- Email: [email protected]
Two-stage extraction of antitumor, antioxidant and antiacetylcholinesterase compounds from Ganoderma lucidum fruiting body
J. of Supercritical Fluids 91 (2014) 53–60
Contents lists available at ScienceDirect
The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu
Two-stage extraction of antitumor, antioxidant and antiacetylcholinesterase compounds from Ganoderma lucidum fruiting body Darija Cör a , Tanja Botic´ a , Zˇ eljko Knez a,∗ , Urˇska Batista b , Andrej Gregori c,d , Franc Pohleven e , Tonica Bonˇcina f a University of Maribor, Laboratory for Separation Processes and Product Design, Faculty of Chemistry and Chemical Engineering, Smetanova 17, 2000 Maribor, Slovenia b Institute for Biophysics, Faculty of Medicine, Lipiˇceva 2, 1000 Ljubljana, Slovenia c Institute for Natural Sciences, Ulica bratov Uˇcakar 108, 1000 Ljubljana, Slovenia d MycoMedica d.o.o., Podkoren 72, 4280 Kranjska Gora, Slovenia e University of Ljubljana, Biotechnical Faculty, Department of Wood Technology, Roˇzna Dolina, Cesta VIII/34, 1000 Ljubljana, Slovenia f University of Maribor, Faculty of Mechanical Engineering, Smetanova 17, 2000 Maribor, Slovenia
a r t i c l e
i n f o
Article history: Received 6 March 2014 Received in revised form 15 April 2014 Accepted 16 April 2014 Available online 24 April 2014 Keywords: Ganoderma lucidum ESEM Antitumour Antioxidant Antiacetylcholinesterase
a b s t r a c t Ganoderma lucidum has been used in oriental medicine for its contribution to vitality and longevity. None of them report about targeted antitumor activity against adenocarcinoma cells, or antioxidant and antiacetylcholinesterase activity for potential application in treatment of Alzheimer’s and other neurodegenerative diseases. To date, there are a few studies available concerning supercritical carbon dioxide extraction of biologically active compounds from G. lucidum fruiting body. In our study, two stage extractions of biologically active compounds from G. lucidum were performed. First, supercritical carbon dioxide was used as extraction solvent. Next, the same material was used for hot water isolation of biologically active polysaccharides. Cytotoxicity, antioxidant and antiacetylcholinesterase activity were tested for all obtained extracts. Additionally, the effect of extraction process conditions on the biological activity of extracts was assessed. © 2014 Published by Elsevier B.V.
1. Introduction Ganoderma lucidum is a mushroom belonging to the family of Ganodermataceae, and has been used in oriental medicine because of its beneficial effect on vitality and longevity [1]. It was also used for prevention and treatment of various human diseases such as chronic hepatitis, nephritis, hypertension, bronchitis and tumorigenic affections [2]. Recently, the study of oxidative stress, especially in human body, has become a subject of great interest. Oxidative stress is implicated in the development of many neurodegenerative diseases including Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and Alzheimer’s disease (AD) [3]. One of the important strategies for treating AD is to control the levels of acetylcholine in the brain through the inhibition of acetylcholinesterase (AChE) [4].
∗ Corresponding author. Tel.: +386 2 2294402; fax: +386 2 2527 774. E-mail addresses: [email protected], [email protected] (Zˇ . Knez). http://dx.doi.org/10.1016/j.supflu.2014.04.006 0896-8446/© 2014 Published by Elsevier B.V.
Many compounds extracted from medicinal mushrooms exhibit antioxidant properties [5,6]. G. lucidum contains different types of biologically active compounds, which boost the immune system and exhibit antitumour, antimicrobial, antiinflammatory, antioxidant and acetylcholinesterase inhibitory actions [4,5,7,8]. G. lucidum can also be used as a sleep-promoting agent as described in Pharmacopaedia of China [9]. These active compounds belong to the classes of triterpenoids and polysaccharides, oils and fats, inorganic ions and sterols [10]. They are mainly located in the mycelium and the fruiting body of this fungus. Since the resistant fungal cell walls are difficult to break in order to access the active compounds [5], different methods such as soaking, physical smashing, ultrasounds, high pressure and enzyme hydrolysis have been used for cell rupture [11–14]. Fu et al. [15] proposed supercritical carbon dioxide (SCCO2 ) as an alternative method for breaking cell walls. Only a few studies cover the extraction of biologically active compounds from G. lucidum fruiting body using SCCO2 [16–19]. However none of them reports about G. lucidum SCCO2 extracts
54
D. Cör et al. / J. of Supercritical Fluids 91 (2014) 53–60
and their targeted antitumor activity against adenocarcinoma cells, antioxidant activity evaluated using sensitive photochemiluminescence or antiacetylcholinesterase activity for potential treatment of neurodegenerative diseases. Moreover, the effect of SCCO2 extraction conditions on the biological activity of extracts obtained from G. lucidum was not yet studied. In the present study, SCCO2 has been used, as solvent, to obtain G. lucidum extracts rich in active compounds. The morphological changes of the material after cell breaking by SCCO2 were observed by scanning electron microscopy (SEM). In a second stage, extraction of the same material was performed, using hot water to obtain active polysaccharides. Cytotoxicity towards normal and adenocarcinoma cells and antioxidant and antiacetylcholinesterase activity of the SCCO2 and polysaccharidic extracts were then investigated. In addition, the effect of process conditions applied during SCCO2 extraction on the biological activity exhibited by crude extracts from G. lucidum was also evaluated. 2. Materials and methods 2.1. Materials G. lucidum fruiting bodies (G. lucidum material) were obtained from MycoMedica d.o.o. (Podkoren, Slovenia). Carbon dioxide (CO2 ) (PubChem CID: 280) purity 99.5% vol. was delivered by Messer (Ruˇse, Slovenia) and ethanol abs. (PubChem CID: 702) purity 99.9% vol. was supplied by Carlo Erba (Italy). 2.2. Material preparation G. lucidum material was lyophilized, crushed using liquid nitrogen and milled (particle size was between 1.6 and 2.0 mm). 8 g of material were then loaded in a high-pressure extractor, which was subsequently filled with SCCO2 . A static-breaking process was used to achieve breaking of cell walls. The exposure pressure was 300 bar at room temperature. Afterwards the autoclave was rapidly depressurised and the dynamic-cycle extraction procedure with SCCO2 followed. 2.3. Environmental scanning electron microscopy (ESEM) observation ESEM was used to observe morphological changes that occurred in material during the static SCCO2 high-pressure treatment. ESEM, microscope Quanta FEI 200 3D in environmental mode, provides a relatively new technology for imaging hydrated materials without specimen preparation and conductive coating. The cells were mounted on aluminium stubs and direct observed in their native state. The pressure chamber was around 83 Pa and an accelerating voltage for imaging was 15 kV. 2.4. Supercritical extraction G. lucidum material was extracted using SCCO2 as described by Hadolin et al. [20]. The extraction experiments were performed in a semi-continuous high-pressure flow-up apparatus (Pmax = 400 bar, Tmax = 100 ◦ C and Vmax = 60 mL) (Fig. 1). They were carried out in cycles at pressures of 250 bar and 300 bar and temperatures of 40 ◦ C and 50 ◦ C. 8 g of 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 biological activities. 2.5. Extraction of polysaccharides The polysaccharidic extracts were obtained by hot water extraction and precipitation with ethanol. After SCCO2 extraction, G. lucidum material was collected and hot water extraction performed. 5 g of material were extracted with 100 mL of distilled water at 85 ◦ C for 6 h under stirring. The crude hot water extracts were filtered and finally concentrated under vacuum to V = 10 mL using a rotary evaporator. Polysaccharides were separated as ´ et al. [21]. Briefly, 30 mL of described before by Skalicka-Wozniak ethanol was added to concentrated hot water extracts and polysaccharides were precipitated overnight at +4 ◦ C. The precipitated polysaccharides were collected after centrifugation (Eppendorf 5804 R refrigerated centrifuge) at 3100 × g for 10 min, and extraction yield calculated. The polysaccharidic extracts of residue of raw materials after SCCO2 extraction were re-dissolved in water and subjected to determination of total polysaccharide content and in vitro tests to confirm their biological activity. 2.6. Determination of polysaccharide content Polysaccharide content was determined using a phenolsulphuric acid method and d-glucose as standard [22]. Polysaccharidic extract of residue of raw materials after SCCO2 extraction solutions (0.05 mg/mL) were prepared in distilled water. 1 mL of solution was mixed with 1 mL of 5% aqueous phenol solution and 5 mL of concentrated sulphuric acid. The mixture was stirred for 30 min. The total sugar was determined by UV–vis based on the standard curve for glucose (0.0047–0.15 mg/mL) at a wavelength of 490 nm. The absorbance was measured using Varian, Cary 50 Probe UV-VIS spectrophotometer (±0.0001). The results were expressed as g of glucose equivalents per g of dry weight of polysaccharidic extract. 2.7. Determination of antioxidant activity using the photochemiluminescence method The analyses were carried out using the Photochem analytical system (Analytik Jena, Germany). This system uses the photochemiluminescence method for the determination of antioxidant activity water- and lipid-soluble compounds (ACWKit and ACLKit; Jena AG Company) and allows quantification of the antioxidant state. The results were compared with the calibration curve, and thus quantified and expressed in equivalents of standard; i.e. ascorbic acid for the water-soluble and trolox for lipid-soluble compounds. Sample solutions with concentration 10 mg/mL were prepared by dissolving polysaccharidic extracts of residue of raw materials after SCCO2 extraction into water and SCCO2 extracts into methanol. The samples were then analyzed according to prescribed apparatus procedure. The antioxidant activity of polysaccharidic extracts of residue of raw materials after SCCO2 extraction was reported as g of ascorbic acid per g of extract while antioxidant activity of SCCO2 extracts was reported as g of Trolox standard per g of extract. 2.8. Evaluation of cytotoxic activity Obtained extracts were dissolved in dimethyl sulfoxide (DMSO) and further subjected to cytotoxic activity testing. The HUVEC
D. Cör et al. / J. of Supercritical Fluids 91 (2014) 53–60
55
Fig. 1. Diagram of semi-continuous high-pressure flow up apparatus.
