
==== Front
J Toxicol
J Toxicol
jt
Journal of Toxicology
1687-8191
1687-8205
Wiley

10.1155/2024/5539386
Research Article
Ethanolic Extract of Mangifera indica Protects against CCl4-Induced Hepatotoxicity via Antioxidant Capabilities in Albino Rats
https://orcid.org/0000-0003-3439-096X
Kabbashi Ahmed Saeed ahmedsak88@gmail.com
1
https://orcid.org/0000-0001-8955-4138
Eltawaty Salwa Abdulla 1
https://orcid.org/0000-0003-0172-8362
Ismail Amar Mohamed 1
https://orcid.org/0000-0002-0682-0312
Elshikh Ahmed Abdelhhafiz 2
https://orcid.org/0000-0002-3813-3656
Alrasheid Ayat Ahmed 3
Elmahi Rawan Ahmed 4
https://orcid.org/0000-0002-7773-995X
Koko Waleed S. 5
Osman Elbadri Elamin 6
1 Department of Biomedical Science Faculty of Pharmacy Omar Al-Mukhtar University, Al-Bayda, Libya
2 Department of Botany Faculty of Science and Technology Omdurman Islamic University, Omdurman, Sudan
3 Department of Pharmacognosy Faculty of Pharmacy University of Medical Sciences and Technology, Khartoum, Sudan
4 Department of Histopathology Faculty of Medical Laboratory Sciences International University of Africa, Khartoum, Sudan
5 Department of Biology College of Science Qassim University, Qassim 51452, Saudi Arabia
6 Department of Microbiology Faculty of Pure and Applied Science International University of Africa, Khartoum, Sudan
Academic Editor: Phuping sucharitakul

2024
27 8 2024
2024 55393868 2 2024
1 8 2024
17 8 2024
Copyright © 2024 Ahmed Saeed Kabbashi et al.
2024
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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.
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pmc1. Introduction

Herbal remedies often include plant antioxidants such as polyphenols, flavonoids, and phenolic compounds, which act as free radical scavengers to protect against damage caused by oxidative stress [1]. The liver is the initial line of defense in the body's protective system as it detoxifies and eliminates toxic and foreign substances [2, 3]. Previous studies have shown that oxidative stress, inflammation, and toxic compounds contribute to hepatocellular damage [4]. In experimental models, CCl4 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 CCl4. 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-inflammatory 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. The 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 effects of the leaves, bark, fruit peel and flesh, roots, and flowers of mango trees [11], hepatoprotective [12], radioprotective [13], cell migration [14], antidiarrheal [15], anticancer [16], and antimicrobial activities [17]. The primary components of the pulp include water, carbohydrates, organic acids, lipids, minerals, colors, tannins, vitamins, and flavoring 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. Therefore, this study was conducted to investigate the antioxidant and hepatoprotective effects of the ethanolic extract of M. indica seeds on CCl4-induced hepatotoxicity in rats.

2. Materials and Methods

2.1. Chemicals and Reagents

Oxford Lab Fine Chem LLP (India) sells carbon tetrachloride (CCl4). Liquid paraffin (Oxford Lab Fine Chem LLP, India), 10% formalin (Novochem Engineering, India), the standard drug silymarin (Sigma-Aldrich), ethanol (SIGMA-ALDRICH, Germany), xylene (Scien TEST-bioKEMIX GmbH, Germany), kits for liver chemistry (Humana, Germany), DPPH (Chemos GmbH and Co.KG. Germany), and hematoxylin and eosin (H&E) (Santa Cruz Biotechnology, Inc., USA).

2.2. Collection of Plant Materials

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

2.3. 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. The supernatant was removed by random shaking for 24 hours at room temperature. The supernatant was filtered at 55°C by rotary evaporation at a low pressure. Each residue was weighed, and the yield percentage was calculated. The 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.

2.4. Antioxidant Activity (DPPH Assay) of the M. indica Extract

The 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 modifications as previously described [20]. The DPPH radical was converted to purple diphenyl-picrylhydrazine. The 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. The percentage of antioxidant activity was calculated using the following formula:(1) DPPH radical scavenging %=100−Ac−AtAc×100,

where At = absorbance value of the test compound; Ac = absorbance value of the control.

