Hepatoprotective Potential of Mesembryanthemum forsskalii Fruits Extract Against Carbon Tetrachloride-Induced Liver Toxicity in Mice

1Department of Biology, College of Science, Jouf University, Sakaka, KSA 2Department of Zoology, Faculty of Science, Tanta University, Tanta, Egypt 3Department of Zoology, Faculty of Science, Port Said University, Port Said, Egypt 4Department of Zoology, Faculty of Science, Fayoum University, Fayoum, Egypt Article Information Received 30 September 2022 Revised 15 November 2022 Accepted 04 December 2022 Available online 30 January 2023 (early access)


INTRODUCTION
T oxins, infectious agents, and serum inflammatory mediators are the main reasons leading to the loss of functional liver capacity (Edwards and Wanless, 2013). Experimental animals that expose to CCl 4 exhibit different morphological alterations in livers (Li et al., 2019;Owojuyigbe et al., 2020;Begum et al., 2022). Fibrosis, focal inflammation, cell infiltration, hepatic cell degeneration O n l i n e

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raised serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and diminished IL-10, IL-12 (Elshopakey and Elazab 2021). 1.25 ml/kg BW of CCl 4 induced hepatotoxicity related parameters in rats (Al-Yahya et al., 2013). CCl 4 in 2 ml/kg BW induced oxidative stress and hepatotoxicity in mice, where a marked alteration in histology and marked inflammation was induced in CCl 4 treated mice (Ullah et al., 2020). Many drugs are now available for the treatment of liver failure disease; however, the majority showed many serious side effects besides their cost-effectiveness. Therefore, several studies have been focused on finding alternative natural sources including plants to replace conventional drugs in the last two decades (Katiyar et al., 2012). Many desert medicinal plants showed therapeutic and hepatoprotective effects against CCl 4 -induced hepatotoxicity in mice and rats (Wahid et al., 2016;Ammar et al., 2022;Ayoka et al., 2022;Doudach et al., 2022;Nwaechefu et al., 2022;Ouassou et al., 2021). The methanolic extract of Corrigiola telephiifolia roots showed hepatoprotective potentiality through improving the blood biochemical parameters as well as enhancement of the hepatic tissues in CCl 4 induced hepatotoxicity in mice (Doudach et al., 2022). Liver enzymes in rats treated with the methanol extract of Cajanus cajan at 100 and 200 mg/ kg BW showed lower levels of liver enzymes compared to CCl 4 treated group (Nwaechefu et al., 2022). The extract of C. cajan also reduced the inflammatory cell infiltration, hepatic fibrosis and necrosis and severe hepatocytes apoptosis induced by CCl 4 (Nwaechefu et al., 2022). The aqueous extract of Caralluma europaea stem showed hepatoprotective potential in CCl 4 induced hepatotoxicity rats (Ouassou et al., 2021). The ethanolic flower extract of Salix subserrata reduced the liver enzymes in CCl 4 treated rats and also ameliorated the negative impact of CCl 4 on liver tissues (Wahid et al., 2016). Naringenin isolated from Citrus sinensis ameliorated the increased levels of liver enzymes induced by CCl 4 as well as attenuated the pathological changes in liver tissues. Naringenin also enhanced the expression of Bcl-2 (Ammar et al., 2022). CCl 4 induced hepatotoxicity in rats through destruction of the liver tissues. The administration with the alkaloid fraction of the Vitex doniana showed protective effect in rats through improving various blood parameters and also restoration of the normal appearance of the destructive liver tissues induced by CCl 4 (Ayoka et al., 2022).
Desert plants that are growing wildly in the kingdom of Saudi Arabia (KSA); have remarkable medicinal and commercial value to local communities (Aati et al., 2019). Among these plants, Mesembryanthemum forsskalii (family Aizoaceae) which has multiuse and is considered a rich source of natural products and bioactive secondary metabolites that have not been well exploited until now (Moawad et al., 2016;El-Amier et al., 2021). It was reported that M. forsskalii has more than 14 unsaturated fatty acids representing about 5.6% dominated by linoleic and oleic acids and also saturated fatty acids dominated by palmitic acid (Bilel et al., 2020). In addition to the fatty acids, significant amounts of carbohydrates, protein, and amino acids were also detected in seeds of M. forsskalii (Al-Jassir et al., 1995). In the northern part of KSA, the seeds of M. forsskalii are used as a traditional food for the public due to its high content of carbohydrates, protein, and fats (Najib et al., 2004;Abdel-Hamid et al., 2021).
Seeds oil extract of M. forsskalii showed antifungal activity against different fungal strains such as Penicillium chrysogenum, P. slilacinus, P. oxalicum, Fusarium oxysporum, and Aspergillus fumigatus, A. niger, A. flavus, A. carneus, Alternaria alternata, Rhizopus oryzae, Cladosporium cladosporioides and Paecilomyces lilacinus (Bilel et al., 2020). The effect of seeds oil extract of M. forsskalii was more prominent on P. chrysogenum, A. fumigatus, A. flavus and A. carneus (Bilel et al., 2020). Samh seeds diets decreased the lipid peroxidation in streptozotocin (STZ)-induced diabetic of Wistar Albino rats (Al-Faris et al., 2010). The nanoparticle prepared using M. forsskalii seeds showed antimicrobial activity against Gram positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) as well as antifungal activity against Candida albicans. The nanoparticles also showed anticancer activity against LoVo cancer cell lines with low IC 50 (28.3 µg/ml) (Aabed and Mohammed, 2021). The biological activity of one species of Mesembryanthemum (M. nodiflorum) showed an analgesic effect and significant inhibitory activity against colon, cervix, liver and melanocyte carcinoma. Moreover, the ethanolic extract of the aerial parts of M. nodiforum had a significant hypoglycemic effect, anti-oxidant, anti-inflammatory and hepatoprotective activity (El-Hawary et al., 2020). Some of the secondary metabolites of M. forsskalii such as flavonoids, alkaloids, saponins, and phenolics are proven to act as potent antioxidant agents (Bilel et al., 2020;El-Amier et al., 2021). Hence, it may contribute individually or in combination (synergistic effect) to protective capability against liver toxicity. Many researchers demonstrated that drugs of herbal origin may have potential antioxidant activities which may contribute to the protective action of liver toxicity if added as supplementary medications (Hamzawy et al., 2015;Singh et al., 2016). So far, there is no previous study had evaluated the phytochemical and biological activity of M. forsskalii. Accordingly, the main objective of our study is to evaluate the hepatoprotective potency of the aqueous methanol extract of M. forsskalii O n l i n e F i r s t A r t i c l e fruits against hepatotoxicity induced by CCl 4 .