(human umbilical vein endothelial) and CaCo-2 (human colon adenocarcinoma) cell lines were used. The HUVEC cells were grown in advanced Eagle’s minimal essential medium (MEM; Gibco, Invitrogen, UK) and the CaCo-2 cells in advanced RPMI 1640 (Gibco), both at 37 ◦ C in a CO2 incubator (5% CO2 , 95% air; at 95% relative humidity). Both of these culture media were supplemented with 2 mmol/L l-glutamine (Gibco), 100 U/mL penicillin (Gibco), 100 g/mL streptomycin (Gibco) and 5% foetal bovine serum (Gibco). For the in vitro cytotoxicity assays, the cells were plated in 96well microtiter plates (Costar, USA) at 10000 cells/well. After a 3-h incubation, the DMSO-dissolved extracts (or DMSO as a control) prepared in medium without serum, were added to the cells at a final concentration of 0.2 mg/mL up to 0.5 mg/mL, and the incubations were carried out for 1 h in the CO2 incubator. The cells were then washed once with medium, and 100 l fresh medium with foetal bovine serum was added for a further 48 h. The cytotoxicity was determined using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) test. MTS (CellTiter 96 Aqueous Reagent, Promega, USA) was added to the cultured cells in each well (20 l). After 1 h, the absorbance at 490 nm was measured using a microplate reader (Bio-Tek Instruments Inc., USA). The absorbance corresponded to the amount of soluble formazan produced, which is directly proportional to the number of viable cells. The viability (%) is expressed as the ratio between absorbance at 490 nm of the treated and control cells. The data are presented as means ± SD of 3 independent experiments. The differences were analyzed using Student’s t-tests on the two populations, with p < 0.01 considered significant.
2.9. Antiacetylcholinesterase activity Antiacetylcholinesterase (anti-AChE) activity was measured according to the Ellman method [23], 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, Cary 50 Probe) at 405 nm. Inhibition of acetylcholine (AChE) was monitored for 5 min
for each extract. The final concentration of the extract was 1 mg/mL. The effect of the pure solvents (e.g. methanol for SCCO2 extracts and water for polysaccharidic extracts of residue of raw materials after SCCO2 extraction) on the assay was also monitored. The solvent used for the preparation of extract samples did not exceed 5% of the total volume of the reaction mixture. All readings were corrected for their appropriate blanks, and every measurement was repeated at least two times. 3. Results and discussion 3.1. Efficiency of cell wall breaking procedure Before dynamic extraction with SCCO2 , G. lucidum material was exposed to pressurized gas at 300 bar, followed by rapid depressurization (5 s) in order to achieve rupture of cell walls. The untreated and treated G. lucidum material was than examined by ESEM to investigate the morphological changes of the cell walls during the breaking process. The results are presented in Fig. 2. Rupture of cell walls was reported to be the essential step before isolation of biologically active compounds from G. lucidum, due to better access of solvent into the material [15]. G. lucidum material exposed to SCCO2 at a pressure of 300 bar showed morphological changes (Fig. 1c and d). Under ESEM, the intact cells were ovateoblong or ovoid with truncated apex or blunt taper. There were some sinuous depressions or mini holes on the surface of the cell walls. The processed cells showed morphological changes. Some cells had crevices and gaps and some were completely broken. It can be observed that SCCO2 pressurization and rapid depressurization had a strong impact on cell walls as the desired ruptures were achieved. 3.2. Supercritical extraction After cell wall breaking procedure, dynamic extraction with SCCO2 followed. In the present study 8 g of G. lucidum material have been used and the extraction performed under the conditions specified in Table 1. Extraction yield was also calculated (Table 1).
Table 1 SCCO2 extraction conditions and extraction yield. T (◦ C)
P (bar)
t (h)
CO2 (kg/m3 )
mGanoderma extract (g)
Qv (LCO2 h
40 40 50 50
250 300 250 300
4.2 4.5 2.7 4.2
879.7 910.3 834.0 870.9
0.147 0.166 0.118 0.154
2.4 3.2 3.6 3.6
T, temperature; P, pressure; t, time; mGanoderma extract , mass of extract; CO2 , CO2 density; Qv , flow rate; , extraction yield.
−1
)
(%) 1.84 2.07 1.47 1.92
56
D. Cör et al. / J. of Supercritical Fluids 91 (2014) 53–60
Fig. 2. ESEM micrographs of G. lucidum cells. (a and b) Untreated G. lucidum material (magnification 1250× and 5000×). (c and d) G. lucidum material after exposure to SCCO2 at 300 bar followed by rapid depressurization (magnification 2500× and 4000×). Rupture cells are indicated with white arrows.
Extraction Yeld /(%)
2.5
2.0
1.5
1.0
40°C, 300 bar 50°C, 250 bar 50°C, 300 bar 40°C, 250 bar
0.5
0.0 0.0
1.0
2.0
3.0
4.0
5.0
S/F (kgCO2/kgraw material) Fig. 3. SCCO2 extraction yield of G. lucidum fruiting body as a function of solvent to feed ratio in kg CO2 per kg of raw material.
The flow rate of SCCO2 solvent was between 2.4 and 3.6 L h−1 . The extraction times, necessary to reach a constant extraction yield varied from 2.7 to 4.5 h, due to the different operating pressures and temperatures. From the obtained results it can be observed, that higher operating pressure at constant temperature (T = 40 ◦ C) resulted in higher extraction yield (1.84% and 2.07% at pressure 250 bar and 300 bar, respectively) (Table 1 and Fig. 3). Hsu et al. [16]
investigated SCCO2 extraction of G. lucidum at 100 bar and temperature of 40 ◦ C and reported an extraction yield of 0.12%, which fits in the trend observed in our investigation. The shape of the extraction curves on Fig. 3 shows that at the beginning of the extraction the main mechanism is the mass transfer from the particle surface. After that diffusion becomes the controlling mechanism until the maximum and constant extraction yield is being reached. The highest extraction yield was 2.074% obtained at 40 ◦ C and 300 bar, where the solvent density was 910 kg/m3 . The lowest yield was 1.47% obtained at 50 ◦ C and 250 bar, with solvent density 834 kg/m3 . These results show that the amount of the extract is related to solvent power, which is a function of density. Solvation power depends on its density, which increases with pressure at constant temperature and decreases with temperature at constant pressure [24]. The effect of temperature on the extraction yield, at constant pressure, can be followed by two mechanisms: (1) an increase in the process temperature increases the solubility because to solute vapour pressure; (2) the temperature increase reduces the solubility due to decrease in the solvent’s density. These two opposing effects result in the crossover of solubility isotherms [25]. Based on results shown in Fig. 3 it can be suggested that crossover could have happened.
D. Cör et al. / J. of Supercritical Fluids 91 (2014) 53–60
57
Table 2 Yield (%) of hot water extraction and precipitation from G. lucidum residue material previously extracted using SCCO2 at different conditions. Polysaccharidic extract (PE) of raw material after SCCO2 extraction
Extraction yield of hot water extraction (%)
PE250 bar , 40 ◦ C PE250 bar , 50 ◦ C PE300 bar , 40 ◦ C PE300 bar , 50 ◦ C
4.758 2.906 2.380 2.274
3.3. Extractions of polysaccharides 3.3.1. Extraction yield After SCCO2 extraction, hot water extraction and precipitation of polysaccharides from the residue of G. lucidum material followed. The results are summarized in Table 2. Extraction yields of hot water extraction of residue material ranged from 2.274% to 4.758%. In residue material being extracted with SCCO2 at 250 bars and 40 ◦ C probably highest content of hot water soluble polysaccharides remain (e.g. 4.758%). These variations in extraction yield of polysaccharides could be due to the degradation of compounds when higher temperature and pressure were applied. G. lucidum has a complex chemical composition, which contains various active compounds. It was reported that the major bioactive compounds located in the fruiting bodies of G. lucidum are polysaccharides and triterpenoids; however other active compounds also present in small amounts are phenols, sterols, fats and oils, amino acids, vitamins, nucleosides [1,26–29]. Compounds such as polysaccharides as well as phenols or flavonoids can be obtained from the G. lucidum by hot water extraction [8]. 3.3.2. Determination of polysaccharide content Quantitative determination of total polysaccharides in polysaccharidic extracts of residue of raw materials after SCCO2 extraction obtained from G. lucidum was performed using a phenol-sulphuric acid method. The linear calibration curve with the equation y = 2.7472x + 0.0012 and in concentration range 0.0047–0.150 mg/mL was prepared with standard of glucose. The quantities of total polysaccharides in polysaccharidic extracts are shown in Fig. 4 and are expressed as mg of glucose eq. on g of dry polysaccharidic extract. The amount of polysaccharides in the extracts varies between 17.385 and 24.443 mg/g, depending on the conditions used during SCCO2 extraction. ´ et al. [21] also evaluated the polysaccharide Skalicka-Wozniak content in fruiting bodies of G. lucidum after hot water extraction. The polysaccharide content of 36 tested samples varied between
Fig. 4. Quantification of total polysaccharides in polysaccharidic extract of residue of raw materials after SCCO2 extraction. Data expressed as mg of glucose eq. per g of G. lucidum fruiting body polysaccharidic extract. The standard deviation is expressed as ±value, from two replicates, where two independent experiments were performed.
