INTRODUCTION:

Ganoderma lucidum is a tough, woody Basidiomycetes mushroom widely reported to be of immense importance in trado-medicine and as a home remedy for various ailment among the ancient Chinese and other Asian countries where it is known as reishi, Ling Chih, Ling Zhi among others (Mau et al., 2002; Boh et al., 2007). The fruiting body and mycelium of the fungus contain protein, carbohydrates, fiber, vitamins, minerals in varying amount and according to the specie (Borchers et al. 1999; Orole, 2016), and it is readily available in tablets, extracts, injections, spore, and granule forms for consumption (Wang et al., 2007; Zhao et al., 2012).

Of late, researchers have continued to come up with different importance to which the mushroom could be put confirming the therapeutic respect early Chinese medical practitioners adduced to the fungus which resulted from the array of compounds confined within it.

G. lucidum is a lameless fungus containing alkaloids with capacity to stimulate the nervous systems, blood pressure, act as pain relievers, coupled with antimicrobial properties (Orole et al., 2016); saponins with capacity to lower cholesterol level in the blood and reduce the risk of getting intestinal cancer (Alli and Adanlawon, 2013); phenols – are anti-oxidative crystalline aromatic organic compounds (Mesuda et al., 2008), exhibiting antimicrobial action against Gram-positive bacteria by interfering with enzymes involved in the production of energy, and by denaturing bacterial proteins at high concentrations (Roman et al., 2010; Tiwari et al., 2009); and flavonoids with anti-carcinogenic and anti-mutagenic activities which acts by resisting tumour initiation and other stages of cancer formation (Okwu, 2004; Uruquiaga and Leighton, 2000). Flavonoids present in the fungus possesses anti-inflammatory, anti-oxidant, antiviral and anti-carcinogenic potentials.

Metabolites and compound present in plant materials and parts have been reported to be of immense therapeutical importance to man, animals and plants. Extraction of the constituent compounds is dependent on a host of factors among which is temperature, time, and very critical, the solvent system. Orole (2016) explained that G. lucidum contain 1-docosanol an alcohol with reportedly cytotoxic and hemolytic activities against viral cell wall, and Katz et al. (1991) added that the alcohol exhibits no toxic, mutagenic, or teratogenic properties thus giving it a clean bill in usage. Other compounds of medicinal importance present in the fungus among others include 1-Octadecene with anticancer, antioxidant and antimicrobial activities (Lee et al., 2007; Mishra and Sree, 2007), Silane, diethylheptyloxyoctadecyloxy- a phytosterol possessing cholesterol lowering potentials (Jones, 1999), 1-docosene, 1-octadecene, (E)-5-eicosene, 1-nonadecene with capacities to prevent the growth of bacteria (Yogeswari et al., 2012; Yassa et al., 2009), benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, octadecyl ester with inhibitory activities at different concentrations to green algae and bacteria. The study is to compare the activities of crude extract of Ganoderma lucidum on serum marker enzymes and parameters of ochratoxin A administered albino rats as part of an ongoing study of the activities of the fungus on animal models however, further investigative studies to better understand the mechanisms involved in its action is on-going.

MATERIAL AND METHODS:

Collection and Identification of Plant Materials

Fresh fruiting bodies of Ganoderma lucidum were harvested, cleaned, shade dried, and authenticated in the Department of Botany, Federal University Lafia, Nigeria. Identification of the fungus macroscopically was based on colour, odour, morphology, and the cap of the mushroom; microscopic examinations of spores on a glass slide using lacto phenol in cotton blue was also done before it was ground into cottony form, and kept in the refrigerator for further testing.

Extraction Procedure

Extraction was carried out by blending and pulverizing the mushroom in a warring blender after which 400 g of the pulverized fungus was weighed and soaked in 2000 mL of methanol (98% BDH) at a ratio of 1:5 (powder: solvent). The mixtures were kept in corked container, left for 48 hr at room temperature on an electric shaker, then filtered using vacuum pump. The filtrate was kept in a rotary evaporator to allow the methanol to evaporate to 10% of the original volume. The final concentrations to dryness was done by evaporating to dryness in water bath at 60^oC.