MATERIALS AND METHODS
Plant collection and extract preparation M. forsskalii was collected from Al-Adare region in the Aljouf district in the Northern part of KSA. Fruits were separated from the rest of the plant, dried at room temperature, and grounded into powder using an electrical grinder. Powdered materials were macerated in aqueous methanol until exhaustion for 24h. The solvent was evaporated under vacuum at 45 o C using a rotary evaporator to obtain the aqueous methanolic crude extract for further study (Mahalel, 2015).

Determination of the LD 50 of M. forsskalii extract
A total number of 30 adult male mice of eight weeks old (25-30g) were divided into 5 groups of 6 mice each. These groups were injected with a single dose of plant extract at 3, 3.5, 4, 4.5, and 5g/kg of plant extract, respectively (Dhanarasu et al., 2016). Mice were monitored for 24h to assess the acute toxicity of M. forsskalii extract. LD 50 of the extract was calculated using the arithmetic method of Finney (Finney, 1985). According to the following formula: LD 50 = Dm -∑ (z*d)/n Where Dm is the highest dose that kills all animals. z is mean of dead animals between two successive groups. d is the constant factor between two successive groups and n is number of animals per each group.

Carbon tetrachloride (CCl 4 ) preparation
CCl 4 of 100 % concentration and olive oil were obtained from Algomhoria Company, Egypt. CCl 4 dissolved in olive oil was given by intraperitoneal (i.p) injection in a dose of 0.8 ml/Kg body weight of mice twice weekly for three weeks.