Fig. 5. Antioxidative activity of G. lucidum fruiting body SCCO2 extracts. Data are expressed as equivalent of Trolox in mg per g of SCCO2 extract. The standard deviation is expressed as ±value, from two replicates, where two independent experiments were performed.
18.450 and 112.820 mg/g. Our results are in the range of results ´ obtained by Skalicka-Wozniak et al. [21]. 3.4. Antioxidant activity The antioxidant activity of G. lucidum SCCO2 and polysaccharidic extract of residue of raw materials after SCCO2 extraction are presented in Figs. 5 and 6, respectively. The highest antioxidant activity of SCCO2 extracts was recorded at 50 ◦ C and pressure 300 bar: e.g. 14.517 mg Trolox per g of dry SCCO2 extract (Fig. 5). Furthermore, it can be observed that antioxidant activity increases with increasing SCCO2 extraction temperature at constant pressure, and as well with increasing pressure at constant temperature. The antioxidant activity of polysaccharidic extracts of residue of raw materials after SCCO2 extraction is presented in Fig. 6. The highest antioxidant activity, of 4.467 mg ascorbic acid per g of dry polysaccharidic extract, was obtained at pressure of 300 bar and temperature of 50 ◦ C. The SCCO2 extraction conditions have the same influence on the antioxidant activity of polysaccharidic extract of residue of raw materials after SCCO2 extraction as observed for SCCO2 extracts: higher antioxidant activity was measured after exposure to higher temperature and pressure. The antioxidant activity of extracts depends on SCCO2 extraction conditions. In addition, it can be observed, that compounds both with antioxidant activity can be extracted with SCCO2 and with hot
Fig. 6. Antioxidative activity of G. lucidum fruiting body polysaccharidic extract of residue of raw materials after SCCO2 extraction. Data are expressed as equivalent of ascorbic acid in mg per g of dry polysaccharidic extract. The standard deviation is expressed as ±value, from two replicates, where two independent experiments were performed.
58
D. Cör et al. / J. of Supercritical Fluids 91 (2014) 53–60
Fig. 8. Acetylcholinesterase (AChE) inhibitory activities of SCCO2 extracts from G. lucidum fruiting body. Data are expressed as percentage (%) of AChE inhibition. Data are means ± SD from two replicates, where two independent experiments were performed. Fig. 7. Effect of 0.2 mg/mL concentration of G. lucidum fruiting body SCCO2 extracts on viability of HUVEC and CaCo-2 cell lines (% of control) after 1 h treatment. Data are shown as mean values ± SD for at least three separate experiments.
water. Two stage extractions enable us to extract from G. lucidum both nonpolar and polar compounds with antioxidant activity. 3.5. Cytotoxic activity The cytotoxic activity of G. lucidum SCCO2 and polysaccharidic extracts of residue of raw materials after SCCO2 extraction dissolved in DMSO was tested on HUVEC (human umbilical vein endothelial) and CaCo-2 (human colon adenocarcinoma) cell lines. The tested concentrations ranged from 0.2 to 0.5 mg/mL. Polysaccharidic extracts of residue of raw materials after SCCO2 extraction did not show any activity towards adenocarcinoma cells (results are not presented), while strong and targeted activity towards adenocarcinoma cells was noticed after incubation with SCCO2 extracts (Fig. 7 and Table 3). The results indicate that the compounds from G. lucidum possessing antitumor activity towards adenocarcinoma cells have nonpolar nature, as they were isolated from the starting material only with SCCO2 . It was observed that SCCO2 extracts had strong antitumor activity, even when the lowest concentrations were applied, e.g. 0.2 mg/mL, resulting in viabilities of adenoma cancer cells between 20.8% and 65.4% after only 1 h of incubation (Fig. 7). The variation in cytotoxicity depended on the process conditions used for SCCO2 extraction. The viability of the HUVEC (normal) cells remains higher than 92%. At the given concentration no significant negative effect on the HUVEC cells was observed, therefore results indicate that SCCO2 extracts show targeted cytotoxic activity towards adenocarcinoma cells solely. To our best knowledge this is the first report about targeted antitumor activity of SCCO2 extracts towards adenocarcinoma cells with no effect on normal cells as such. All results for tested concentrations of SCCO2 extracts and their cytotoxic activity towards the cell lines used in this study are summarized in Table 3. In general it was noticed that higher concentrations of SCCO2 extract resulted in greater cytotoxic action towards adenocarcinoma cells; however also cytotoxicity towards normal (HUVEC) cells increased. SCCO2 extracts obtained at a pressure of 250 bar and temperature of 40 ◦ C gave the best results during cytotoxicity testing. However, they also showed significant influence on the viability of the normal cells. In order to avoid high cytotoxicity towards normal cells and still preserve the high cytotoxic activity towards adenocarcinoma cells G. lucidum extraction with SCCO2 should be performed at 300 bars and 40 ◦ C. SC CO2 at defined conditions can selectively extract certain group of chemical compounds and by that affect on composition of the extract obtained. SC CO2 extract
obtained at p = 250 bar and T = 50 ◦ C probably contained different group of chemical compounds that those obtained at p = 300 bar and T = 40 ◦ C therefore higher bioactivity was recorded (Fig. 7 and Table 3). It is thus demonstrated that both the pressure and the temperature applied during extraction with SCCO2 , clearly influence the composition of G. lucium extracts and their cytotoxic activities towards adenocarcinoma cells. 3.6. Antiacetylcholinesterase activity Inhibition of acetylcholinesterase (AChE) has been investigated for both SCCO2 and G. lucidum polysaccharidic extracts. The results of the extracts AChE inhibitory activities are presented in Figs. 8 and 9, where extract concentrations of 1 mg/mL were tested. The AChE inhibition by SCCO2 G. lucidum extracts was between 7.33% and 22.54% (Fig. 8), while the inhibition by polysaccharidic extracts of residue of raw materials after SCCO2 extraction was from 5.12% to 19.24% (Fig. 9). Hasnat et al. [8] studied AChE inhibitory activity of hot water extracts obtained from fruiting body of G. lucidum. The authors observed 50% inhibition of AChE using an extract concentration of 1 mg/mL. Their result is much higher than in present study; however they used crude hot water extracts that contained, beside polysaccharides, also phenols and flavonoids known to inhibit AChE in higher degrees [30]. Up to date there are no reports about the anti-AChE activity of SCCO2 extracts obtained from G. lucidum. The same trend can be noticed for antioxidant and AChE inhibitory activity of extracts obtained at conditions applied. This may indicate that the compounds with antioxidant activity extracted from G. lucidum also elicit anti-AChE activity. The
Fig. 9. Acetylcholinesterase (AChE) inhibitory activities of polysaccharidic extract of residue of raw materials after SCCO2 extraction from G. lucidum fruiting body. Data are expressed as percentage (%) of AChE inhibition. Data are means ± SD from two replicates, where two independent experiments were performed.
D. Cör et al. / J. of Supercritical Fluids 91 (2014) 53–60
59
Table 3 Effect of higher concentrations (0.2–0.5 mg/mL) of SCCO2 G. lucidum extracts on viability of HUVEC and CaCo-2 cell lines (% of control) after 1 h treatment. Data are shown as mean values ± SD for at least three separate experiments. SCE conditions
Cell lines
0.2 mg/mL
250 bar 40 ◦ C 250 bar 50 ◦ C 300 bar 40 ◦ C 300 bar 50 ◦ C
HUVEC CaCo-2 HUVEC CaCo-2 HUVEC CaCo-2 HUVEC CaCo-2
92.5 35.3 93.8 49.5 93.9 20.8 92.7 65.4
± ± ± ± ± ± ± ±
11.4 12.3 12.2 13.3 12.9 8.7 12.1 11.9
same observations were made by in Hasant et al. [9] for hot water extracts. The effect of SCCO2 extraction conditions on SCCO2 anti-AChE activity and polysaccharidic extracts of residue of raw materials after SCCO2 extraction was also evaluated. From Fig. 8 it can be clearly observed that at both applied pressures (250 and 300 bar) the AChE inhibitory activity of SCCO2 extracts increases with increasing extraction temperature. In both cases, e.g. SCCO2 and polysaccharidic extracts of residue of raw materials after SCCO2 extraction the best AChE inhibitory activity was recorded when SCCO2 extraction conditions were 300 bar and 50 ◦ C (Figs. 8 and 9). Overall, the results indicate that G. lucidum contains different nonpolar and polar compounds with anti-AChE activity since both SCCO2 and polysaccharidic extracts of raw materials after SCCO2 extraction showed potential to inhibit AChE during bioassay. Additionally, the 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. G. lucidum extracts with anti-AChE activity could be used as support treatment in patients diagnosed with Alzheimer or other neurodegenerative diseases.