Animal Experimental Design

Forty two (42) healthy inbred Albino rats weighing between 248 - 302 g were selected for the study. The animals obtained from Plant Science and Biotechnology Department, Adekunle Ajasin University, Akungba-Akokos were kept in 12 h light and 12 h dark conditions and provided animal feed and water ad libitum at temperature 25±2^oC till the end of the study. Animal feed were prepared by Grand Cereals Limited, a subsidiary of UAC of Nigeria plc, Jos, Plateau State, Nigeria. The feed was confirmed to be free of mycotoxin before use. Maintenance of the animals was in conformity with laid down policy of “Guide for care and use of laboratory animals in research and teaching” by the National Academy of Science, published by the National Institute of Health (NIH) publication 86-23 (1985 revised).

The animals were divided into six groups of seven animals each, allowed to acclimatize for two weeks (14 days) before the start of the experiment.

The protocol is described thus:

Group 1:

Negative Control – Animals were given water alone

Group 2:

Positive Control – Animals were treated with 100 mg G. lucidum per kg body weight

Group 3:

Animals were treated with 1 µg ochratoxin A per kg bw of animals only

Group 4:

Animals were treated with 0.5 µgochratoxin A per kg bw of animals only

Group 5:

Animals were treated with 1 µg OTA per kg bw + 100 mg/kg body weight G. lucidum

Group 6:

Animals were treated with 0.5 µg OTA per kg bw + 300 mg/kg body weight G. lucidum

Animals in groups 3, 4, 5, and 6 were first treated with ochratoxin A, observed for behavioral changes for 3 h after which they were treated with G. lucidum extract based on body weight.

Preparation and administration of ochratoxin A and G. lucidum extract

Ochratoxin A was obtained from Trilogy Analytical Laboratory, USA. OTA was dissolved in sterile water and volume and dose adjusted to animal weight. Dosages were administered intraperineally per at 1 and 0.5 µg per kg body weight of animal once but before that, the animals were fasted for 12 h though the animals had access to water. The weight of the animals were monitored and recorded every three day from the start of the experiment. Ten milligram per one milliliter (10mg/ml) of extract was prepared by adding 2000mg extract in 200ml of sterile distilled water. 1 mL extract solution contains 10 mg extract translating into 300/1000 x weight of animal. Extract administration was done at days 1, 3, and 5 at 0070 hour in the morning. Oral administration with the aid of an intubator was adopted to force feed (gavage) the animals.

Serum and organ collection

Finally at day seven (7), the animals were weighed and subsequently euthanized. Blood samples were collected from the tail vein of animals per group into plain tubes, and the animals sacrificed by cervical dislocation, the liver and kidney removed, washed with water and blotted with filter paper then weighed individually. A big lobe of the liver and a half of the kidney were then fixed in 4% formaldehyde while the remaining portion was kept for biochemical tests.

Biochemical parameters

The blood samples according to groups were individually centrifuged at 1085xg for 15 min at 4oC to get serum content and analyzed using kit produced by RANDOX group of company.

a) Urea test

To 10 µL of sample was added 100 µL of urease reagent I, followed by 2.5 mL each of urease reagents II and III then incubated for 25 min at 37^oC and read at 546 nm.

b)Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) Tests

100 µL of serum sample in a test tube was added 500 µL of AST or ALT reagent I and incubated for 30 min at 37^oC in a water bath. Thereafter, 5 mL of NaOH was added and read at 546 nm.

c) Alkaline phosphatase test

To two test tubes, 1 mL of distilled water was added followed by, one drop of alkaline phosphatase substrate and incubated for 5 min at 37^oC. Five milliliter (5 mL) of color developer was added and incubated at 37^oC for 5 min again and read.

d)Total protein test

To 40 µL of serum sample was added 20 mL of protein reagent, incubated for 30 min at 37^oC then read at 546 nm.

e)Albumin test

In the test, 3 mL of albumin reagent was added to 10 µL of serum sample, incubated for 5min at 37^oC then read at 630 nm.

Ochratoxin A in Serum Samples

The residue of ochratoxin A in serum samples were extracted and analyzed using HPLC according to the technique of Grajewski et al. (2012). To 1ml serum were added 5ml ratio 1:1 mixture of 0.2M magnesium chloride with 0.1M hydrochloric acid and 3ml chloroform and the mixture shaken for 30 min at 200 rpm then centrifuge at 4500rpm for 15 min at 10oC after which the water phase was removed and dried at 40^oC using nitrogen. The dry extracts was re-dissolved in 300 µL methanol left for 3 min and 2 mL phosphate buffered saline (PSB) added. The extract was filtered using immunoaffinity column (IAC) produced by R-Biopharm Rhone Limited, Glasgow, USA; allowed to pass through the column by gravity followed by 20 mL PBS at a flow rate of 5 mL/min, dried by passing air through the column and eluted with 1.5 mL acetic acid:methanol (2:98 v/v), then back-flushed thrice to ensure complete elution and collected into 3 mL vial bottles. 1.5 mL distilled water was allowed through the column and collected in sample vial by vortexing. One hundred microliter of the eluate was injected into the HPLC system to quantify ochratoxin concentration.

