1 Introduction

Plants have been harnessed as medicines since the early centuries. Remedies were initially taken in the form of crude drugs such as tinctures, elixirs, compresses, powders and other herbal formulations (Alsarhan et al., 2012). Statistics about the origin of drugs that were launched in the last twenty five years revealed that both natural products and semi-synthetic compounds, derived from natural origin, comprised 34% of all new chemical entities, while 18% of them were synthetic mimics of natural compounds (Gurib-Fakim, 2006). However, many of these herbs and natural supplements have not been thoroughly evaluated, and their safety as well as effectiveness may not yet have been proven (Dunnick and Nyska, 2013). Pharmacological interest in the efficacy and safety of the herbal medicines has grown during the past decade because of the realization that many people are self-medicated using these agents (Calixto, 2005; Firenzuoli and Gori, 2007). However, the use of herbal products should be based on scientific origin; otherwise, they would be useless and unsafe (Mir et al., 2013). Furthermore, the irrational use of these herbal products may cause serious toxicity in humans (Parthasarathi and Olsson, 2004). Unfortunately, many people underestimate the toxicity of natural products and do not realize that these agents could be as toxic as, if not more than, synthetic drugs. A typical example for a toxic herbal product is the leaves of Atropa belladonna and Digitalis purpurea, which show severe systemic toxicity if taken orally (Mir et al., 2013).

In this regard, Cratoxylum arborescens Blume Guttiferae family (Fig. 1) is a traditional medicine and its natural range of distribution includes Malaysia, South Burma, Sumatra and Borneo. Due to its geographically wide-ranging natural habitat, it is often referred to by several Malay colloquial names depending on the region or locality where it is found. In Sabah, Serangan is synonymous with Geronggang. In Sarawak, it is locally called by different names depending on the community (Jensen and Zwieniecki, 2013). This plant is used traditionally as a cure for fever, cough, diarrhea and other ailments (Srithi et al., 2009). Phytochemicals which have been reported to be found in Cratoxylum are xanthones (Iinuma et al., 1996a; José et al., 1998), and some of these xanthones have been shown to exhibit significant pharmacological activities (Jiang et al., 2004). α-Mangostin (AM) (Fig. 2) is one of the major xanthones isolated from the stem bark of the plant (El-Seedi et al., 2009). AM has been reported to possess a wide spectrum of biological activities such as anti-inflammatory (Chairungsrilerd et al., 1996; Shankaranarayan et al., 1979; Tewtrakul et al., 2009), cardioprotective (Devi Sampath and Vijayaraghavan, 2007), anti-tumor (Akao et al., 2008; Chitchumroonchokchai et al., 2013), antidiabetic (Ryu et al., 2011), antibacterial (Iinuma et al., 1996b; Negi et al., 2008; Sundaram et al., 1983), antifungal (Kaomongkolgit et al., 2009), antioxidant (Jung et al., 2006; Márquez-Valadez et al., 2009), antiparasitic (Obolskiy et al., 2009) and can also act as well as anti-obesity (Jiang et al., 2010; Quan et al., 2012) agents. Therefore, the purpose of this study is to investigate the safety of the pure compound of AM and to evaluate its effect on the extent of tissue damages in mouse liver and kidney.

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Figure 1 Cratoxylum arborescens (Guttiferae); (A) the appearance of overall tree. (B) The flowers and leaves.

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Figure 2 Chemical structure of AM.

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2 Materials and methods

3 Results

3.1

3.1 General observations

Ingestion of AM at any tested dose did not cause mortality in mice. There was no abnormal behavior or physical appearance among these animals. In all cases, their feces were dry and dark. The average of daily food and water intake among the groups were similar throughout this study. On the other hand, this natural compound did not show any cytotoxic activities against normal liver cells (WRL-68) (Fig. 3).

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Figure 3 Effect of the AM on WRL-68 cell lines determined by MTT assay. Cells treated with the compound at different concentration (IC₅₀ 65 μg/ml).

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3.4

3.4 Histopathological evaluation

At autopsy, macroscopic examinations of the liver and kidney of the mice did not show any abnormality in gross appearance and weight as a result of the compound consumption (data not shown). In addition, we did not detect any damage in their gastrointestinal tracts, the potential and direct target for toxic effects of ingested foods. Results from the gross examination were also confirmed by histopathological assessment. AM did not produce any significant histological changes in the organ tissues of any of the animals (Fig. 4). Hence, no necrosis, inflammation or cirrhosis was observed.

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Figure 4 Effect of AM on histological sections of the liver and kidney in mice. (1A and B) Normal control. (1C and D) Mice treated with 100 mg/kg of AM. (1E and F) Mice treated with 1000 mg/kg of AM. There is no significant difference in the architecture of the livers and kidneys between the treated and control groups (Hematoxylin and Eosin stain, 20× magnifications).

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4 Discussion

AM, as a natural compound, possesses various biological properties (Pedraza-Chaverri et al., 2008). However, despite several evidences of its pharmacological properties and therapeutic benefits, there have been no reported toxicological evaluations performed on this compound in vivo.

In vitro cytotoxicity assay can be used to predict the toxicity of chemical substances (van der Laan et al., 2012; West et al., 2010). Different results have previously been reported, depending on the test agent used and the cytotoxicity assay employed (Weyermann et al., 2005). MTT assay, being the most sensitive in detecting cytotoxic event and causing a cytotoxicity assay based on mitochondrial respiratory activity, would give early signs of toxicity following exposure to a mitochondrial toxicant (Fotakis and Timbrell, 2006).

Cell cultures derived from humans and animals have been used during the past 30 years in determining the cytotoxic effect caused by natural compounds. Permanent cell lines and primary cells have been used as models (Sabaliauskas et al., 2011).