4. Conclusion The present study provides that SCCO2 extracts elicited significant targeted activity towards adenocarcinoma cells and had antioxidant and anti-AChE activity. The G. lucidum material, which remained after SCCO2 , was rich in water soluble polysaccharides eliciting antioxidant and antiacetylcholinesterase activity, but not showing antitumor activity. It was observed that the extracts with the highest antioxidant activity elicited also the strongest AChE inhibitory activity. In the case of cytotoxicity this relation was not observed. Extraction conditions show important influence on the properties of extracts with high biological activity. It can be concluded that SCCO2 can be used to obtain G. lucidum extracts rich in compounds with strong and targeted antiadenocarcinoma activity. G. lucidum material after SCCO2 extraction can be reused to obtain polysaccharides with antioxidant and antiacetylcholinesterase activity. Therefore G. lucidum represents a rich source of natural compounds with different polarities, for potential treatment of adenocarcinoma, Alzheimer’s and other neurodegenerative diseases as well as degradation of radical oxygen species.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgments The authors gratefully acknowledge the Slovenian Research Agency (Research Project L2 – 4124). The authors wish to thank Dr. Elena Markoˇciˇc for editorial assistance with the manuscript.
0.3 mg/mL 55.2 ± 13.8 0 39.0 ± 17.4 0 68.8 ± 17.7 7.2 ± 3.9 70.3 ± 12.4 0
0.4 mg/mL
0.5 mg/mL
50.1 ± 10.5 0 10.0 ± 5.2 0 48.4 ± 16.7 0 40.1 ± 10.9 0
17.4 ± 7.2 0 2.3 ± 2.1 0 32.6 ± 10.3 0 13.6 ± 8.5 0
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, pp. 251. [3] S.A. Heleno, L. Barros, A. Martins, M.J.R.P. Queiroz, C. Santos-Buelga, ICFR, Ferreira Fruiting body, spores and in vitro produced mycelium of Ganoderma lucidum from Northeast Portugal: a comparative study of the antioxidant potential of phenolic and polysaccharidic extracts, Food Research International 46 (2012) 135–140. [4] D.K. Lahiri, M.R. Farlow, N.H. Greig, K. Sambamurti, Current drug targets for Alzheimer’s disease treatment, Drug Development Research 56 (2012) 267–281. [5] F. Liu, V.E.C. Ooi, S.T. Chang, Free radical scavenging activities of mushroom polysaccharide extracts, Life Sciences 60 (1997) 763–771. [6] H.Y. Fu, D.E. Shieh, C.T. Ho, Antioxidant and free radical scavenging activities of edible mushrooms, J. of Food Lipids 9 (2002) (2002) 35–43. [7] R. Russell, M. Paterson, Ganoderma – a therapeutic fungal biofactory, Phytochemistry 67 (2006) 1985–2001. [8] 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. [9] X.Y. Cui, S.Y. Cui, J. Zhang, Z.J. Wang, B. Yu, Z.F. Sheng, X.Q. Zhang, Y.H. Zhang, Extract of Ganoderma lucidum prolongs sleep time in rats, J. Ethnopharmacology 139 (2012) 796–800. [10] S. Mekkawy, M.R. Meselhy, N. Nakamura, Anti-HIV-1 and anti-HIV-1 protease substances from Ganoderma lucidum, Phytochemistry 49 (2007) 1651–1657. [11] J.H. Ni, B.S. Song, H.B. Li, Y.S. Xie, Breaking of the sporoderm of Ganoderma lucidum and its effect on the extraction yield of polysaccharides, Chinese Traditional Herbal Drugs 33 (2002) 422. [12] Q. Li, Y. Li, P. Zhang, Z.Q. Xing, P.L. Kang, The effect on the distill of amylose in glossy Ganoderma sporule powder treated with ultrasonic and thawing and freezing test of liquid nitrogen, J. of Liaoning University 31 (2004) 81–82. [13] H. Wu, S.Q. Zhang, J.J. Zhu, Broken Ganoderma lucidum spores by breakingextracting method, J. of Jilin University 34 (2004) 312–315. [14] Y.Z. Xie, S.Z. Li, A. Yee, D.P. La Pierre, Z. Deng, D.Y. Lee, Ganoderma lucidum inhibits tumor cell proliferation and induces tumour cell death, Enzyme and Microbial Technology 40 (2006) 177–185. [15] Y.J. Fu, W. Liu, Y.G. Zu, X.G. Shi, Z.G. Liu, G. Schwarz, T. Efferth, Breaking the spores of the fungus Ganoderma lucidum by supercritical CO2 , Food Chemistry 112 (2009) 71–76. [16] R.C. Hsu, B.H. Lin, C.W. Chen, The study of supercritical carbon dioxide extraction for Ganoderma lucidum, Industrial & Engineering Chemistry Research 40 (2001) 4478–4481. [17] J. Zhang, J.C. Duan, Z. Liang, W.B. Zhang, L.H. Zang, Y.S. Huo, Y.K. Zhang, Analysis of triterpenoids in fruiting bodies of Ganoderma lucidum with off-line supercritical fluid extraction – high performance liquid chromatography system, Chinese J. of Analytical Chemistry 34 (2006) 447–450. [18] T.Q. Chen, J.G. Wu, Y.B. Wu, H.Y. Wang, F.H. Mao, J.Z. Wu, Supercritical fluid CO2 extraction, simultaneous determination of total sterols in the spore lipids of Ganoderma lucidum by GC–MS/SIM methods, Chemistry of Natural Compounds 48 (2012) 657–658. [19] M.S. Lin, Z.R. Yu, Y.M. Weng, Study of continuous extraction process utilizing supercritical fluid for G. lucidum, National Resource of Sustainable Development II 524–527 (2012) 2310–2315. ˇ [20] M. Hadolin, M. Skerget, Zˇ . Knez, High pressure extraction of vitamin E-rich oil from Silybum marianum, Food Chemistry 74 (2001) 355–364. ´ [21] K. Skalicka-Wozniak, J. Szypowski, R. Ło´s, M. Siwulski, K. Sobieralski, K. Głowniak, A. Malm, Evaluation of polysaccharides content in fruit bodies and their antimicrobial activity of four Ganoderma lucidum (W. Curt.: Fr.) P. Karst. strains cultivated on different wood type substrates, Acta Societatis Botanicorum Poloniae 81 (2012) 17–21. [22] M. DuBois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substance, Analytical Chemistry 28 (1956) 350–356. [23] G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochemical Pharmacology 7 (1961) 88–95.
60
D. Cör et al. / J. of Supercritical Fluids 91 (2014) 53–60
[24] G. Brunner, Gas Extraction: An Introduction to Fundamentals of Supercritical Fluids and the Application to Separation Processes, vol. 4, Steinkopff, Darmstadt, 1994. [25] E.M.Z. Michielin, L.F.V. Bresciani, L. Danielski, R.A. Yunes, S.R.S. Ferreira, Composition profile of horsetail (Equisetum giganteum L.) oleoresin: comparing SFE and organic solvents extraction, J. of supercritical fluids 33 (2005) 131–138. [26] H.C. Wang, S.Y. Sun, Analysis on the amino acids and trace elements in wild and artificial cultivation Ganoderma lucidum collected from Taishan, Traditional Chinese Medicine 12 (1990) 14. [27] M.W. Zhao, C.G. Wang, P. Bao, Z.T. Xing, W.Q. Liang, J.Q. Yu, X.D. Shang, D.Y. Men, N. Wang, Y.J. Pan, Triterpenes and polysaccharide contents of liquid culturing
mycelium of different Ganoderma lucidum, Edition Fungus Chinese 22 (2002) 43–46. [28] A.V. Avtonomova, L.M. Krasnopol’skaya, V.N. Maksimov, Optimization of nutrient medium for submerged cultivation of Ganoderma lucidum (Curt.: Fr.) P. Karst, Microbiology 75 (2006) 148–153. [29] J. Lu, J.Z. Qin, P. Chen, X. Chen, Y.Z. Zhang, S.J. Zhao, Quality difference study of six varieties of Ganoderma lucidum with different origins, Frontiers in Pharmacology 3 (2012) 7. [30] I. Orhan, M. Kartal, F. Tosun, B. Sener, Screening of various phenolic acids and flavonoid derivatives for their anticholinesterase potential, Zeitschrift für Naturforschung C 62 (2007) (2007) 829–832.