Experimental design
Based on the LD 50 value, low and high doses of M. forsskalii extract were used in the study as a 100 and 500 mg/kg BW, respectively. Six experimental groups of mice (6 mice/group) were divided as follows: Group 1 (Gp1): Mice were administered with 200 µl of olive oil and served as vehicle control; Gp2: mice were injected intraperitoneal (i.p) with CCl 4 as 0.8 ml/kg BW twice a week for three weeks(3 days intervals); Gp3: mice had administered orally with M. forsskalii extract as 100 mg/kg BW, 7 times/ 15 day(every other day); Gp4: mice had injected with CCl 4 as in Gp2, then followed by administration with M. forsskalii extract as in Gp3 (100 mg/kg BW); Gp5: mice had administered with M. forsskalii extract as 500 mg/kg BW (7 times/15 day, day after day) and Gp6: mice had injected i.p with CCl 4 as in Gp2, then followed by administration with M. forsskalii extract as in Gp5.

Determination of the total body weights and liver relative weight
The body weight of mice was assessed at the beginning of the experiment and at the end of the experiment after treatment with CCl 4 and/or M. forsskalii extract. The significant difference between the body weight before and after treatment was assessed. After decapitation, livers were removed, dried by blotting paper, and weighed on a digital balance. To calculate the relative liver weight, the liver weight was divided by the total body weight x 100.

Biochemical analysis
Twenty-four-hours after the last treatment, mice were sacrificed. The blood was collected from the orbital plexus in heparinized glass tubes from each mouse, allowed to stand for 30 min at room temperature, and centrifuged at 3000 rpm for 15 min to separate the blood serum samples. The separated serum was used for the estimation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) using the protocol described by Ahmadi et al. (2011) and Habibi et al. (2015). Total serum protein was determined by the method of Biuret (Perez Gutierrez et al., 2011). Albumin was determined using the method of Webster (Webster, 1977). The contents of cholesterol and triglycerides were also determined using commercial kits. All the above estimations were performed using standard autopak kits using a spectrophotometer.

Histological examinations
Liver tissues were sliced into small pieces and then immersed in neutral formalin buffered (10%) for 24h. The fixed tissues were processed routinely, embedded in paraffin (to get paraffin sections 4-5 μm), sectioned, deparaffinized, and dehydrated using the standard techniques (Suvarna et al., 2018). Sections were then stained with hematoxylineosin and studied for histopathological changes. During the microscopic examination representative photos were captured (Nikon TE 2000-U microscope (NIKON, Tokyo, Japan).

Immunohistochemical studies
Representative sections of the formalin-fixed, paraffin-embed liver tissue from different groups were used for P53 and Bcl-2 expression investigation. Tissues were deparaffinized in xylene and rehydrated in descending ethanol series. Antigen retrieval was accomplished through microwave irradiation of the sections in 10 mM sodium citrate buffer. After microwave antigen repair, the sections Hepato-Protective Activity of Mesembryanthemum forsskalii Fruits O n l i n e

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were incubated at 4°C overnight with rabbit anti-P53 and anti-Bcl-2 antibody (1:100, dilution), followed by incubation at 37°C in PV6001 for 30 min. The bound immune complexes were developed by the addition of 3, 3′-diaminobenzidine tetrahydrochloride (DAB) substrate and the nuclei were stained with hematoxylin (Sigma-Aldrich). The sections were incubated with a normal goat serum as a negative control. Samples were viewed by using Nikon TE 2000-U microscope (NIKON, Tokyo, Japan). Immunohistochemical positivity was evaluated by proportion and staining intensity for each protein.

Statistical analysis
Analysis of variance (ANOVA) using Minitab (ver. 12.21) was used to evaluate the significant difference in blood parameters in control and treated groups of mice. ANOVA was also used to assess the difference between the relative body weight before and after treatment and also the relative liver weight in the control and treated groups.

