Anguera, M. (1996) Diseños. En: Fernández-Ballesteros, R. (Ed.) Evaluación de programas. Una guía práctica en ámbitos sociales, educativos y de salud. Editorial Síntesis. Madrid. Advanced Strategic Management Consultants (1999). How to Measure (and Prove) the Success of Your Organization (and its use of technology) in Fulfilling Purpose, Mission and Values. Presented at the Strategy Institute Conference: Leveraging the Power of Technology and the Internet for Non-Profits, Toronto.
Zdravljeizgljiva.hrInternational Journal of Medicinal Mushrooms, 15(5): 435–448 (2013) Cytotoxicity of Blended Versus Single Medicinal
Mushroom Extracts on Human Cancer Cell Lines:
Contribution of Polyphenol and Polysaccharide
Ksenija Durgo,1 Mladen Koncar,1 Drazenka Komes,1 Ana Belscak-Cvitanovic,1 Jasna Franekic,1
Ivan Jakopovich,2* Neven Jakopovich,2 & Boris Jakopovich2
1Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia; and 2Dr Myko San–Health from Mushrooms Company, Zagreb, Croatia *Address all correspondence to: Ivan Jakopovich, Dr Myko San–Health from Mushrooms Company, Miramarska 109, 10 000 Zagreb, Croatia; ABSTRACT: The use of mushrooms contributes to human nutrition by providing low lipid content of lipids and high
dietary fiber content, as well as significant content of other biologically active compounds such as polysaccharides, minerals, vitamins, and polyphenolic antioxidants. This study aimed to determine the content of polyphenols and polysaccharides, as well as the cytotoxic and antioxidative properties of several medicinal mushroom preparations. The content of total phenols and flavonoids of preparations of blended mushroom extracts (Lentifom, Super Polyporin, Agarikon, Agarikon Plus, Agarikon.1, and Mykoprotect.1) was evaluated quantitatively by using ultraviolet–visible spectroscopy spectrophotometric methods. The antioxidant capacity of the preparations was evaluated using the ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and ferric reducing/antioxidant power assays. The content of water-soluble polysaccharides was determined using a specific gravimetric method, based on ethanol precipitation. To determine cytotoxic effects of single and blended mushroom extracts, MTT (3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide) and neutral red assays were conducted using human small cell lung cancer, lung adenocarcinoma, colon cancer, and brain astrocytoma cancer cells. The obtained results suggest that due to the significant content of beneficial polyphenolic antioxidants and soluble polysaccharides, use of these mushroom preparations is beneficial in maintaining good health, as well as in the prevention and adjuvant biotherapy of various human pathological aberrations. These results reveal that these extracts exhibit different cytotoxic effects on tumor cells originating from different tissues. In addition, the comparison of investigated blended mushroom extracts with three well-known commercial mushroom products derived from single mushroom species or single mushroom compounds shows that blended mushroom extracts exhibit significantly stronger cytotoxic effects on human tumor cell lines.
KEY WORDS: medicinal mushrooms, cancer, colon cancer, small cell lung carcinoma, lung adenocarcinoma, brain
astrocytoma, human tumor cell lines, cytotoxicity, polysaccharides, polyphenols, antioxidants
ABBREVIATIONS: ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid); Caco2, human colon cancer
cell line; CCTG-1, human astrocytoma cell line; FRAP, ferric reducing/antioxidant power; GAE, gallic acid equiva-
lent; H69V, human small cell lung carcinoma cell line; MEM, Minimum Essential Medium Eagle growth medium
(Sigma-Aldrich); MGN, immunobran MGN-3 arabinoxylan compound; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; NR, neutral red assay (3-amino-7-dimethylamino-2-methylphenazine hydrochloride);
PSK, polysaccharide-K (Krestin); PSP, polysaccharide peptide; ROS, reactive oxygen species; SKLU-1, human lung
adenocarcinoma cell line; TFC, total flavonoid content; TPC, total phenol content
cancer in humans and other mammals.1–3 A vast number of experimental studies, beginning in the Millennial experience of traditional medicine and United States in the late 1950s and followed mainly more than 60 years of scientific research have con- by Japanese and Chinese researchers in the 1970s clusively proven that medicinal mushrooms inhibit and 1980s, determine whether the antitumor activity 1045-4403/13/$35.00 2013 Begell House, Inc. www.begellhouse.com Durgo et al.
of some mushrooms exists and if so, what the tu- were also determined. Therefore, as part of ongoing mor inhibition rates and complete regression rates research on medicinal mushrooms and their bioac-are. Research began on animal and human tumor tive compounds and biological activities, commer-cell lines and animal models, followed by human cially available preparations of several medicinal trials and clinical studies.1–4 Following the human mushrooms were screened for their antioxidant clinical trials of single mushroom compounds, the properties and biological activity on human tumor first official antitumor drugs from medicinal mush- cell lines using an array of in vitro assays.
