Alleviating effect of Ficus racemosa in high-fat-high-fructose diet-induced non-alcoholic fatty liver disease

INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD) is a diverse hepatic state caused by irregular fat deposition in hepatocytes with or without inflammation. It embraces a range of liver diseases, include hepatic steatosis, steatohepatitis, fibrosis, cirrhosis, and liver failure.^[1] As per the American Association for the Study of Liver Diseases guidelines, NAFLD is histological, which is categorized into two main categories such as fatty liver (simple steatosis) and steatohepatitis (NASH). The first one serves as a benign condition, whereas the development of this condition in the later stage has severe complications such as liver inflammation, hepatocellular injury, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).^[2] NAFLD is associated with metabolic syndromes, such as obesity, type 2 diabetes mellitus (T2DM), hyperlipidemia, and hypertension. It is related to the highest rate of mortality as a consequence of the augmented risk of HCC and cardiovascular diseases.^[3]

At present, NAFLD is one of the most popular chronic liver diseases, particularly in developed countries, with a prevalence of 25–30%, which is expected to increase due to unhealthy diet patterns rich in high-carbohydrates and high-fat foods. Intake of saturated fats and trans-fatty acids; lipogenic sugars such as fructose and sucrose and insulin resistance (IR) play a significant role in the pathogenesis of NAFLD.^[4,5] NAFLD affects all ages and ethnicities. In the adult population, the prevalence of the disease ranges between 20 and 30% and rises up to 47%.^[4,6] The prevalence of NAFLD is growing parallelly with the worldwide increase epidemic of obesity.^[7]

Based on the available in vivo studies, the first mechanism based “two-hit” hypothesis for the pathogenesis of NAFLD was considered. As per this hypothesis, accumulations of fat droplets in hepatocytes act as the “first hit,” and further progression leads to activation of inflammatory cascades and development of fibrosis act as a “second hit.”^[8] In the development and progression of human NAFLD, multiple parallel factors are involved, which increase the complexity of the disease. Therefore, currently the new “multiple mechanism hit” hypothesis has replaced older “Two-hit” prediction.^[9] Accumulation of a high amount of lipids in the liver leads to lipotoxicity and chronic liver inflammation. A higher level of leptin hormone is also observed in patients with obesity and NAFLD, which is generally known as leptin resistance state. Therefore, it may have some role in the pathogenesis of NAFLD.^[10]

Increased prevalence and negative pathological outcomes of NAFLD show a monumental economic burden for many countries. Still, up to now, no effective treatment is available for the disease.^[11] Lifestyle changes serve as a cornerstone of management for NAFLD and obesity; however, only a couple of patients can achieve adequate weight loss and very few can maintain the body weight for the long term.^[7] Today, other primary treatment approaches for NAFLD include anti-obesity drugs, lipid-lowering agents, insulin-sensitizing drugs, antioxidants, surgical interventions, and in the last stage liver injury and liver transplantation. Antidiabetic and hypolipidemic drugs, which improve IR and lower serum lipid level, significantly affect the disease and slow down the progression of symptoms.^[12]

Pathogenesis of NAFLD is multifactorial; therefore, none of the monotargeted treatment approaches have provided satisfactory results and due to various side effects, these treatments are not tolerated by patients in the long term.^[13] Traditional herbal medicines in various countries have provided a good source of natural substances for the management of chronic disorders, including metabolic syndrome, T2DM, gastrointestinal complications, and hyperlipidemia. At present, the number of researches, which focus on herbal extracts is increasing, and many of these researchers have discovered potent multi-targeted herbal products against NAFLD.^[14] Hence, the multipronged therapeutic nature and safety of herbal medicine are essential for their use in treating NAFLD.

Ficus racemosa Linn. (Moraceae), commonly known as cluster Figure (gular), is an evergreen, medium to large-sized tree lacking primary aerial roots. It is widely distributed all over India, Australia (northern), and other parts of Asia.^[15] All parts (leaves, fruits, bark, latex, and sap of the root) of this plant are considered medicinally important in mentioned in ancient scriptures of Ayurveda.^[16] It has been used for 1000 years in Ayurvedic medicinal system for the treatment of jaundice, diabetes, dysentery, biliary disorders, diarrhea, and inflammatory conditions.^[17] A broad range of chemical compounds were isolated from stem bark of this plant such as glycosides; sterols (β-sitosterol, stigmasterol, leucoanthocyanin, α-amyrin acetate, lupeol, and lupeol acetate); flavonoids (naringenin, quercetin, kaempferol, and baicalein); tannins (ellagic acid); and triterpenoids and gluanol acetate.^[18,19] An extract of the bark of this plant possessed various pharmacological actions including hypolipidemic, antioxidant, anti-inflammatory, antidiarrheal, antibacterial, and antifungal and is closely related to prevention, treatment, and cure of obesity and diabetes.^[20,21]

