Mitigative effect of Momordica cymbalaria fruit extract against sodium fluoride induced hepatotoxicity in Wistar male albino rats

Introduction

Fluoride (F^‐) is the element that does not occur in the elemental state in nature because of its high reactivity [1]. It accounts for about 0.3 g/kg of the Earth’s crust and estimated to be the 13th most abundant element. It exists only in the form of fluorides in several minerals such as fluorspar, cryolite, and fluorapatite [2]. At low concentrations, it is useful for normal mineralization of bone, dentin, and functions of teeth enamel. It is considered as the most productive prophylactic element as well as the treatment agent for dental caries and osteoporosis [3], [4].

Fluorosis is a public health related problem that is endemic all over the globe, including India. At the permissible levels (F^‐≤1.0 ppm), all ingested amount excreted in the urine but when it crosses the permissible levels, it causes dental, skeletal, and non-skeletal fluorosis in both animals as well as in human beings [5], [6]. The primary sources of fluoride intake include drinking water, dental products, food, processed beverages, tea, fluorinated pharmaceuticals, pesticides, and mechanically deboned meat [7], [8]. Referring to a common salt of fluoride as sodium fluoride (NaF), the lethal dose for most adults is estimated to be 5–10 g [9]. At acidic pH, ingested fluorides combine with hydrogen ions to form hydrogen fluoride which was passively entered in the circulation through the passive diffusion process and interferes with the major metabolic pathways [10].

Liver is the one the major affected organ for potential fluoride toxicity after the kidney. Earlier studies have clearly shown that intake of high concentration of fluoride in the drinking water causes biochemical changes in the liver by alterations in the synthetic, metabolic, detoxification, excretion, homeostasis, increased lipid peroxidation, disturbed antioxidant defense system, and storage functions may cause hepatotoxicity. Also, histopathological changes reported in the liver after fluoride exposure included necrosis, infiltration of mononuclear cells, swelling of Kupffer cells, extensive vacuolization, hyperemia, ultra structural changes in hepatocytes, and increased apoptosis [11], [12], [13], [14], [15], [16].

The plant Momordica cymbalaria, which is a vine of the Momordica genus originated from the Indian states of Andhra Pradesh, Tamil Nadu and Maharashtra, belongs to the Cucurbitaceae family. M. cymbalaria is economical, nutritional-rich, and also contains flavonoids and phenolic compounds as major secondary metabolites. It has been valued for its edible fruits, roots, and leaves as a vegetable [17]. It was already proved for its various medicinal purpose such as cardioprotective effect [18], anti-diabetic, and hypoglycemic activity [19], anti-diarrheal [20], anti-ulcer [21], anti-microbial [22], anti-implantation [23], anti-ovulatory [24], antiangiogenic, anticancer, and anti-tumor [25], [26], and nephroprotective [27] activity. The study aims to see the mitigative effect of hydroalcoholic extract of M. cymbalaria fruits [HAEMC] against NaF induced hepatotoxicity and oxidative stress in male Wistar albino rats.

Materials and methods

Results

Effect on liver serum biomarkers level

Excessive consumption of fluoride increases the blood serum levels of ALT (125.0 ± 9.85), AST (99.6 ± 6.65), ALP (73.3 ± 3.83), direct and total bilirubin (0.40 ± 0.04 and 3.44 ± 0.30) and decreased levels of total protein (6.40 ± 0.17) and albumin (2.093 ± 0.07) were observed in toxic control when compared to the normal control (35.19 ± 2.40, 38.4 ± 3.09, 34.0 ± 2.43, 0.14 ± 0.01, 1.25 ± 0.05, 7.36 ± 0.16, and 3.925 ± 0.08, respectively). After treatment with HAEMC showed at a dose of 200 mg (81.99 ± 3.64, 75.2 ± 2.80, 51.6 ± 2.69, 0.35 ± 0.03, 2.43 ± 0.16, 6.66 ± 0.031 and 3.215 ± 0.10) and 400 mg (61.15 ± 0.94, 52.3 ± 2.95, 36.6 ± 2.41, 0.21 ± 0.01, 1.85 ± 0.09, 7.94 ± 0.15 and 3.822 ± 0.14, respectively) dependent recovery from NaF induced hepatotoxicity. No significant variations were observed in HAEMC control (37.03 ± 1.46, 32.5 ± 1.78, 31.4 ± 2.01, 0.40 ± 0.04, 3.44 ± 0.30, 6.40 ± 0.17, and 2.093 ± 0.07, respectively) when compared to normal control. All values are specified in Figure 1.