Contents lists available at ScienceDirect
The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu
Two-stage extraction of antitumor, antioxidant and antiacetylcholinesterase compounds from Ganoderma lucidum fruiting body Darija Cör a , Tanja Botic´ a , Zˇ eljko Knez a,∗ , Urˇska Batista b , Andrej Gregori c,d , Franc Pohleven e , Tonica Bonˇcina f a University of Maribor, Laboratory for Separation Processes and Product Design, Faculty of Chemistry and Chemical Engineering, Smetanova 17, 2000 Maribor, Slovenia b Institute for Biophysics, Faculty of Medicine, Lipiˇceva 2, 1000 Ljubljana, Slovenia c Institute for Natural Sciences, Ulica bratov Uˇcakar 108, 1000 Ljubljana, Slovenia d MycoMedica d.o.o., Podkoren 72, 4280 Kranjska Gora, Slovenia e University of Ljubljana, Biotechnical Faculty, Department of Wood Technology, Roˇzna Dolina, Cesta VIII/34, 1000 Ljubljana, Slovenia f University of Maribor, Faculty of Mechanical Engineering, Smetanova 17, 2000 Maribor, Slovenia
a r t i c l e
i n f o
Article history: Received 6 March 2014 Received in revised form 15 April 2014 Accepted 16 April 2014 Available online 24 April 2014 Keywords: Ganoderma lucidum ESEM Antitumour Antioxidant Antiacetylcholinesterase
a b s t r a c t Ganoderma lucidum has been used in oriental medicine for its contribution to vitality and longevity. None of them report about targeted antitumor activity against adenocarcinoma cells, or antioxidant and antiacetylcholinesterase activity for potential application in treatment of Alzheimer’s and other neurodegenerative diseases. To date, there are a few studies available concerning supercritical carbon dioxide extraction of biologically active compounds from G. lucidum fruiting body. In our study, two stage extractions of biologically active compounds from G. lucidum were performed. First, supercritical carbon dioxide was used as extraction solvent. Next, the same material was used for hot water isolation of biologically active polysaccharides. Cytotoxicity, antioxidant and antiacetylcholinesterase activity were tested for all obtained extracts. Additionally, the effect of extraction process conditions on the biological activity of extracts was assessed. © 2014 Published by Elsevier B.V.
1. Introduction Ganoderma lucidum is a mushroom belonging to the family of Ganodermataceae, and has been used in oriental medicine because of its beneficial effect on vitality and longevity [1]. It was also used for prevention and treatment of various human diseases such as chronic hepatitis, nephritis, hypertension, bronchitis and tumorigenic affections [2]. Recently, the study of oxidative stress, especially in human body, has become a subject of great interest. Oxidative stress is implicated in the development of many neurodegenerative diseases including Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and Alzheimer’s disease (AD) [3]. One of the important strategies for treating AD is to control the levels of acetylcholine in the brain through the inhibition of acetylcholinesterase (AChE) [4].
∗ Corresponding author. Tel.: +386 2 2294402; fax: +386 2 2527 774. E-mail addresses: [email protected], [email protected] (Zˇ . Knez). http://dx.doi.org/10.1016/j.supflu.2014.04.006 0896-8446/© 2014 Published by Elsevier B.V.
Many compounds extracted from medicinal mushrooms exhibit antioxidant properties [5,6]. G. lucidum contains different types of biologically active compounds, which boost the immune system and exhibit antitumour, antimicrobial, antiinflammatory, antioxidant and acetylcholinesterase inhibitory actions [4,5,7,8]. G. lucidum can also be used as a sleep-promoting agent as described in Pharmacopaedia of China [9]. These active compounds belong to the classes of triterpenoids and polysaccharides, oils and fats, inorganic ions and sterols [10]. They are mainly located in the mycelium and the fruiting body of this fungus. Since the resistant fungal cell walls are difficult to break in order to access the active compounds [5], different methods such as soaking, physical smashing, ultrasounds, high pressure and enzyme hydrolysis have been used for cell rupture [11–14]. Fu et al. [15] proposed supercritical carbon dioxide (SCCO2 ) as an alternative method for breaking cell walls. Only a few studies cover the extraction of biologically active compounds from G. lucidum fruiting body using SCCO2 [16–19]. However none of them reports about G. lucidum SCCO2 extracts
54
D. Cör et al. / J. of Supercritical Fluids 91 (2014) 53–60
and their targeted antitumor activity against adenocarcinoma cells, antioxidant activity evaluated using sensitive photochemiluminescence or antiacetylcholinesterase activity for potential treatment of neurodegenerative diseases. Moreover, the effect of SCCO2 extraction conditions on the biological activity of extracts obtained from G. lucidum was not yet studied. In the present study, SCCO2 has been used, as solvent, to obtain G. lucidum extracts rich in active compounds. The morphological changes of the material after cell breaking by SCCO2 were observed by scanning electron microscopy (SEM). In a second stage, extraction of the same material was performed, using hot water to obtain active polysaccharides. Cytotoxicity towards normal and adenocarcinoma cells and antioxidant and antiacetylcholinesterase activity of the SCCO2 and polysaccharidic extracts were then investigated. In addition, the effect of process conditions applied during SCCO2 extraction on the biological activity exhibited by crude extracts from G. lucidum was also evaluated. 2. Materials and methods 2.1. Materials G. lucidum fruiting bodies (G. lucidum material) were obtained from MycoMedica d.o.o. (Podkoren, Slovenia). Carbon dioxide (CO2 ) (PubChem CID: 280) purity 99.5% vol. was delivered by Messer (Ruˇse, Slovenia) and ethanol abs. (PubChem CID: 702) purity 99.9% vol. was supplied by Carlo Erba (Italy). 2.2. Material preparation G. lucidum material was lyophilized, crushed using liquid nitrogen and milled (particle size was between 1.6 and 2.0 mm). 8 g of material were then loaded in a high-pressure extractor, which was subsequently filled with SCCO2 . A static-breaking process was used to achieve breaking of cell walls. The exposure pressure was 300 bar at room temperature. Afterwards the autoclave was rapidly depressurised and the dynamic-cycle extraction procedure with SCCO2 followed. 2.3. Environmental scanning electron microscopy (ESEM) observation ESEM was used to observe morphological changes that occurred in material during the static SCCO2 high-pressure treatment. ESEM, microscope Quanta FEI 200 3D in environmental mode, provides a relatively new technology for imaging hydrated materials without specimen preparation and conductive coating. The cells were mounted on aluminium stubs and direct observed in their native state. The pressure chamber was around 83 Pa and an accelerating voltage for imaging was 15 kV. 2.4. Supercritical extraction G. lucidum material was extracted using SCCO2 as described by Hadolin et al. [20]. The extraction experiments were performed in a semi-continuous high-pressure flow-up apparatus (Pmax = 400 bar, Tmax = 100 ◦ C and Vmax = 60 mL) (Fig. 1). They were carried out in cycles at pressures of 250 bar and 300 bar and temperatures of 40 ◦ C and 50 ◦ C. 8 g of 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 biological activities. 2.5. Extraction of polysaccharides The polysaccharidic extracts were obtained by hot water extraction and precipitation with ethanol. After SCCO2 extraction, G. lucidum material was collected and hot water extraction performed. 5 g of material were extracted with 100 mL of distilled water at 85 ◦ C for 6 h under stirring. The crude hot water extracts were filtered and finally concentrated under vacuum to V = 10 mL using a rotary evaporator. Polysaccharides were separated as ´ et al. [21]. Briefly, 30 mL of described before by Skalicka-Wozniak ethanol was added to concentrated hot water extracts and polysaccharides were precipitated overnight at +4 ◦ C. The precipitated polysaccharides were collected after centrifugation (Eppendorf 5804 R refrigerated centrifuge) at 3100 × g for 10 min, and extraction yield calculated. The polysaccharidic extracts of residue of raw materials after SCCO2 extraction were re-dissolved in water and subjected to determination of total polysaccharide content and in vitro tests to confirm their biological activity. 2.6. Determination of polysaccharide content Polysaccharide content was determined using a phenolsulphuric acid method and d-glucose as standard [22]. Polysaccharidic extract of residue of raw materials after SCCO2 extraction solutions (0.05 mg/mL) were prepared in distilled water. 1 mL of solution was mixed with 1 mL of 5% aqueous phenol solution and 5 mL of concentrated sulphuric acid. The mixture was stirred for 30 min. The total sugar was determined by UV–vis based on the standard curve for glucose (0.0047–0.15 mg/mL) at a wavelength of 490 nm. The absorbance was measured using Varian, Cary 50 Probe UV-VIS spectrophotometer (±0.0001). The results were expressed as g of glucose equivalents per g of dry weight of polysaccharidic extract. 2.7. Determination of antioxidant activity using the photochemiluminescence method The analyses were carried out using the Photochem analytical system (Analytik Jena, Germany). This system uses the photochemiluminescence method for the determination of antioxidant activity water- and lipid-soluble compounds (ACWKit and ACLKit; Jena AG Company) and allows quantification of the antioxidant state. The results were compared with the calibration curve, and thus quantified and expressed in equivalents of standard; i.e. ascorbic acid for the water-soluble and trolox for lipid-soluble compounds. Sample solutions with concentration 10 mg/mL were prepared by dissolving polysaccharidic extracts of residue of raw materials after SCCO2 extraction into water and SCCO2 extracts into methanol. The samples were then analyzed according to prescribed apparatus procedure. The antioxidant activity of polysaccharidic extracts of residue of raw materials after SCCO2 extraction was reported as g of ascorbic acid per g of extract while antioxidant activity of SCCO2 extracts was reported as g of Trolox standard per g of extract. 2.8. Evaluation of cytotoxic activity Obtained extracts were dissolved in dimethyl sulfoxide (DMSO) and further subjected to cytotoxic activity testing. The HUVEC
D. Cör et al. / J. of Supercritical Fluids 91 (2014) 53–60
55
Fig. 1. Diagram of semi-continuous high-pressure flow up apparatus.
