it is noted that a significant rise (p<0.05) in leptin levels in the obese rats than in normal rats. The leptin level is found to be positively connected with the proportion of fat stores in white adipocytes [45, 46]. Thus, elevated leptin concentration in serum is a marker of body fat mass increase. At normal conditions, the circulating leptin signals the high energy reserves of the periphery to the hypothalamus thereby regulating energy metabolism by suppressing appetite. However, in obese conditions, elevated leptin levels do not seem to do this role because of leptin resistance. It was proposed that raised leptin levels in obesity can cause leptin receptors desensitization in the hypothalamus, thereby disturbing the downstream regulation of energy consumption and body weight [47]. Elevated leptin levels were found to be significantly reduced with ASEDB intake, in proportionate to the dose. This indicates that intake of ASEDB is effective in refining changes in fat mass and leptin levels, affected by HFD intake.Being overweight and obese have a strong association with T2DM and this may be because of insulin resistance (IR) [48]. In obese conditions, enlarged adipocytes secrete comparably more proinflammatory cytokines (leptin, TNF-α, and IL-6), which causes IR [32, 49]. The resulting IR in obesity thus affects the liver or muscle cells’ insulin-dependent glucose intake and contributes to elevated blood glucose levels. The β-cells of the pancreas continually synthesize and secrete insulin in response to increased blood glucose levels, which in turn contributes to hyperinsulinemia. IR, hyperglycemia, and hyperinsulinemia are frequently connected with obesity [50]. The obtained high levels of both blood glucose and serum insulin in the current study also confirmed hyperglycemia and hyperinsulinemia in obese rats. However, in ASEDB-treated rats, the levels of glucose and insulin seemed to be significantly (p<0.05) reduced at a higher dose. The reduced level of leptin observed in ASEDB intake might have improved insulin sensitivity, which is well correlated with the reduced levels of blood glucose and insulin in ASEDB-treated animals.Dyslipidemia is one of the major concerns of obesity as it significantly escalates the risk of CVD, T2DM, and certain types of cancer. Mounting evidence suggests that IR is the most probable link between obesity and obesity-associated metabolic dyslipidemia [51]. The main patterns in patients with dyslipidemia are their high concentrations of lipid profiles. In the current study, we also encountered significantly (p<0.01) elevated levels of TC, TG, LDL-C, VLDL-C, FFA, and reduced levels of HDL-C in obese rats than in the control group. Elevated FFAs in an obese state are mainly an outcome of the enhanced release of FFA from adipose fat mass due to the resistance of adipocytes to the anti-lipolysis deed of insulin and the inability of adipocytes to reutilize FFAs efficiently [52]. Hypertriglyceridemia in obesity is partly due to increased liver FFA fluxes and their accumulation as TG. This causes elevated synthesis of large VLDL by hepatocytes, which interrupts the lipolysis of chylomicrons due to competition for lipoprotein lipase (LPL). Under these circumstances of increased concentration and delayed clearance of VLDL, cholesterol ester transfer protein (CETP) promotes the interchange of cholesterol esters in HDL and LDL with TGs in VLDL. This exchange also results in increased cholesterol ester-enriched VLDL particles and reduced HDL-C concentrations [53]. The administration of ASEDB ameliorated dyslipidemia by substantially reverting the altered levels of these lipid profiles to normal in a dose-based manner. The observed reversion of lipid levels may be due to the actions of phytoconstituents present in the extracts in the enhancement of satiety as well as suppression of digestion and absorption of dietary triglycerides through inhibition of lipase activity.Hepatic steatosis (fatty liver) is a pathological condition due to the deposition of large fat mass in the liver which frequently occurs in patients with obesity. Although liver steatosis is a multifactorial condition, IR is a prime requirement for hepatocellular fat accumulation. IR in obesity may result in boosted lipolysis of TG in the adipose tissue and it leads to more accessibility of FFAs for an enhanced synthesis and storage of triglycerides in liver cells [54]. Our data showed elevated levels of hepatic TGs in HFD-fed control rats, thereby suggesting the development of a fatty liver. However, the supplementation of ASEDB in addition to HFD bettered the fatty liver by overturning the elevation of liver TG. ALT and AST, the hepatic marker enzymes, were also noted to raise in the HFD control rats and this raise may be attributed to fatty liver and oxidative stress-induced hepatic necro-inflammatory injury upon continuous feeding of HFD. Elevated levels of AST and ALT are the common index of fatty liver and associated hepatotoxicity [55]. Whereas, these raised hepatic enzymes were considerably (p<0.05) restored in the ASEDB-administered rats, suggesting the recovery of necro-inflammatory injury, caused by HFD-induced fatty liver.Excess intakes of fat-rich diets are capable of generating reactive oxygen species (ROS) due to altered oxygen metabolism. Excessive lipid peroxidation and ROS will utilize the body’s antioxidant enzymes and vitamins [56, 57], resulting in the depletion of these protective substances and leading to oxidative stress and ROS-mediated cellular damage. Chronic oxidative stress is an important factor in the pathogenesis of obesity and its related morbidities [58]. In the current study, we witnessed higher content of malondialdehyde (MDA) which is the common biomarker for oxidative stress and end product of lipid peroxidation [59], in liver tissues of obese rats than in standard laboratory diet-fed normal rats, indicating HFD-induced oxidative stress in obese rats. ASEDB administration, however, prevented lipid peroxidation which was evidenced by a substantially diminished quantity of MDA in the liver of the treated rats. The level of glutathione (GSH), an important cytosolic antioxidant, was found to be markedly depleted in obese rats than the control group; however, in ASEDB-treated rats, the concentration of GSH was restored significantly (p<0.05). The depletion of GSH in obese rats could be understandable because whenever the cells experience oxidative stress, GSH could be utilized in large amounts to directly quench the ROS such as lipid peroxides. Cells also have another set of powerful antioxidant enzymes named SOD and CAT to respond to the superoxide anions which are generated during oxidative damage. SODs respond to these superoxide anions (O2•−) by converting them into H2O2 while CAT converts this toxic H2O2 into O2 and H2O [60]. We detected significantly diminished levels of both antioxidant enzymes in obese control rats than in control and the reduction may owe to rapid and more consumption of these enzymes storage, in counteraction to the free radicals produced during the progression of obesity. The levels of SOD and CAT were found to be reversed in ASEDB-treated groups suggesting its free radical scavenging efficacy. Restoration of all these antioxidant systems, both enzymatic and nonenzymatic, might be associated with the effective participation of natural phytoconstituents particularly flavones and polyphenols in the test drug in scavenging the free radicals.


## 5. Conclusions

Taken together, the study represents that ASEDB possesses formidable antiobesity benefits byin-vitro (3T3-L1 adipocytes) and in-vivo (HFD-fed obese rats). The potentiality of ASEDB might be associated with the reduction of food intake, blockade of adipocyte differentiation, enhancement of lipolysis, decrease in intestinal fat absorption, and amelioration of dyslipidemia through regulated fat metabolism and improvement of antioxidant grade arbitrated by the defensive phytoconstituents present in it. In addition, exploring the mRNA expression of adipogenic proteins, and fatty acid oxidations, as well as in-depth analysis of adipocytokines and ghrelin levels are warranted to understand the detailed mechanism of action. It is also indispensable to isolate and characterize the lead molecules responsible for antiobesity action to develop a promising pure drug to combat obesity.



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*Source: 6251200-2023-02-10.xml*