LD 50 of M. forsskalii fruit extract
The estimation of the median lethal dose (LD 50 ) in the experimental animals was carried out according to Finney, 1985. The result showed that the LD 50 of M. forsskalii extract was 3.625 g/kg BW (Table I). Effect of M. forsskalii extract on the body weight and relative liver weight The total body weight of mice was reduced significantly after CCl 4 treatment. The weight was not significantly changed after treatment with either M. forsskalii alone or after CCl 4 followed by 100 mg/kg BW from M. forsskalii extract (Gp5) and increased significantly after CCl 4 followed by 500 mg/kg BW from M. forsskalii extract (Gp6) ( Table II).
The relative liver weight of mice was increased significantly after CCl 4 treatment (Gp2) compared to vehicle control (Gp1). The reduction of relative liver weight was observed after treatment with CCl 4 followed by M. forsskalii extract (either 100 or 500 mg/kg BW) (Gp5 and Gp6). Low or high doses of M. forsskalii after CCl 4 treatment (Gp5 and Gp6) reduced liver weight observed in treated mice (Gp2). There was no significant change between the relative weights of vehicle control (Gp1) and that of CCl 4 followed by 100 or 500 mg/kg BW (Gp5 and Gp6) (Table II).

Biochemical changes
Treatment of the animals with CCl 4 (Gp2) resulted in a significant increase in ALT compared to the vehicle control (Gp1). Both low dose (100 mg/kg BW) (Gp5) and high dose (500 mg/kg BW) (Gp6) significantly reduced the levels of ALT in the treated mice compared to the CCl 4 treated mice (Gp2) (p<0.05). The reduction of the ALT content was more prominent after treatment with the low dose (100 mg/kg BW). No significant difference was observed in the animal group which received 100 mg/kg BW alone (Gp3) and the vehicle control group (Gp1). But the treatment of mice with 500 mg/kg BW alone (Gp4) significantly increased the content of ALT compared to the vehicle group (Gp1) ( Table III).
CCl 4 administration to the mice increased AST levels (Gp2) when compared to the vehicle group (Gp1). Treatment of mice with a high dose (500 mg/kg BW) after CCl 4 treatment (Gp6), enhanced the AST content, whereas treatment with a low dose (100 mg/kg BW) (Gp5) reduced the AST content in the serum comparing to the CCl 4 treated group (Gp2) to a level almost normal as indicated by that of the vehicle control group (Gp1) indicating that low dose of M. forsskalii has improved the liver enzymes.
Although M. forsskalii extract at any dose has not significantly changed the albumin and the total protein levels, protein content was elevated after treatment with low or high doses (Gp5 and Gp6) compared to that of CCl 4 treated group (Gp2). Albumin content also was elevated after the administration of 500 mg/kg BW from M. forsskalii (Gp6) but the elevation of the content was not significant (Table III).
Even the CCl 4 treated mice (Gp2) showed lower cholesterol content than vehicle control (Gp1), the content of cholesterol in blood serum showed a significant increase after treatment with M. forsskalii either with low or high doses (Gp5 and Gp6), respectively comparing to CCl 4 treated group (Gp2) ( Table III).
A high dose of M. forsskalii (Gp6) had significantly increased the content of triglycerides compared to CCl 4 treated group (Gp2), whereas a low dose (Gp5) elevated the content of triglycerides compared to the CCl 4 treated group (Gp2) but the increase was not significant (Table III).

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Hepato-Protective Activity of Mesembryanthemum forsskalii Fruits  Table II. Effect of M. forsskalii fruits extract on the total body weight and relative liver weight of mice.

