Cytotoxicity of Blended Versus Single Medicinal Mushroom Extracts on Human Cancer Cell Lines: Contribution of Polyphenol and Polysaccharide Content
- 1. International Journal of Medicinal Mushrooms, 15(5): 435–448 (2013) 4351045-4403/13/$35.00 © 2013 Begell House, Inc. www.begellhouse.com Cytotoxicity of Blended Versus Single Medicinal Mushroom Extracts on Human Cancer Cell Lines: Contribution of Polyphenol and Polysaccharide Content Ksenija Durgo,1 Mladen Koncar,1 Drazenka Komes,1 Ana Belscak-Cvitanovic,1 Jasna Franekic,1 Ivan Jakopovich,2 * Neven Jakopovich,2 & Boris Jakopovich2 1 Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia; and 2 Dr 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; [email protected]. 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 I. INTRODUCTION Millennial experience of traditional medicine and more than 60 years of scientific research have con- clusively proven that medicinal mushrooms inhibit cancer in humans and other mammals.1–3 A vast number of experimental studies, beginning in the United States in the late 1950s and followed mainly by Japanese and Chinese researchers in the 1970s and 1980s, determine whether the antitumor activity
- 2. International Journal of Medicinal Mushrooms Durgo et al.436 of some mushrooms exists and if so, what the tu- mor inhibition rates and complete regression rates are. Research began on animal and human tumor cell lines and animal models, followed by human trials and clinical studies.1–4 Following the human clinical trials of single mushroom compounds, the first official antitumor drugs from medicinal mush- rooms were registered, including PSK (1977), Len- tinan (1985), and Schizophyllan (1986) in Japan, and PSP (1983) in China. Such drugs are limited to the Far Eastern countries, whereas medicinal mushroom supplements are used as oncological therapy support in Western countries.1 New studies address interpretation of molecu- lar mechanisms related to the anticancer effects of medicinal mushrooms. In addition to the immu- nomodulation mainly caused by high-molecular- weight polysaccharides, polysaccharide-protein complexes, and proteins produced during primary metabolism, other compounds (especially low- molecular-weight polysaccharides, polyphenols, flavonoids, triterpenes, tocopherols, and carotenes) and certain secondary metabolic products directly affect cancer cells through interaction with in- tracellular signaling pathways and changing the course of inflammation, cell differentiation, sur- vival, apoptosis, angiogenesis, tumor progression, and so forth.4,5 Since there is a multitude of evidence that single compounds from medicinal mushroom ex- tracts possess certain biological activities, there are tendencies to isolate new, prospective, and synthe- sizable compounds. Most recent studies focus on single compounds or “the best” single mushroom species; however, for many mushroom species, it is not known whether a single compound or a syn- ergy of multiple ingredients causes the observed effects. Empirical evidence and studies6 strongly support the superiority of the blends. In addition, the optimal daily dose and extraction conditions for most compounds remain unknown.7 In this work, the content of bioactive com- pounds of mushroom preparations was elucidated, especially in terms of polyphenol and polysac- charide content. The dosages of active mushroom compounds that provide effective cytotoxic action were also determined. Therefore, as part of ongoing research on medicinal mushrooms and their bioac- tive compounds and biological activities, commer- cially available preparations of several medicinal mushrooms were screened for their antioxidant properties and biological activity on human tumor cell lines using an array of in vitro assays. II. MATERIALS AND METHODS A. Medicinal Mushroom Extracts Dr Myko San–Health from Mushrooms Com- pany (Zagreb, Croatia) supplied their commer- cial blended mushroom products, which included four liquid extracts (Lentifom, Super Polyporin, Agarikon, and Agarikon Plus) and two in tablet form (Agarikon.1 and Mykoprotect.1). Lentifom is a proprietary extract blend of 3 medicinal mush- room species, whereas Super Polyporin contains 7, Agarikon contains 8, and Agarikon Plus contains 10 mushroom species, including the most well- known ones such as Lentinus edodes, Ganoderma lucidum, Trametes versicolor, Grifola frondosa, and Agaricus brasiliensis (=blazei ss. Heinem.). Agarikon.1 is made from L. edodes, G. lucidum, A. brasiliensis, G. frondosa, and Pleurotus ostrea- tus, with 750 mg of mushroom polysaccharides per tablet (the standard daily dosage is three tablets per day). Mykoprotect.1 is made