|Year : 2014 | Volume
| Issue : 3 | Page : 139-145
Toxicological assessment of Pleurotus ostreatus in Sprague Dawley rats
Krishnamoorthy Deepalakshmi, Sankaran Mirunalini
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Chidambaram, Tamil Nadu, India
|Date of Submission||03-Mar-2013|
|Date of Acceptance||23-Mar-2014|
|Date of Web Publication||16-May-2014|
Source of Support: UGC Major Research Project., Conflict of Interest: None
| Abstract|| |
Objective: To evaluate toxicological and histopathological assessment of Pleurotus ostreatus, an oyster mushroom in Sprague Dawley rats. Materials and Methods: Toxicity assessment was carried out by acute (72 h) and sub acute toxicity (28 days) studies and also its effects on hepatic marker enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT) and renal markers enzymes (urea, uric acid, and creatinine) in blood serum and liver histology of experimental Sprague Dawley rats was performed. Results: The studies indicated that the LD 50 value was found to be >5,000 mg/kg body weight (b.wt). The body weight and general behaviors of animals were observed throughout the experimental period and at end of the study, organ weight, biochemical parameters of blood (serum) as well as liver histology indicated that no toxic clinical symptoms or histopathological changes were observed in experimental Sprague Dawley rats. Conclusion: The above studies clearly indicated that the P. ostreatus extract had high margin of safety.
Keywords: Acute toxicity, hepatic markers, Pleurotus ostreatus, renal markers, subacute toxicity
|How to cite this article:|
Deepalakshmi K, Mirunalini S. Toxicological assessment of Pleurotus ostreatus in Sprague Dawley rats. Int J Nutr Pharmacol Neurol Dis 2014;4:139-45
|How to cite this URL:|
Deepalakshmi K, Mirunalini S. Toxicological assessment of Pleurotus ostreatus in Sprague Dawley rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2014 [cited 2020 Jan 18];4:139-45. Available from: http://www.ijnpnd.com/text.asp?2014/4/3/139/132665
| Introduction|| |
Mushrooms have received a remarkable interest in recent decades as functional foods and as source materials for drug development.  The major contributory factors to this growing interest include rising cost of orthodox medications, low therapeutic index of synthetic compounds, and the growing incidence of drug resistance among the pathogens especially in developing countries with very weak economic indices.  Moreover, the active principles from natural sources have contributed significantly to the development of new drugs from herbal plants and mushrooms for the treatment of various diseases. , Unfortunately, there is limited scientific evidence regarding safety and efficacy to back up the continued therapeutic application of these remedies. The rationale for their utilization has rested largely on long-term clinical experience.  Conversely, worldwide revolution for the improvement of patient safety is gaining momentum; hence, the drug safety for the subject become even more prominent in the present day scenario.  Therefore, closely associated with screening of plant extracts for their activities against microorganism or disease conditions is the need to know their toxic potentials. 
Macrofungi have been used as flavorful foods and as health nutritional supplements since Greek and Roman days.  Basidiomycetes have been widely studied over the past 30 years in terms of their polysaccharide composition and therapeutic applications.  Among the various edible species Pleurotus ostreatus is now rank second among the mushroom consumptioners in the world.  Generally, the P. ostreatus contains approximately 100 of different bioactive compounds of each having their own outstanding medical effects.  Furthermore, public is enjoying widespread use of mushroom for treatment of several ailments, but still little is known about their toxicity and safety issue which are always a concern. Hence, evaluation of toxic properties of a substance is crucial when considering for public health protection because exposure to chemicals can be hazardous and results to adverse effects of human being. The toxicity assessments includes acute, subacute, and chronic effects. 
Thus, the present study aims to determine the toxicity assessments of ethanolic extract P. ostreatus using an acute oral toxicity test in animal models. The acute and subacute toxicity testing was carried out on animals based on the Organization for Economic Cooperation and Development (OECD) guidelines. 
| Materials and methods|| |
Chemicals and acids were of certified analytical grade and purchased from S D Fine Chemicals, Mumbai or HiMedia Laboratories Pvt Ltd, Mumbai, India.
