Anti-obesity effects and underlying molecular mechanisms of the ethanolic extract of figs from Ficus hispida using high fat-fed wister rats

Obesity is a known risk factor for many chronic diseases and a substantial threat to public health. We investigated the effects of figs sourced from Ficus hispida on a high fat-fed experimental rat model. We found that a 500-mg dose of ethanolic extract of figs (EFH) reduced oxidative stress markers nitric oxide (NO), malondialdehyde (MDA), and advanced oxidation protein products (AOPP), which were increased in high fat-fed rats. Antioxidant enzymes superoxide dismutase (SOD), catalase, reduced glutathione (GSH), and myeloperoxidase (MPO), found elevated in high fat-fed rats, were also normalized to nearly regular levels by fig treatment. Administration of EFH further reduced fat deposition and expression of adipogenic genes leptin, fatty acid synthase (FAS), peroxisome proliferator-activated receptor gamma (PPARγ), and sterol regulatory element-binding protein-1c (SERBP-1c). Our results suggest that figs have significant effects on reducing oxidative stress and mitigating obesity-associated liver and adipose tissue abnormalities via suppressing adipogenesis. Thus, we propose that F. hispida has potential benefits in reducing obesity.


Introduction
Obesity, a metabolic disease, has emerged as a global public health concern over the past few decades.Despite ongoing global awareness campaigns, the obesity rate continues to escalate among people of all ages worldwide.Obesity occurs when the adipocytes of the body lead to hypertrophy or hyperplasia [1].Obesity shows a distinct positive correlation with increased morbidity and decreased life expectancy [2].Sedentary lifestyles, socioeconomic and environmental changes [3], decreased physical activity, psychological issues, and the consumption of energy-rich meals [4] contribute to the onset of obesity.According to a study, 1.1 billion adults worldwide and about 10 % of the children are obese [5].A study by the World Health Organization (WHO) revealed that over 1 billion individuals are overweight, and about 300 million suffer from obesity [6].Obesity possesses detrimental consequences on lipid levels, cholesterol, and triglycerides.It leads to complications like fatty liver [7], type 2 diabetes [8], dyslipidemia [9], coronary heart diseases [10], musculoskeletal disturbance, particularly osteoarthritis [11], and risk of cancers [12].Non-alcoholic fatty liver disease development and progression depend on the pathologic accumulation of lipid droplets within hepatocytes [13,14], and this can progress to steatohepatitis, cirrhosis, and end-stage liver disease over time [15,16].Dysregulation of hepatic bioactive mediators and cytokines such as adiponectin, leptin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) influence body weight homeostasis, lipid levels, fibrinolysis, and inflammation [17].Despite these complications, obesity is the most prevalent preventable cause of death [18].
Treatment of obesity is complicated due to ambiguous etiology.Diverse integrative and complementary approaches are available for treating obesity, including surgical procedures, dietary programs, lifestyle changes, and pharmacological therapies [3].Given their non-intrusive nature, pharmacological approaches are often favored [3].However, many anti-obesity drugs have been discontinued due to substantial side effects.These include fenfluramine, dexfenfluramine, phentermine, diethylpropion, mazindol, and rimonabant [19,20].Therefore, there is an urge for novel therapies from plants or other natural resources to manage obesity and its comorbidities with few to no adverse effects.
Phytochemicals have been recognized as a financially viable substitute for current anti-obesity medications [21].Several studies have revealed that plant extracts have hypoglycemic, anti-obesity and anti-inflammatory activities [19,21].Medicinal plants of the genus Ficus have been reported to show anti-inflammatory, antidiabetic, antitumor, and hepatoprotective activities [22,23].F. hispida is used as a traditional remedy in different countries, including India, China, Australia, Sri Lanka, and Myanmar.It has been used to treat anemia, diarrhea, ulcer, diabetes, cancer, and inflammation [22,24].Raw figs have high levels of carbohydrate, pectin, flavonoids, terpenoids, alkaloids, and vitamins.Some flavonoids, such as naringenin, have been shown to inhibit HMG CoA reductase and Acyl-CoA cholesteryl acyltransferase (ACAT) activities in diet-induced obesity models in rats, indicating that the flavonoids present in figs might play a role in the anti-lipidemic effect [25].
In 2015, M. Selvakumar and colleagues conducted research on the ethanolic extract of Ficus religiosa and found that it significantly reduced both body weight and organ weight in rats, highlighting its potential anti-obesity effects [26].In 2016, Mopuri and Islam conducted an in-vitro study demonstrating the antioxidant, antidiabetic, and anti-obesogenic effects of ethanolic extract of Ficus carica [9].
Given that other varieties of figs have been studied previously, the present study focused on the ethanolic extract of F. hispida fruits, rich in polyphenols and flavonoids, to investigate its effects on alleviating obesity-associated inflammation in the liver and adipose tissue.We used high fat-fed obese Wister rats to determine the lipid-lowering effects of figs by examining biochemical parameters, histology, and expression of adipogenic genes.

