Aflatoxins (AFs) are group of difuranocoumarins toxic secondary metabolites produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius, which contaminate various foodstuff and feed. The most predominant major AFs that contaminate food and feed commodities include AFB₁, AFB₂, AFG₁ and AFG₂ and their hydroxylated metabolites, namely AFM₁ (Ráduly et al. 2020). Aflatoxin B₁ is the most potent hepatotoxin, mutagen, teratogen, immunosuppressive and carcinogen amongst other major groups of AFs in nature (Zarev et al. 2020). When ingested, it is bio-transformed in the liver primarily by CYP3A4 and CYP1A2 isoforms that are members of the cytochrome P450 (CYP) superfamily of drug metabolising enzymes and generate a highly reactive species, namely AFB₁-exo-epoxide and other metabolites (Bedard & Massey 2006; Rushing & Selim 2019). These reactive species bind to the macromolecules causing toxicity and mutations, which lead to lipid peroxidation, necrosis, cell damage, cell death, deoxyribonucleic acid (DNA) lesions, carcinogenicity and other genetic diseases (Bedard & Massey 2006; Zarev et al. 2020).

Aflatoxin B₁ deleteriously affects global public health as it is the major contributor to the worldwide occurrence of hepatocellular carcinoma (HCC). The toxin also works synergistically with hepatitis B and C viruses to significantly increase the risk of HCC far above either factor individually (Rushing & Selim 2019). Surgery and liver transplant offer limited treatment options as they are only useful in the treatment of early stages of HCC. Other non-surgical options such as chemo- and radio-therapies are only successful in patients with localised liver tumour (Darvesh, Aggarwal & Bishayee 2012). Therefore, research that focuses on the development of alternative preventative and therapeutic strategies may lead to valuable findings that may be effective in the control of HCC. Although the principal toxic target of AFB₁ is the liver, several reports showed that exposure to AFB₁ has adverse renal effects as it increases the levels of creatinine and urea in animal experiments (El-Mahalaway 2015; Yilmaz et al. 2018). Previous studies also revealed that oxidative stress resulting from AFB₁ consumption leads to myocardial cell membrane destruction manifested by a marked increase in lactate dehydrogenase (LDH) activity (Mannaa et al. 2014).

The biosynthesis and activation of AFs can be attenuated using bioactive compounds present in herbal products. Plant extracts have been studied for their antifungal growth inhibition and mycotoxin detoxification. Several studies have been conducted on the protective effects of plant extracts and its bioactive compounds against AFB₁-induced hepatotoxicity (Choi et al. 2010; Soni et al. 1997; Tang et al. 2007). Curcumin and ellagic acid are examples of phytochemicals isolated from plants and they inhibited hepatocarcinogenicity in rat and chicken models (Gowda et al. 2008; Soni et al. 1997). These compounds are known for their potent free radical scavenging ability and exert their anticarcinogenic effect primarily through prevention of oxidative stress (Darvesh et al. 2012). Furthermore, phytochemicals such as lycopene and quercetin have been reported to inhibit biotransformation of AFB₁ toxicity by inducing detoxification enzymes (Choi et al. 2010; Tang et al. 2007).

Monanthotaxis caffra (Annonaceae) is a shrub occurring in evergreen forests of Eastern Cape, KwaZulu-Natal and Mpumalanga provinces of South Africa (National Research Council 2008). This plant species has been used in traditional medicine against tumours, microbial and parasitic infections in veterinary and human health (Mulholland et al. 2000; Okhale et al. 2016). Methanolic leaf extract of M. caffra has been also reported for its antigenotoxic properties against AFB₁-induced genotoxicity (Makhuvele et al. 2018a). Furthermore, Crotepoxide, a known antitumour compound and other polyoxygenated cyclohexane derivative, 5,6-diacetoxy1-benzoyloxymethyl-1,3-cyclohexadiene were isolated from M. caffra (Makhuvele et al. 2018b; Mulholland et al. 2000). The in vivo hepatoprotective effects of M. caffra against mycotoxin-induced toxicity has never been explored. Therefore, this study aimed to investigate the protective effects of methanolic leaf extracts of M. caffra (MLEMC) against AFB₁-induced hepatotoxicity on rats.