In some in vivo studies, it has been demonstrated that administration of high-fat-high-fructose diet (HFFD) causes IR, hepatic steatosis, and diminishes the inherent antioxidant defense system which results in excessive generation of reactive oxygen species (ROS) and fatty infiltration. This leads to a reduction of the hepatocyte populace and liver damage in rats.^[22,23]

In the context of the information as mentioned above regarding the biological activities of F. racemosa bark, the present study has been performed to evaluate the effect of F. racemosa bark extract in the NAFLD animal model induced by administration of HFHF diet

MATERİALS AND METHODS

RESULTS

Biochemical estimation results

After 10 weeks of study, liver index was showed significant different for disease control group compared to normal control. FRE, 400 mg/ kg dose showed significant reduction in liver index compared to disease control rats [Figure 1]. After 10 weeks, results observed from biochemical parameters were shown significant (P < 0.05) elevation in ALT [Figure 2a], AST [Figure 2b], Cholesterol [Figure 3a], reduction in HDL [Figure 3b], increase in LDL [Figure 4a], Triglyceride [Figure 4b] and VLDL [Figure 5] levels in disease control group compared to normal control group rats. In FRM treatment group (100, 200, and 400 – mg/kg), reduction in AST and ALT levels was observed significantly, which was reduced in dose, dependent on the manner compared to the disease control group. However, in TG, TC, LDL, and VLDL levels significant (P < 0.05) reduction was observed only in a medium and high dose of FRM (200 and 400 – mg/kg) and standard drug-treated rats, but not in low dose FRM (100 mg/kg) treated rats compared to disease control rats. HDL level was seen to significantly increase in medium and high dose of FRM (200 and 400 – mg/kg) treated rats compared to disease control group. There was a significant increase in the serum leptin level (P < 0.05) in HFHF diet-fed rats compared to normal control rats. While in high dose, FRM treated rats significant (P < 0.05) reduced leptin was observed compared to disease control rats [Figure 6].

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Figure 1:

Effects of methanolic extract of Ficus racemosa (FRM) on liver index (liver-body wt ratio) DC= disease control; FRE-1= 100mg/kg; FRE-2= 200mg/kg; FRE-3= 400mg/kg; Std= 20mg/kg Atorvastatin ^#Significantly different from normal control group, P < 0.05; *Significantly different from disease control group, P < 0.05.

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Figure 2:

Effects of methanolic extract of Ficus racemosa (FRE) on serum Aspartate transferase activity (a) and serum Alanine transferase activity (b) DC= disease control; FRE-1= 100mg/kg; FRE-2= 200mg/kg; FRE-3= 400mg/kg; Std= 20mg/kg Atorvastatin #Significantly different from normal control group, P < 0.05; *Significantly different from disease control group, P < 0.05.

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Figure 3:

Effects of methanolic extractof Ficus racemosa (FRM) on serum cholesterol (a) and HDL level (b) DC= disease control; FRE-1= 100mg/kg; FRE-2= 200mg/kg; FRE-3= 400mg/kg; Std= 20mg/kg Atorvastatin ^#Significantly different from normal control group, P < 0.05; *Significantly different from disease control group, P < 0.05.

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Figure 4:

Effects of methanolic extract of Ficus racemosa (FRM) on serum triglyceride (a) and LDL (b) level DC= disease control; FRE-1= 100mg/kg; FRE-2= 200mg/kg; FRE-3= 400mg/kg; Std= 20mg/kg Atorvastatin ^#Significantly different from normal control group, P < 0.05; *Significantly different from disease control group, P < 0.05.

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Figure 5:

Effects of methanolic extract of Ficus racemosa (FRM) on serum VLDL level DC= disease control; FRE-1= 100mg/kg; FRE-2= 200mg/kg; FRE-3= 400mg/kg; Std= 20mg/kg Atorvastatin #Significantly different from normal control group, P < 0.05; *Significantly different from disease control group, P < 0.05.