rooms were registered, including PSK (1977), Len- tinan (1985), and Schizophyllan (1986) in Japan, II. MATERIALS AND METHODS
and PSP (1983) in China. Such drugs are limited
to the Far Eastern countries, whereas medicinal A. Medicinal Mushroom Extracts
mushroom supplements are used as oncological
therapy support in Western countries.1
Dr Myko San–Health from Mushrooms Com- New studies address interpretation of molecu- pany (Zagreb, Croatia) supplied their commer- lar mechanisms related to the anticancer effects of cial blended mushroom products, which included medicinal mushrooms. In addition to the immu- four liquid extracts (Lentifom, Super Polyporin, nomodulation mainly caused by high-molecular- Agarikon, and Agarikon Plus) and two in tablet weight polysaccharides, polysaccharide-protein form (Agarikon.1 and Mykoprotect.1). Lentifom complexes, and proteins produced during primary is a proprietary extract blend of 3 medicinal mush-metabolism, other compounds (especially low- room species, whereas Super Polyporin contains 7, molecular-weight polysaccharides, polyphenols, Agarikon contains 8, and Agarikon Plus contains flavonoids, triterpenes, tocopherols, and carotenes) 10 mushroom species, including the most well-and certain secondary metabolic products directly known ones such as Lentinus edodes, Ganoderma affect cancer cells through interaction with in- lucidum, Trametes versicolor, Grifola frondosa, tracellular signaling pathways and changing the and Agaricus brasiliensis (=blazei ss. Heinem.). course of inflammation, cell differentiation, sur- Agarikon.1 is made from L. edodes, G. lucidum, vival, apoptosis, angiogenesis, tumor progression, A. brasiliensis, G. frondosa, and Pleurotus ostrea-and so forth.4,5 tus, with 750 mg of mushroom polysaccharides per Since there is a multitude of evidence that tablet (the standard daily dosage is three tablets per single compounds from medicinal mushroom ex- day). Mykoprotect.1 is made from L. edodes and tracts possess certain biological activities, there are G. lucidum, and contains 850 mg of mushroom tendencies to isolate new, prospective, and synthe- polysaccharides per tablet (the standard daily dos- sizable compounds. Most recent studies focus on age is three tablets per day).
single compounds or "the best" single mushroom For comparison, we included three commer- species; however, for many mushroom species, it cial single mushroom products (extracts produced is not known whether a single compound or a syn- in a similar manner as described below): PSP ergy of multiple ingredients causes the observed (from T. versicolor; KunShan Long-Teng Biotech effects. Empirical evidence and studies6 strongly Manufacture, KunShan City, China), ImmunoBran support the superiority of the blends. In addition, MGN-3 (from L. edodes; Daiwa Pharmaceutical, the optimal daily dose and extraction conditions Tokyo, Japan), and β-glucan (from Saccharomy-for most compounds remain unknown.7 ces cerevisiae; Transfer Point Inc. Columbia, SC, In this work, the content of bioactive com- pounds of mushroom preparations was elucidated, We extracted 50 g of dried mushroom fruit- especially in terms of polyphenol and polysac- ing bodies in 1 L of boiling water for 24 h using a charide content. The dosages of active mushroom Dr Myko San–Health from Mushrooms Company compounds that provide effective cytotoxic action proprietary production method. Insoluble matter International Journal of Medicinal Mushrooms Cytotoxic Effects of Medicinal Mushroom Extracts on Human Cancer Cells was removed by forcing the solution (in suspen- no-2-methylphenazine hydrochloride), and MTT sion form) through a filter press, and then concen- (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-trating it 4-fold. For tablet preparations, hot water lium bromide) were obtained from Sigma-Aldrich extract was precipitated with ethanol and dried.
(Steinheim, Germany). Growth media (MEM and To measure dry matter content, we used the RPMI 1640, respectively), fetal bovine serum, and Association of Official Analytical Chemists meth- penicillin-streptomycin solution were purchased od8 of drying the material in an oven at 105°C until from Gibco Co. (Carlsbad, CA, USA).
constant weight was achieved. The moisture con-tent was determined by the weight difference be- C. Determination of Polyphenolic
fore and after drying, whereas the dry matter was Compounds
the ratio of the final to the initial sample weight percentage.
Total phenol content (TPC) of medicinal mush- To determine the phenolic content and anti- room preparations was determined spectropho- oxidant capacity, liquid extracts were used; tablets tometrically according to a modified Lachman were ground using a mortar and pestle and dis- method.9 To determine total flavonoid content solved in distilled water (1 g/25 mL).
(TFC), these compounds were precipitated using To determine the cytotoxic effects, the liquid formaldehyde, which reacts with C-6 or C-8 on extracts and dissolved tablets were evaporated 5,7-dihydroxy flavonoids. The condensed products until dry and dissolved in corresponding growth of these reactions were removed by filtration and medium (MEM and RPMI 1640, respectively). remaining nonflavonoid phenols were determined Following the manufacturers' recommendations according to the previously mentioned procedure for treatment doses, the prescribed amounts of for the determination of TPC. Flavonoid content bioactive compounds were determined and used was calculated as the difference between total phe-in order to prepare the extract concentrations that nol and nonflavonoid content. Gallic acid was used correspond to 0.1, 1, 10, and 100 times of the pre- as the standard and the results were expressed as scribed amounts.
milligrams of gallic acid equivalents (GAE) per li- The recommended dosages for Dr Myko San ter. All measurements were performed in triplicate.