Statistical Analysis

Tukey HSD All-Pairwise Comparisons Tests at 5% was used to compare the means. The result of ochratoxin A quantity were expressed as means and Standard Error of Means (SEM).

RESULTS AND DISCUSSION :

The blood urea content ranged between 10.8±6.5 mg/dL and 20.2±5.8 mg/dL (Table 1). Animals given water alone gave the highest value while animals in the group exposed to 100 mg/kg bw extract gave the lowest value. The results were significantly different from each other at p=0.05. Alkaline phosphatase value for animals in Group 6 had value of 98.3±2.9 U/L. Animals treated to 1000 ng/kg bw concentration of OTA had value of 104±12.7 U/L while the control group recorded 73.3±4.5 U/L. The data were significantly different from each other.

DISCUSSION :

The animals were sacrificed on day 7 due to the short half life of about 100 h of ochratoxin A in the blood, and excretion and elimination time of 5 – 6 days (Storen et al., 1982). Animals first treated with OTA at different concentrations and subsequently administered G. lucidum at 100 mg/kg bw by ingestion showed recovery as seen in the AST, alkaline phosphatase, and ALT levels. The report is in agreement with findings of Lin (2005) and You and Lin (2002) showing the fungus protects the macrophages from oxidative damage in vivo (mice) and prevent morphological changes to the mitochondria and endoplasmic reticulum, and that the mushroom also rehabilitate damaged mitochondrial membrane in the process which alludes to the protective effect of the mushroom (Aydin et al., 2010). Lin (2005) also proposed another mechanism of healing employed by the mushroom to be rehabilitative which involves the supplementation of the membrane potential previously damaged by oxidative insult which may account for reduction in the values of AST, ALP, and ALT as seen in the OTA administered and later G. lucidum treated animals when compared with the groups treated to OTA alone. Another argument postulated the scavenging of radicals (superoxide anion) as the mechanism behind the protective covering the plant extracts.

Urea content of the pre-treatment set were in the normal range except the last group of animal first administered 100mg/kg bw G. lucidum extract then intoxicated with 500ng/kg body weight. In the set treated with G. lucidum extract, the albumin content within the group were slightly reduced which could be that the mushroom prevented formation of urea. The study observed increment in globulin level at both ochratoxin A concentration administered to the animals which agrees with the suggestions of Galtier (1991) and Rahimtula and Chong (1991) that at mM concentrations, toxicity results in DNA strand breakages, adduct formation and reactive oxygen species formation resulting in increase globulin levels in the serum acting at a compensatory level. G. lucidum also confers therapeutic and healing advantages to the treated animals by suppressing protein synthesis directed by OTA influenced proliferating signals pathways and degrading enzymes in order to restore normalcy to the cells as seen in the AST and ALP levels (Suarez-Arrayo et al., 2013).

In the set of animals administered G. lucidum, sharp reduction in the body weights were recorded two days (48 h) after administering the extract; the downward trend continued till the animals were sacrificed. Two possible reasons adduced to the weight losses recorded by G. lucidum treated groups were that the bitter taste of the fungus could cause withdrawal from food by the animals sensing it could be incorporated into their feed, and the other explanation centers around sedation and induced hypnotic sleep effect of the fungus, leading to subsequent reduced food intake thus reduced weight (Chu et al., 2007). Tang et al. (2005) and Soo (1996) reported the capacity of the fungus to induce sleep in humans though at varying concentrations. The work in addition agrees with earlier study by Chu et al. (2007) that moderate doses of the fungus extract do not bring about inactivity after a single dose as seen in the present study but that, resulting effects are noted after 3 days of treatment in weight reduction in the animals treated with the extract. The work of Honda et al. (1988) that continued treatment of animals with the extract bring about more weight loss effects agrees with findings in the present study. Animals treated to OTA alone also showed reduced weight loss which agrees with the study of Abdel-Aziz et al. (2010) who attributed the recorded weight loss to DNA breakage, inhibition of protein synthesis and gluconeogenesis (Petzinger and Ziegler, 2000).