from L. edodes and G. lucidum, and contains 850 mg of mushroom polysaccharides per tablet (the standard daily dos- age is three tablets per day). For comparison, we included three commer- cial single mushroom products (extracts produced in a similar manner as described below): PSP (from T. versicolor; KunShan Long-Teng Biotech Manufacture, KunShan City, China), ImmunoBran MGN-3 (from L. edodes; Daiwa Pharmaceutical, Tokyo, Japan), and β-glucan (from Saccharomy- ces cerevisiae; Transfer Point Inc. Columbia, SC, USA). We extracted 50 g of dried mushroom fruit- ing bodies in 1 L of boiling water for 24 h using a Dr Myko San–Health from Mushrooms Company proprietary production method. Insoluble matter
- 3. Volume 15, Number 5, 2013 Cytotoxic Effects of Medicinal Mushroom Extracts on Human Cancer Cells 437 was removed by forcing the solution (in suspen- sion form) through a filter press, and then concen- trating it 4-fold. For tablet preparations, hot water extract was precipitated with ethanol and dried. To measure dry matter content, we used the Association of Official Analytical Chemists meth- od8 of drying the material in an oven at 105°C until constant weight was achieved. The moisture con- tent was determined by the weight difference be- fore and after drying, whereas the dry matter was the ratio of the final to the initial sample weight percentage. To determine the phenolic content and anti- oxidant capacity, liquid extracts were used; tablets were ground using a mortar and pestle and dis- solved in distilled water (1 g/25 mL). To determine the cytotoxic effects, the liquid extracts and dissolved tablets were evaporated until dry and dissolved in corresponding growth medium (MEM and RPMI 1640, respectively). Following the manufacturers’ recommendations for treatment doses, the prescribed amounts of bioactive compounds were determined and used in order to prepare the extract concentrations that correspond to 0.1, 1, 10, and 100 times of the pre- scribed amounts. The recommended dosages for Dr Myko San products were developed during the company’s 23 years of operation. Early dosages were determined from published research and later refined through systematic follow-up and evaluation of product use in several thousand patients. Cancer patients today receive 3.5–7 g/d of active medicinal mush- room compounds. B. Chemicals Analytical grade Folin–Ciocalteu, formic acid, potassium peroxydisulfate, sodium carbonate, formaldehyde, ferric chloride hexahydrate, ferrous sulfate heptahydrate, ethanol, and hydrochloric acid were supplied by Kemika (Zagreb, Croatia). Trolox (6-hydroxy-2,5,7,8-tetramethylchromane- 2-carboxylic acid), ABTS (2,2-azino-bis(3-eth- ylbenzthiazoline-6-sulphonic acid)diammonium salt), neutral red (NR) (3-amino-7-dimethylami- no-2-methylphenazine hydrochloride), and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- lium bromide) were obtained from Sigma-Aldrich (Steinheim, Germany). Growth media (MEM and RPMI 1640, respectively), fetal bovine serum, and penicillin-streptomycin solution were purchased from Gibco Co. (Carlsbad, CA, USA). C. Determination of Polyphenolic Compounds Total phenol content (TPC) of medicinal mush- room preparations was determined spectropho- tometrically according to a modified Lachman method.9 To determine total flavonoid content (TFC), these compounds were precipitated using formaldehyde, which reacts with C-6 or C-8 on 5,7-dihydroxy flavonoids. The condensed products of these reactions were removed by filtration and remaining nonflavonoid phenols were determined according to the previously mentioned procedure for the determination of TPC. Flavonoid content was calculated as the difference between total phe- nol and nonflavonoid content. Gallic acid was used as the standard and the results were expressed as milligrams of gallic acid equivalents (GAE) per li- ter. All measurements were performed in triplicate. D. Determination of Antioxidant Capacity 1. Ferric Reducing/Antioxidant Power The ferric reducing/antioxidant power (FRAP) as- say was carried out according to a standard pro- cedure by Benzie and Strain.10 The FRAP assay is based on the reduction of the Fe3+ -2,4,6-tripyridyl- S-triazine complex (Fe3+ -TPTZ) to the ferrous form (Fe2+ ). Antioxidant activity of the samples was monitored by measuring the change of absorption at 593 nm. FRAP reagent was prepared by mix- ing acetic buffer, TPTZ, and FeCl3 ×6⋅H2 O (20 mM water solution) at a ratio of 10:1:1. Briefly, 3.8 mL of FRAP reagent was added to a 200 µL volume of the sample. After 4 min, the absorbance of blue coloration was measured against a blank sample. All measurements were performed in triplicate.