products were developed during the company's 23
years of operation. Early dosages were determined D. Determination of Antioxidant Capacity
from published research and later refined through
systematic follow-up and evaluation of product 1. Ferric Reducing/Antioxidant Power
use in several thousand patients. Cancer patients
today receive 3.5–7 g/d of active medicinal mush-
The ferric reducing/antioxidant power (FRAP) as- room compounds.
say was carried out according to a standard pro- cedure by Benzie and Strain.10 The FRAP assay is B. Chemicals
based on the reduction of the Fe3+-2,4,6-tripyridyl- S-triazine complex (Fe3+-TPTZ) to the ferrous form Analytical grade Folin–Ciocalteu, formic acid, (Fe2+). Antioxidant activity of the samples was potassium peroxydisulfate, sodium carbonate, monitored by measuring the change of absorption formaldehyde, ferric chloride hexahydrate, ferrous at 593 nm. FRAP reagent was prepared by mix-sulfate heptahydrate, ethanol, and hydrochloric ing acetic buffer, TPTZ, and FeCl ×6⋅H O (20 mM acid were supplied by Kemika (Zagreb, Croatia). water solution) at a ratio of 10:1:1. Briefly, 3.8 mL Trolox (6-hydroxy-2,5,7,8-tetramethylchromane- of FRAP reagent was added to a 200 µL volume 2-carboxylic acid), ABTS (2,2-azino-bis(3-eth- of the sample. After 4 min, the absorbance of blue ylbenzthiazoline-6-sulphonic acid)diammonium coloration was measured against a blank sample. salt), neutral red (NR) (3-amino-7-dimethylami- All measurements were performed in triplicate. Volume 15, Number 5, 2013
Durgo et al.
Aqueous solutions of FeSO ×7H O (100–1000 precipitates collected by centrifugation (10,000 mmol/L) were used for the calibration and the re- rpm for 15 min at 20°C) were solubilized in de-sults are expressed as millimoles of Fe(II) per liter.
ionized water and lyophilized to obtain the crude polysaccharides. The tablet preparations were pri- 2. ABTS Radical Scavenging Assay
marily ground to obtain a fine powder and were then dissolved in distilled water as described above The Trolox equivalent antioxidant capacity in order to obtain liquid samples. The content of (TEAC) was also estimated by the ABTS radical soluble polysaccharides was determined according cation decolorization assay.11 This method is based to the previously described procedure. Polysaccha-on the scavenging of stable blue-green ABTS radi- ride yield was expressed as milligrams per gram of cal cations (ABTS+), which are formed either by dry matter of sample. chemical or enzymatic oxidation of ABTS several hours prior to the analysis. The improved tech- F. Human Cell Lines
nique for the generation of ABTS•+ applied here in-volves direct production of the blue-green ABTS•+ The cytotoxic effect of mushroom extracts was chromophore through the reaction between ABTS examined on four human cancer cell lines: hu-and potassium persulfate. Stock solutions of ABTS man colon cancer cells (Caco-2), human lung ad-(7 mM) and potassium peroxydisulfate (140 mM) enocarcinoma cells (SKLU-1), human small lung in water were prepared and mixed together to a carcinoma cells (H69V), and human astrocytoma final concentration of 2.45 mM potassium per- cells (CCTG-1). All human cancer cell lines were oxydisulfate. The mixture was left to react for purchased from the European Collection of Cell 12–16 h at room temperature in the dark. Prior to Cultures/Sigma-Aldrich. Cancer cell lines were the analysis, the ABTS radical solution was diluted grown as monolayer cultures in MEM (Caco-2 and with ethanol to an absorbance of 0.70 (±0.02) at SKLU-1) and RPMI (H69V and CCTG-1) media 734 nm. All measurements were performed as fol- (Gibco) and were supplemented with 10% fetal bo- lows: 20 µL of the sample was added to 2.0 mL vine serum (Gibco). Growth medium served as the of the ABTS radical solution, and the absorbance negative control.
readings were taken after exactly 6 min against the
appropriate reagent blank instead of the sample. G. Cytotoxicity Assay
The results, obtained from triplicate analyses, were
expressed as Trolox equivalents and derived from NR and MTT assays were used to determine cy-a calibration curve determined for this standard totoxic effects of single mushroom extracts and (100–1000 µmol/L). mushroom blends in order to define whether ac- tive compounds destabilize the membrane or in- E. Determination of Soluble
hibit mitochondrial activity. Cells were seeded in microtiter plates at a concentration of 6×103 cells/well. After 24-hour incubation, cells were treated The content of soluble polysaccharides was deter- with mushroom extracts for 72 hours. After treat- mined according to a modified method of Wei et ment, the MTT and NR assays were performed al.12 Briefly, liquid mushroom extracts were centri- as described by Babich and Borenfreund13 and fuged (10,000 rpm for 15 min at 20°C) to collect Mishchish et al.,14 respectively. The absorbance the supernatant, which was subsequently concen- intensity was measured at 432 nm and 540 nm, re-trated by vacuum evaporation until approximately spectively, using a microplate reader (Cecil Instru-35° Brix was reached. The obtained concentrate ments Ltd., Cambridge, UK). Each concentration was precipitated by the addition of two volumes of was tested in quadruplicate and each experiment ethanol to a final concentration of 75% (v/v). The was repeated three times.
