ACETYLCHOLINESTERASE INHIBITION ACTIVITY OF THE GANODERMA LUCIDUM EXTRACTS OBTAINED USING SUPERCRITICAL CARBON DIOXIDE Tanja Botić, Darija Cör, Željko Knez* Laboratory for Separation Processes and Product Design, Faculty of Chemistry and Chemical Engineering University of Maribor, Slovenia *zeljko.knez@um.si ABSTRACT In present study acetylcholinesterase inhibition activity of the Ganoderma lucidum (G. lucidum) extracts obtained using supercritical carbon dioxide (SCCO2) was evaluated. Many studies on G. lucidum are being carried out, and there are data that support its positive health benefits, including anticancer effects, blood glucose regulation, antioxidant, antimicrobial effects, and protection against liver and gastric injury however very little is known about its potential to inhibit acetylcholinesterase. Acetylcholinesteras inhibitors have been widely used in the treatment of neurodegenerative disease such as Alzheimer’s. Results of study have shown that extracts in concentration of 1 mg/mLwere able to inhibit acetylcholinesterase, furthermore activity of extracts depended on process parameters used for their obtainment with SCCO2. INTRODUCTION G. lucidum (Fr.) Krast (Lingzhi), is a member of the mushroom family (lamellae basidiomycete of the family Polyporaceae) and has been used in complementary medicine for over 2000 years. The mycelia, fruiting body and spores of G. lucidum contain approximately 400 different bioactive compounds, which include triterpenoids, polysaccharides, nucleotides, sterols, steroids, fatty acids, proteins/peptides, together with trace elements which have been reported to have a number of pharmacological effects such as immunomodulation, antitumor, anti-atherosclerotic , anti-inflammatory, antimicrobial, hypolipidemic, hepatoprotective, anti-diabetic, anti-androgenic, anti-angiogenic, anti-herpetic, antioxidative and radical-scavenging, anti-aging, hypoglycemic and anti-ulcer properties. Recently, the study of oxidative stress, particularly in human body, has become a subject of great interest. Many compounds extracted from medicinal mushrooms show to have antioxidant properties. Oxidative stress is also implicated in the development of many neurodegenerative diseases including Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and Alzheimer’s disease (AD). One of the important strategies for AD treatment is to control the levels of acetylcholine in the brain through the inhibition of acetylcholinesterase (AChE) . In the present study SCCO2 has been used, as solvent, to obtain G. lucidum extracts rich in active compounds. Herein, we evaluated the AChE inhibitory activities. Moreover, the effect of SCCO2 extraction parameters conditions on the AChE inhibitory activity of extracts obtained from G. lucidum was studied. G. lucidum material was extracted using SCCO2. The extraction experiments were performed in a semi-continuous high-pressure flow-up apparatus (Pmax = 400 bar, Tmax = 100 ◦C and Vmax = 60 mL). They were carried out in cycles at pressures of 250 bar and 300 bar and temperatures of 40 ◦C and 50 ◦C. Milled G. lucidum material was filled into the extractor. The temperature was regulated and maintained at constant value (±0.5 ◦C; LAUDA DR.R. WOBSER GmbH & Co. KG, Lauda Königshofen, Germany). Next the liquefied CO2 was pumped, using high-pressure pump (ISCO syringe pump, model 260D, Lincoln, Nebrasca), through the preheating coil into the extractor. The SCCO2 extract was collected in a glass trap at atmospheric conditions (P = 1 bar and T = 25 ◦C). The flow rate of CO2 was 0.15 kg/h and was measured with flow- metre (ELSTER HANDEL GmbH, Mainz, Germany). The extraction time was approximately 3.5 h. The collected extract was weighed (±0.1 mg) and the extraction yield calculated. The SCCO2 extracts were then subjected to in vitro tests to confirm their AChE inhibitory activities. AChE inhibition activity was measured according to the Ellman method using acetylthiocholine iodide (1 mM) as the substrate in 100 mM potassium phosphate buffer, pH 7.4, at 25 °C, and electric eel AChE as the source of enzyme (6.25 U/mL, Sigma). Hydrolysis of acetylthiocholine iodide was followed on a Kinetic Microplate Reader (Varian, USA) at 405 nm. Fig. 1 Scheme of semi-continuous high-pressure flow up apparatus. RESULTS Up to date there are no reports about the anti-AChE activity of SCCO2 extracts obtained from G. lucidum. Inhibition of acetylcholinesterase (AChE) has been investigated for SCCO2 extracts obtained from G. lucidum fruiting body. The results of the extracts AChE inhibitory activities is presented in Fig 3. where extract concentrations of 1 mg/mL were tested. The AChE inhibition by SCCO2 G. lucidum extracts was between 7.33% and 22.54 % . Results indicate that the best conditions for SCCO2 extraction of compounds with anti-AChE activity from G. lucidum should be 300 bars and 50 °C. CONCLUSION Overall, the results indicate that G. lucidum contains different polar compounds with anti-AChE activity. Extracts of raw material after SCCO2 extraction showed potential to inhibit AChE during bioassay. G. lucidum extracts with anti-AChE activity could be used as support treatment in patients diagnosed with Alzheimer or other neurodegenerative diseases. REFERENCES [1] Z.B. Lin, Modern Research of Ganoderma lucidum, 2nd ed. Beijing Medical University Press, Beijing, 2007, pp.284-309. [2] S.S. Kim, Y.S. Kim, Korean mushrooms. Yupoong Publishing Co., Seoul, Korea. 1990, p.251. [3] D.K. Lahiri, M.R. Farlow, N.H. Greig, K. Sambamurti, Current drug targets for Alzheimer's disease treatment. Drug Development Research 56 (2012) 267-81. [4] M. Hasnat, M. Pervin, B. Lim, Acetylcholinesterase Inhibition and in Vitro and in Vivo Antioxidant Activities of Ganoderma lucidum Grown on Germinated Brown Rice. Molecules 18 (2013) 6663-6678. 1 – gas cylinder 2 – high pressure pump 3 – one-way valve 4 – HPLC pump 5 – heating coil 6 – autoclave 7 – thermo bath 8 – temperature indicator and regulator 9 – manometer 10 – sampling trap 11 – flow-meter Fig. 2 SCCO2 extraction yield of G. lucidum fruiting body as a function of solvent to feed ratio in kg CO2 per kg of raw material. Fig. 3 Acetylcholinesterase (AChE) inhibitory activities of SCCO2 extracts (c= 1 mg/mL) from G. lucidum fruiting body. Data are expressed as percentage (%) of AChE inhibition. MATERIALS AND METHODS G. lucidum fruiting bodies (G. lucidum material) were obtained from MycoMedica d.o.o., (Podkoren, Slovenia). Acetycholinesterase inhibition