Our study revealed that based on MTT assay, AM low toxic effect to normal liver cells (WRL-68) in a dose-dependent manner with high IC₅₀ value 65 μg/ml (Fig. 3) compared to cisplatin (Sigma Aldrich), a known cytotoxic agent as a positive control against WRL-68 with low IC₅₀ value 5.1 μg/ml (Abdelwahab et al., 2013).

Toxicologically, an acute toxicity investigation is the initial step toward toxicological analysis of chemical or pharmaceutical materials (Andersen and Krewski, 2009).

In relation to this, the Environmental Protection Agency Health Effects Test Guidelines, OPPTS 870.3050 (2000) and OECD Guidelines for Testing of Chemicals, Section 407 (1998) as well as the United States have prioritized rats as the standard rodent species. The mouse has been one of the main mammalian species used for preclinical purposes, ranging from pharmacology to safety assessments. The use of rodents as models in safety evaluations is currently required in both pharmaceutical and chemical international guidelines (Suckow et al., 2012). The Food and Drug Administration Guidelines for Toxicological Principles for the Safety Assessment of Food Ingredients, Redbook 2000, Chapter IV.C.3.a (Redbook, 2000) elucidated that short term toxicity studies are conducted with rats and mice. Thus, both male and female mice that are healthy and have not been assigned to previous experimental procedures ought to be utilized in acute toxicity studies.

Results of the present study not only provide scientific evidences to evaluate the safety of AM using an acute toxicity study design in mice; they are also useful in determining the dose range of this pure compound for subsequent studies on mice, such as carcinogenicity, antioxidant, antimicrobial and anti-parasitic studies. Moreover, this is the first time that AM has been investigated in an acute oral toxicity in mice. In addition, the advantages of using mice for research purposes are numerous. Firstly, in spite of their clear physical differences, mice and human genes are approximately 99% identical. In a biological context, genes in the mouse and human function in virtually the same way. Secondly, the genome of mouse is easily manipulated through various genetic engineering technologies for different experiments. Thirdly, mice are relatively small in size and their ease of maintenance reduces research costs. Fourthly, their accelerated lifespan (1 mouse year = 30 human years) allows all life stages to be studied. Fifthly, their short gestation time (3 weeks) and large litter size quickly provide a large sample population and enable rapid genetic and pathophysiologic characterizations. Finally, they can be easily handled with practice (Suckow et al., 2012). Thus, the mouse is an ideal model organism for various experiments.

The exposure of animals to chemicals toxicologically is divided into four categories: acute, subacute, subchronic and chronic. Acute toxicity is known as the toxicity produced by a pharmaceutical when it is administered in one or more doses during a period not exceeding 24 h. In the case of rodents, they should be monitored for 14 days after pharmaceutical administration. All clinical signs, mortalities, time of onset, duration, and reversibility of toxicity should be recorded. Gross necropsies should be performed on all animals, including those that were sacrificed moribund, found dead, or terminated at 14 days (Bidlack, 2002). In the present study, ICR mice of both genders were administered oral gavage with AM suspension at single doses of 100, 500 and 1000 mg kg for 14 days. Throughout the study, treatment with AM did not produce any mortality or toxicity in the mice regardless of gender. No treatment-related adverse effects were found on body weight, organ weight, food and water consumption, serum biochemistry and oxidative stress status biomarkers.

Prior to implementing our study design, an analysis done on previous research papers on AM studies in an acute oral toxicity had found a valuable body of literature reported by Hutadilok-Towatana et al. (2010). They reported that mice treated with a single intragastric dose of 2 and 5 g/kg body weight of hydroethanolic extracts from the fruit pericarp of mangosteen upon acute toxicity for 14 days did not show any mortality or toxicity. Moreover, for the purpose of subchronic toxicity study, the mangosteen extracts at 400, 600 and 1200 mg/kg body weight were administered by oral gavage to male and female rats daily for 12 weeks. In all instances, consumption of the extract showed no effect on behavior, food and water intake, growth or health status of these animals. In both sexes, hematology values monitored throughout the study period did not alter from those of the control. After the 12-week period, no significant dose-related differences in blood biochemical parameters were detected among the female groups, whereas in all male groups, dose-variation increases were observed in direct bilirubin compared with the control. However, neither gross necropsy nor histopathological examination of their livers as well as other internal organs revealed any abnormal appearances. However, AM in the present study was isolated from the stem bark of C. arborescens. Therefore, the literature reported by Hutadilok-Towatana et al. (2010) has already included a good in vivo toxicological design; nevertheless, there is a difference in objective between the literature and our study due to the use of hall extract rather than the pure AM compound.

Single doses effect of AM on the kidney and liver function was carried out to evaluate the nephrotoxicity and hepatotoxicity of this compound on both male and female mice. In the evaluation, the kidney and liver cells were damaged, and creatinine and blood urea nitrogen together with ALT, ALP and GGT enzymes leaked out, causing the blood levels of these chemical components and enzymes to rise. Increased levels of these chemical components and enzymes in blood are considered good indicators for kidney and liver cell damage. In this present study, our results showed no significant changes in the levels of serum electrolytes, total protein, albumin, globulin, creatinine and BUN as well as conjugated bilirubin, total bilirubin ALT, AST and ALP.

In addition, our findings also indicated that there were no nephrocellular and hepatocellular injuries in mice gavage with 100, 500 and 1000 mg/kg AM. This was later confirmed by histological examinations of tissue sections using H&E staining of light microscope and MDA levels in the kidney and liver tissues of the animals. Results from this study suggested that single oral doses of AM at 100–1000 mg/kg have no ill effect on the kidney and liver tissues of both male and female ICR mice.