CYTOTOXICITY EFFECT OF SELECTED MEDICINAL MUSHROOMS ON BV2 MICROGLIAL CELLS Neeranjini Nallathamby1, Sri Nurestri Abd. Malek1, Murali Naidu2, Wong Kah – Hui2, Pamela David2 and Vikinewary Sabaratnam1 1
Mushroom Research Centre & Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia; 2Mushroom Research Centre & Department of Anatomy ,University of Malaya, 50603 Kuala Lumpur, Malaysia E-mail:
[email protected]
Abstract Microglial cells are the resident macrophages of the brain and spinal cord, and thus act as the first and main line of active defence in the central nervous system against assaults including inflammation. In the search for safe antiinflammatory agents, mushrooms are being actively sought. This study was conducted to determine the cytotoxic effects of various solvent extracts of three culinary and medicinal mushrooms - Lignosus rhinocerotis (Cooke) Ryvarden, Cordyceps militaris (L.) Link and Hericium erinaceus (Bull.:) Pers, if any, to BV2 microglial cells. Cytotoxic effects were assessed by MTT assay. All fractions tested were not toxic to the BV2 cells at concentrations tested. Ethanol extracts (100% - 191.77%) showed 35%-90% higher proliferation of BV2 cells when compared to hot aqueous extracts (19.38% - 98.22%). The extract concentrations tested were 0.1ug/ml to 1000ug/ml. The ethanol extracts were further fractionated to hexane, ethyl acetate and aqueous fraction. These fractions were also tested for cytotoxic effects to the BV2 cells. The hexane fractions of the three mushrooms tested showed the highest proliferation (ranging 100%- 200.16%) effects on the microglial cell line, followed by ethyl acetate fraction (92.06 %-167.41%). However, the aqueous fraction (2.00% - 93.21%) showed toxic effects to the cells in a dose dependent manner. Keywords: microglial cells, medicinal mushrooms, MTT, culinary mushrooms, solvent fractions
1.Introduction Microglia is the resident macrophages immune cells of the brain and spinal cord in the central nervous system (CNS) (Aloisi, 2001). They are distributed in large non-overlapping regions throughout the brain and spinal cord (Kreutzberg, 1995; Bushong, 2001). Microglia is constantly scavenging the CNS for damaged neurons, plaques, and infectious agents and plays an active role in host defence and tissue repair in the brain (Gehrmann, 1995). In response to stress or injury, they assume a pro-inflammatory phenotype that can lead to profound neuronal damage if uncontrolled or dysregulated (Block et al., 2007). Since this process must be done quickly to prevent potentially fatal damage, microglia is extremely sensitive to even small pathological changes in the CNS (Dissing-Olesen, 2007). The greatest attribute of mushrooms, besides their taste, is their peculiar healing properties. Medicinal mushrooms have been appreciated and used since ancient times by the Chinese and Egyptians. These mushrooms were related with longevity and immortality (Hobbs, 1995). Today, there are over 270 identified fungal species with known therapeutic properties. These include anti-oxidants, hypotensives, hypocholesterolemics, liver protectants, immune suppressants, anti-fibrotics, as well as anti-inflammatory, anti-diabetic, anti-viral and anti-microbial properties (Robert, 201; Halpern et al. 2002). They also contain bioactive metabolites capable of helping to revitalize and modulate our immune system (Eliaz, 2010). These
biological response modifiers help activate macrophages and T cells, and produce cytokines, interleukins and tumor necrosis factors. The aim of this study was to access the cytotoxic effects, if any, of three popular medicinal mushrooms namely Lignosus rhinocerotis (Cooke) Ryvarden (locally known as tiger milk mushroom), Cordyceps militaris (L.) Link and Hericium erinaceus (Bull.:Fr.) Pers.(locally known as monkey head mushroom). 2. Materials and Methods The freeze dried sclerotia of Lignosus rhinocerotis and fruit bodies of Cordyceps militaris and Hericium erinaceus were purchased from the local industry. Freeze dried mushroom powder was extracted using hot water and solvent. Mushroom powder was soaked in distilled water (1:20) and left to shake overnight at 150rpm. It was then subjected to double boiling for 30min. Extracts were centrifuged at 5000 rpm at 40C for 15 min and filtered to give a clear supernatant. The aqueous extracts were evaporated to small volume. For ethanol extracts, 100g of freeze dried sample was soaked in ethanol and water at a ratio of 8:2 (1 L) at room temperature. The solvent-containing extract was then filtered and the process was repeated three times. The filtrates were combined and the excess solvent was evaporated under reduced pressure using a rotary evaporator. The crude ethanol extract was then extracted with n-hexane and concentrated in vacuo. The hexane insoluble residue was further partitioned with mixture of ethyl acetate and water (1:2) and evaporated. The ethyl acetate insoluble residue considered as aqueous-soluble fraction. Each extract was tested for cytotoxicity to BV 2 microglial cells using MTT assay based on the initial protocol described by Dudhgaonkar et al ( 2009) with some modifications. 