Figure 1:

Effect of HAEMC treatment on serum liver profile against NaF induced toxicity. The values are represented as mean ± SEM. Statistical analysis performed using one way ANOVA followed by post hoc Dunnett’s multiple comparison test. ^ap<0.001, ^bp<0.01, and ^cp<0.005 vs. toxic control; ^dp<0.001, ^ep<0.01, and ^fp<0.005 vs. normal control.

Effect on lipid peroxidation and antioxidant profile of liver

Effect of HAEMC on the liver lipid peroxidation, reduced glutathione, and catalase levels were specified in Figure 2. There was a significant increase in the lipid peroxidation (5.24 ± 0.78) and decrease in the levels of reduced glutathione (4.82 ± 0.48) and catalase (0.24 ± 0.06) levels observed in toxic control when compared to the normal control (1.32 ± 0.35, 13.2 ± 0.85, and 1.18 ± 0.10). Treatment with HAEMC showed significant recovery from lipid peroxidation (3.17 ± 0.25 and 1.61 ± 0.15) and increased levels of reduced glutathione (8.89 ± 0.60 and 13.0 ± 0.80) and catalase (0.39 ± 0.10 and 0.81 ± 0.04) in a dose-dependent manner when compared to the NaF control. No significant variations observed in the tissue levels of lipid peroxidation (1.13 ± 0.91) and reduced glutathione (13.9 ± 0.46) and catalase (1.35 ± 0.20) in plant control when compared to the normal control.

Figure 2:

Effect of HAEMC treatment on oxidative stress markers against NaF induced hepatotoxicity. The values are represented as mean ± SEM. Statistical analysis performed using one way ANOVA followed by post hoc Dunnett’s multiple comparison test. ^ap<0.001, ^bp<0.01, and ^cp<0.005 vs. toxic control; ^dp<0.001, ^ep<0.01, and ^fp<0.005 vs. normal control.

Histopathological studies

Histopathological changes of the HAEMC on liver tissue specified in Figure 3. In this, normal control liver tissue showed prominent hepatocytes with preserved cytoplasm, hepatic artery, portal tract, central vein, and normal blood vessels. The toxic control (NaF, 100 ppm) tissue showed distortion of hepatocytes, central vein and blood vessels, necrosis, and edema. The HAEMC control liver tissue is similar to the normal architecture of the liver tissue. Treatment control-I (HAEMC 200 mg) showed moderate destruction of hepatocytes, central vein, decreased fibrosis, and edema. Treatment control-II (HAEMC 400 mg) showed mild damage of hepatocytes, central vein, and decreased fibrosis and edema.

Figure 3:

Histopathological studies of liver (magnification 10×).

Discussion

The fluoride acts as the most productive agent for dental caries [33]. At the permissible levels (F^‐≤1.0 ppm), all ingested amount excretes in the urine. But when it crosses the permissible levels, it causes dental, skeletal, and non-skeletal fluorosis in both animals and human beings [34].

