ORIGINAL RESEARCH published: 09 August 2016 doi: 10.3389/fphar.2016.00246 Sub-Acute Toxicity Study of Tiger Milk Mushroom Lignosus tigris Chon S. Tan Cultivar E Sclerotium in Sprague Dawley Rats Boon-Hong Kong 1, Nget-Hong Tan 1, Shin-Yee Fung 1* and Jayalakshmi Pailoor 2 1 Medicinal Mushroom Research Group, Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, 2 Department of Pathology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Lignosus also known as “Tiger Milk Mushroom,” is classified in the family Polyporaceae and mainly consumed for its medicinal properties in Southeast Asia and China. The sclerotium is known as the part with medicinal value and often used by the natives to treat a variety of ailments. Lignosus tigris Chon S. Tan, one of the species of the Malaysia Tiger Milk mushroom, has recently been successfully cultivated in laboratory. Earlier studies have demonstrated the L. tigris cultivar E sclerotia exhibited beneficial biomedicinal properties. This study evaluated the potential toxicity of L. tigris E sclerotia Edited by: Adolfo Andrade-Cetto, in a 28-day sub-acute oral administration in Sprague Dawley (SD) rats. L. tigris E sclerotial National Autonomous University of powder was administered orally at three different doses of 250, 500, and 1000 mg/kg to Mexico, Mexico the SD rats once daily, consecutively for 28-days. Body weight of the rats was recorded Reviewed by: and general behavior, adverse effects, and mortality were observed daily throughout the Benedict Green, United States Department of experimental period. At the end of the experiment, blood hematology and biochemistry, Agriculture-Agricultural Research relative organ weights, and histopathological analysis were performed. Results showed Service, USA Manoj Gajanan Kulkarni, that there were no mortality nor signs of toxicity throughout the 28-day sub-acute University of KwaZulu-Natal, South toxicity study. Oral administration of the L. tigris E sclerotial powder at daily dose up Africa to 1000 mg/kg had no significant effects in body weight, relative organ weight, blood *Correspondence: hematological and biochemistry, gross pathology, and histopathology of the organs. Shin-Yee Fung [email protected] L. tigris E sclerotial powder did not cause any treatment-related adverse effect in the rats at different treatment dosages up to 1000 mg/kg. As the lethal dose for the rats Specialty section: is above 1000 mg/kg, the no-observed-adverse-effect level (NOAEL) dose is more than This article was submitted to Ethnopharmacology, 1000 mg/kg. a section of the journal Keywords: Lignosus tigris, tiger milk mushroom, sclerotium, sub-acute toxicity, Sprague-Dawley rats Frontiers in Pharmacology Received: 09 June 2016 Accepted: 25 July 2016 INTRODUCTION Published: 09 August 2016 Citation: Lignosus spp. belonging to the Polyporaceae family has been long valued for its medicinal and Kong B-H, Tan N-H, Fung S-Y and functional properties. The species of the Lignosus mushroom including L. dimiticus Ryvarden, L. Pailoor J (2016) Sub-Acute Toxicity ekombitii Douanla-Meli, L. goetzii (Henn.) Ryvarden, L. hainanensis B. K. Cui, L. sacer (Afzel. ex Study of Tiger Milk Mushroom Lignosus tigris Chon S. Tan Cultivar E Fr.) Ryvarden and L. rhinocerotis (Cooke) Ryvarden. L. rhinocerotis (commonly known as the Tiger Sclerotium in Sprague Dawley Rats. Milk Mushroom) is utilized by the local communities in Southeast Asia and China as traditional Front. Pharmacol. 7:246. medicine. The sclerotium is the part with medicinal values which is used as general tonic and doi: 10.3389/fphar.2016.00246 natural remedy for various ailments. Frontiers in Pharmacology | www.frontiersin.org 1 August 2016 | Volume 7 | Article 246 Kong et al. Sub-Acute Toxicity of Lignosus tigris In Peninsular Malaysia, the mushroom sclerotia was utilized E sclerotial powder did not exhibit acute toxicity at the dose of by Orang Asli (Semai, Temuan, and Jakun) for treating variety 2000 mg/kg. In this study, we examined the sub-acute toxicity of illnesses including coughs, asthma, food poisoning, breast of the L. tigris cultivar E to investigate the potential toxicity swelling, arthritis, edema, liver diseases, and cancer (Lee et al., of repeated exposure of the mushroom sclerotial powder in 2009; Ismail, 2010). Local Malay and Chinese traditional Sprague-Dawley rats. practitioners have used it to treat leukemia, cervical cancer, intestinal ulcer disease, kidney disease, body swelling, fever, asthma, and consumed as general tonic (Tan, 2009). In Hong MATERIALS AND METHODS Kong and China, the mushroom sclerotial is a costly traditional medicine utilized for the treatment of gastric ulcers and liver- Cultivation and Preparation of L. tigris E related diseases, such as hepatitis and cancer (Wong and Cheung, Sclerotial Powder 2009). Recent scientific studies have proved that L. rhinocerotis Wild type of L. tigris Chon S. Tan specimen was collected from a ◦ ′ ◦ ′ has a wide spectrum of bioactive properties. An in vitro study tropical forest in Lata Iskandar (4 17.46 N 101 34.41 E), Pahang, on the antioxidant activity revealed that the mushroom sclerotial Malaysia (Tan et al., 2013). The fungus identity was verified extracts exhibited potent antioxidant activity (Yap et al., 2013). by PCR amplification coupled with sequence analysis of the Lee et al. (2012) demonstrated that the cold water extract of internal transcribed spacer (ITS) region (Yap et al., 2014b). Live L. rhinocerotis sclerotium exhibited significant anti-proliferative culture of the fungus was prepared inoculating a small portion activity against human breast (MCF-7), and lung (A549) (50 mg) of clean sclerotial tissue in potato dextrose agar (PDA) adenocarcinoma cells. The polysaccharides of the L. rhinocerotis supplemented with 100 µg/L of streptomycin. Pure and healthy sclerotium were reported to have immunomodulatory effect by mycelium culture (200 mg) was then inoculated in sterilized 100g activating human innate immune cells (Wong et al., 2011). The of brown rice with 100 ml of distilled water in a polypropylene sclerotial extracts of the L. rhinocerotis have also been reported container. The inoculated brown rice was incubated in dark at ◦ to have acute anti-inflammatory activity and antimicrobial 24 C for 3 months. The sclerotia grown from the surface of rice properties against bacteria (Mohanarji et al., 2012; Lee et al., media were then harvested and freeze dried. The freeze dried 2014). It also has the ability to enhance neurite outgrowth in sclerotia were ground into powder using 0.2 µm mesh filter. The rat adrenal pheochromocytoma (PC-12) cells (Eik et al., 2012). nutritional and amino acid compositions of the sclerotial powder Owing to its wide ethnomedicinal usages and supportive data have been analyzed in our previous study. The sclerotium is rich on its medicinal properties, L. rhinocerotis is potentially to be in carbohydrate, protein, dietary fibers, minerals and contains developed as health supplement. Toxicity assessments including considerable amount of essential amino acids and bioactive sub-acute, chronic, fertility, teratogenic, and mutagenic studies components, such as phenolics, terpenoids, and glucan (Kong on L. rhinocerotis sclerotial powder all indicated it is safe for et al., 2016). human consumption (Lee et al., 2011, 2013). Recently, a new species of Lignosus mushroom collected Experimental Animals from the tropical forest in the state of Pahang, Malaysia has A total of 80 (40 males and 40 females) Sprague-Dawley been identified and named Lignosus tigris Chon S. Tan (Tan (SD) rats aged 7–8 weeks old were obtained from the Animal et al., 2013). The sclerotia of this mushroom have also been Experimental Unit, University of Malaya. The experimental rats used as traditional medicine by the local community. It is not were housed under the conditions with a 12 h of light/dark distinguished from L. rhinocerotis due to its similarity in gross ◦ cycle and temperature adjusted between 22 and 25 C with morphologies (Tan et al., 2013). Yap et al. (2014a) revealed that 60 ± 10% of humidity. Rats were given free access to standard the wild type L. tigris possess good nutritive value and exhibited sterile rodent diet (Altromin #1324 Rodent Maintenance Diet, potent antioxidant activity. Recently, L. tigris mushroom has been Denmark) and water. The experimental procedures were successfully cultivated by Ligno Biotech (Malaysia). approved by the Institutional Animal Care and Use Committee, In our earlier study, the sclerotia of the L. tigris cultivar University of Malaya (UM IACUC—Ethics reference no. 2013- E has been shown to have high carbohydrate, protein and 09-17/MOL/R/TNH). dietary fiber content (Kong et al., 2016). The sclerotia also contain high amount of potassium which is an essential electrolytes to maintain body function. Substantial amount of Dose Formulation and Administration essential amino acids was found in the sclerotial protein. It The sub-acute toxicity study was performed according to also possesses potent superoxide anion scavenging activity. The the guidelines of Organization for Economic Co-operation mushroom’s sclerotial extracts show high contents of glucan and Development (OECD) for testing of chemicals, TG 407 and terpenoid, compounds which may exhibit anticancer and (adopted—3 October 2008; OECD, 2008). SD rats were assigned immunomodulatory properties (Huang et al., 2012; Kong et al., into four groups; a control group and 3 treatment groups (n = 10; 2016). The study suggested that L. tigris E sclerotia is a promising 5 males and 5 females). L. tigris E sclerotial powder was dissolved medicinal mushroom and may also be a good source of functional in distilled water and administered orally on a daily basis for 28- food and nutraceutical. Therefore, the toxicity data of the days at single doses of 250, 500, and 1000mg/kg with the volume mushroom is essential before it being developed as health not exceeding 1 ml/100 g. Rats in control group received only supplement. Acute toxicity assessment has indicated that L.