Data are presented as mean ± standard deviation. Differences between groups were determined using one-way analysis of variance (ANOVA). The standardised residuals were tested for deviations from normality using Dunnett’s, t-test and Shapiro–Wilk’s test. Least significant difference (LSD) test was used to determine statistical significance between the means of treated groups and the controls. The data were considered significant at p < 0.05.

Human and animals are exposed to different xenobiotic substance such as aflatoxins that cause deleterious effects on the biomolecules and cellular membrane by inducing oxidative damage (El-Agamy 2010). The liver is an essential organ of human and animals, which is involved in metabolism of xenobiotics (Colakoglu & Donmez 2012). Aflatoxin B₁ is a well-known hepatotoxic agent. It induces cellular and tissue damage to the hepatocytes, thus resulting in aflatoxicosis, HCC, necrosis, cirrhosis, etc. (Shan 2020; Zarev et al. 2020). Aflatoxin B₁ mediates these various deleterious effects through the induction of oxidative stress (El-Agamy 2010). Methanolic leaf extracts of M. caffra ameliorated AFB₁-induced genotoxicity in in vitro antigenotoxic assays (Makhuvele et al. 2018b). Due to this antigenotoxic effect of MLEMC, we hypothesised that MLEMC will ameliorate AFB₁-induced hepatotoxicity in rats. Therefore, we further investigated the protective effects of MLEMC against AFB₁-induced hepatotoxicity in rats.

In our study, AFB₁ intoxication significantly increased the serum levels of the liver enzymes AST and ALT in comparison with untreated control group, which implies that AFB₁ caused cellular damage to the liver because of the release of these enzymes, which are normally located in the cytoplasm of the liver cells, in the blood stream. The higher standard deviation observed in the AFB₁ intoxicated group were because of the two rats which did not respond to AFB₁, their serum levels were not afffected by administration of AFB₁. In general, AST and ALT are released into the blood stream when there is a liver damage or loss of cell membrane intergrity. These serum enzymes are considered as common biomarkers for the diagnosis of liver damage (Amacher 1998). Alanine aminotransferase and AST were normal in healthy control group whilst co-adminsteration of AFB₁+curcumin and AFB₁+MLEMC (100 mg/kg, 200 mg/kg and 300 mg/kg) normalised the serum enzymes ALT and AST. No significant difference was observed between the MLEMC and curcumin-treated groups in comparison with the healthy control group. This is a clear indication that MLEMC and curcumin ameliorated the toxic effect of the AFB₁, by inhibiting AFB₁-induced liver cell injury damage and consequently lowering the AST and ALT levels in the blood. Curcumin was used as a positive control in this study as it is renowned for its protective effects against AFB₁-induced hepatocarcinogenesis and hepatotoxicity in rodents, broilers and ducklings by lowering the levels of serum markers and lipid peroxidation (Chuang et al. 2000; Gowda et al. 2008; Mathuria & Verma 2008). Methanolic leaf extracts of M. caffra demonstrated protective effect against AFB₁-induced toxicity at all tested concentrations. However, the highest concentration of MLEMC showed high reduction comparably with the lower concentration, although no significant difference was observed.

An increase in LDH levels in rats intoxicated with AFB₁ only was observed as compared with untreated control. Increased LDH activity is one of the major events involved in liver damage after AFB₁ administration, as it is only released in the blood stream when the liver is injured (Choi et al. 2010; Nayak & Sashidhar 2010). However, this increase in LDH activity was not observed in the healthy control and in rats treated with different concentrations of AFB₁+MLEMC. A significant decrease in the LDH level in rats treated with curcumin was observed, which is in line with literature reports (Nayak & Sashidhar 2010).