Extract preparation
Green fruits of F. hispida were sourced from Khulna, Bangladesh in premonsoon season.After a three-week period of drying under shade, the figs were crushed and immersed in 80 % ethanol for four days with periodic stirring for better yield.Subsequently, the content was passed through the Whatman No-1 filter paper and the solvent was removed through rotary evaporation.The residual solvent was evaporated through incubation at room temperature for 12 h and the extract was refrigerated for further investigations [27].

Determination of total phenolic content
The total phenolic content was determined using the Folin-Ciocalteu assay.In various test tubes, 300 μl of the EFH was transferred at different concentrations, and 1.2 ml of a 10 times diluted Folin-Ciocalteau reagent solution was thoroughly mixed with each.After 3 min of incubation at room temperature, 1.2 ml of sodium carbonate solution (7.5 % w/v) was added.The mixtures were kept in the dark for 1 h at the room temperature before taking the absorbance at 765 nm using a spectrophotometer against blank [28].

Determination of total flavonoid content
The AlCl 3 method was employed to estimate the total flavonoid content of the extracts [29].To 0.5 ml of the extract solution, we added methanol (1.5 ml), 10 % AlCl 3 (0.1 ml), 1 M potassium acetate (0.1 ml), and distilled water (2.8 ml).The reaction mixture was incubated for 30 min at room temperature, followed by spectrophotometric measurement at 415 nm.

Animal experimentation
Forty-two Wister rats, aged 6-8 weeks and weighing between 250 and 270 g, were sourced from the Animal House of North South University.Six rats were housed in each polycarbonate cage with optimum laboratory conditions (25 ± 2 • C, 55 % relative humidity, 12:12-h dark/light cycle).All rats were provided with free access to water and a standard laboratory diet.The experimental protocols received approval from the Ethical Committee of North South University.There were seven groups (six rats per group): Group I (CON): rats received regular diet, Group II (HF): rats received high fat (HF) diet, Group III (CON + FH): rats received control The daily food and water intake and body weight were documented during the treatment period of 56 days.On day 57, rats were sacrificed after anesthesia through peritoneal ketamine injection.The whole liver was collected and gently washed with PBS (pH 7.4), blotted with Kimwipes and weighed while the inguinal adipose tissues were collected and processed.Both liver and adipose tissues were divided into two parts-one part was fixed in 10 % formalin (pH 7.4) and used for histology, and the other part was stored at − 20 • C for subsequent investigations.Blood samples from the abdominal aorta were collected into citrate buffer-containing tubes and kept on ice.The samples were centrifuged at 8000 rpm for 15 min at 4 • C and the supernatant plasma was transferred to 1.5 mL Eppendorf tubes and stored at − 20 • C until further analysis.

Oral glucose tolerance test (OGTT)
An oral glucose tolerance test (OGTT) was conducted twice, first at day 0 and again at day 57.The rats were fasted for 12 h before the test, and only drinking water was administered.The glucose load was provided with dextrose (2 g/kg body weight).Blood samples were obtained from the tail vein for the measurement of blood glucose concentration at 30 min intervals for 120 min using a glucometer (Accu-Chek Active, Roche Diabetes Care GmbH, Germany).

Plasma biochemistry analysis
The biochemical tests for measuring total cholesterol, HDL, LDL, triglycerides, ALP, AST, ALT, uric acid, and creatinine were performed in plasma samples using kits following standard protocols of the manufacturers (Human Lab, Germany).