International Journal of Medicinal Mushrooms Cytotoxic Effects of Medicinal Mushroom Extracts on Human Cancer Cells TABLE 1. Composition and Antioxidant Activity of Blended Mushroom Extracts
(mg/g dry matter)
H. Statistical Analysis
in Table 2. According to the obtained results, the content of soluble polysaccharides correlated Statistical analysis was performed using SPSS well only with the cytotoxic effect exerted in the software (version 8.0, SPSS Inc., an IBM Com- SKLU-1 cell line (r SKLU/polysaccharides pany, Chicago, IL, USA). A one-way analysis of lowed by a low correlation in the astrocytoma cell variance was employed to determine whether the line (r = 0.415). A higher correla- means obtained with various groups differed sig- tion was observed between the polyphenolic com- nificantly from each other. Significance was estab- pounds, antioxidant capacity, and cytotoxic effects, lished using the Schaffer and Tukey post hoc tests. thus linking the potential cytotoxic effects to poly- A probability level <0.05 was considered signifi- phenolic compounds, rather than polysaccharides. cant. All data are expressed as means ± standard TFC and antioxidant potential of examined extracts deviations (SD) of the values obtained by three in- showed good correlation to the H69V cell line rH69V/ABTS = 0.718, and rH69V/FRAP = 0.568). A high correlation coefficient was obtained III. RESULTS
in the case of total flavonoid and polyphenol com-pounds and astrocytoma cells (rastroMTT/TPC, rastroMTT/TFC Table 1 presents the dry matter content and bio- = 0.805 and r = 0.766). Medium correlation active composition of six mushroom preparations. was obtained between SKLU-1 and CaCo2 cells The highest content of soluble polysaccharides was (NR cytotoxicity test; rSKLU-1/CaCo2 = 0.571) as well detected in tablet products Agarikon.1 and Myko- as CaCo2 and H69V (MTT assay and NR cytotoxic- protect.1. The highest content of total polyphenols ity test, respectively: rCaCo2/H69V = 0.571 and rCaCo2/H69V and flavonoids was measured in the Agarikon Plus = 0.683).
mushroom preparation (908.33 mg GAE/L) fol- Human colon cancer cells were resistant to lowed by Agarikon (839.17 mg GAE/L), where- the majority of investigated extracts at the recom- as Lentifom exhibits the lowest content of total mended daily doses. The higher concentrations phenols and flavonoids (339.58 mg GAE/L). The of Agarikon.1 and Agarikon Plus (10× and 100×) ranking of mushroom preparations based on their significantly decreased cell survival. A slight cyto-antioxidant potential corresponds to those obtained toxic effect at the therapeutic dose was observed for the TPC, which is confirmed by a high correla- after treatment with Agarikon and Agarikon.1. Su-tion obtained between the results (r = 0.966 per Polyporin decreased cell survival in a dose-re and rTPC/FRAP = 0.954).
sponse manner, with observable cytotoxic effects Correlation coefficients (r) of all evaluated at the therapeutic dose (Figure 1).
bioactive and cytotoxic parameters are displayed Agarikon influenced membrane permeability Volume 15, Number 5, 2013
Durgo et al.
International Journal of Medicinal Mushrooms Cytotoxic Effects of Medicinal Mushroom Extracts on Human Cancer Cells FIGURE 1. Comparison of human colon cancer cell (Caco-2) survival measured by the MTT assay (A) and the NR
at the therapeutic dose after treatment of human destabilized cell membranes. Agarikon.1 and adenocarcinoma cells. No effect on mitochondri- Mykoprotect.1 strongly decreased cell survival al dehydrogenase activity was observed. On the at the therapeutic dose, affecting both mitochon- contrary, Agarikon Plus inhibited mitochondrial drial activity and membrane selectivity. In addi-activity at the therapeutic dose and caused a loss tion, Super Polyporin caused cytotoxic effects in of membrane selectivity to some extent (Figure 2). dose-dependent manner. Cytotoxicity was detect- Super Polyporin decreased membrane stability af- ed on both levels, with decreased membrane and ter treatment of SKLU-1 cells with the therapeutic mitochondrial activity (Figure 3). Lentifom and dose.
Super Polyporin had strong cytotoxic effects on Agarikon Plus decreased mitochondrial de- human astrocytoma cells (Figure 4). All investi- hydrogenase activity of small cell lung carcinoma gated extracts showed significant cytotoxic effects cells in a dose-response manner, while it slightly at concentrations 10 or 100 times higher than the Volume 15, Number 5, 2013
Durgo et al.