3. Result and Discussion Freeze dried mushroom powders (Lignosus rhinocerotis, Cordyceps militaris and Hericium erinaceus were subjected to two different extraction methods: ethanol extraction and hot aqueous extraction. The cytotoxicity results are shown in Table 1. Each extract was subjected to MTT assay to test the cytotoxicity effect to BV 2 cell line. The ethanol extract of all three medicinal mushroom tested showed higher proliferation of cells compared to their respective hot aqueous water extracts. The ethanolic extracts were further fractionated to hexane, ethyl acetate and aqueous fractions. The cytotoxic effects of these fractions to BV 2 cell line tested using the MTT assay. Both hexane and ethyl acetate fractions of all three mushrooms showed high proliferation of BV2 cells compared to their aqueous fractions. The hexane fraction of all three mushrooms tested gave the highest proliferation of the BV 2 cells. Ethanolic extract of the tiger milk mushroom supported BV2 cells proliferation at all concentrations tested. The highest proliferation of 125.8% was recorded at the concentration of 0.1ug/mL. Whereas the hot aqueous extract had toxic effects to the cells as proliferation of BV2 cells at all concentrations tested was less than control (100%). Previous publication (Lee et.al, 2012) also reported the cold water extract showed a decrease of cell viability with the increase of extract concentration correlating with the current study. Hexane and ethyl acetate fractions, however, were not toxic at all concentration tested. However, at 0.1 ug/mL of hexane fraction the best proliferation was 117.64% (Table 1). The ethanol extract of Cordyceps militaris induced proliferation of BV2 cells at the lowest concentration tested and there was a gradual decrease in proliferation as the concentrations of the extract was increased (Table1). The highest proliferation of 132.77% was recorded at 0.1ug/mL. Toxic effects were observed at concentrations above 10ug/mL possibly due to excess of extracts in the wells. the extracts at high concentration has been report to have toxic and apoptosis effect on various cell line due to the content of cordycepin(Kim et. al, 2001 & Hellinida et.al,2008).The hot aqueous extract did not triggered proliferation of BV2 cells compared to the control. Hexane fractions also promoted proliferation at all concentrations tested except at 1000ug/mL which was toxic to the cells. The highest proliferation of 123.14% was recorded 1ug/mL. The concentrations ethyl acetate fraction tested showed linear decreasing effects on proliferation. The aqueous fraction, however, was toxic at to the cells at all concentrations tested. This phenomenon was also reported by
Wol et.al (2010), whereby the hot water extract showed cytotoxicity effect at concentrations from78 to 1250 ug/mL in RAW 264.7 cells. The ethanol extracts of H.erinaceus supported very high proliferation of BV2 cells compared to hot aqueous extracts (Table 1). The highest proliferation was 191.77 % at 1000ug/mL of extract. Fractions tested showed that hexane fraction was the best proliferating fraction and followed by ethyl acetate fraction. However, the aqueous fraction did not promote proliferation. The highest proliferation at 1000ug/ml of hexane fraction of H. erinaceus was 200.61%. 5. Conclusions All extracts of selected mushroom were not cytotoxic to BV2 cells except for aqueous extract of C. militaris. Hexane extract of H.erinaceus promoted maximum of a two- fold BV2 cell proliferation compared to all extracts tested. Acknowledgements The author thanks University of Malaya for grants PV007-2012A, TA039-2012A, J-21001-76536 and Ministry of Higher Education for High Impact Research (HIR) grant, F-000001-21001. References [1] Aloisi, F.2001. Immune function of microglia, Glia (36):165–179. [2] Block, M.L. Zecca, L. and Hong, J.S. 2007.Microglia-mediated neurotoxicity: uncovering the molecular mechanisms, Nat. Rev. Neurosci. 8 : 57–69. [3] Bushong, EA., Martone, ME., Jones, YZ., and Ellisman, MH. 2002. "Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains". J. Neurosci. 22 (1): 183–92 [4] Dissing-Olesen, L., Ladeby, L., Nielsen, HH., Toft-Hansen, H., Dalmau, I. and Finsen, B. 2007. "Axonal lesion-induced microglial proliferation and microglial cluster formation in the mouse". Neuroscience 149 (1): 112–122. [5] Dudhgaonkar, M., Thyagarajan, A. and Sliva, D. 2009. Suppression of the inflammatory response by the triterpenes isolated from the mushroom Ganoderma lucidum. International Immunopharmacology (9) : 1272-1280. [6] Gehrmann, J., Matsumoto, Y. Kreutzberg, GW., 1995. "Microglia: intrinsic immuneffector cell of the brain". Brain Research Reviews 20 (3): 269–287. [7] Halpern, Georges, M., and Miller, AH.2002. Medicinal Mushrooms. New York: M. Evans and Co. [8] Hellinida Thomadaki, Chris Milto Tsiapalis and Andreas Scorilas.2008.The effect of the polyadenylation inhibitor cordycepin on human Molt-4 and Daudi leukaemia and lymphoma cell lines. Cancer Chemother Pharmacology. 61:703-711 [9] Hobbs, C. 1995. Medicinal Mushrooms: An Exploration of Tradition, Healing and Culture. Santa Cruz, CA: Botanica Press. [10] Isaac Eliaz. 2010. The Healing Power of Medicinal Mushrooms Immune Support For Cancer, Colds and Lifelong Health. Better Health Publication. Santa Rosa, CA.