P. ostreatus mushrooms were collected in and around areas of Udhagamandalam, Nilgiri district, Tamil Nadu. The plant was taxonomically identified and authenticated by Dr V Venkatesalu, Associate Professor, Department of Botany, Annamalai University. A voucher specimen (no: 233) was deposited in the Herbarium of Botany, Department of Botany, Annamalai University.
The fresh fruiting bodies of P. ostreatus were dried in shade conditions and the dried materials were pulverized in a blender to get coarse powder. For P. ostreatus fruiting bodies' ethanolic extraction, 5 g of the powder was extracted with 100 mL of 95% ethanol using a Soxhlet apparatus. The solvent was evaporated under reduced pressure and controlled temperature (40-50°C). The ethanolic extracts were redissolved in ethanol for the antioxidant activity.  A dark, semisolid material (yield 6 g) obtained was stored at 4°C until use. A known amount of the residual extracts were suspended in distilled water and was orally administrated to the animals by gastric intubation.
Animals and diet
Six-weeks-old, female Sprague Dawley rats, weighing approximately 130 -150 g were obtained from National Institute of Nutrition, Hyderabad and maintained in the Central Animal House, Rajah Muthiah Medical College and Hospital, Annamalai University. All the rats were acclimatized for a week under standard husbandry conditions. The rats were housed in polypropylene cages (45 × 24 × 15 cm), maintained at room temperature (27 ± 2°C), in 12 h light/12 h dark conditions. The animals were fed on a standard pellet diet (Amrut Laboratory Animal Feed, Mysore Feed Limited, Bangalore, India) and water ad libitum was available to the animals throughout the experimental period and replenished daily. The standard pellet diet comprised of 21% protein, 5% lipids, 4% crude fiber, 8% ash, 1% calcium, 0.6% phosphorous, 3.4% glucose, 2% vitamin, and 55% nitrogen free extract (carbohydrate) and it provides metabolizable energy of 3,600 kcal/kg.
Animal handling and experimental procedure were approved by the Institutional Animal Ethical Committee of Rajah Muthiah Medical College (Reg. No: 160/1999, Proposal number: 947 CPCSEA), and the experiments were performed in accordance with the "Guide for the case and use of laboratory animal" (National Institutes of Health (NIH), 1985) and committee for the purpose of control and supervision on experimental animals (Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)).
| Experimental Design|| |
Acute toxicity study
The acute toxicity of P. ostreatus was evaluated in rats using the up and down procedure of OECD guidelines - 423 (adopted - December, 2001) with minor modifications (OECD, 2000). In accordance with the limit test, single dose of P. ostreatus (5,000 mg/kg body weight (b.wt)) was orally administered to three female Sprague Dawley rats through gastric intubation. The animals were observed continuously for 72 h for any signs of behavioral changes and mortality.
Subacute toxicity study
Female Sprague Dawley rates of (130-150 g) were divided into five groups of six animals each and were housed under the same conditions as described above. The ethanolic extract of P. ostreatus was administrated for 28 days at doses of 250, 500, 750, and 1,000 mg/kg b.wt, respectively. The control animals received 0.5 mL of the vehicle alone. Toxic manifestations and mortality were monitored daily till the end of the experimental period; all the rats were kept overnight fasting and anesthetized using ketamine chloride (24 mg/kg b.wt) by intramuscular injection and sacrificed by cervical decapitation between 8.00 am to 10.00 am. Blood was collected in clean dry test tube and serum were used for various biochemical estimations. Liver tissues were removed, cleared off blood, and immediately transferred to ice-cold containers containing 0.9% NaCl. The tissues were homogenized in an appropriated buffer and used for the estimation of various biochemical parameters.