FIGURE 2. Comparison of human lung adenocarcinoma cells (SKLU-1) survival measured by the MTT assay (A)
and the NR method (B).
therapeutic concentration. The cytotoxic effect of tive stress–related diseases, consequences of ion-medicinal mushroom blends was stronger than that izing radiation) are similar for both edible and in-of single mushroom species extracts.
edible mushrooms.15–18 Polysaccharides are the best known and most potent mushroom-derived antitumor and immuno-modulating substances.19 On the other hand, plant The use of edible and inedible mushroom prepara- polyphenolic antioxidants are an important group tions in medicinal purposes as a supportive therapy of secondary metabolites because of their contribu-is well known and documented.15 The compounds tion to human health and their multiple biological in the majority of mushrooms with biological ac- effects, such as antioxidant activity, antimutagenic tivity against infections (both viral and microbial, and/or anticancerogenic activities, and anti-inflam-high blood pressure, cancer development, oxida- matory action. However, there is a lack of data re- International Journal of Medicinal Mushrooms Cytotoxic Effects of Medicinal Mushroom Extracts on Human Cancer Cells FIGURE 3. Comparison of human small cell lung carcinoma cell (H69V) survival measured by the MTT assay (A)
and the NR method (B).
garding their content in mushrooms, especially in saccharides (e.g., 6.8% in L. edodes versus 16.0% blended mushroom extracts.
in T. fuciformis16) (Table 1), confirming the effec- This work determined the content of soluble tive polysaccharide extraction procedure devel- polysaccharides, total polyphenols, and flavo- oped by the producer. According to the findings of noids and the antioxidant capacity of six blended Song and van Griensven,21 the polysaccharide con- mushroom extracts, four liquid extracts (Agarikon, tent of three mushroom mixtures ranges from 1.2 Agarikon Plus, Lentifom, and Super Polyporin) to 15.0 mg glucose equivalent/mL (mg GE/mL), and two tablet products (Agarikon.1 and Myko- which is higher than the mushroom blends evaluat- ed in our study.21 The same authors also evaluated Compared with single mushroom species, the the TPC of several medicinal mushroom species blended mushroom preparations analyzed in this and their mixtures. Their results showed high vari-study contained a higher content of soluble poly- ability of polyphenolic content in the same mush- Volume 15, Number 5, 2013
Durgo et al.
FIGURE 4. Comparison of human astrocytoma cell (CCTG-1) survival measured by the MTT assay (A) and the NR
room species derived from different geographical tween the liquid and tablet preparations can also be origins; the TPC of their three mushroom mixtures observed in the content of soluble polysaccharides, ranged from 213.8 to 647.8 µg GAE/mL, which since the tablet preparations exhibit almost double is in accordance with the results obtained in this the content compared to the liquid ones. It is well study. However, the results indicate that both phe- known that one of the main characteristics of poly- nolic content and polysaccharides depend primar- phenols is their propensity to form complexes with ily on the composition of mushroom blends.21 proteins, polysaccharides, and alkaloids (e.g., caf- The results displayed in Table 1 also reveal a feine).22–24 Therefore, in tablet preparations with higher content of total phenols in liquid mushroom higher polysaccharide content, the formation of blends, rather than in the tablets. This may be due polyphenol-polysaccharide complexes may have to the use of other substances during tablet form- occurred, leading to underestimation of total phe- ing process, which may interfere in the analytical nols in these mushroom blends. However, this determination of polyphenols. The difference be- could also be attributed to the mushroom composi- International Journal of Medicinal Mushrooms Cytotoxic Effects of Medicinal Mushroom Extracts on Human Cancer Cells tion (species) in each evaluated mushroom blend. ma cells and colon cancer cells in a dose-depen-In addition, there were no proteins or peptides in dent manner, whereas it caused a slight decrease the examined extracts because these compounds in cell survival in other cell lines. Polysaccharide-were degraded during mushroom extract prepara- rich extracts, Agarikon.1 and Mykoprotect.1, were tion (unpublished data). Therefore, it can be con- the most effective through both mitochondrial cluded that polyphenols, flavonoids, and polysac- and membrane activity against small cell lung charides play a crucial role in biological activity of carcinoma cells and human lung adenocarcinoma the examined extracts.
cells. Agarikon.1 decreased cell survival of small High correlation coefficients observed be- cell lung carcinoma and human lung adenocarci- tween the TPC and antioxidant capacities of mush- noma cells by 80%. Super Polyporin and Myko-room blends indicate that antioxidant activity of protect.1 also inhibited growth of small cell lung the examined mushroom preparations is attributed carcinoma and human lung adenocarcinoma cells to the polyphenolic compounds, rather than poly- at 50% and 30%, respectively. Single pharmaceuti- saccharides. Although Song and van Griensven21 cal compound PSP decreased cell survival at 20% determined a high correlation among the polyphe- (Figure 3), pointing out that mushroom blends nols and polysaccharides of different medicinal have stronger cytotoxic effects compared with the mushroom extracts (R=0.82), our study revealed single compound.
no connection between these compounds (Table 2). A possible explanation of observed cytotoxic According to Lu and Ding,25 naturally derived an- effects lies in the fact that polysaccharide extracts tioxidant compounds of Coprinus comatus include of various mushrooms are highly active in reactive total phenols, flavonoids, tocopherols, and poly- oxygen species (ROS) generation; however, there saccharides. The results of our study revealed no is evidence that some 1,4 glucans cannot induce correlation between the content of polysaccharides ROS.12,21 ROS mediate in signal transduction and and antioxidant capacity of mushroom blends.