[11] Kreutzberg, GW.,1995. "The First Line of Defense in Brain Pathologies". Drug Research 45 (1): 357– 360. [12]M. L. Lee, N. H. Tan, S. Y. Fung, C. S. Tan and S. T. Ng. 2012.The Antiproliferative Activity of Sclerotia of Lignosus rhinocerus (TigerMilkMushroom). Evidence-Based Complementary and Alternative Medicine [13] Mi-Nam Kim, Cheng-Bi Cui, Duek-Sik Lee and Seong-Shi Ham.2001.Cytotoxicity and Antigenotoxic Effects of Cordyceps militaris Extracts. Journal of Korean Society Food Science and Nutrition. 30(5): 921-927 [14] Rogers,R. 2011. The Fungal Pharmacy: the Complete Guide to Medicinal Mushrooms and Lichens of North America. Berkeley, CA: North Atlantic Books. [15] Wol Soon Jo, Yoo Jin Choi, Hyoun Ji Kim, Jae Yun Lee,Byung Hyouk Nam, Jae Dong Lee, Sang Wha Lee, Su Yeong Seo and Min Ho Jeong .2010.The anti-inflammatory effects of water extract from Cordyceps militaris in murine macrophage. Mycobiology 38(1):46-51.
Table 1 Percentage of cell proliferation with various mushroom extracts Extract concentrations 10ug/ml 100ug/ml
Mushroom
0.1ug/ml
1.0 ug/ml
1000ug/ml
Tiger milk Ethanol extract Hot water extract Hexane fraction Ethyl acetate fraction Aqueous fraction
125.48±0.06 83.27±0.28 117.64±0.08 100±0.04 63.15±0.28
117.20±0.05 71.65±0.42 115.19±0.03 103.05±0.04 79.59±0.08
117.15±0.05 90.12±0.36 113.77±0.05 100.54±0.10 85.87±0.10
118.35±0.08 82.96±0.02 107.84±0.068 111.32±0.01 84.92±0.02
104.30±0.13 82.96±0.02 100±0.04 92.98±0.04 95.20±0.04
Cordyceps Ethanol extract Hot water extract Hexane fraction Ethyl acetate fraction Aqueous fraction
132.77±0.04 68.59±0.08 115.41±0.07 108.38±0.02 73.079±0.396
117.25±0.042 83.53±0.08 123.14±0.04 96.24±0.05 72.46±0.15
93.06±0.10 82.91±0.06 122.59±0.01 63.64±0.07 87.97±0.26
75.81±0.04 70.76±0.02 92.98±0.08 138.27±0.05 63.10±0.10
52.48±0.48 19.38±0.15 66.30±0.05 66.74±0.03 2.00±0.003
Monkey head Ethanol extract Hot water extract Hexane fraction Ethyl acetate fraction Aqueous fraction
112.51±0.53 98.22±0.07 166.35±0.02 134.37±0.27 93.03±0.09
127.31±0.65 88.25±0.03 170.63±0.12 165.13±0.26 101.84±0.18
184.09±0.05 90.25±0.11 193.49±0.08 167.58±0.24 55.16±0.06
175.70±0.05 93.73±0.01 167.41±0.21 162.35±0.20 90.16±0.05
191.77±0.07 93.73±0.01 200.61±0.05 161.51±0.04 93.21±0.05