CONCLUSION :

Ganoderma lucidum from the foregoing has the capacity to heal and restore back to normalcy enzymes in the serum and kidney produced in response to foreign substances in the body. While the fungus has challenging factor as regards animal withdrawal from food which could be resolved with further works so as not to alter its efficacy, it also needed further work to streamline the potentials of the fungus in modifying its actions to tackle reactions elicited by disease conditions such as cancer, infectious diseases, and so on.

REFERENCES :

1. Abdel-Aziz, K. B., Faraag, I.M., Tawfek, N.S., Nada, S.A., Amra, H.A., Darwish, H.R. Saccharomyces cereviciae amelio -rates oxidativestress, genotoxicity and spermato-toxic effects induced by Ochratoxin A in male Albino Mice. New York Science Journal, 2010, 3(11).

2. Alli, S.Y.R., Adanlawo, I.G. Tissue Lipid Profile of Rats Administered Saponin Extract from the Root of Bitter Kola, Advances in Biochemistry. 2013, 1(1): 1-4.

3. Aydin, S., Aytac, E., Uzun, H., Altug, T., Mansur, B., Saygili, S., Buyukpinarbasili, N., Sariyar, M. Effects of Ganoderma lucidum on obstructive jaundice-induced oxidative stress. Asian J Surg. 2010, 33 (4) :173- 80.

4. Boh, B., Berovic, M., Zhang, J., Zhi-Bin, L. Ganoderma lucidum and its pharmaceutically active compounds. Bio-technol. Annu. Rev. 2007, 13: 265–301.

5. Borchers, A.T., Stern, J.S., Hackman, R.M., Keen, C.L., Gershwin, M.E. Mushrooms, tumors, and immunity. Proc Soc Exp Biol Med. 1999, 221(4) : 281–293.

6. Chu, Q.P., Wang, L.E., Cui, X.Y., Fu, H.Z., Lin, Z.B., Lin, S.Q., et al. Extract of Ganoderma lucidum potentiates pentobarbital-induced sleep via a GABAergic mechanism. Pharmacol Biochem Behav. 2007, 86: 693–698.

7. Galtier, P. Pharmacokinetics of ochratoxin A in animals. In: Castegnaro M, Plestina R, Dirheimer G, Chernozemsky IN, Bartsch H, editors. Mycotoxins, endemic nephropathy and urinary tract tumors. IARC, Lyon: 1991, 187-200.

8. Honda, K., Komoda, Y., Inoué, S. Sleep-promoting effects of Ganoderma extracts in rats: comparison between long-term and acute administrations. Tokyo Ika Shika Daigaku Iyo Kizai Kenkyusho Hokoku. (1988)

9. Jones, J.P. Cholesterol-lowering action of plant sterols. Current Atherosclerosis Reports. 1999, 1(3): 230–235.

10. Katz, D.H., Marcelletti, J.F., Khalil, M.H., Pope, L.E., Katz, L.R. Antiviral activity of 1-docosanol, an inhibitor of lipid-enveloped viruses including herpes simplex. Proc. Natl. Acad. Sci. USA. 1991, 88: 10825-10829, Medical Sciences

11. Lee, S.H., Chang, K.S., Su, M.S., Huang, Y.S., Jang, H.D. Effects of some Chinese medicinal plant extracts on five different fungi, Food Control. 2007, 18: 1547-1554.

12. Lin, Z.B. Cellular and molecular mechanisms of immuno-modulation by Ganoderma lucidum. J Pharmacol Sci. 2005.

13. Masuda, T., Yamada, K., Akiyama, J., Someya, T., Odaka, Y., Takeda, Y., Tori, M., Nakashima, K., Maekawa, T., Sone, Y. Antioxidation mechanism studies of caffeic acid: dentification of antioxidation products of methyl caffeate from lipid oxidation. Journal of Agricultural and Food Chemistry. 2008, 56: 5947–5952.

14. Mau, J.L., Lin, H.C., Chen, C.C. Antioxidant properties of several medicinal mushrooms. J Agric Food Chem. 2002, 50 (21) : 6072-7

15. Mishra, P.M., Sree, A. Antibacterial Activity and GCMS Analysis of the Extract of Leaves of Finlaysonia obovata (A Mangrove Plant), Asi. J. Pl. Sci. 2007, 6: 168-172.

16. Okwu, D.E. Phytochemicals and vitamin content of indigenous spices of SoutheasternNigeria. J. Sustain. Agric. Environ. 2004, 6 (1) : 30-37.