regulation of diverse processes, such as phagocyte Tablets contain the highest content of polysac- activation, cell proliferation, and migration, and fi- charides, while Agarikon and Agarikon Plus pos- nally apoptosis. Molecular damage caused by ROS sess the highest content of polyphenols and flavo- in normal cells, induces repair mechanisms, while noids. The comparison of cancer cell line survival in tumor cells ROS activate cell death processes after treatment with the six blends and single com- through apoptosis.22 Shnyreva et al.5 found that pounds reveals a strong cell type–specific cytotox- ROS-dependent activation of apoptotic cell death ic effect. Agarikon was the most effective against is crucial to antitumor activity.
human lung adenocarcinoma cells (SKLU-1) at the In this work, we showed that Agarikon and therapeutic dose. This effect was detected through Agarikon Plus, which have high polyphenol and changes in membrane active transport. This com- flavonoid content, decrease cell survival of hu- pound caused decreased mitochondrial activity man lung adenocarcinoma cells by 70% and in colon cancer cells (Caco2) and small cell lung 30%, respectively, at the daily recommended dose carcinoma cells (H69V) at the therapeutic dose to (Figure 2). Astrocytoma cells were most sensitive some extent.
to Super Polyporin and Lentifom, which inhibited Agarikon Plus inhibited mitochondrial dehy- astrocytoma cell growth by 30% (Figure 4). These drogenase activity in human lung adenocarcinoma blends contain the lowest levels of polysaccha-cells and colon cancer cells, while astrocytoma rides, polyphenols, and flavonoids. It is possible cells were susceptible to Lentifom at the therapeu- that the synergistic effect of all three major groups tic dose through mitochondrial activity depriva- of compounds decreased astrocytoma cell survival. In addition, Lentifom is rich in lentinan polysac- Super Polyporin inhibited both mitochondrial charide, which has been proven to inhibit cancer and membrane activity of small cell lung carcino- cell growth. Lentinan causes release of cytokines, Volume 15, Number 5, 2013
Durgo et al.
nitric oxide, and other cell signal messengers, po- glucans showed a direct antioxidant effect in vitro.4 tentiating immune cells.4 In in vivo conditions, β-glucans rapidly enter the Human colon cancer cells seem most resis- small intestine, where they are partially degraded tant to cytotoxic effects of the examined blends to smaller units and transferred to the bone marrow and single mushroom extracts. Super Polypo- and endothelial reticular system. These units are rin decreased cell survival by 40% at the thera- released from macrophages and are consequently peutic dose, while isolated compounds, PSP and taken up by circulating granulocytes, monocytes, β-glucan, decreased cell survival by 30% and 40%, and dendritic cells, inducing humoral and cell- respectively (Figure 1). Lentifom also decreased mediated immunity that eliminates tumor cells and colon cancer cell survival, but this was only note- worthy at concentrations higher than the daily rec- Smina et al.31 isolated triterpenes from Gano- ommended dose.
derma lucidum that cause its strong antioxida- There are various events responsible for the tive activity with insignificant toxicity. Grifolin, a biological effects of different mushrooms and their secondary metabolite from Albatrellus confluens, selected components. Postemsky et al.26 detected causes deprivation of kinases and G1 arrest.32
that Grifola gargal extract inhibits the mutagenic
effect of standard promutagen DMBA in the fruit V. CONCLUSIONS
fly (Drosophila melanogaster). The extract's bio- active compounds modify detoxification in vivo, At the recommended doses, the most effective preventing the promutagen from metabolizing into blended extracts were as follows: Super Polyporin active intermediates that can damage cellular mac- was effective against colon cancer cells (Caco2), romolecules (including DNA). The study excluded human lung adenocarcinoma cells (SKLU-1), analysis of the responsible compound(s).26 Joseph small cell lung carcinoma cells (H69 V) and hu-et al.18 isolated a polysaccharide–protein complex man astrocytoma cells (CCTG-1). Agarikon and from Phellinus rimosus that prevents gamma radia- Agarikon Plus were most effective against human tion–induced oxidative stress. The mushroom also lung adenocarcinoma cells (SKLU-1). Lentifom contains important immunomodulatory polysac- was effective against human lung adenocarci- charides as well as a protein-bound polysaccharide noma cells (SKLU-1), small cell lung carcino- that inhibits cell matrix-degrading enzyme produc- ma cells (H69 V), and human astrocytoma cells tion, tumor cell-induced platelet aggregation and (CCTG-1). Agarikon.1 and Mykoprotect.1 were cell mobility, and angiogenesis by modulating cy- effective against small cell lung carcinoma cells tokine production, which explain its antimetastatic (H69 V).