(human umbilical vein endothelial) and CaCo-2 (human colon adenocarcinoma) cell lines were used. The HUVEC cells were grown in advanced Eagle’s minimal essential medium (MEM; Gibco, Invitrogen, UK) and the CaCo-2 cells in advanced RPMI 1640 (Gibco), both at 37 ◦ C in a CO2 incubator (5% CO2 , 95% air; at 95% relative humidity). Both of these culture media were supplemented with 2 mmol/L l-glutamine (Gibco), 100 U/mL penicillin (Gibco), 100 g/mL streptomycin (Gibco) and 5% foetal bovine serum (Gibco). For the in vitro cytotoxicity assays, the cells were plated in 96well microtiter plates (Costar, USA) at 10000 cells/well. After a 3-h incubation, the DMSO-dissolved extracts (or DMSO as a control) prepared in medium without serum, were added to the cells at a final concentration of 0.2 mg/mL up to 0.5 mg/mL, and the incubations were carried out for 1 h in the CO2 incubator. The cells were then washed once with medium, and 100 l fresh medium with foetal bovine serum was added for a further 48 h. The cytotoxicity was determined using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) test. MTS (CellTiter 96 Aqueous Reagent, Promega, USA) was added to the cultured cells in each well (20 l). After 1 h, the absorbance at 490 nm was measured using a microplate reader (Bio-Tek Instruments Inc., USA). The absorbance corresponded to the amount of soluble formazan produced, which is directly proportional to the number of viable cells. The viability (%) is expressed as the ratio between absorbance at 490 nm of the treated and control cells. The data are presented as means ± SD of 3 independent experiments. The differences were analyzed using Student’s t-tests on the two populations, with p < 0.01 considered significant.
2.9. Antiacetylcholinesterase activity Antiacetylcholinesterase (anti-AChE) activity was measured according to the Ellman method [23], 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, Cary 50 Probe) at 405 nm. Inhibition of acetylcholine (AChE) was monitored for 5 min
for each extract. The final concentration of the extract was 1 mg/mL. The effect of the pure solvents (e.g. methanol for SCCO2 extracts and water for polysaccharidic extracts of residue of raw materials after SCCO2 extraction) on the assay was also monitored. The solvent used for the preparation of extract samples did not exceed 5% of the total volume of the reaction mixture. All readings were corrected for their appropriate blanks, and every measurement was repeated at least two times. 3. Results and discussion 3.1. Efficiency of cell wall breaking procedure Before dynamic extraction with SCCO2 , G. lucidum material was exposed to pressurized gas at 300 bar, followed by rapid depressurization (5 s) in order to achieve rupture of cell walls. The untreated and treated G. lucidum material was than examined by ESEM to investigate the morphological changes of the cell walls during the breaking process. The results are presented in Fig. 2. Rupture of cell walls was reported to be the essential step before isolation of biologically active compounds from G. lucidum, due to better access of solvent into the material [15]. G. lucidum material exposed to SCCO2 at a pressure of 300 bar showed morphological changes (Fig. 1c and d). Under ESEM, the intact cells were ovateoblong or ovoid with truncated apex or blunt taper. There were some sinuous depressions or mini holes on the surface of the cell walls. The processed cells showed morphological changes. Some cells had crevices and gaps and some were completely broken. It can be observed that SCCO2 pressurization and rapid depressurization had a strong impact on cell walls as the desired ruptures were achieved. 3.2. Supercritical extraction After cell wall breaking procedure, dynamic extraction with SCCO2 followed. In the present study 8 g of G. lucidum material have been used and the extraction performed under the conditions specified in Table 1. Extraction yield was also calculated (Table 1).
Table 1 SCCO2 extraction conditions and extraction yield. T (◦ C)
P (bar)
t (h)
CO2 (kg/m3 )
mGanoderma extract (g)
Qv (LCO2 h
40 40 50 50
250 300 250 300
4.2 4.5 2.7 4.2
879.7 910.3 834.0 870.9
0.147 0.166 0.118 0.154
2.4 3.2 3.6 3.6
T, temperature; P, pressure; t, time; mGanoderma extract , mass of extract; CO2 , CO2 density; Qv , flow rate; , extraction yield.
−1
)
(%) 1.84 2.07 1.47 1.92
56
D. Cör et al. / J. of Supercritical Fluids 91 (2014) 53–60
Fig. 2. ESEM micrographs of G. lucidum cells. (a and b) Untreated G. lucidum material (magnification 1250× and 5000×). (c and d) G. lucidum material after exposure to SCCO2 at 300 bar followed by rapid depressurization (magnification 2500× and 4000×). Rupture cells are indicated with white arrows.
Extraction Yeld /(%)
2.5
2.0
1.5
1.0
40°C, 300 bar 50°C, 250 bar 50°C, 300 bar 40°C, 250 bar
0.5
0.0 0.0
1.0
2.0
3.0
4.0
5.0
S/F (kgCO2/kgraw material) Fig. 3. SCCO2 extraction yield of G. lucidum fruiting body as a function of solvent to feed ratio in kg CO2 per kg of raw material.
The flow rate of SCCO2 solvent was between 2.4 and 3.6 L h−1 . The extraction times, necessary to reach a constant extraction yield varied from 2.7 to 4.5 h, due to the different operating pressures and temperatures. From the obtained results it can be observed, that higher operating pressure at constant temperature (T = 40 ◦ C) resulted in higher extraction yield (1.84% and 2.07% at pressure 250 bar and 300 bar, respectively) (Table 1 and Fig. 3). Hsu et al. [16]
investigated SCCO2 extraction of G. lucidum at 100 bar and temperature of 40 ◦ C and reported an extraction yield of 0.12%, which fits in the trend observed in our investigation. The shape of the extraction curves on Fig. 3 shows that at the beginning of the extraction the main mechanism is the mass transfer from the particle surface. After that diffusion becomes the controlling mechanism until the maximum and constant extraction yield is being reached. The highest extraction yield was 2.074% obtained at 40 ◦ C and 300 bar, where the solvent density was 910 kg/m3 . The lowest yield was 1.47% obtained at 50 ◦ C and 250 bar, with solvent density 834 kg/m3 . These results show that the amount of the extract is related to solvent power, which is a function of density. Solvation power depends on its density, which increases with pressure at constant temperature and decreases with temperature at constant pressure [24]. The effect of temperature on the extraction yield, at constant pressure, can be followed by two mechanisms: (1) an increase in the process temperature increases the solubility because to solute vapour pressure; (2) the temperature increase reduces the solubility due to decrease in the solvent’s density. These two opposing effects result in the crossover of solubility isotherms [25]. Based on results shown in Fig. 3 it can be suggested that crossover could have happened.
D. Cör et al. / J. of Supercritical Fluids 91 (2014) 53–60
57
Table 2 Yield (%) of hot water extraction and precipitation from G. lucidum residue material previously extracted using SCCO2 at different conditions. Polysaccharidic extract (PE) of raw material after SCCO2 extraction
Extraction yield of hot water extraction (%)
PE250 bar , 40 ◦ C PE250 bar , 50 ◦ C PE300 bar , 40 ◦ C PE300 bar , 50 ◦ C
4.758 2.906 2.380 2.274
3.3. Extractions of polysaccharides 3.3.1. Extraction yield After SCCO2 extraction, hot water extraction and precipitation of polysaccharides from the residue of G. lucidum material followed. The results are summarized in Table 2. Extraction yields of hot water extraction of residue material ranged from 2.274% to 4.758%. In residue material being extracted with SCCO2 at 250 bars and 40 ◦ C probably highest content of hot water soluble polysaccharides remain (e.g. 4.758%). These variations in extraction yield of polysaccharides could be due to the degradation of compounds when higher temperature and pressure were applied. G. lucidum has a complex chemical composition, which contains various active compounds. It was reported that the major bioactive compounds located in the fruiting bodies of G. lucidum are polysaccharides and triterpenoids; however other active compounds also present in small amounts are phenols, sterols, fats and oils, amino acids, vitamins, nucleosides [1,26–29]. Compounds such as polysaccharides as well as phenols or flavonoids can be obtained from the G. lucidum by hot water extraction [8]. 3.3.2. Determination of polysaccharide content Quantitative determination of total polysaccharides in polysaccharidic extracts of residue of raw materials after SCCO2 extraction obtained from G. lucidum was performed using a phenol-sulphuric acid method. The linear calibration curve with the equation y = 2.7472x + 0.0012 and in concentration range 0.0047–0.150 mg/mL was prepared with standard of glucose. The quantities of total polysaccharides in polysaccharidic extracts are shown in Fig. 4 and are expressed as mg of glucose eq. on g of dry polysaccharidic extract. The amount of polysaccharides in the extracts varies between 17.385 and 24.443 mg/g, depending on the conditions used during SCCO2 extraction. ´ et al. [21] also evaluated the polysaccharide Skalicka-Wozniak content in fruiting bodies of G. lucidum after hot water extraction. The polysaccharide content of 36 tested samples varied between
Fig. 4. Quantification of total polysaccharides in polysaccharidic extract of residue of raw materials after SCCO2 extraction. Data expressed as mg of glucose eq. per g of G. lucidum fruiting body polysaccharidic extract. The standard deviation is expressed as ±value, from two replicates, where two independent experiments were performed.