The activity of both serum aspartate aminotranferase (AST) and alanine aminotransferase (ALT) were assayed by using the diagnostic kit based on the method of Reitman and Frankel, 1957.  Serum alkaline phosphatase (ALP) was estimated using Kind and King, 1954.  The serum gamma-glutamyl transferase (GGT) was assayed according to the method of Rosalki and Rau 1972.  Estimation of renal functional markers such as urea, uric acid, and creatinine were determined by Fawcett and Scott 1960, Caraway 1955, and Jeffe 1886. ,,
For histopathological study, three rats from each group were perfused with physiological saline, followed by formalin (10% formaldehyde). The liver tissues were excised immediately and fixed in 10% formalin. The liver tissues were sliced and embedded in paraffin wax, 3-5 μm thick sections were cut in a rotary microtome and were stained with hematoxylin and eosin. The specimens were evaluated with a light microscope. All histopathological changes were examined by the pathologist.
Statistical analysis was performed using Statistical Package for Social Sciences (SPSS) software, version 11.5. The values were analyzed by one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT). All these results were expressed as mean ± standard deviation (SD) for six rats in each group: P < 0.05 were considered as significant.
| Results|| |
The body weight and food and water consumption of rats were found to be unaffected by the treatment of P. ostreatus extract. No mortality and no significant changes in general behavior of rats were observed to the maximum dose level of 5,000 mg/kg b.wt of orally administered P. ostreatus for 72 h treatment. Hence, the LD50 was estimated to be > 5,000 mg/kg b.wt.
In order to evaluate the adverse effect of repeated daily exposure of P. ostreatus, subacute toxicity study was carried out. To determine dose-related toxic effects, doses of 250, 500, 750 and 1,000 mg/kg b.wt of P. ostreatus extract were administered for the experimental duration of 28 days. P. ostreatus extract at the different doses did not produce any significant changes in animals, as evidenced by the absence of toxic syndromes without any changes in water/food ingestion and general behaviors.
Effect of P. ostreatus on body weight and organ weight changes
The body and organ weight of control and experimental rats were shown in [Table 1]. Oral administration of P. ostreatus was found to change the body weights of all the rats, but this was not statistically significant. The observed weight gain in the P. ostreatus treated animals shown that the administrated P. ostreatus does not have any untoward action that affect the growth of the animals. Moreover, morphological observation in vital organ like liver indicated that there was no sign of any inflammation or toxicity in both control as well as in the P. ostreatus treated groups. Thus, the P. ostreatus treated group revealed no significant difference in body weight or weight of liver organ. The present result clearly showed that the P. ostreatus does not produce any toxicological effects on the body and organ weight.
Effect of P. ostreatus on biochemical analysis
The activities of hepatic marker enzymes in control and experimental groups were shown in [Table 2]. However, no such deleterious changes were found in serum of liver marker enzymes (ALT, AST, ALP, and GGT) of P. ostreatus treated groups when compared with the control rats. The current results clearly indicated that treatment with P. ostreatus did not induce any harmful biochemical effects on the animals [Table 3] showed the effect of P. ostreatus on renal functional markers. The level of serum urea, uric acid, and creatinine of P. ostreatus treated rats showed no significance changes like control rats.
|Table 1: Effect of Pleurotus ostreatus on body weight and liver weight to body weight ratio of control and experimental animals |
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|Table 2: Effect of Pleurotus ostreatus on hepatic marker enzymes in serum of control and experimental animals |
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|Table 3: Effect of Pleurotus ostreatus on renal marker enzymes in serum of control and experimental animals |
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Effect of P. ostreatus on histopathology
Histopathological evaluation was carried out to characterize the biological response factors. [Figure 1] shows the histopathological examination of liver tissue. The assessment of histopathology of liver showed normal architecture implied no detrimental changes or morphological alterations in control and P. ostreatus treated animals. This indicates that P. ostreatus did not exert any toxic effect on the animals.