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Figure 6:

Effects of methanolic extract of Ficus racemosa (FRM) on serum leptin level DC= disease control; FRE-1= 100mg/kg; FRE-2= 200mg/kg; FRE-3= 400mg/kg; Std= 20mg/kg Atorvastatin #Significantly different from normal control group, P < 0.05; *Significantly different from disease control group, P < 0.05.

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Estimation of oxidative markers

HFHF diet-fed rats exhibited higher MDA level [Figure 7a] in liver tissue homogenate compared to normal control group. In FRM treated groups, the reduction in MDA level observed, but it was not statistically significant. 400 mg/kg FRM and atorvastatin treated rats showed significant (P < 0.05) reduction in MDA levels compared to the disease control group. Antioxidant markers such as GSH [Figure 7b], SOD [Figure 8a], and CAT [Figure 8b] activity were observed significantly (P < 0.05) to be reduced in disease control rats liver compared to normal control rats. However, 400 mg/kg FRM and atorvastatin treated rats showed significant (P < 0.05) elevation in GSH, SOD, and CAT enzymes activity compared to the disease control group.

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Figure 7:

Effects of methanolic extract of Ficus racemosa (FRM) on Reduced glutathione (a) level in rat liver DC= disease control; FRE-1 = 100 mg/kg; FRE-2 = 200 mg/kg; FRE- 3 = 400 mg/kg; Std = 20 mg/kg Atorvastatin (b) Malondialdehyde ^#Significantly different from normal control group, P < 0.05; *Significantly different from disease control group, P < 0.05.

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Figure 8:

Effects of methanolic extract of Ficus racemosa (FRM) on Superoxide Dismutase (a) and Catalase (b) level in rat liver DC= disease control; FRE-1= 100mg/kg; FRE-2= 200mg/kg; FRE- 3= 400mg/kg; Std= 20mg/kg Atorvastatin ^#Significantly different from normal control group, P < 0.05; *Significantly different from disease control group, P < 0.05.

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Histopathology assessment in liver tissue

Histology of control group livers [Figure 9a] appeared to be normal. In contrast, the liver from HFHF diet-fed rats [Figure 9b] showed fatty changes in the hepatocyte with macro- and micro-vesicular fat droplets. The liver section of low and moderate dose FRM treatment [Figure 9c and d] showed lesser microvacuoles disposition compared to the disease control group and high dose FRM [Figure 9e] and standard treated rats [Figure 9f] significantly preserved normal hepatic morphology.

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Figure 9:

Photomicrograhs of liver sections (H & E staining, 20X). Liver section of (a) Normal control, (b) Disease control, (c) FRE 100mg/kg (d) FRE 200mg/kg (e) FRE 400mg/kg (f) Atorvastatin 20mg/kg.

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Overall 400 mg/kg was considered as optimum dose of F. racemosa for the treatment of NAFLD.

DISCUSSION

Herbal medicines are blooming rapidly as an alternative to primary healthcare, even in developed countries. Due to its multi-targeted therapeutic nature, the effectiveness of many hepatoprotective herbal plants was wildly explored in NAFLD condition.^[29] The present study was the first to investigate the effectiveness of FRM bark in in vivo NAFLD condition. NAFLD is a multifactorial disease; therefore, the development of ideal animal model for NAFLD that would mimic the natural history and metabolic characteristics of human disease condition is challenging. Evidence suggests that dietary models are more closely reflected in the NAFLD condition compared to other animal models for NAFLD.^[30,31] Along with the previous result of over-nutrition and sedentary lifestyle, recent evidence suggests that diet high in fructose also increases the risk development of NAFLD.^[32] A combination of high-fat and high-fructose diet increases delivery of fatty acids through portal circulation and increase de novo lipogenesis.^[33] This leads to the accumulation of fat in the liver and increases the vulnerability to further events such as oxidative stress and steatohepatitis, although the progression of hepatic steatosis to steatohepatitis required lengthy feeding period.^[34] Our aim was to investigate the efficacy of the plant extract in the initial phase of the NAFLD spectrum. Research suggests that a high-calorie diet leads to body weight gain and elevation liver index (liver/body weight ratio) due to accumulation of fat pads mostly in the abdominal area.^[35] The same was seen in the HFHF diet-fed groups in this study. It is reported that FRM treatment produces a reduction in body fat disposition in a moderate manner and similar results were observed in the present study. The liver size was also seen to be increased in HFHF fed rats while significant reduction was seen in high dose of FRM and standard treated group. Admission of HFHF diet is associated with hepatocellular damage and microvesicular steatosis. Elevated hepatic marker enzymes (ALT and AST) suggest chances of presence of hepatic cell damage. The increased level of these enzymes was seen in the disease control group. Treatment with different doses of FRM significantly prevents the increased activity of these enzymes. FRM conserves cell integrity and prevents cell damage in the liver to maintain the survival of functionally active cells. This result is comparable with the research reported by Faiyaz and Asna (2010) who observed the hepatoprotective activity of FRM in CCl₄- treated rats.^[36] This effect could be due to the presence of bergenin in FRM, which is reported to show notable hepatoprotective action. Fat accumulation, mainly hepatic TG filtration, is considered the first step in the development of a disease, even though the mechanism of NAFLD is still under exploration and this fat accumulation in the liver results from dysregulation of TGs and FFAs.^[37] As mentioned earlier, fructose is a lipogenic compound; it plays a role in the elevation of hepatic TGs level through generating substrates for DNL.^[33] In the present study, treatment with HFHF for 10 weeks produces significantly elevation TGs, TC, and LDL and reduction in HDL level. The amount of these serum lipid markers was found significantly reversed in FRM treated group comparable to that of the standard treated group.