Histopathological changes
Liver tissue sections were either stained with H and E (Fig. 1) or immune stained for the detection of the expression levels of the cell cycle regulator and tumor suppressor protein, P53 (Fig. 2), and the expression levels of the anti-apoptotic Bcl-2 (Fig. 3). The histological observations of liver tissues support the results obtained from serum enzyme assays. Negative control tissue sections exhibited no apparent pathological alterations (Fig. 1A). No cavitation, necrosis, or fibrosis was found in control sections. In contrast, sections from CCl 4 -only treated mice displayed apparent cavitation in broad areas, necrosis with inflammation, and loss of cellular boundaries (Fig. 1B). On the other hand, liver sections treated with low and high dose of M. forsskalii extract (100mg and 500mg/kg BW, respectively) separately showed no marked variation in the histological architecture of the liver tissue (Fig. 1C,   D). Interestingly, the broad cavitation in the liver was attenuated in mice treated with M. forsskalii extract during the experimental periods (Fig. 1E, F). The administration of M. forsskalii extract resulted in less cavitation in the liver. Importantly, the remaining cavitation level and the range of the necrotic cells in the 100 mg/kg BW-treated group (Fig. 1E) were lower than that in the 500 mg/kg BW-treated group (Fig. 1F).
Liver sections prepared from M. forsskalii -treated groups displayed less fibrosis as compared to the CCl 4only control group. This group's liver sections showed regeneration of hepatocytes, almost toward near-normal liver architecture and possessing higher hepatoprotective action. The improvement in the 100 mg/kg group (Fig.  1E) was more obvious in comparison to that of the 500 mg/kg group (Fig. 1F). We also examined the distribution of fibrosis in different liver regions (from the central vein region to hepatic portal veins). Both doses of the extract reduced apparent liver injury caused by CCl 4 , compared to the group of mice that were treated with CCl 4-only.
The immunostaining study revealed that CCl 4 had enhanced the expression of the cell cycle regulator and necro-apoptotic driver, P53 in the liver tissues (Fig. 2B)  ( Fig. 2E, F) but the effect was more prominent when the extract was administered at a low dose (Fig. 2E). There was no appreciated change in the expression level in P53 under the extract treatment when administered to normal animals as represented in (Fig. 2C, D) if compared to the negative control ( Fig. 2A). On the other side, the expression level of the tumor-promoting and cytoprotective molecule, Bcl-2, was significantly enhanced in the liver section of animals challenged by CCl 4 (Fig. 3B). Interestingly, this elevated expression had been reverted after treatment with the plant extract at both doses (Fig. 3E, F). Similarly, the expression of Bcl-2 in the liver from normal animals did not show detectable changes before and after the administration of the extract neither at a low dose nor a higher one (Fig. 3C, D) when compared to the negative control (Fig. 3A).

DISCUSSION
The change in dietary habits as well as the chemoprevention show considerable effective strategies against oxidative stress and are the main focus of the area of research these days (Lee and Park, 2003). CCl 4 is a known, reliable, and commonly used chemical to induce liver damage through oxidative degradation in the adipose tissue which resulted in fatty infiltration of the hepatocytes (Pal et al., 2014;Wang et al., 2019;Begum et al., 2022) and increased permeability, acute toxicity and may be hepatic necrosis (Naji et al., 2017). Increasing relative liver weight and the level of liver enzymes such as AST and ALT in the serum is the first clue to liver toxicity (Tsai et al., 2009;Bahashwan et al., 2015). The current investigation was undertaken to evaluate the possible protective effect of M. forsskalii extract against carbon tetrachloride-induced hepatotoxicity and oxidative stress in mice. CCl 4 causes acute hepatocyte injuries and altered membrane integrity and as a result enzymes in hepatocytes leak out (Hu et al., 2000). However, after treatment with M. forsskalii, the pathological increases in AST and ALT were significantly restored at least after treatment with a low dose. These results indicate that M. forsskalii can protect against CCl 4 -induced hepatocyte injuries. Extract from M. forsskalii fruits showed the protective capability against treatment through the restoration of the liver enzymes level to their normal values and restoration of the normal relative weights. The protective potentiality of M. forsskalii against CCl 4 -induced hepatotoxicity may be attributed to the presence of some secondary metabolites well known as an antioxidant and radical scavenging agents such as flavonoids, saponins, and phenolics.
Many plants are used as hepatoprotective natural sources against CCl 4 -induced hepatotoxicity. For instance, the aqueous ethanol extract of the pods of Acacia senegal O n l i n e