|Figure 1: Histopathological evaluation of liver tissue in control and experimental animals of Pleurotus ostreatus subacute toxicity study. (a) Control animals, (b) P. ostreatus treated with 250 mg/kg body weight (b.wt)), (c) P. ostreatus treated with 500 mg/kg b.wt, (d) P. ostreatus treated with 750 mg/kg b.wt, (e) P. ostreatus treated with 1,000 mg/kg b.wt. All groups showing normal lobular architecture with central vein and radiating hepatic cords. No other histological changes were noted in all groups of specimens|
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| Discussion|| |
Phytotherapeutic products from medicinal plants have become universally popular, particularly in developing countries, and some have been mistakenly regarded as safe just because they are a natural source. However, there is a lack of proven scientific studies on the toxicity and adverse effect of these remedies.  An edible mushroom P. ostreatus has long been known to be endowed with beneficial and diverse activity. Therefore, the present study was carried out to elucidate the acute and subacute toxicity profiles, including histological evaluation of P. ostreatus in female Sprague Dawley rats. The toxicity studies are useful parameter to investigate the therapeutic index of drug and xenobiotics.  At present, the following chemical labeling and classification of acute systemic toxicity based on oral LD 50 values are recommended by the OECD (Paris, France): Very toxic, ≤5 mg/kg; toxic, >5 and ≤50 mg/kg; harmful, >50 and ≤500 mg/kg; and no label, >500 and ≤2,000 mg/kg.  According to the OECD guidelines, the P. ostreatus extract appraisal no adverse effects were observed in female animals administrated with up to 5,000 mg/kg b.wt. This demonstrating the safety median acute toxicity value (LD 50 ) was estimated to be greater than 5,000 mg/kg b.wt, respectively.  Earlier reports have shown that if the median lethal dose of a test substance is three times more than the minimum effective dose, the substance is considered as good aspirant for the further studies.  Therefore, P. ostreatus extract indicate that it does not cause any toxicity and safe for oral use for the management of several diseases.
Nevertheless such acute toxicity data are of limited clinical application since cumulative toxic effects do occur even at very low doses. Hence, subacute and chronic toxicity studies are almost always invaluable in evaluating the safety profile of phytomedicine.  This probably explains why some authors have suggested that subchronic toxicity data are inevitable to predict the hazard of long-term, low-dose exposure to a particular compound. 
In subacute toxicity studies, the body weight and internal organ weight changes serve as an indicator of adverse side effects since animals that survive cannot lose more than 10% of the initial body weight.  In general, toxic nature of the drug leads to abnormalities in body weight.  Nevertheless, the evaluation of the chronic toxicity at doses of 250, 500, 750, and 1,000 mg/kg b.wt of P. ostreatus ethanolic extract showed increased in body weight. Moreover, the increased in body weight was not significantly different from that of the control. Hence, we could substantiate that the P. ostreatus ethanolic extract indicate the improvement in nutritional state of animals. Earlier reports suggest that the P. ostreatus had a rich content of protein and the superior quality of this mushroom may be because of this genus contain complete proteins with the well distribution of essential amino acids, as well as nonessential amino acids, this might be the key factor for the improved body weight of the rats.  The growth response effect could be a result of increased food and water intake.  Organ weight also is an important index of physiological and pathological status in animals. The relative organ weight was fundamental to diagnose whether the organ was exposed to the injury or not.  However, the observed results in vital organ-like liver indicated that there were no sign of any inflammation or no significant differences in organ weight in both control as well as in P. ostreatus extract treated animals. Hence, it can be suggested that P. ostreatus ethanolic extract is virtually nontoxic.
Generally, hepatic cells take part in a variety of metabolic actions and restrain a host of enzymes. The biological role of transaminase (AST and ALT), ALP, and GGT concerned with the interconversion of highly important metabolite. The enzyme serves as an index of liver cell injury.  However, any elevation pertaining to these enzymes indicate their outflow into the blood stream due to damage in liver parenchymal cells. Thus, liver cell damage is characterized by a rise in plasma enzymes (AST, ALT, ALP, and GGT).  AST is a liver function test (LFT) and is used to monitor damage to liver parenchymal cells. Elevated level of AST is a sign of serious liver damage. ALT is another enzymes associated with liver parenchymal cells. ALT is more specific indicator of liver damage than the AST, as the AST may also be elevated in diseases affecting other organs. ALT and AST is commonly measured clinically as a part of diagnostic LFT, to determine liver health. 