17. Orole, O.O.GC-MS Evaluation, Phytochemical, and Nutritional Screening of Ganoderm A lucidum. Journal of Applied Biology and Biotechnology. 2016, 12(2): 1-7.

18. Orole, O.O., Adejumo, T.O., Orole, R.T. Antifungal Activity of Nine Medicinal Plants against Aspergillus species from Cocoa Beans Theobroma cacao). Journal of Agriculture and Ecology Research International. 2016, 2: 1-11.

19. Petzinger, E., Ziegler, K. Ochratoxin A from a toxicological perspective. J. Vet. Pharmacol. Therap. 2000, 23: 91-98.

20. Rahimtula, A.D., Chong, X.Alterations in calcium homeostasis as a possible cause of ochratoxin A nephrotoxicity. In Myco-toxins, Endemic Nephropathy and Urinary Tract Tumours (M. Castergano, R. Plestina, G. Dirheimer, I. N. Chermozemsky, and H. Bartsch, Eds.). IARC Scientific Publication 1991,115. International Agency for Research on Cancer, Lyon, France.

21. Roman, M., Iveta, H., Vladimír, F., Šmi drkal, J. Antimicrobial and Antioxidant Properties of Phenolic Acids Alkyl Esters. Czech J. Food Sci. 2010, 28(4): 275–279.

22. Soo, T.S. Effective Dosage of the Extract of Ganoderma Lucidum in the Treatment of Various Ailments AGRIS: International Information System for the Agricultural Science and Technology; World Society for Mushroom Biology and Mushroom Products 1996, 177-185.

23. Storen, O., Holm, H., Stormer, F.C. Metabolism of ochratoxin A by rats. Appl Environ Microbiol. 1982, 44(4): 785-789.

24. Suarez-Arroyo, I.J., Rosario-Acevedo, R., Aguilar-Perez, A., Clemente, P.L., Cubano, L,A., Serrano, J., et al. Anti-Tumor Effects of Ganoderma lucidum (Reishi) in Inflammatory Breast Cancer in In Vivo and In Vitro Models. PLoS ONE. 2013, 8(2): e57431. doi:10.1371/journal.pone.0057431

25. Tang W., Gao, Y., Chen, G., Zhou, S. A randomized, double-blind and placebo-controlled study of a Ganoderma lucidum polysaccharide extract in neurasthenia. J Med Food, 2005, 8(1): 53-58.

26. Tiwari, B.K.,Valdramidis, V.P., O’Donnell, C.P., Muthukuma-rappan, K., Bourke, P., Cullen, P.J. Application of natural antimicrobials for food preservation. J. Agric. Food Chem. 2009, 57: 5987–6000.

27. Uruquiaga, I., Leighton, F. Plant polyphenol antioxidants and oxidative stress. Biological Research. 2000, 33: 159-165.

28. Wang, X., Liu, R., Sun, J., Guan, S., Yang, M., Bi, K., Guo, D. HPLC method for the determination and pharmacokinetic studies of four triterpenoids in rat plasma after oral administration of Ganoderma lucidum extract. Biomed. Chromatogr. 2007, 21: 389-396.

29. Yassa, N., Masoomi, F., Rohani-Rankouhi, S.E., Hadjia-khoondi, A. Chemical composition and antioxidant activity of the extract and essential oil of Rosa damascena from Iran, Population of Guilan. DARU J. Pharm. Sci. 2009, 17: 175-180.

30. Yogeswari, S., Ramalakshmi, S., Neelavathy, R., Muthumary, J. Identification and comparative studies of different voltaile fractions from Monochaetia kansensis by GC-MS. Global J Pharmacol. 2012, 6(2): 65-71.

31. You, Y.H., Lin, Z.B. Protective effects of Ganoderma lucidum polysaccharides peptide on injury of macrophages induced by reactive oxygen species. Acta Pharmacol Sin. 2002, 23(9): 787-791.

32. Zhao, H., Zhang, Q., Zhao, L., Huang, X., Wang, J., Kang, X. Spore Powder of Ganoderma lucidum Improves Cancer-Related Fatigue in Breast Cancer Patients Undergoing Endocrine Therapy: A Pilot Clinical Trial. Evid Based Complement Alternat Med. Cui XY, et al. Extract of Ganoderma lucidum prolongs sleep time in rats. J Ethno-pharmacol. (2012)

Received on 07.07.2016 Modified on 16.07.2016

Res. J. Pharmacology & Pharmacodynamics.2016; 8(4): 151-156.

DOI: 10.5958/2321-5836.2016.00027.6