Ethanolic Extract of Mangifera indica Protects against CCl4-Induced Hepatotoxicity via Antioxidant Capabilities in Albino Rats

Objective To investigate the antioxidant and hepatoprotective effects of ethanolic Mangifera indica (M. indica) seed extract on carbon tetrachloride (CCl4)-induced hepatotoxicity in albino rats. Methods Forty-eight albino rats weighing (100–150 g) were used for hepatoprotective and toxicity experiments. Antioxidant activity was determined using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay. The toxicity of M. indica seeds on the liver was evaluated by examining wellness parameters, body weight, and liver histological sections. The protective effects of 50 mg/kg and 100 mg/kg of seed extract on CCl4-induced hepatotoxicity were investigated by evaluating hematological, renal, and liver function parameters, body weight, and liver histological sections. Results The antioxidant activity of the M. indica ethanolic extract was (92 ± 0.03 RSA %) compared with (91 ± 0.01 RSA %) of propyl gallate, and the IC50 was (8.3 ± 0.01 µg/ml) and (14.1 ± 0.01 µg/ml). No changes were observed in the health indicators, body weights, and liver histological sections following oral administration of 50 mg/kg and 100 mg/kg of M. indica seed extracts. Treatment with M. indica seed extract significantly reduced alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), blood sugar, and urea levels compared with those in the CCl4-treated group. Conclusion The IC50 of the M. indica ethanolic extract was 8.3 µg/ml, and the M. indica extract is a potential source of natural antioxidants that protect against CCl4-induced hepatotoxicity.


Introduction
Herbal remedies often include plant antioxidants such as polyphenols, favonoids, and phenolic compounds, which act as free radical scavengers to protect against damage caused by oxidative stress [1].Te liver is the initial line of defense in the body's protective system as it detoxifes and eliminates toxic and foreign substances [2,3].Previous studies have shown that oxidative stress, infammation, and toxic compounds contribute to hepatocellular damage [4].In experimental models, CCl 4 is commonly used to induce hepatocellular damage and promote hepatotoxicity [5,6], leading to liver enzyme leakage [7].Antioxidants can help reduce the risk of liver disorders by preventing oxidative damage caused by free radicals such as trichloromethyl free radicals and reactive oxygen species (ROS) generated by CCl 4 .Herbal remedies have been used for centuries to treat various illnesses [8].Natural products are an important source of many medications, including anticancer drugs, anti-infammatory agents, antioxidants, and detoxifying agents [9].M. indica referred to as mango is a plant in the Anacardiaceae family widely used in pharmacology, ethnomedicine, and phytochemistry.Te M. indica tree is used in traditional medicine to treat various diseases, and it contains polyphenols, terpenes, sterols, carotenoids, vitamins, and amino acids [10].Several studies have demonstrated the pharmacological efects of the leaves, bark, fruit peel and fesh, roots, and fowers of mango trees [11], hepatoprotective [12], radioprotective [13], cell migration [14], antidiarrheal [15], anticancer [16], and antimicrobial activities [17].Te primary components of the pulp include water, carbohydrates, organic acids, lipids, minerals, colors, tannins, vitamins, and favoring compounds, and it also contains large amounts of vitamins A, C, and D. According to [18], the ester and carbonyl types constitute the characteristic scent that evolves throughout the ripening process.However, the possible antioxidant and hepatoprotective properties of M. indica seed extracts have been poorly studied, and the large number of discarded mango seeds encourages their use because of their high productivity and low cost.Terefore, this study was conducted to investigate the antioxidant and hepatoprotective efects of the ethanolic extract of M. indica seeds on CCl 4 -induced hepatotoxicity in rats.

Collection of Plant Materials.
Fresh and high-quality M. indica seeds were collected from Khartoum State between January and May 2019.Te M. indica was accurately identifed and confrmed by the taxonomists at the Herbarium of Medicinal and Aromatic Plants and Traditional Medicine Research Institute (MAPTMRI) in Khartoum, Sudan.Te seeds were air-dried in a well-ventilated area in the shade, and the powder was prepared for extraction.

Preparation of Crude
Extract.M. indica seeds were prepared using an overnight maceration process [19], and 50 g of crushed seeds were macerated in 500 mL of 80% ethanol for three days at room temperature.Te supernatant was removed by random shaking for 24 hours at room temperature.Te supernatant was fltered at 55 °C by rotary evaporation at a low pressure.Each residue was weighed, and the yield percentage was calculated.Te glass vial containing the extract was carefully sealed and stored at 4 °C.After drying, the extract was stored in a deep freezer (Virtis, USA) for 48 h before use.