Preparation of tissue samples
Liver tissues were homogenized into ten volumes of phosphate buffer.The homogenates were centrifuged at 10,000 rpm for 20 min at 4 • C and the supernatant was used for further analysis as described below.

Determination of MPO activity
We prepared the o-Dianisidine solution by adding 5 ml of concentrated HCl to 97 ml of distilled water and then mixing 1 ml of the HCl solution with 20 mg of o-Dianisidine.In 96-well plates, 290 μL of peripheral blood smears were combined with 10 μL of tissue/ plasma.Subsequently, 3 μL of H 2 O 2 and 3 μL of o-Dianisidine solutions were introduced.The absorbance of the final solution was recorded at 460 nm [30].

Determination of MDA
Tissue homogenate and plasma samples were obtained at a ratio of 2:5 in an Eppendorf tube.Glacial acetic acid and PBS were added to it.The mixture was incubated at room temperature for 15 min, and TBA solution was added to the sample at a ratio of 1:1, sample and TBA.After 30 min of hot water inoculation, the Eppendorf tube was cooled.The absorbance of the supernatant was read at 490 nm on a 96-well plate [28].

Determination of NO
Tissue homogenates or plasma samples were taken at a ratio of 2:5 with PBS in 96-well plates and mixed with 0.1 mL sulfanilamide.

Determination of AOPP
Determination of AOPP levels was performed by a modified Witko-Sarsat method [31].Tissue homogenate/plasma was taken at a ratio of 1:2 with PBS.50 μL potassium iodide and acetic acid were added.Following a 2-min incubation at room temperature, the absorbance was measured at 405 nm [32].

Determination of catalase
Catalase activities were determined using the previously described method by Mueller [33] with minor modifications.A 190 μL phosphate buffer was added to the 10 μL tissue homogenate/plasma sample.The mixture was then combined with a 100 μL H2O2 solution.The absorbance was determined at 240 nm at different intervals.

Determination of SOD
The activity of the superoxide dismutase enzyme was measured according to the modified approach reported by Misra [35].Briefly, a 10 μL tissue homogenate or plasma sample was integrated with 90 μL PBS. 100 μL of adrenaline injection was introduced into the solution, and absorbance was recorded at 480 nm.

Histopathological examination
We prepared 5-mm paraffin sections from formalin-fixed tissue blocks.We performed hematoxylin/eosin staining to investigate the tissue structure and infiltration of inflammatory cells.Histopathological evaluation was performed using three samples from each group by two independent observers.Photographs of all stained sections were captured using a light microscope (Olympus SZ61, Japan) at a magnification of 20x.

RNA extraction and reverse-transcriptase polymerase chain reaction (PCR)
We extracted total RNAs from the adipose tissue homogenates using the Trizol reagent (Sigma-Aldrich, USA) and RNAs were reverse transcribed to cDNAs using a DNA synthesis kit (Promega, USA).Semi-quantitative PCR was performed with specific primer sets (Table 1) for leptin, FAS, PPARγ, and SREBP-1c.PCR products were then run into the gel electrophoresis machine and gel images were captured with a gel documentation system (Axygen®).

Statistical analysis
Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls post hoc test.P-values <0.05, 0.01, 0.001 are considered statistically significant.

Effect of EFH on the total body weight and liver wet weight
We observed a considerable increase in body weight in the HF group than the control group.(Fig. 1a, p < 0.001).There were no appreciable changes in body weight in the control and CON + EFH groups.Compared with HF rats, a substantial body weight reduction was observed when EFH was administered.The higher dose (500 mg/kg) of EFH had the most significant effect and exhibited results identical to the standard drug, orlistat (Fig. 1a).
Fig. 1b shows that the liver wet weight of the HF group was significantly higher (Fig. 1b, p < 0.001) than that of the control group.No difference in the liver wet weight between the control and the CON + EFH groups was observed.Compared to the HF group, all treatment groups showed a decrease in the wet weight of the liver; however, the effect of EFH at a higher dose was nearly identical to that seen with orlistat (Fig. 1b).