Creatinine, a major marker of kidney function, is the final metabolite of creatine conversion. Increased creatinine as an index of impaired kidney function because of chronic exposure to AFB₁ was reported in chickens and rats (El-Mahalaway 2015; Valchev et al. 2014). In this study, significant changes were not observed in the serum level of creatinine in rats treated with a single dose of AFB₁, which is in line with previous reports where nephrotoxicity was observed only in animals having chronic exposure to AFB₁ (El-Mahalaway 2015; Valchev et al. 2014).

The result of biochemical analysis of the protective effects of MLEMC was also confirmed by the histopathological investigations. Aflatoxicosis is characterised by hydropic changes, vacuolar degeneration, bile duct proliferation and lymphoplasmacytic infiltration in exposed hepatocytes. However, the histopathological effects of AFB₁ are directly proportional to the concentration and exposure time to AFB₁ (Do & Choi 2007; Yaman, Yener & Celik 2016). Aflatoxin B₁ intoxication caused mild to severe hepatocellular injury accompanied by bile duct proliferation, hydropic changes and lymphoplasmacytic infiltrate in the hepatocytes of exposed rats. This histological effect of AFB₁ on rat hepatocytes has been reported in literature (Yaman et al. 2016). Minimal degree of the above-mentioned histological characteristics in rats exposed to co-administered AFB₁+curcumin and AFB₁+MLEMC were observed in this study, thus implying that curcumin and MLEMC had recuperative effects against AFB₁-induced acute toxicity. Curcumin has been reported in literature to possess ameliorative effects on the histology of the liver and other organs (El-Agamy 2010; Gowda et al. 2008).

Members of the genus Monanthotaxis are known to contain essential oils (Parmena, Mgina & Joseph 2012), alkaloids, flavonoids and cyclohexane epoxides (Mulholland et al. 2000). Chemical analysis of the 80% extract of M. caffra revealed it is rich in cyclohexane epoxide derivatives including crotepoxide and 5,6-diacetoxy1-benzoyloxymethyl-1,3-cyclohexadiene and a mixture of two related benzoyloxy cyclohexidiene derivatives (Makhuvele et al. 2018b). Bioassay-guided fractionation of the extract using VITOTOX genotoxicity assay yielded the two antimutagenic compounds crotepoxide and 5,6-diacetoxy1-benzoyloxymethyl-1,3-cyclohexadiene. In addition to their antimutagenic effects against aflatoxin B₁-induced mutagenicity in Ames and Vitotox assays, the antitumour or anticarcinogenic properties of these polyoxygenated cyclohexane derivatives have been reported in literature. Crotepoxide inhibited the expression of tumour necrosis factor (TNF) regulated gene products involved in anti-apoptosis such as Bcl-2, Bcl-XL, cyclin D1, Cox-2, Bax, Bid, c-Myc, MMP-9 and VEFG, etc. Furthermore, crotepoxide also inhibited the tumours by preventing the activation of genes that are involved in tumorigenesis at gene levels (Alonso-Amelot 2016). The compound also possessed antitumour properties against various carcinoma in rats and mice (Parmena et al. 2012; Shing & Tam 1996; Starks et al. 2012). The protective effects of these plant extracts against AFB₁ hepatotoxicity may be due to the presence of the above-mentioned compounds, which are found in Annonaceae plant species.

The present study showed that pre-treatment of rats with MLEMC protected hepatocytes from AFB₁-induced hepatotoxicity as evidenced by the normalisation of the serum enzyme levels of AST, ALT and LDH and the reduction of hepatocellular lesions. This effect was comparable to that produced by curcumin a known hepatoprotective agent against AFB₁-induced toxicity. This hepatoprotective effects results from the synergistic effects of the compounds present in the MLEMC. Our results suggest that MLEMC can be considered as potential natural agent for prevention of AFB₁-induced hepatotoxicity. Further studies on long time treatment of MLEMC against AFB₁-induced hepatotoxicity in rats are recommended.