Antioxidant Activity (DPPH Assay) of the M. indica
Extract.Te antioxidant activity was evaluated using the stable radical, 2.2 di-(4-tretoctylphenyl)-1-picrylhydrazyl stable free radical (DPPH), which is the ability to donate hydrogen or scavenge free radicals, with minor modifcations as previously described [20].Te DPPH radical was converted to purple diphenyl-picrylhydrazine.Te extracts and standards were then added to the appropriate microplate wells containing DPPH and incubated at 37 °C for 30 min while maintaining the level of DPPH at 300 mM.After incubation, the reduction in the absorbance at 517 nm was measured using an ELISA reader spectrophotometer.Te percentage of antioxidant activity was calculated using the following formula: where At � absorbance value of the test compound; Ac � absorbance value of the control.Te IC 50 of the extract was calculated using the EZ-Fit Enzyme Kinetic Program (Perrella Scientifc Inc, U.S.A).

Experimental Animals and M. indica Extract Toxicity.
Albino rats weighing 100-150 g were used to assess the subchronic toxicity of the M. indica extract.Te rats were housed in an environment with a humidity of 40-50%, a 12hour light/dark cycle, and a room temperature of 22-24 °C for 14 days.Te rats were divided randomly into three groups of six rats each.Group 1 untreated group served as the control group, Group 2 received 50 mg/kg/day of M. indica extract, and Group 3 was administered 100 mg/kg/ day for 14 days.Te rats were fasted overnight for 18 hours.After anesthesia, rats were sacrifced and clinical signs were recorded.Blood samples were collected, and serum was obtained after centrifugation at 3000 rpm.All animal experiments were performed following the principles of the Declaration of Helsinki.

Estimation of Biochemical Parameters.
Serum AST, ALT, and ALP activities were determined using kinetic methods, while protein, urea, albumin, and calcium levels were evaluated using a fully automated chemistry analyzer.Tissue specimens from the liver, kidneys, heart, spleen, and brain were carefully removed, weighed, fxed in 10% formal saline, and prepared for histopathological analyses.Te relative organ weight of each animal was calculated using the following formula: Visual observations of mortality and changes in physical appearance and behavior (sleepiness, salivation, and lethargy) were recorded.

Experimental Animals and M. indica Extract Hepatoprotective
Activity.Tis study aimed to evaluate hepatotoxicity induced by CCl 4 -induced liver injury in rats.Tirty adult albino rats weighing 100-150 g were divided into fve groups of six rats each.Group 1 was treated with olive oil 0.2 ml/kg/3 times a week for 10 days.Group 2 was treated intraperitoneally with CCl 4 0.2 ml/kg dissolved in equal volume olive oil (V/V).Group 3 was treated with 100 mg/kg M. indica extract and 0.2 ml/kg/day of CCl 4 .Group 4 received 50 mg/kg of the extract and 0.2 ml/kg 3 times a week for 10 days.Group 5 received CCl 4 0.2 ml/kg and silymarin suspended in 5% acacia mucilage.After ten days, the rats were fasted and euthanized and blood samples were collected.Serum was obtained after centrifugation at 3000 rpm.A fully automated analyzer was used to measure the biochemical parameters.Te livers were removed, fxed in 10% formal saline, embedded in parafn wax, sectioned at 5 µm thickness, and stained with H & E using Mayer's hemalum.All experiments were conducted in accordance with the Declaration of Helsinki.

Statistical Analysis.
Statistical Package for (SPSS) software (version 21.0 (SPSS ver.21.0, Inc., Chicago, IL, USA) was used for the statistical analysis.Mean ± standard error of the mean (SEM) and percentage (%) were used to represent the data.Independent t-tests and analysis of variance (ANOVA) were used to compare group means.Diferences were considered statistically signifcant at a p value of 0.05.

Te Efect of M. indica Extract on Clinical Signs and
Behaviors.Te acute oral toxicity of the ethanolic extract of M. indica was examined after 14 days.As shown in Table 2, no toxic symptoms were observed and signs of depression and mortality were reported throughout the study period.Additionally, no treatment-related faws or overt clinical indications of toxicity, stress, or changes in appearance or behavior were observed.Moreover, the liver histological analysis of the extract-treated and control groups revealed no signifcant diferences.3 and Figure 1, the body weight gain of the group treated with M. indica extract 100 mg/kg was signifcantly decreased (6.15%) compared to the control group (8.47%), and in contrast, no change was observed in the group treated with 50 mg/kg (8.33%).

Hepatoprotective Activity of M. indica Extract.
Compared to the baseline, CCl 4 therapy caused a substantial increase in liver size (2.10 g) and reduced BWG % (0.74%); in contrast, treatment with 100 mg/kg and low 50 mg/kg extracts resulted in BWG % reduction (4.00% and 2.31%) and no change in liver size (1.65 g and 1.68 g), respectively, as indicated in Table 4 and Figure 2.