The IC50 of the extract was calculated using the EZ-Fit Enzyme Kinetic Program (Perrella Scientific Inc, U.S.A).

2.5. 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. The rats were housed in an environment with a humidity of 40-50%, a 12-hour light/dark cycle, and a room temperature of 22–24°C for 14 days. The 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. The rats were fasted overnight for 18 hours. After anesthesia, rats were sacrificed 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.

2.6. 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, fixed in 10% formal saline, and prepared for histopathological analyses. The relative organ weight of each animal was calculated using the following formula:(2) Relative organ weight=organ weight gbody weight of the animal on sacrifice day g×100.

Visual observations of mortality and changes in physical appearance and behavior (sleepiness, salivation, and lethargy) were recorded.

2.7. Experimental Animals and M. indica Extract Hepatoprotective Activity

This study aimed to evaluate hepatotoxicity induced by CCl4-induced liver injury in rats. Thirty adult albino rats weighing 100–150 g were divided into five 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 CCl4 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 CCl4. Group 4 received 50 mg/kg of the extract and 0.2 ml/kg 3 times a week for 10 days. Group 5 received CCl4 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. The livers were removed, fixed in 10% formal saline, embedded in paraffin 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.

2.8. 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. Differences were considered statistically significant at a p value of 0.05.

3. Results

3.1. Antioxidant Activity of M. indica Extract

The results of DPPH radical scavenging activity show that the M. indica extract had a higher antioxidant activity (92 ± 0.03 RSA% than propyl gallate (91 ± 0.01 RSA%) and the lowest IC50 (8.3 ± 0.01 µg/ml) and (14.1 ± 0.01 µg/ml), respectively, presented in Table 1.

3.2. The Effect of M. indica Extract on Clinical Signs and Behaviors

The 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 flaws 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 significant differences.

3.3. The Effect of M. indica Extract on Body Weight

As shown in Table 3 and Figure 1, the body weight gain of the group treated with M. indica extract 100 mg/kg was significantly 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%).

3.4. Hepatoprotective Activity of M. indica Extract

Compared to the baseline, CCl4 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.

3.5. The Effect of M. indica Extract on Biochemical Markers

The biochemical profile was investigated using a fully automated chemistry analyzer after treatment with 50 and 100 mg/kg/day for 10 days. Treatment with 100 mg/kg of M. indica extract significantly reduced ALT (130 ± 1.37 U/l), AST (149 ± 2.20 U/l), ALP (141 ± 1.86 U/l) activities, blood glucose levels (74.1 ± 1.10 mg/dl), and blood urea level (35.9 ± 3.05 mg/dl) when compared with the CCl4-induced liver cells damage group ALT (153 ± 0.76 U/l), AST (165 ± 2.24 U/l), ALP (187 ± 3.81 U/l), and blood glucose (95.0 ± 1.09 mg/dl) and 61.2 ± 0.09 mg/dl), respectively. In contrast, silymarin significantly decreased the activities of ALT (120 ± 0.71 U/l), AST (142 ± 2.62 U/l), ALP (128 ± 2.10 U/l), blood glucose levels (73.2 ± 1.05 mg/dl), and urea (36.4 ± 1.23 mg/dl), respectively, are presented in Table 5.

3.6. Histopathological Findings of M. indica Extract

To assess the possible protective effects of M. indica on liver cells damage induced by CCL4, histopathological analysis of the liver tissue was performed. The results indicated that CCl4 treatment led to the emergence of fatty liver cells, necrosis, hyperplasia, infiltration, and inflammation, in contrast to the normal liver cells treated with both low and high doses of M. indica (Figures 3(a), 3(b), 3(c), 3(d), 3(e), and 3(f)).

4. Discussion

In tropical regions, M. indica has traditionally been used as a medicinal plant, and scientific studies have confirmed its positive effects on health [21]. This study aimed to investigate the antioxidant and hepatoprotective properties of M. indica ethanolic seed extracts against CCl4-induced liver damage in rats. This study revealed that the M. indica extract protects against CCl4-induced hepatotoxicity and is nonharmful. The protective effects 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 could serve as a natural source of antioxidants that protect against CCl4-induced cell damage.