Fluoride exhibits diverse metabolic, structural, and functional toxic effects. The toxic changes depend on the type of cell, duration, and concentration of exposure. It can bind to inhibit the function of Na+/K+-ATPase leading to ATP depletion, alterations in the cell membrane potential, and inactivate function of various key enzymes involved in the glycolysis and the Krebs cycle [35]. At micromolar levels, fluoride inhibits the synthesis and/or secretion of protein, and thus various signaling pathways are involved in the cell multiplication and death, such as mitogen-activated protein kinase, p53, activator protein-1, and nuclear factor kappa pathways. At millimolar concentrations fluoride inhibits various enzymes, including phosphatases [36], [37], [38].

Earlier studies discussed the relationship between fluoride and free radical production in various biological systems in natural as well as experimental cases of fluoride toxicity [16]. Antioxidants and chelating agents reduce the stress and/or toxicity against fluoride intoxication [7]. However, long term exposure to vitamins and minerals may not be safe due to their accumulation. Therefore, the study aimed not only mitigation of the fluoride induced toxicity in the liver but also safe, economical, and secure availability in nature. M. cymbalaria fruits are nutritional and unexplored plant material for the treatment of fluoride induced hepatotoxicity and oxidative stress.

Earlier studies clearly reported the variation in the extraction yield depends on the solvent nature and chemical nature of the sample [39]. Based on the percentage of yield obtained, selected 30:70 proportion of hydroalcoholic extract is used for further studies. The phytochemical constituents of HAEMC found to be a good source of alkaloids, glycosides, steroids, flavonoids, phenols, and tannins as secondary metabolites. The acute toxicity test of HAEMC showed tolerance at 2000 mg/kg b. wt, p.o. All the animals in the treatment groups were normal in alertness and behavior up to 72 h of post administration. No mortality observed until the completion of the study.

The fluoride exposed rats showed a significant decrease in the body, it might be due to atrophic gastritis and poor absorption in the GIT by disturbed digestibility and decreased appetite was contributed to excessive breakdown of macromolecules causing weight loss. The decreased liver weight observed due to the degeneration of hepatocytes and altered antioxidant systems, which was the primary factor in fluoride toxicity [40]. The improved body and liver weights were found at the end of the study after treatment with HAEMC.

The abnormal increase in the concentration of aminotransferases (AST and ALT) and ALP levels in serum and altered protein metabolism was a clear indication of abnormal function of the hepatic tissue. Previous studies reported that a significant increase in AST, ALT, ALP, direct and total bilirubin levels and decreased levels of total protein and albumin in rats after NaF-intoxication [41], [42]. The increase in the serum bilirubin levels reflects the severity of hepatotoxicity [43]. Data obtained in the present study also revealed that disturbed liver function in toxic control group reflected by increased levels of AST, ALT, ALP, and direct and total bilirubin and decreased levels of total protein and albumin. Treatment with HAEMC at a dose of 200 and 400 mg/kg b. wt showed normalized serum biomarkers and also increased total protein and albumin levels in a dose-dependent manner. The present results correlate with the findings of the Prakash et al. [44].

Fluoride induces hepatotoxicity by augmenting the free radical formation, which reacts with the polyunsaturated fatty acids to initiate lipid radical chain reaction leading to damage of the cell membrane. It can also reduce the functional capacity of antioxidant system in the hepatocytes cells [45]. The administered HAEMC showed a significant reduction in the lipid peroxidation level and a significant increase in the levels of reduced glutathione and catalase in a dose-dependent manner. These findings have coincided with the results of Perera et al. [12]. Histopathological studies also strongly supported the mitigative effects of HAEMC against fluoride induced hepatotoxicity and oxidative stress.

Conclusion

In conclusion, the present study has proven that the hydroalcoholic extract of M. cymbalaria fruit’s effect on NaF induced hepatotoxicity by decreasing the level of free radical production and also by improving the liver function. We have also concluded that the present results strongly support further application in drug development for the treatment of fluoride toxicity. However, further detailed studies are necessary for a better understanding of changes in the molecular level and exact mechanism involved in the free radical scavenging activity of the plant material.