Mushroom materials
The X. nigripes materials were produced by solid-state culture system. The authenticity of X. nigripes species was confirmed by Prof. Airong Song, Qingdao Agricultural University (Shandong, China), and its culture specimen (no. KJ-XN-07-1) was deposited at Kang Jian Biotech Corp., Ltd. (Nantou County, Taiwan), whereas its ribosomal RNA/internal transcribed spacer sequences were deposited in the National Center for Biotechnology Information GenBank database (no. KJ627786).
Preparation of X. nigripes standardized extract
The commercial X. nigripes extract composing of fungal mycelium and fruiting bodies (Kang Jian Biotech Corp., Ltd.) was prepared by boiling water at 95 °C for 2 h. The decoction was filtered and concentrated under vacuum to produce a thick concentrated extract, which was subjected to spray-drying to obtain the dried powder, followed by passing through a 60-mesh sieve to obtain a final powdered product (XNE); this standardized extract contained about 5.5% of water soluble β-linked polysaccharides with molecular weight greater than 10 kDa, and composing of 86.6% glucose, 6.8% mannose and 6.6% galactose.
Animals
Three-week-old male and female Sprague-Dawley rats were purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan), they were maintained under standard laboratory conditions (12 h light/dark cycle, a temperature of 22 ± 2 °C and a relative humidity of 55 ± 5%) with free access to standard pellet food (Oriental Yeast Co., Ltd., Tokyo, Japan) and water. Ethical approval for use of animals was obtained from the Institutional Animal Ethical Committee, with the protocol approval number 105–59. The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, MD, USA, 1996).
Experimental design
The subchronic toxicity study was carried out according to the protocol described by the OECD guideline 408 for testing chemicals [20]. Rats were allowed to acclimatize to the laboratory conditions and the gavage procedures for 15 days. Healthy animals were then selected, weighed and randomly divided into four groups, namely control group, low-, medium- and high-dose groups, with each group contained 20 animals (10 males and 10 females). Rats of the treatment groups were intragastrically (orally) administered with XNE at 20 mg/kg/day (recommended intake), 1000 mg/kg/day (50 times the recommended intake) and 2000 mg/kg/day (100 times the recommended intake) daily for 90 days, and the dosing volume was 5 mL/kg body weight; the control group received the same volume of distilled water (vehicle) for the same duration.
Visual observations for mortality, behavioral patterns, physical appearance and symptoms of illness for all animals were performed throughout the experimental period. Food intake was recorded daily, while body weights of the animal were measured every 3 days. The dose received by each animal was calculated based on the individual animal body weight, and adjusted according to the subsequent changes in body weight.
At the end of the experimental period, all rats were euthanized with carbon dioxide after overnight starvation (about 15 h), blood and organ samples were then collected for hematological and biochemical measurements, and histopathological examination.
Relative organ weight
Following blood collection, liver, kidney, heart, lungs, spleen, stomach, testicles, ovary, brain, and pancreas of all animals were carefully dissected free of connective tissue and fat, and then weighed and observed for abnormalities. The relative organ weight of each animal was calculated as follows: Relative organ weight = Absolute organ weight (g)/Body weight of the animal on sacrifice day (g) × 100.
Hematology and serum biochemistry
For the hematological investigation, whole blood was collected in ethylenediamine-tetraacetic acid (EDTA) tubes and processed immediately without any delay. The parameters measured were white blood cells (WBC), red blood cells (RBC), haemoglobin, lymphocytes, monocytes, haematocrit, mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), platelet count, neturophils, basophils, eosinophils, prothrombin time and activated partial thromboplastin time (APTT) using a hematology analyzer MEK-6318 K (Nihon Kohden Corp., Tokyo, Japan).
For biochemical assay, blood without anticoagulant were centrifuged at 3000×g at 5 °C for 15 min. Serum samples were then collected for measuring biochemical parameters comprising aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine, total protein, albumin, total bilirubin and glucose (GLU), as well as serum electrolytes such as sodium (Na), potassium (K), calcium (Ca), chloride (Cl) and phosphorous (P) using an automated biochemistry analyzer (Cobas Integra® 400 plus, Roche, Basel, Switzerland).
Histopathological studies
All organ and tissue samples were fixed in 10% neutral buffered formalin, dehydrated in graded ethanol, cleared in xylene, embedded in paraffin, and then sectioned at about 3–5 μm thickness, followed by staining with hematoxylin-eosin (H & E) dye. The microscopic features of the organs of male and female XNE-treated rats were compared with that of the control group.
The following tissues were examined microscopically: adrenal gland, brain, esophagus, bone femur (including marrow), cervix, heart, small intestine (duodenum, jejunum, and ileum), large intestine (cecum, colon, and rectum), kidney, liver, lung, lymph nodes (mesenteric), ovary, pancreas, pituitary gland, parathyroid gland, prostate gland, sciatic nerve, spinal cord, spleen, stomach, testicles, thymus, thyroid gland, trachea, urinary bladder and uterus.
Statistical analysis
All data are expressed as mean ± standard deviation (SD). Statistical significances between control and treated groups were determined by one-way analysis of variance (ANOVA), followed by post-hoc Duncanʼs multiple range tests. Difference was considered significant when P-value was < 0.05.