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U. Mahalel et al. was used efficiently as a hepatoprotective agent against hepatotoxicity induced by CCl 4 in rats through the restoration of the blood serum enzymes and bilirubin and also restoration of the architecture of the liver through the presence of normal hepatic cords, absence of necrosis and fatty infiltration through histology of liver sections (Pal et al. 2014;Azubuike et al. 2018). It was reported that leaves extract of Pyrenacantha staudtii has a protective effect against CCl 4 induced liver toxicity and damage, as well as CCl 4 induced elevations in the liver enzymes were significantly reduced by 750 mg/kg and 1500 mg/kg BW of the plant extract (Anosike et al., 2008). As well as (Ouassou et al., 2021) found that Caralluma europaea stem extract (250 mg/kg BW) had a significant hepatoprotective effect by ameliorating CCl 4 -induced alterations of some biochemical parameters.
In line with the results of the current study, the blood parameters were improved in CCl 4 induced animals after administration of different plant extracts such the flowers extract of S. subserrata (Wahid et al., 2016), roots extract of C. telephiifolia (Doudach et al., 2022) and the extract of C. cajan (Nwaechefu et al., 2022). In accordance with our results, the inflammatory cell infiltration, hepatic fibrosis, necrosis and severe hepatocytes apoptosis induced by CCl 4 were ameliorated by the extract of C. cajan (Nwaechefu et al., 2022). Administration of S. subserrata extract ameliorated the negative impact of CCl 4 on liver tissues, where the appearance of the liver tissues was restored to the normal appearance post-administration of S. subserrata extract (Wahid et al., 2016). Naringenin from C. sinensis ameliorated the negative impact of CCl 4 on liver tissue appearance. Moreover, it enhanced the expression of Bcl-2 (Ammar et al., 2022). The alkaloids extract of V. doniana restored the normal appearance of the destructive liver tissues induced by CCl 4 (Ayoka et al., 2022).
The lower level of cholesterol in CCl 4 treated mice than that of normal (vehicle control) may be attributed to loosing of an animal's appetite after CCl 4 treatment compared to other groups which could be concluded from the amounts of food remaining in the CCl 4 treatment group comparing to other groups. It seems that animals hardly eat after treatment with CCl 4 . It was reported that feed intake was significantly reduced after CCl 4 administration compared with the constant feed intake by the control group (Uemitsu and Nakayoshi, 1984). The effect of decreasing cholesterol and the nonsignificant changes in the content of triglycerides, protein, and albumin in CCl 4 treated mice may also be attributed to the decrease in the number of hepatocytes due to liver damage by CCl 4 and consequently decrease in the liver capacity to synthesize these metabolites (Bhandarkar and Khan, 2003). The significant increase in cholesterol and triglycerides after treatment with either high or low doses from M. forsskalii indicates the preliminary evidence for the protective role of M. forsskalii. This may refer to the restoring of the liver ability to synthesize these metabolites after its retardation by the CCl 4 effect.
It is well established that CCl 4 induces liver damage through necrosis. Immunostaining results from our study showed that CCl 4 alone induced relative up-regulation of P53 and down-regulation in the expression of Bcl-2 in the liver tissues. Importantly, unlike high dose, administration of the M. forsskalii at a low dose significantly restored this effect by the downregulation of P53 and upregulation of Bcl-2 expression. CCl 4 is reported to induce hepatic damage as a result of metabolic conversion of the radicals through lipid peroxidation and disturbance of the activities of the antioxidant enzymes (Adesanoye and Farombi, 2010), induce oxidative stress, and cause liver injury by the formation of free radicals (Manna et al., 2006). On the other hand, carbon tetrachloride causes noticeable toxicity by enhancing liver lipid peroxidation as found by increased concentrations of hepatic malondialdehyde (Poli, 1993;Dalton et al., 2009). Results from the current study have not included any evidences for the antioxidant activity of the M. forsskalii extract, the antioxidant activity of each fraction from the same fruits should be evaluated.
In conclusion, the administration of a low dose of M. forsskalii (100 mg/kg BW) improved the liver function through restoration of liver enzymes, reducing P53 expression, and increasing the Bcl-2 expression to the normal levels after CCl 4 treatment. However, 500 mg/kg BW prominently showed no beneficial effect after CCl 4 induced toxicity except enhancing Bcl-2 expression; even it exerts some toxic effect on the liver enzymes in mice.

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Hepato-Protective Activity of Mesembryanthemum forsskalii Fruits 9 took all the possible procedures to reduce mice sufferance.

Statement of conflict of interest
The authors declare no competing interests.