Fig. 5. Antioxidative activity of G. lucidum fruiting body SCCO2 extracts. Data are expressed as equivalent of Trolox in mg per g of SCCO2 extract. The standard deviation is expressed as ±value, from two replicates, where two independent experiments were performed.
18.450 and 112.820 mg/g. Our results are in the range of results ´ obtained by Skalicka-Wozniak et al. [21]. 3.4. Antioxidant activity The antioxidant activity of G. lucidum SCCO2 and polysaccharidic extract of residue of raw materials after SCCO2 extraction are presented in Figs. 5 and 6, respectively. The highest antioxidant activity of SCCO2 extracts was recorded at 50 ◦ C and pressure 300 bar: e.g. 14.517 mg Trolox per g of dry SCCO2 extract (Fig. 5). Furthermore, it can be observed that antioxidant activity increases with increasing SCCO2 extraction temperature at constant pressure, and as well with increasing pressure at constant temperature. The antioxidant activity of polysaccharidic extracts of residue of raw materials after SCCO2 extraction is presented in Fig. 6. The highest antioxidant activity, of 4.467 mg ascorbic acid per g of dry polysaccharidic extract, was obtained at pressure of 300 bar and temperature of 50 ◦ C. The SCCO2 extraction conditions have the same influence on the antioxidant activity of polysaccharidic extract of residue of raw materials after SCCO2 extraction as observed for SCCO2 extracts: higher antioxidant activity was measured after exposure to higher temperature and pressure. The antioxidant activity of extracts depends on SCCO2 extraction conditions. In addition, it can be observed, that compounds both with antioxidant activity can be extracted with SCCO2 and with hot
Fig. 6. Antioxidative activity of G. lucidum fruiting body polysaccharidic extract of residue of raw materials after SCCO2 extraction. Data are expressed as equivalent of ascorbic acid in mg per g of dry polysaccharidic extract. The standard deviation is expressed as ±value, from two replicates, where two independent experiments were performed.
58
D. Cör et al. / J. of Supercritical Fluids 91 (2014) 53–60
Fig. 8. Acetylcholinesterase (AChE) inhibitory activities of SCCO2 extracts from G. lucidum fruiting body. Data are expressed as percentage (%) of AChE inhibition. Data are means ± SD from two replicates, where two independent experiments were performed. Fig. 7. Effect of 0.2 mg/mL concentration of G. lucidum fruiting body SCCO2 extracts on viability of HUVEC and CaCo-2 cell lines (% of control) after 1 h treatment. Data are shown as mean values ± SD for at least three separate experiments.
water. Two stage extractions enable us to extract from G. lucidum both nonpolar and polar compounds with antioxidant activity. 3.5. Cytotoxic activity The cytotoxic activity of G. lucidum SCCO2 and polysaccharidic extracts of residue of raw materials after SCCO2 extraction dissolved in DMSO was tested on HUVEC (human umbilical vein endothelial) and CaCo-2 (human colon adenocarcinoma) cell lines. The tested concentrations ranged from 0.2 to 0.5 mg/mL. Polysaccharidic extracts of residue of raw materials after SCCO2 extraction did not show any activity towards adenocarcinoma cells (results are not presented), while strong and targeted activity towards adenocarcinoma cells was noticed after incubation with SCCO2 extracts (Fig. 7 and Table 3). The results indicate that the compounds from G. lucidum possessing antitumor activity towards adenocarcinoma cells have nonpolar nature, as they were isolated from the starting material only with SCCO2 . It was observed that SCCO2 extracts had strong antitumor activity, even when the lowest concentrations were applied, e.g. 0.2 mg/mL, resulting in viabilities of adenoma cancer cells between 20.8% and 65.4% after only 1 h of incubation (Fig. 7). The variation in cytotoxicity depended on the process conditions used for SCCO2 extraction. The viability of the HUVEC (normal) cells remains higher than 92%. At the given concentration no significant negative effect on the HUVEC cells was observed, therefore results indicate that SCCO2 extracts show targeted cytotoxic activity towards adenocarcinoma cells solely. To our best knowledge this is the first report about targeted antitumor activity of SCCO2 extracts towards adenocarcinoma cells with no effect on normal cells as such. All results for tested concentrations of SCCO2 extracts and their cytotoxic activity towards the cell lines used in this study are summarized in Table 3. In general it was noticed that higher concentrations of SCCO2 extract resulted in greater cytotoxic action towards adenocarcinoma cells; however also cytotoxicity towards normal (HUVEC) cells increased. SCCO2 extracts obtained at a pressure of 250 bar and temperature of 40 ◦ C gave the best results during cytotoxicity testing. However, they also showed significant influence on the viability of the normal cells. In order to avoid high cytotoxicity towards normal cells and still preserve the high cytotoxic activity towards adenocarcinoma cells G. lucidum extraction with SCCO2 should be performed at 300 bars and 40 ◦ C. SC CO2 at defined conditions can selectively extract certain group of chemical compounds and by that affect on composition of the extract obtained. SC CO2 extract
obtained at p = 250 bar and T = 50 ◦ C probably contained different group of chemical compounds that those obtained at p = 300 bar and T = 40 ◦ C therefore higher bioactivity was recorded (Fig. 7 and Table 3). It is thus demonstrated that both the pressure and the temperature applied during extraction with SCCO2 , clearly influence the composition of G. lucium extracts and their cytotoxic activities towards adenocarcinoma cells. 3.6. Antiacetylcholinesterase activity Inhibition of acetylcholinesterase (AChE) has been investigated for both SCCO2 and G. lucidum polysaccharidic extracts. The results of the extracts AChE inhibitory activities are presented in Figs. 8 and 9, where extract concentrations of 1 mg/mL were tested. The AChE inhibition by SCCO2 G. lucidum extracts was between 7.33% and 22.54% (Fig. 8), while the inhibition by polysaccharidic extracts of residue of raw materials after SCCO2 extraction was from 5.12% to 19.24% (Fig. 9). Hasnat et al. [8] studied AChE inhibitory activity of hot water extracts obtained from fruiting body of G. lucidum. The authors observed 50% inhibition of AChE using an extract concentration of 1 mg/mL. Their result is much higher than in present study; however they used crude hot water extracts that contained, beside polysaccharides, also phenols and flavonoids known to inhibit AChE in higher degrees [30]. Up to date there are no reports about the anti-AChE activity of SCCO2 extracts obtained from G. lucidum. The same trend can be noticed for antioxidant and AChE inhibitory activity of extracts obtained at conditions applied. This may indicate that the compounds with antioxidant activity extracted from G. lucidum also elicit anti-AChE activity. The
Fig. 9. Acetylcholinesterase (AChE) inhibitory activities of polysaccharidic extract of residue of raw materials after SCCO2 extraction from G. lucidum fruiting body. Data are expressed as percentage (%) of AChE inhibition. Data are means ± SD from two replicates, where two independent experiments were performed.
D. Cör et al. / J. of Supercritical Fluids 91 (2014) 53–60
59
Table 3 Effect of higher concentrations (0.2–0.5 mg/mL) of SCCO2 G. lucidum extracts on viability of HUVEC and CaCo-2 cell lines (% of control) after 1 h treatment. Data are shown as mean values ± SD for at least three separate experiments. SCE conditions
Cell lines
0.2 mg/mL
250 bar 40 ◦ C 250 bar 50 ◦ C 300 bar 40 ◦ C 300 bar 50 ◦ C
HUVEC CaCo-2 HUVEC CaCo-2 HUVEC CaCo-2 HUVEC CaCo-2
92.5 35.3 93.8 49.5 93.9 20.8 92.7 65.4
± ± ± ± ± ± ± ±
11.4 12.3 12.2 13.3 12.9 8.7 12.1 11.9
same observations were made by in Hasant et al. [9] for hot water extracts. The effect of SCCO2 extraction conditions on SCCO2 anti-AChE activity and polysaccharidic extracts of residue of raw materials after SCCO2 extraction was also evaluated. From Fig. 8 it can be clearly observed that at both applied pressures (250 and 300 bar) the AChE inhibitory activity of SCCO2 extracts increases with increasing extraction temperature. In both cases, e.g. SCCO2 and polysaccharidic extracts of residue of raw materials after SCCO2 extraction the best AChE inhibitory activity was recorded when SCCO2 extraction conditions were 300 bar and 50 ◦ C (Figs. 8 and 9). Overall, the results indicate that G. lucidum contains different nonpolar and polar compounds with anti-AChE activity since both SCCO2 and polysaccharidic extracts of raw materials after SCCO2 extraction showed potential to inhibit AChE during bioassay. Additionally, the 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. G. lucidum extracts with anti-AChE activity could be used as support treatment in patients diagnosed with Alzheimer or other neurodegenerative diseases.