The DPPH assay was performed to assess the antioxidant properties of M. indica ethanolic extract. The results demonstrated that the ethanolic extract of M. indica exhibited the highest radical scavenging capacity and the lowest IC50 value compared with propyl gallate. These 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 inflammation [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. The study revealed no noticeable discrepancies between the groups that received M. indica and the control for any of the examined aspects. These data confirm 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].

The M. indica extract exhibited efficacy in safeguarding against CCl4-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 CCl4-treated group indicated the effectiveness of the M. indica extract. Elevated transaminase activity indicates that CCl4 triggers the release of ROS, which causes hepatocellular damage, and higher ALP activity suggests hepatobiliary damage. These findings were further confirmed by the presence of fatty liver and necrosis in the liver tissues of the CCl4-treated group. Increased blood urea levels suggested impaired renal function. The 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 CCl4. 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. This suggests that M. indica exhibits significant antioxidant effects 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].

The effects of the M. indica extract on liver histological sections were examined. Histological sections of the group treated with M. indica seed extract showed hepatoprotective effects. The results indicated that the CCl4-treated group exhibited fatty liver, necrosis, hyperplasia, infiltration, and inflammation, 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 CCl4-induced liver injury [29, 30]. However, one study reported that the administration of aqueous and ethanolic extracts of M. indica stem bark significantly increases AST and ALT activities [31]. The limitations of this study include the exclusion of ammonia, acute inflammatory markers, and antioxidant enzymes, which provide insights into the protective mechanisms of M. indica against hepatocellular damage, and the lack of clinical data.

5. Conclusion

In this study, we evaluated the antioxidant and hepatoprotective effects of the M. indica seed extract against CCl4-induced toxicity. M. indica exhibits antioxidant activity and protects against CCl4-induced hepatotoxicity, with an IC50 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 CCl4-induced hepatocellular damage. Based on these findings, M. indica appears to be a safe, effective, 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.

Acknowledgments

The authors gratefully acknowledged the Medicinal and Aromatic Plants and Traditional Medicine Research Institute (MAPTMRI), National Center for Research, and Department of Microbiology, Faculty of Medical Laboratory Sciences, International University of Africa, Khartoum, Sudan.

Abbreviations

ALP: Alkaline phosphatase

ALT: Alanine aminotransferase

AST: Aspartate aminotransferase

CCl4: Carbon tetrachloride

DPPH: 2,2-diphenyl-1-picrylhydrazyl

IC50: Half maximal inhibitory concentration

M. indica: Mangifera indica

MAPTMRI: Medicinal and aromatic plants and traditional medicine research institute

ROS: Reactive oxygen species.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors' Contributions

A.S.K., A.A.A., and A.M.I. conceptualized the study; A.S.K. and A.M.I. proposed and designed compounds; A.S.K., A.A.E., R.A.E., A.A.A., and S.A.E. conducted experiments; A.M.I, E.E.O., and A.S.K. wrote the original draft; and A.S.K., A.A.E., S.A.E., W.S.K, R.A.E., A.A.A., A.M.I, and E.E.O wrote, reviewed, and edited the study. This work has been reviewed and approved by all authors.

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

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

Figure 3 Microscopic examination of liver tissue morphology: (a) liquid paraffin control-treated group exhibits normal hepatocyte, (b) silymarin (10 mg/kg) as hepatoprotective drug control, (c) M. indica extract (50 mg/kg) combined with CCl4 exhibits recovery of normal hepatocyte, (d) M. indica extract (100 mg/kg) induced with CCl4 shows the significant recovery of normal hepatocyte, (e) CCl4 (0.2 mg/kg) induced toxicity, and (f) treatment with M. indica extract alone (100 mg/kg).

Table 1 Percentage radical scavenging activity (%RSA) and IC50 (mg/ml) of M. indica extract using DPPH assay.

