Preclinical toxicological evaluations of the sclerotium of Lignosus rhinocerus (Cooke), the Tiger Milk mushroom

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Abstract

Ethnopharmacological relevance

Lignosus rhinocerus (Tiger Milk mushroom) is distributed in South China, Thailand, Malaysia, Indonesia, Philippines and Papua New Guinea. In Malaysia, it is the most popular medicinal mushroom used by the indigenous communities to relieve fever, cough, asthma, cancer, food poisoning and as a general tonic. In China, this mushroom is an expensive traditional medicine used to treat liver cancer, chronic hepatitis and gastric ulcers. The sclerotium of the mushroom is the part with medicinal value. This rare mushroom has recently been successfully cultivated making it possible to be fully exploited for its medicinal and functional benefits. The present study was carried out to evaluate the chronic toxicity of the sclerotial powder of Lignosus rhinocerus cultivar (termed TM02), its anti-fertility and teratogenic effects as well as genotoxicity.

Materials and methods

Sprague Dawley rats (10 rats/group/sex) were fed orally with 250, 500 and 1000 mg/kg of sclerotial powder of TM02. The sclerotial powder was orally administered once daily and consecutively for 180 days. At the completion of the oral feeding period, analysis of hematological and clinical biochemical parameters, urine profiles, organ weight as well as histopathological analysis were carried out. The effect of the sclerotial powder on fertility and its possible teratogenicity were examined by feeding rats orally with 100 mg/kg sclerotial powder consecutively for 7–8 weeks. Genotoxicity was evaluated by Ames test using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA.

Results

The results showed that oral administration of the sclerotial powder of the Lignosus rhinocerus cultivar at daily dose of up to 1000 mg/kg for 180 days had no adverse effect on the general clinical observations, body weight, hematology, clinical biochemistry, urinalysis, absolute organ weight as well as relative organ weight, nor induced histological changes in the organs. Oral administration of 100 mg/kg sclerotial powder of the Lignosus rhinocerus for 7–8 weeks did not affect the fertility of the rats nor induce teratogenic effect on their offspring. Lignosus rhinocerus sclerotial powder up to 5000 μg/plate in the presence and absence of metabolic activation did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Conclusion

Our results showed that the no-observed-adverse-effect level (NOAEL) dose of the sclerotial powder of Lignosus rhinocerus in 180-day chronic toxicity study is more than 1000 mg/kg. Oral feeding of the sclerotial powder at 100 mg/kg did not induce adverse effect on rats' fertility nor causing teratogenic effect on their offspring. In the reverse mutation Ames test, the sclerotial powder at all tested concentration did not show any genotoxicity.

Introduction

Lignosus rhinocerus (Synonym Lignosus rhinocerotis (Cooke) Ryvarden), commonly known as Tiger Milk mushroom, belongs to the Polyporaceae family. Its geographical distribution is only in the tropical rainforest in the region of South China, Thailand, Malaysia, Indonesia, Philippines and Papua New Guinea (Tan et al., 2012). In Malaysia, it is also known as ‘Cendawan susu rimau’ and is the most popular medicinal mushroom used by the indigenous communities of Peninsular Malaysia to relieve fever, cough, asthma, cancer, food poisoning and as a general tonic (Lee et al., 2009). In China, the sclerotium of the mushroom is an expensive traditional medicine used for treatment of liver cancer, chronic hepatitis and gastric ulcers (Wong and Cheung, 2008). Phylogenetic analysis indicated that the mushroom is closely related to Ganoderma lucidum and Trametes versicolor, the two most popular medicinal mushrooms used in Asia (Tan et al., 2010).

The sclerotium of Lignosus rhinocerus has been demonstrated to exhibit anti-proliferative activity. Lai et al. (2008) demonstrated that sclerotial polysaccharides from Polyporus rhinocerus (synonym of Lignosus rhinocerus) exhibited antiproliferative effects on several kinds of leukemic cell lines. Wong et al. (2009) reported that the hot water extract of Polyporus rhinocerus exhibited immunomodulatory effect by stimulating human innate immune cells. Our studies demonstrated that the cold water extract of Lignosus rhinocerus sclerotium exhibited direct cytotoxicity on human breast carcinoma (MCF-7) and human lung carcinoma (A549) cell lines (Lee et al., 2012a). It has also been demonstrated that the sclerotial extracts of Lignosus rhinocerus exhibited anti-acute inflammatory activity in an animal model study (Lee et al., 2012b). Gao et al. (2009) showed that the non-digestible carbohydrates may function as novel prebiotics. Recently, aqueous sclerotial extract of Lignosus rhinocerus was reported to contain neuroactive compounds that stimulated neurite outgrowth in PC-12 cell line (Eik et al., 2012).

In view of its wide ethno-botanical usages as well as proven in vitro anti-proliferative and anti-inflammatory activities, Lignosus rhinocerus sclerotium may be used as a health supplement. It is therefore necessary to carry out in depth safety evaluation of the sclerotium. Earlier studies to evaluate the subacute toxicity of the sclerotial powder of Lignosus rhinocerus cultivar (termed TM02) demonstrated its no-observed-adverse-effect level (NOAEL) was higher than 1000 mg/kg in a 28 days animal studies using rats. In the present study, we carried out a 180 day chronic toxicity study of the sclerotial powder, as well as evaluating its possible anti-fertility and teratogenic effects and genetoxicity.

Section snippets

Preparation of Lignosus rhinocerus sclerotial powder

Sclerotial powder of the Lignosus rhinocerus cultivar TM02 was provided by Ligno Biotech Sdn. Bhd. (Selangor, Malaysia). The fungus was identified by internal transcribed spacer (ITS) regions of the ribosomal DNA (Tan et al., 2010). The sclerotial powder was freeze-dried and milled into powder using 0.2 mm sieve. The powder is light brown, dry fluffy powder with milk like taste.

Animals

Sprague Dawley (SD) rats aged 5 weeks old (male and female) were supplied by Chenur Supplier (Selangor, Malaysia). The

Body weight and general clinical observations

Oral administration of the sclerotial powder of Lignosus rhinocerus (cultivar TM02) at all the doses tested did not produce any treatment related abnormality in the rats, and there were no death observed. Fig. 1, Fig. 2 show the body weight of male and female rats treated with various doses of the sclerotial powder, and that of the control group for 180 days (26 weeks). The net body weight gain of the all the treated groups were not significantly different from the control animals (data not

Discussion

Changes in body weight have been used as an indicator of adverse effects of drugs and chemicals (Hilaly et al., 2004, Mukinda and Eagles, 2010). In the present studies, the similar growth pattern as shown by body weight (Fig. 1, Fig. 2) and body weight gain (data was not shown) for the treated and the control groups indicated that oral administration of sclerotial powder of Lignosus rhinocerus (TM02) at a daily dose of up to 1000 mg/kg and for 180 days had no adverse effect on the growth of the

Conclusions

Our results showed that there are no treatment-related chronic toxicity in rats of both sexes following the long term (180-days) oral administration of 250, 500 and 1000 mg/kg of Lignosus rhinocerus (TM02) sclerotial powder, as shown by the clinical observations, body weight gain, hematological analysis, clinical biochemistry, urinalysis, absolute organ weight as well as relative organ weight, and histological examinations of the organs. Thus, the no-observed-adverse-effect level (NOAEL) dose of

Acknowledgements

This research is supported by MOHE-UM HIR E20040-20001 and PV054-2011B from University of Malaya. The authors would like to express gratitude to staff from Laboratory Animal Center, Faculty of Medicine, University of Malaya for their assistance.

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