EFH's effect on oral glucose tolerance test
The hypoglycemic effect of the EFH was assessed by OGTT.The first OGTT test that was done before starting the experiment showed an initial blood glucose level of around 5 mmol/L (Fig. 2a) in all the seven groups.After 30 min of feeding glucose to the rats, the blood sugar level of the rats raised, and the count was within 10-12 mmol/L which gradually decreased and again returned close to basal value after 2 h for all the 7 groups of rats (Fig. 2a).The second OGTT performed at the end of the treatment showed that after 30 min of feeding glucose, the blood glucose level was around 11-15 mmol/L (Fig. 2b) in all groups except the control group, however, Table 1 Primer sequences.

Genes
Forward Reverse A.T. Shama et al.  blood glucose level dropped slowly to the basal level in 2 h after administering the glucose solution in all groups except the HF group.
In HF group, the blood glucose level was around 9-10 mmol/L after 2 h (Fig. 2b).EFH showed a dose-dependent effect in lowering blood glucose level, and the orlistat exhibited the highest hypoglycemic activity after 2 h (Fig. 2c).The control and the CON + EFH groups exhibited almost the same effect.

The physiological effect of EFH on liver enzymes
The plasma AST level in the HF rats was substantially higher (p < 0.01) than that in the control group (Fig. 3a).The control and the CON + EFH groups, on the other hand, demonstrated almost a similar AST level.Three treatment doses significantly decreased the plasma AST compared to the HF group.However, the plasma AST levels in the medium and high-dose groups were nearly identical, while the decrease was less in the low-dose group.The orlistat-treated rats exhibited an AST level similar to the level observed in highdose EFH treatment group.In all groups, similar results were observed for ALP (Fig. 3b).
The HF group's plasma ALT level was considerably (p < 0.001) higher than the control group (Fig. 3c).However, EFH administration lowered the elevated level of ALT significantly in a dose-dependent manner.The orlistat treatment produced a similar effect as observed with the high dose of EFH while there was no significant difference in the ALT level between the control and CON + EFH groups.

Effect of EFH on the plasma lipid profile
The HF rat group had significantly elevated levels of total cholesterol (p < 0.001), triglycerides (p < 0.001), and LDL (p < 0.001) compared to the control group (Fig. 4).However, after administering EFH, there was a dose-dependent reduction in LDL (p < 0.01), triglyceride (p < 0.01), and total cholesterol (p < 0.05) levels.No significant changes in lipid markers were noticed between the control and CON + EFH groups.Remarkably, the HDL values in each group were similar (Fig. 4d).Also, it is noteworthy that no significant difference in the HDL level was observed by the high dose of EFH and orlistat.

EFH's effect on oxidative stress indicators in liver and plasma
The MDA, NO, and AOPP levels were considerably greater in the liver of HF rats compared to the other groups (Fig. 5).Upon treatment with EFH, the MDA (Fig. 5a), NO (Fig. 5b) and AOPP (Fig. 5c) levels were remarkably decreased dose-dependently.The low EFH dose did not produce any considerable effects on MDA and NO levels in liver while medium and high doses had powerful effects.No changes in the biomarkers' levels were observed between the control and CON + EFH groups.Almost the same effect was seen in rats treated with a high dose of EFH and in rats treated with orlistat.We further checked MDA, NO and AOPP levels in the plasma and observed similar results (Fig. 5d-f).

EFH's effects on cellular antioxidants and myeloperoxidase in plasma and liver tissue
In contrast to the control rats, the activities of enzymatic antioxidants SOD (Fig. 6a), GSH (Fig. 6b), and catalase (Fig. 6c) were considerably lowered in the liver of HF rats compared to the control.EFH-treated rats displayed a dose-dependent increase in all three parameters.HF rats also showed a markedly increased level of lipid peroxidation in MPO compared to control rats (Fig. 6d).This level was strongly diminished by EFH, in a dose-independent manner.EFH treatment in control rats did not show any appreciable change in these markers while orlistat worked robustly in enhancing SOD, GSH and catalase activities while decreasing MPO activity.We further checked the EFH effect on these enzymes in plasma, and found similar effects except in the MPO activity, which was decreased in a  dose-dependent manner (Fig. 6e-h).No significant difference was observed between the control and CON + EFH groups.The impact of orlistat was superior to the high dose of EFH.