Lipid dysregulation in HFHF diet fed rats was connected to the induction of oxidative stress in hepatocytes which favors a disease progression. Hypothesis for progressive cycle for NAFLD progression suggests the involvement of ROS formation, metabolic disease, and inflammation.^[32] ROS-induced oxidative stress produces MDA as a byproduct. Results of the current study showed increased level of MDA and diminished levels of GSH, SOD, and CAT in HFHF-diet-fed rats liver tissue homogenate. GSH, CAT, and SOD enzymes protect liver from free radicals and reflect detoxification activity of the liver. Decreased level of GSH was found in the disease control group, which is relatable with other study results of impaired antioxidant defense activity of fructose-fed rats.^[38] Treatment with antioxidants was reported to improve NAFLD conditions. Plant’s phenolic compounds play a great role as primary free-radical terminators (antioxidants). The phytochemical screening test found the presence of phenolic compounds (mainly tannins and flavonoids) in FRM, and this evidence is also supported by a phytochemical screening article of F. racemosa. A high dose of FRM treated rats showed enhanced antioxidant defense activity significantly compared to disease control group.

Higher level of leptin in serum was observed in the disease control rats. Studies suggest that leptin resistance may have a functional role to play in the pathogenesis of NAFLD.^[10,39] Leptin is the main hormone, which is secreted from adipose tissue, and elevation in leptin level shows an excess amount of fat in the liver and intestine of animals.^[40] Fructose ingestion also plays a role in leptin resistance.^[41] Leptin resistance leads to higher leptin level in the serum, which may result in IR and development of NAFLD.^[39] Improvement in serum leptin level prevents hepatic steatosis in animal models by affecting both lipid and glucose metabolism.^[25] This result was observed in rats treated with high dose FRM. Thus, we hypothesized that constituents present in FRM might have a role in the reduction of the increased levels of leptin in the serum. Histopathological studies showed that HFHF diet induces fat droplets accumulation in hepatocytes with macrovesicular and microvesicular fatty changes, which is the same as expressed in other studies.^[35,42] It is possible that increased liver weight in also associated with higher number of fat vacuoles in the hepatocytes. Research finding of Poudya et al. (2010) also supported these fatty changes in the liver by admission of high carbohydrate and high-fat-diet in the rats. These histological changes are corelatable with elevated lipid and liver function markers.^[43] Improvement in hepatic architect is considered as a major sign of positive improvement in NAFLD. This was observed in high dose FRM treated groups, which is explained by reduced level of lipids and TG levels in the serum. In earlier study, F. racemosa had suggested improvements in functional and structural markers in diabetic neuropathy model.^[44]

CONCLUSION

This study indicated the preventing effect of F. racemosa bark against HFHF diet-induced NAFLD possibly through a combination of antidiabetic, antioxidant, and hypolipidemic effects. These results rationalized its use in NAFLD conditions. Further, active phytoconstituents isolation from the extract for experimental in vivo and clinical studies is required to establish the mechanism(s) responsible for pharmacological action.