ALP is a hydrolase enzyme that eliminated in the bile. It hydrolyses monophosphate at an alkaline pH. ALP is particularly present in cells, which line the biliary ducts of the liver. Generally, heptotoxicity leads to elevation of normal values due to the body's inability to excrete it through bile due to the congestion or obstruction of the biliary tract.  Upgrade in level of ALP with little or no increase in ALT is primarily a biomarker of hepatobiliary effects and cholestasis.  GGT or transpeptidase (GGTP) is an enzyme which is found in liver, kidney, and pancreatic tissues, the enzyme concentration being low in liver as compared to kidney.  It catalyzes transfer of γ-glutamyl groups to amino acids and short peptides. It is more useful clinically when compared to ALP. ALP is more sensitive, but much less specific than GGT. The comparison of the two enzymes helps in determining the occurrence of bone or liver injury. Hence, GGT is a specific indicator of bile duct lesions in rat liver. 
Owing to our results, there were no significant differences in the serum AST, ALT, ALP, and GGT levels, which reveal that P. ostreatus did not affect liver functions or metabolism. It is now established that excess lipid accumulation in the liver cause fatty changes and ultimately contains stains such as lovastatin, which works to reduce cholesterol. In addition, isolated β-glucan from P. ostreatus lowered the serum cholesterol concentration in hypercholesterolemic rats.  Thus, from the above finding it is evident that usage of P. ostreatus did not induce any harmful biochemical effects on the animals.
Kidney is an important organ actively involved in maintaining homeostasis of the body by reabsorbing important material and excreting waste products.  Furthermore, kidney functional markers such as urea, uric acid, and creatinine are the main indicators of renal dysfunction. Indeed, creatinine is known as a good indicator of renal function. Any rise in creatine levels observation is only which marked damage to functional nephrons.  In our study, there was an insignificant difference in urea, uric acid, and creatine levels between the treated and the control group probably indicate that the extract did not interfere with the renal capacity to excrete the metabolites. Moreover, the above study is in line with Jaganathan et al., that the Tridham, a siddha medicine does not induce any harmful and adverse effects on the biochemical parameters of Wistar albino rats. 
In general, the histopathology analysis collaborated with the results of body weight and organ weight. The P. ostreatus ethanolic extract did not cause toxicity towards the organs as there was no structural damage to the organs of liver, kidney, and lungs of the rat. The liver is the main target organ of acute toxicity, which was exposed to foreign substances, being absorbed in intestines, and metabolized to other compounds which may or may not be hepatotoxic to the rats.  In our study, the liver histopathology analysis showed the normal hepatocyte architecture and did not cause any alteration to the structure of the liver cells between the controls and treated. The above results were in line with the study by Akanmu et al., 2004 revealed no necrosis, inflammatory reaction, fibrosis, or local fatty degradation observed in liver and arrangements of cell structure almost similar to that of the rats in control groups.  The observation of the current study portrays that the oral administration of the ethanolic extract of P. ostreatus did not cause any transience nor altered the biochemical and histopathological indices was not harmful at the level tested and can be safely used as a therapeutic agents.
| Conclusion|| |
In conclusion, the present investigation demonstrates the nontoxic nature of the ethanolic extract of P. ostreatus was evident from the acute and subacute toxicity assessments conducted as per OECD guidelines. Based on 28 days repeated dose toxicity study suggests the P. ostreatus as relatively safe, as it did not cause either mortality or produce severe toxicological effects on selected body organs, biochemical indices, and histological markers of rats. Consequently, P. ostreatus could be safe up to the dose of 5,000 mg/kg b.wt of the animals. Owing to this scientific appraisal, it can be concluded that the ethanolic extracts of the P. ostreatus had a high margin of safety as it did not induce any toxicological effects.
| Acknowledgments|| |
The authors would like to acknowledge for the Financial Support from UGC Major Research Project (F. NO: G7/17342/2012 (SR)) to carry out the work successfully.
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[Table 1], [Table 2], [Table 3]