Effect of EFH on renal indicators
The HF group had considerably higher plasma uric acid and creatinine levels than those in the control group (Fig. 7).There was no remarkable difference in these parameters between the control and CON + EFH groups.The increased serum uric acid and creatinine levels were significantly reduced by EFH treatment at varying degrees.The low and medium doses of EFH had almost identical effects on lowering the uric acid level (Fig. 7a), however, the EFH showed a dose-dependent effect in lowering the creatinine level (Fig. 7b).The standard drug orlistat showed an effect which was very similar to the high dose of the EFH.

Histopathological observation
Rats on a high-fat diet had higher levels of fat deposition in their liver (Fig. 8a-c) and adipose (Fig. 8h-j) tissues compared to the control group as determined by H&E staining.Tissue necrosis was evident in hepatic tissues of the HF group.The size of adipocytes also increased remarkably in this group.The tissues of the control rats presented regular distribution of fat with normal morphology.Treatment with EFH decreased fat deposition in the liver tissues (Fig. 8d-f) and also reduced the size of adipocytes (8k-m) No change in cell morphology was observed between control and CON + EFH groups (Fig. 8a, b and h, i).The high dose of EFH and the standard drug orlistat produced almost similar effects (Fig. 8f, g and m, n).

The expression of adipogenic genes
The results obtained from RT-PCR analysis present that the genes related to fat cell formation -leptin, proliferator-activated receptor gamma (PPARγ), peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS) and sterol regulatory element-binding protein-1c (SERBP-1c) -were strongly upregulated in HF rats as compared to the control group (as seen in Fig. 9).However, the administration of EFH reversed the expression of these genes.Furthermore, PPARγ expression was significantly reduced by the high dose of EFH.

GC-MS
A total of 87 compounds were detected in the EFH using gas chromatography and mass spectroscopy (GC-MS).The components,     The GC-MS chromatogram obtained for the ethanolic EFH is presented in Fig. 10.Saturated and unsaturated fatty acids, which contain even and odd carbon numbers were found in monocarboxylic form.Monoand poly-hydroxy monocarboxylic and epoxy fatty acids, made up the majority of the hydrolysable and soluble components in the EFH.However, Pitchaipillai, M. et al. studied the composition of volatile organic compound of the leaves of F. hispida by GC-MS, and reported Stigmasterol as the principal constituent [36].

Discussion
In this study, we used a high fat-fed obese rat model and investigated the lipid-lowering effect of EFH.We observed that high fat-fed rats gained weight with increased levels of TG, HDL, and LDL as was reported in other studies [37,38].High levels of these biomarkers can be indicative of dyslipidemia, which is a risk factor for cardiovascular diseases [39].The high-fat rats further exhibited altered levels of several biomarkers such as SOD, GSH, AOPP, catalase, NO, MDA, uric acid, and creatinine.However, upon treatment with the EFH, these markers changed to almost normal levels.
The OGTT test showed an increased level of blood glucose in high fat-fed rats, which subsided when the rats were co-administered with EFH after 60, 90, and 120 min.A similar result was delineated by a previous study which revealed that F. hispida was effective in  lowering blood glucose level in alloxan-induced diabetic rats [40].Similarly, treatment with the EFH lowered cholesterol, TG, and LDL levels, similar to that of the standard drug, Orlistat.Notably, these observations are partially consistent with the previous findings of Hossain and colleagues [41].Their study exhibited a marked hypolipidemic action of the F. hispida fruits in a hypercholesterolemic rat model, reducing TG and LDL.Mandal and colleagues have also demonstrated the anti-hyperlipidemic effect of F. hispida leaves in rat models.They reported a significant reduction in serum lipid parameters such as total cholesterol, triglycerides, VLDL, and LDL while an increase in HDL by the F. hispida in tyloxapol-induced hyperlipidemic model [42].In this study, we did not observe any increase in the HDL level.We hypothesize that the extract primarily functions by inhibiting 3-hydroxyl-3-methyl-glutaryl-coenzyme A (HMG CoA) reductase activity leading to significant reduction in LDL cholesterol but not the HDL cholesterol [43].SOD, catalase and GSH are enzymes that play a crucial role in protecting cells from oxidative stress by neutralizing free radicals [44].AOPP, MDA and NO are markers of oxidative stress, and elevated levels of these biomarkers indicate an increased oxidative damage to cells and tissues.However, NO is also a signaling molecule that plays roles in many physiological processes, including vasodilation, immune function, and neurotransmission [45].In this study, HF rats displayed decreased levels of SOD, GSH, catalase, while increased levels of MDA, NO, and AOPP.The EFH treatment reversed their levels to nearly normal values.Thus, the anti-lipid peroxidation and free radical scavenging activities of the EFH are apparent, which may mitigate the abnormalities in liver of FH-treated rats.This finding aligns with the results obtained in a previous study conducted by M. Arunsundar and T. S. Shanmugarajan [46].
We further observed that EFH administration resulted in a significant hindrance against the increase of ALT, AST, and ALP in rats, suggesting that EFH has hepatoprotective activity, consistent with a past study [42].It was also revealed that EFH treatment reduced plasma uric acid and serum creatinine levels, two most common renal function indicators [47], suggesting its potential impacts on kidney health.
It has been demonstrated that leptin is involved in regulating appetite and metabolism [48], while FAS, PPARγ, and SERBP1c are involved in lipid metabolism and adipogenesis [49,50].Previous studies indicated that high-fat-fed animals exhibit elevated leptin, FAS, PPARγ, and SERBP-1c [51].In our research, we also observed similar effects, and treatment with EFH downregulated the expression of adipogenic genes leptin, FAS, PPARγ, and SERBP-1c.The histopathologic data exhibited that the increased size of adipocytes in HF rats was reduced to almost normal size by a high dose of EFH.Fat deposition in liver was also decreased by co-administration of EFH.Both these functions of EFH resulted in decreased body weight.Taken together, we propose that EFH exerts its anti-obesity effects by suppressing adipogenesis.