Treatments	Solvent	%RSA∗ ± SD (DPPH)	IC50 ± SD (µg/ml)	
Mangifera indica	Ethanolic	92 ± 0.03	8.3 ± 0.01	
Propyl gallate	Std	91 ± 0.01	14.1 ± 0.01	
Key: RSA∗: radical scavenging activity, (n; 3), DPPH: 2, 2, diphenyl-1-picrylhydrazyl, SD: standard division, Std.: standard. IC50: value represents the concentration of the sample required to inhibit 50% of DPPH free radical.

Table 2 The effect of M. indica extract on wellness parameters in study groups.

Name of plant	Observations	Animals/concentrations	
G1/Normal	G2/50 mg/kg	G3/100 mg/kg	
M. indica	Eyes	Normal	Normal	Normal	
Mucous membrane	Normal	Normal	Normal	
Salivation	Normal	Normal	Normal	
Skin and fur	Normal	Normal	Normal	
Lethargy	Absent	Absent	Absent	
Sleep	Normal	Normal	Normal	
Coma	Absent	Absent	Absent	
Convulsion	Absent	Absent	Absent	
Tremors	Absent	Absent	Absent	
Diarrhea	Absent	Absent	Absent	
Mortality	Nil	Nil	Nil	

Table 3 The effect of M. indica on body weight in study groups.

Groups	Body weight (gm)	Change in b. wt.	b. wt. gain or loss (%)	b. wt. gain or loss	
Initial	Final	
Normal	118 ± 0.20	128 ± 1.05	10.0	8.47	Gain	
100 mg/kg	130 ± 0.59	138 ± 0.90	08.0	6.15	Gain	
50 mg/kg	120 ± 1.80	130 ± 0.10	10.0	8.33	Gain	

Table 4 Effect of M. indica extract on body weight, change in body weight (g), body weight, and relative liver weight in CCl4-induced hepatotoxicity.

Groups	Body weight (gm)	Change in b. wt.	b. wt. gain or loss (%)	b. wt. gain or loss	Liver weight (gm)	Relative liver weight (%)	
Initial	Final	
Normal	121 ± 1.20	131 ± 2.00	10.0	8.26	Gain	1.60	1.22 ± 0.11	
CCl4	135 ± 2.00	136 ± 1.70	01.0	0.74	Gain	2.10	1.45 ± 0.50	
100 ml/kg	125 ± 1.50	130 ± 0.90	05.0	4.00	Gain	1.65	1.26 ± 0.20	
50 ml/kg	130 ± 3.60	133 ± 3.10	03.0	2.31	Gain	1.82	1.36 ± 0.30	
Silymarin	139 ± 2.90	146 ± 2.25	07.0	5.03	Gain	1.65	1.13 ± 0.10	
Key: each value represents the mean ± standard error of mean (mean ± SEM).

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

Rats group biochemical parameters	Normal	CCl4	1 ml/kg	0.5 ml/kg	Silymarin	
Mean ± SEM	
Urea (mg/dl)	28.2 ± 0.12	61.2 ± 0.09	40.7 ± 1.07	35.9 ± 1.05	36.4 ± 1.23	
Glucose (mg/dl)	62.1 ± 0.06	95.0 ± 1.09	74.1 ± 1.10	76.4 ± 1.33	73.2 ± 1.05	
Creatinine (mg/dl)	0.79 ± 0.05	0.94 ± 0.07	0.90 ± 0.10	0.79 ± 0.04	0.80 ± 0.06	
Albumin (mg/dl)	3.50 ± 0.07	5.80 ± 1.09	4.50 ± 1.00	3.50 ± 0.96	3.60 ± 0.26	
Total protein (mg/dl)	8.29 ± 0.07	9.98 ± 0.04	8.18 ± 0.11	8.58 ± 0.10	8.21 ± 0.07	
ALT (U/L)	122 ± 2.65	153 ± 0.76	130 ± 1.37	140 ± 1.28	120 ± 0.71	
AST (U/L)	130 ± 2.69	165 ± 2.24	149 ± 2.20	148 ± 1.61	142 ± 2.62	
ALP (U/L)	137 ± 1.81	187 ± 3.81	141 ± 1.86	131 ± 4.54	128 ± 2.10	
Key: values expressed as a mean ± standard error of mean (SEM).
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