activity.27,28 β-glucans from Agaricus brasiliensis In this work, it was determined that the blends (=A. blazei) showed anti-inflammatory, antialler- of mushroom extracts consistently elicit greater gic, and antiasthmatic properties in mouse mod- cytotoxic effects than single species extracts. Anti- els.29 Contrary to those findings, Goncalves et al.30 oxidant activity is not correlated with the polysac-proved that extracts from A. brasiliensis caused charide content of the blends, but is strongly cor-induction of CD4+ T cells, natural killer T cells, related with their TPC. The concentration of any phagocytes, monocytes, and proinflammatory cy- single compound cannot predict the overall cyto- tokines, excreting both local and systemic inflam- toxic effect of the mushroom (or the mushroom mation in mice. One explanation given by the au- blend); the ratio of all components as well as the thors was that the proinflammatory action could be tumor cell type determine the effectiveness of cy- a consequence of applying of whole fruit body ex- totoxicity of the mushroom extract blend.
tract instead of concentrated selected components Many companies worldwide produce dietary of A. brasiliensis, for which an anti-inflammatory supplements from medicinal mushrooms. Most are effect was proven. Some higher Basidiomycetes made from a single mushroom species and usually International Journal of Medicinal Mushrooms Cytotoxic Effects of Medicinal Mushroom Extracts on Human Cancer Cells contain many compounds. Isolated, single com- knowledge support from the Republic of Croatia pound supplements are rare. However, attempts to Ministry of Science, Education, and Sports (proj-register mushroom preparations as dietary supple- ects 058-0000000-3470 and 058-0582261-2246).
ments in Western countries have been almost com-pletely denied.33 Although cancer therapies (such as surgery, 1. Wasser SP. Medicinal mushroom science: history, current chemotherapy, and radiotherapy) can inhibit tumor status, future trends, and unsolved problems. Int J Med growth and prolong patients' life span to some ex- tent, they usually compromise the immune system 2. Hobbs C. Medicinal Mushrooms. 2 ed. Santa Cruz, CA: (often already damaged by the cancer), induce free Botanica Press, 1995.
radical production, and damage genetic material of 3. Halpern GM. Healing Mushrooms. Garden City Park, NY: Square One Publishers, 2007.
healthy cells. Supportive biotherapy, including the 4. De Silva, DD, Rapior S, Fons F, Bahkali AH, Hyde KD. use of effective mushroom extracts, is gaining at- Medicinal mushrooms in supportive cancer therapies: an tention since it reduces side effects and helps over- approach to anti-cancer effects and putative mechanisms come cancer growth.4 of action. Fungal Div. 2012;55:1–35.
The only registered mushroom drugs come 5. Shnyreva AV, Song W, Van Griensven LJLD. Extracts of medicinal mushrooms Agaricus bisporus and Phellinus from the Far East; Western medicine remains in- linteus induce proapoptotic effects in the human leukemia different, and thus medicinal mushrooms are unde- cell line K562. Int J Med Mushrooms. 2010;12:167–75.
rused. Medicinal mushroom research has discov- 6. Ivankovic S, Hirsl N, Jakopovic I, Jurin M. The influence ered many promising compounds that are in need of medicinal mushroom preparations on mouse tumors. of follow-up (e.g., in clinical studies) and develop- Int J Med Mushrooms. 2004;6:107–17.
Chang ST, Wasser SP. The role of culinary-medicinal ment into medical drugs. Contemporary pharma- ceutical drugs are well-defined single compounds mushrooms on human welfare with a pyramid model for human health. Int J Med Mushrooms. 2012;14:95–135.
with completely defined physical and chemical 8. Association of Official Analytical Chemists (AOAC). properties and understood mechanisms of ac- Official Methods of Analysis. 16 ed. Baltimore, MD: tion (pharmacokinetics and pharmacodynamics). AOAC International, 1995. Mushroom extracts contain a mixture of active 9. Lachman J, Hosnedl V, Pivec V, Orsák M. Polyphenols in cereals and their positive and negative role in human and compounds that interact with intracellular signal- animal nutrition. In: Proceedings of Conference Cereals ing pathways, changing the course of inflamma- for Human Health and Preventive Nutrition. Brno, Czech tion, cell differentiation, survival, apoptosis, an- Republic: Mendelova Zemedelska a Lesnicka Univer- giogenesis, and tumor progression.15,16 zita, 1998, p. 118–25.
10. Benzie IF, Strain JJ. The ferric reducing ability of plasma CONFLICTS OF INTEREST
(FRAP) as a measure of ‘antioxidant power': the FRAP assay. Anal Biochem. 1996;239:70–76.
11. Re R, Pellegrini N, Proteggente A, Pannala A, Yang To avoid any doubt in objectivity, the coauthors af- M, Rice-Evans C. Antioxidant activity applying an im- filiated to Dr Myko San–Health from Mushrooms proved ABTS radical cation decolorisation assay. Free Company were not involved in the laboratory mea- Radical Biol Med. 1999;26:1231–37.
surements and have not influenced the results in 12. Wei S, Helsper JPFG, Van Griensven LJLD. Phenolic compounds present in medicinal mushroom extracts gen- erate reactive oxygen species in human cells in vitro. Int J Med Mushrooms. 2008;10:1–15.
13. Babich H, Borenfreund E. Applications of the neutral red cytotoxicity assay to in vitro toxicology (review). ATLA. The authors thank Dr Myko San–Health from 1990;18:129–44. 14. Mickish G, Fajta S, Keilhauer G, Schlick E, Tschada R, Mushrooms Company for the purchase of the hu- Alken P. Chemosensitivity testing of primary human re- man tumor cell lines, growth media, and medici- nal cell carcinoma by tetrazolium based microculture as- nal mushroom preparations. The authors also ac- say (MTT). Urol Res. 1990;18:131–36.