Conclusion
The value of medicinal plants as potential sources of new therapeutic compounds in the development of medicines has been well recognized.From ancient times, figs were a primitive food and various species of figs, either wild or cultivated, are still consumed worldwide.The findings from this study suggest that EFH may have significant anti-obesity effect via alleviating oxidative stress by improving antioxidant enzymes' activities and reducing adipogenesis.Nevertheless, additional research is required to explore the regulatory pathway involved in the pharmacological action of EFH.A comprehensive toxicity study is also warranted to determine its safety for human use as a pharmaceutical product.

Fig. 1 .
Fig. 1.EFH's effect on the body weight (a) and liver weight (b) of rats.High dose of EFH reduced the increased body weight and liver weight.The data is shown as mean ± SD, n = 6.CON, control; EFH, ethanolic extract of figs from F. hispida; HF, high fat; LD, low dose; MD, medium dose; HD, high dose; ns, not significant.Statistical analysis was performed using one-way ANOVA and Newman-Keuls post hoc test.P-values <0.05 (*), 0.01 (**), 0.001(***) are considered statistically significant.

Fig. 2 .Fig. 3 .
Fig. 2. The impact of EFH on the oral glucose tolerance test in rats fed an HF diet (a) prior to treatment and (b) following treatment on day 57.(c) Blood glucose levels at 120 min after treatment.Elevated blood glucose level was significantly decreased by EFH.The data is shown as mean ± SD, n = 6.OGTT: oral glucose tolerance test; CON, control; EFH, ethanolic extract of figs from F. hispida; HF, high fat; LD, low dose; MD, medium dose; HD, high dose; ns, not significant.P-values <0.05 (*), 0.001(***) are considered statistically significant.

Fig. 7 .
Fig. 7. Effect of EFH on the plasma levels of uric acid (a) and creatinine (b) in HF rats.Both uric acid and creatinine levels were reduced by EFH.The data is shown as mean ± SD, n = 6.CON, control; EFH, ethanolic extract of figs from F. hispida; HF, high fat; LD, low dose; MD, medium dose; HD, high dose; ns, not significant.P-values <0.05 (*), 0.01(**) are considered statistically significant.

Fig. 8 .
Fig. 8. Representative images of H&E staining showing the effect of EFH on liver (a-g) and adipose tissues (h-m) in different groups of rats.The fat deposition in liver and size of adipocytes were reduced by EFH treatment.Magnification 20x.H&E, hematoxylin and eosin; CON, control; EFH, ethanolic extract of figs from F. hispida; HF, high fat; LD, low dose; MD, medium dose; HD, high dose.

Table 2
Phytoconstituents present in EFH using GC-MS analysis.