4. Conclusion The present study provides that SCCO2 extracts elicited significant targeted activity towards adenocarcinoma cells and had antioxidant and anti-AChE activity. The G. lucidum material, which remained after SCCO2 , was rich in water soluble polysaccharides eliciting antioxidant and antiacetylcholinesterase activity, but not showing antitumor activity. It was observed that the extracts with the highest antioxidant activity elicited also the strongest AChE inhibitory activity. In the case of cytotoxicity this relation was not observed. Extraction conditions show important influence on the properties of extracts with high biological activity. It can be concluded that SCCO2 can be used to obtain G. lucidum extracts rich in compounds with strong and targeted antiadenocarcinoma activity. G. lucidum material after SCCO2 extraction can be reused to obtain polysaccharides with antioxidant and antiacetylcholinesterase activity. Therefore G. lucidum represents a rich source of natural compounds with different polarities, for potential treatment of adenocarcinoma, Alzheimer’s and other neurodegenerative diseases as well as degradation of radical oxygen species.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgments The authors gratefully acknowledge the Slovenian Research Agency (Research Project L2 – 4124). The authors wish to thank Dr. Elena Markoˇciˇc for editorial assistance with the manuscript.
0.3 mg/mL 55.2 ± 13.8 0 39.0 ± 17.4 0 68.8 ± 17.7 7.2 ± 3.9 70.3 ± 12.4 0
0.4 mg/mL
0.5 mg/mL
50.1 ± 10.5 0 10.0 ± 5.2 0 48.4 ± 16.7 0 40.1 ± 10.9 0
17.4 ± 7.2 0 2.3 ± 2.1 0 32.6 ± 10.3 0 13.6 ± 8.5 0
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, pp. 251. [3] S.A. Heleno, L. Barros, A. Martins, M.J.R.P. Queiroz, C. Santos-Buelga, ICFR, Ferreira Fruiting body, spores and in vitro produced mycelium of Ganoderma lucidum from Northeast Portugal: a comparative study of the antioxidant potential of phenolic and polysaccharidic extracts, Food Research International 46 (2012) 135–140. [4] D.K. Lahiri, M.R. Farlow, N.H. Greig, K. Sambamurti, Current drug targets for Alzheimer’s disease treatment, Drug Development Research 56 (2012) 267–281. [5] F. Liu, V.E.C. Ooi, S.T. Chang, Free radical scavenging activities of mushroom polysaccharide extracts, Life Sciences 60 (1997) 763–771. [6] H.Y. Fu, D.E. Shieh, C.T. Ho, Antioxidant and free radical scavenging activities of edible mushrooms, J. of Food Lipids 9 (2002) (2002) 35–43. [7] R. Russell, M. Paterson, Ganoderma – a therapeutic fungal biofactory, Phytochemistry 67 (2006) 1985–2001. [8] 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. [9] X.Y. Cui, S.Y. Cui, J. Zhang, Z.J. Wang, B. Yu, Z.F. Sheng, X.Q. Zhang, Y.H. Zhang, Extract of Ganoderma lucidum prolongs sleep time in rats, J. Ethnopharmacology 139 (2012) 796–800. [10] S. Mekkawy, M.R. Meselhy, N. Nakamura, Anti-HIV-1 and anti-HIV-1 protease substances from Ganoderma lucidum, Phytochemistry 49 (2007) 1651–1657. [11] J.H. Ni, B.S. Song, H.B. Li, Y.S. Xie, Breaking of the sporoderm of Ganoderma lucidum and its effect on the extraction yield of polysaccharides, Chinese Traditional Herbal Drugs 33 (2002) 422. [12] Q. Li, Y. Li, P. Zhang, Z.Q. Xing, P.L. Kang, The effect on the distill of amylose in glossy Ganoderma sporule powder treated with ultrasonic and thawing and freezing test of liquid nitrogen, J. of Liaoning University 31 (2004) 81–82. [13] H. Wu, S.Q. Zhang, J.J. Zhu, Broken Ganoderma lucidum spores by breakingextracting method, J. of Jilin University 34 (2004) 312–315. [14] Y.Z. Xie, S.Z. Li, A. Yee, D.P. La Pierre, Z. Deng, D.Y. Lee, Ganoderma lucidum inhibits tumor cell proliferation and induces tumour cell death, Enzyme and Microbial Technology 40 (2006) 177–185. [15] Y.J. Fu, W. Liu, Y.G. Zu, X.G. Shi, Z.G. Liu, G. Schwarz, T. Efferth, Breaking the spores of the fungus Ganoderma lucidum by supercritical CO2 , Food Chemistry 112 (2009) 71–76. [16] R.C. Hsu, B.H. Lin, C.W. Chen, The study of supercritical carbon dioxide extraction for Ganoderma lucidum, Industrial & Engineering Chemistry Research 40 (2001) 4478–4481. [17] J. Zhang, J.C. Duan, Z. Liang, W.B. Zhang, L.H. Zang, Y.S. Huo, Y.K. Zhang, Analysis of triterpenoids in fruiting bodies of Ganoderma lucidum with off-line supercritical fluid extraction – high performance liquid chromatography system, Chinese J. of Analytical Chemistry 34 (2006) 447–450. [18] T.Q. Chen, J.G. Wu, Y.B. Wu, H.Y. Wang, F.H. Mao, J.Z. Wu, Supercritical fluid CO2 extraction, simultaneous determination of total sterols in the spore lipids of Ganoderma lucidum by GC–MS/SIM methods, Chemistry of Natural Compounds 48 (2012) 657–658. [19] M.S. Lin, Z.R. Yu, Y.M. Weng, Study of continuous extraction process utilizing supercritical fluid for G. lucidum, National Resource of Sustainable Development II 524–527 (2012) 2310–2315. ˇ [20] M. Hadolin, M. Skerget, Zˇ . Knez, High pressure extraction of vitamin E-rich oil from Silybum marianum, Food Chemistry 74 (2001) 355–364. ´ [21] K. Skalicka-Wozniak, J. Szypowski, R. Ło´s, M. Siwulski, K. Sobieralski, K. Głowniak, A. Malm, Evaluation of polysaccharides content in fruit bodies and their antimicrobial activity of four Ganoderma lucidum (W. Curt.: Fr.) P. Karst. strains cultivated on different wood type substrates, Acta Societatis Botanicorum Poloniae 81 (2012) 17–21. [22] M. DuBois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substance, Analytical Chemistry 28 (1956) 350–356. [23] G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochemical Pharmacology 7 (1961) 88–95.
60
D. Cör et al. / J. of Supercritical Fluids 91 (2014) 53–60
[24] G. Brunner, Gas Extraction: An Introduction to Fundamentals of Supercritical Fluids and the Application to Separation Processes, vol. 4, Steinkopff, Darmstadt, 1994. [25] E.M.Z. Michielin, L.F.V. Bresciani, L. Danielski, R.A. Yunes, S.R.S. Ferreira, Composition profile of horsetail (Equisetum giganteum L.) oleoresin: comparing SFE and organic solvents extraction, J. of supercritical fluids 33 (2005) 131–138. [26] H.C. Wang, S.Y. Sun, Analysis on the amino acids and trace elements in wild and artificial cultivation Ganoderma lucidum collected from Taishan, Traditional Chinese Medicine 12 (1990) 14. [27] M.W. Zhao, C.G. Wang, P. Bao, Z.T. Xing, W.Q. Liang, J.Q. Yu, X.D. Shang, D.Y. Men, N. Wang, Y.J. Pan, Triterpenes and polysaccharide contents of liquid culturing
mycelium of different Ganoderma lucidum, Edition Fungus Chinese 22 (2002) 43–46. [28] A.V. Avtonomova, L.M. Krasnopol’skaya, V.N. Maksimov, Optimization of nutrient medium for submerged cultivation of Ganoderma lucidum (Curt.: Fr.) P. Karst, Microbiology 75 (2006) 148–153. [29] J. Lu, J.Z. Qin, P. Chen, X. Chen, Y.Z. Zhang, S.J. Zhao, Quality difference study of six varieties of Ganoderma lucidum with different origins, Frontiers in Pharmacology 3 (2012) 7. [30] I. Orhan, M. Kartal, F. Tosun, B. Sener, Screening of various phenolic acids and flavonoid derivatives for their anticholinesterase potential, Zeitschrift für Naturforschung C 62 (2007) (2007) 829–832.