Volume 15, Number 5, 2013
Durgo et al.
15. Reis FS, Pereira E, Barros L, Sousa MJ, Martins A, Fer- pounds on the production of laccase isoforms by Copri- reira IC. biomolecule profiles in inedible wild mushrooms nus comatus. Mycoscience. 2010;51:68–74.
with antioxidant value. Molecules. 2011;16:4328–38.
26. Postemsky PD, Palermo AM, Curvetto NR. Protective 16. Petrova RD. New scientific approaches to cancer treat- effects of new medicinal mushroom, Grifola gargal Sing- ment: Can medicinal mushrooms defeat the curse of the er (higher Basidiomycetes) on induced DNA damage century? Int J Med Mushrooms. 2012;14:1–20.
in somatic cells of Drosophila melanogaster. Int J Med 17. Abdullah N, Ismail SM, Amnudin N, Shuib AS, Lau BF. Evaluation of selected culinary-medicinal mushrooms 27. Kobayashi H, Matsunaga K, Oguchi Y. Antimetastatic ef-for antioxidant and ACE inhibitory activities. Evid Based fects of PSK (Krestin), a protein-bound polysaccharide Complement Alternat Med. 2012; 2012:4638–42.
obtained from Basidiomycetes: an overview. Cancer Epi- 18. Joseph J, Panicker SN, Janardhanan KK. Protective ef- demiol Biomarkers Prev. 1995;4:275–81.
fect of polysaccharide-protein complex from a polypore 28. Lavi I, Nimri L, Levinson D, Peri I, Hadar Y, Schwartz mushroom, Phellinus rimosus against radiation-induced B. Glucans from the edible mushroom Pleurotus pulmo- oxidative stress. Redox Rep. 2012;17:22–27.
narius inhibit colitis-associated colon carcinogenesis in 19. Zhang M, Cui SW, Cheung PCK, Wang Q. Antitumor mice. J Gastroenterol. 2012;47:504–18. polysaccharides from mushrooms: a review on their iso- 29. Hetland G, Johnson E, Lyberg T, Bernardshaw S, lation process, structural characteristics and antitumor Tryggestad AM, Grinde B. Effects of the medicinal activity. Trends Food Sci Technol. 2007;18: 4–19.
mushroom Agaricus blazei Murrill on immunity, infec- 20. Guo FC, Williams BA, Kwakkel RP, Verstegen MW. tion and cancer. Scand J Immunol. 2008;68:363–70.
In vitro fermentation characteristics of two mushroom 30. Goncalves JL, Roma EH, Gomes-Santos AC, Aguilar species, an herb, and their polysaccharide fractions, EC, Cisalpino D, Fernandes L R, Vieira AT, Oliveira using chicken fecal contents as inoculum. Poult Sci. DR, Cardoso VN, Teixeira MM, Alvarez-Leite JI. Pro- inflammatory effects of the mushroom Agaricus blazei 21. Song W, Van Griensven LJLD. Pro- and antioxidative and its consequences on atherosclerosis development. properties of medicinal mushroom extracts. Int J Med Eur J Nutr. 2012;51:927–37.
31. Smina TP, Mathew J, Janardhanan KK, Devasagayam 22. Hagerman AE, Butler LG. The specificity of proanthocy- TP. Antioxidant activity and toxicity profile of total trit- anidin-protein interaction. J Biol Chem. 1981;9:4444–97.
erpenes isolated from Ganoderma lucidum (Fr.) P. Karst 23. McManus JP, Davis KG , Beart JE, Gaffney SH, Lilley occurring in South India. Environ Toxicol Pharmacol. TH, Haslam E. Polyphenol interactions. Part 1. Introduc- tion; some observations on the reversible complexation 32. Luo XI, Li W, Yang LF, Xiao LB, Tang M, Dong X, Deng of polyphenols with proteins and polysaccharides. J QP, Bode AM, Liu JK, Cao Y. DAPK1 mediates the G1 Chem Soc. 1985;9:1429–38.
phase arrest in human nasopharyngeal carcinoma cells 24. Spencer CM, Cai Y, Martin R, Gaffney SH, Goulding induced by Grifolin, a potential antitumor natural prod- PN, Magnolato D, Lilley T H, Haslam E. Polyphenol uct. Eur J Pharmacol. 2011;670;427–34.
complexation—some thoughts and observations. Phyto- 33. Jakopovich I. New dietary supplements from medicinal mushrooms: Dr Myko San – A registration report. Int J 25. Lu X, Ding S. Effect of Cu2+, Mn2+ and aromatic com- International Journal of Medicinal Mushrooms
„Moleküle der Gefühle„ Neurobiologische Grundlagen der Sucht K. Hentschel Psychologin FSP Psychotherapeutin SPV ECP Körperpsychotherapeutin EABP Manuskript im Rahmen des Seminars vom Herbst 2001 sowie 2002 an der Fachhochschule Zürich, Hochschule für Soziale Arbeit