Histopathological Findings of M. indica Extract.
To assess the possible protective efects of M. indica on liver cells damage induced by CCL 4 , histopathological analysis of the liver tissue was performed.Te results indicated that CCl 4 treatment led to the emergence of fatty liver cells, necrosis, hyperplasia, infltration, and infammation, in contrast to the normal liver cells treated with both low and high doses of M. indica (Figures 3(a

Discussion
In tropical regions, M. indica has traditionally been used as a medicinal plant, and scientifc studies have confrmed its positive efects on health [21].Tis study aimed to investigate the antioxidant and hepatoprotective properties of M. indica ethanolic seed extracts against CCl 4 -induced liver damage in rats.Tis study revealed that the M. indica extract protects against CCl 4 -induced hepatotoxicity and is nonharmful.Te protective efects of the extracts are attributed to their antioxidant components, such as phenolics, which protect cells from damage [22,23].In line with some studies, our results indicated that the ethanolic extract of M. indica Journal of Toxicology could serve as a natural source of antioxidants that protect against CCl 4 -induced cell damage.
Te DPPH assay was performed to assess the antioxidant properties of M. indica ethanolic extract.Te results demonstrated that the ethanolic extract of M. indica exhibited the highest radical scavenging capacity and the lowest IC 50 value compared with propyl gallate.Tese results align with those of previous studies, which have found that the polyphenols, carotenoids, and tocopherols of M. indica seeds neutralize free radicals, thereby protecting against oxidative stress and the development of diseases, such as cancer, Alzheimer's disease, and infammation [24,25].
In this study, the in vitro cytotoxic activities of M. indica seed methanolic extracts were evaluated and compared to   those of the control.Following a 14-day administration of the M. indica ethanolic seed extract, clinical symptoms, behavior, wellness, and body weights were evaluated.Te study revealed no noticeable discrepancies between the groups that received M. indica and the control for any of the examined aspects.Tese data confrm the safety of this extract.Previous research has also documented a lack of toxic reactions to the stem bark of M. indica in albino rats.However, high-dose treatment with M. indica was found to lead to reduced body weight, which may explain its delayed use in severe illness, weight loss, and improved quality of life in Cuba [26].
Te M. indica extract exhibited efcacy in safeguarding against CCl 4 -induced hepatotoxicity, as evidenced by the notable decreases in ALT, AST, ALP activities, blood glucose, and blood urea levels.Furthermore, the normal hepatocellular histology observed in the extract-treated group compared to that in the CCl 4 -treated group indicated the efectiveness of the M. indica extract.Elevated transaminase activity indicates that CCl 4 triggers the release of ROS, which causes hepatocellular damage, and higher ALP activity suggests hepatobiliary damage.Tese fndings were further confrmed by the presence of fatty liver and necrosis in the liver tissues of the CCl 4 -treated   group.Increased blood urea levels suggested impaired renal function.Te low blood glucose levels observed after the administration of the extract may be attributed to the antioxidant activity of M. indica, which protects against β-cell damage induced by CCl 4 .Several plant products have been shown to protect against and minimize hepatocellular damage caused by the chemical agent [27].Herbal medicine has recently received much attention as a complementary diet and adjuvant therapy for preventing and treating a wide range of diseases such as cancer, cardiovascular diseases, and Alzheimer's disease.Tis suggests that M. indica exhibits signifcant antioxidant efects against chemicals and oxidative stress-induced cellular damage.A previous study on M. indica leaf extracts demonstrated that all parts of M. indica possess considerable antioxidant activity, similar to the leaves [28].Te efects of the M. indica extract on liver histological sections were examined.Histological sections of the group treated with M. indica seed extract showed hepatoprotective efects.Te results indicated that the CCl 4 -treated group exhibited fatty liver, necrosis, hyperplasia, infltration, and infammation, while the liver cells in the M. indica-treated groups appeared healthy.Previous studies on albino rats found that M. indica leaves had hepatoprotective properties against CCl 4 -induced liver injury [29,30].However, one study reported that the administration of aqueous and ethanolic extracts of M. indica stem bark signifcantly increases AST and ALT activities [31].Te limitations of this study include the exclusion of ammonia, acute infammatory markers, and antioxidant enzymes, which provide insights into the protective mechanisms of M. indica against hepatocellular damage, and the lack of clinical data.

Conclusion
In this study, we evaluated the antioxidant and hepatoprotective efects of the M. indica seed extract against CCl 4 -induced toxicity.M. indica exhibits antioxidant activity and protects against CCl 4 -induced hepatotoxicity, with an IC 50 of 8.3 µg/ml.Treatment with the M. indica seed extract decreased the levels of transaminases, ALP, urea, and blood glucose, thereby protecting against CCl 4 -induced hepatocellular damage.Based on these fndings, M. indica appears to be a safe, efective, and promising natural product for use in herbal medicine.Further research is warranted to elucidate the underlying mechanisms by which M. indica protects against cellular damage.

2
Journal of ToxicologyRelative organ weight � (organ weight (g)) (body weight of the animal on sacrifice day (g))× 100.

Figure 1 :
Figure 1: Comparison of body weight gain percentage (BWG %) in study groups.

Figure 2 :
Figure 2: Efect of M. indica extract on body weight gain (%) in study groups.

Table 2 :
Te efect of M. indica extract on wellness parameters in study groups.

Table 3 :
Te efect of M. indica on body weight in study groups.

Table 4 :
Efect of M. indica extract on body weight, change in body weight (g), body weight, and relative liver weight in CCl 4 -induced hepatotoxicity.

Table 5 :
Comparison of biochemical parameters (mean ± SEM) in study groups.