- 4. International Journal of Medicinal Mushrooms Durgo et al.438 Aqueous solutions of FeSO4 ×7H2 O (100–1000 mmol/L) were used for the calibration and the re- sults are expressed as millimoles of Fe(II) per liter. 2. ABTS Radical Scavenging Assay The Trolox equivalent antioxidant capacity (TEAC) was also estimated by the ABTS radical cation decolorization assay.11 This method is based on the scavenging of stable blue-green ABTS radi- cal cations (ABTS+ ), which are formed either by chemical or enzymatic oxidation of ABTS several hours prior to the analysis. The improved tech- nique for the generation of ABTS•+ applied here in- volves direct production of the blue-green ABTS•+ chromophore through the reaction between ABTS and potassium persulfate. Stock solutions of ABTS (7 mM) and potassium peroxydisulfate (140 mM) in water were prepared and mixed together to a final concentration of 2.45 mM potassium per- oxydisulfate. The mixture was left to react for 12–16 h at room temperature in the dark. Prior to the analysis, the ABTS radical solution was diluted with ethanol to an absorbance of 0.70 (±0.02) at 734 nm. All measurements were performed as fol- lows: 20 µL of the sample was added to 2.0 mL of the ABTS radical solution, and the absorbance readings were taken after exactly 6 min against the appropriate reagent blank instead of the sample. The results, obtained from triplicate analyses, were expressed as Trolox equivalents and derived from a calibration curve determined for this standard (100–1000 µmol/L). E. Determination of Soluble Polysaccharides The content of soluble polysaccharides was deter- mined according to a modified method of Wei et al.12 Briefly, liquid mushroom extracts were centri- fuged (10,000 rpm for 15 min at 20°C) to collect the supernatant, which was subsequently concen- trated by vacuum evaporation until approximately 35° Brix was reached. The obtained concentrate was precipitated by the addition of two volumes of ethanol to a final concentration of 75% (v/v). The precipitates collected by centrifugation (10,000 rpm for 15 min at 20°C) were solubilized in de- ionized water and lyophilized to obtain the crude polysaccharides. The tablet preparations were pri- marily ground to obtain a fine powder and were then dissolved in distilled water as described above in order to obtain liquid samples. The content of soluble polysaccharides was determined according to the previously described procedure. Polysaccha- ride yield was expressed as milligrams per gram of dry matter of sample. F. Human Cell Lines The cytotoxic effect of mushroom extracts was examined on four human cancer cell lines: hu- man colon cancer cells (Caco-2), human lung ad- enocarcinoma cells (SKLU-1), human small lung carcinoma cells (H69V), and human astrocytoma cells (CCTG-1). All human cancer cell lines were purchased from the European Collection of Cell Cultures/Sigma-Aldrich. Cancer cell lines were grown as monolayer cultures in MEM (Caco-2 and SKLU-1) and RPMI (H69V and CCTG-1) media (Gibco) and were supplemented with 10% fetal bo- vine serum (Gibco). Growth medium served as the negative control. G. Cytotoxicity Assay NR and MTT assays were used to determine cy- totoxic effects of single mushroom extracts and mushroom blends in order to define whether ac- tive compounds destabilize the membrane or in- hibit mitochondrial activity. Cells were seeded in microtiter plates at a concentration of 6×103 cells/ well. After 24-hour incubation, cells were treated with mushroom extracts for 72 hours. After treat- ment, the MTT and NR assays were performed as described by Babich and Borenfreund13 and Mishchish et al.,14 respectively. The absorbance intensity was measured at 432 nm and 540 nm, re- spectively, using a microplate reader (Cecil Instru- ments Ltd., Cambridge, UK). Each concentration was tested in quadruplicate and each experiment was repeated three times.
- 5. Volume 15, Number 5, 2013 Cytotoxic Effects of Medicinal Mushroom Extracts on Human Cancer Cells 439 H. Statistical Analysis Statistical analysis was performed using SPSS software (version 8.0, SPSS Inc., an IBM Com- pany, Chicago, IL, USA). A one-way analysis of variance was employed to determine whether the means obtained with various groups differed sig- nificantly from each other. Significance was estab- lished using the Schaffer and Tukey post hoc tests. A probability level <0.05 was considered signifi- cant. All data are expressed as means ± standard deviations (SD) of the values obtained by three in- dependent measurements. III. RESULTS Table 1 presents the dry matter content and bio- active composition of six mushroom preparations. The highest content of soluble polysaccharides was detected in tablet products Agarikon.1 and Myko- protect.1. The highest content of total polyphenols and flavonoids was measured in the Agarikon Plus mushroom preparation (908.33 mg GAE/L) fol- lowed by Agarikon (839.17 mg GAE/L), where- as Lentifom exhibits the lowest content of total phenols and flavonoids (339.58 mg GAE/L). The ranking of mushroom preparations based on their antioxidant potential corresponds to those obtained for the TPC, which is confirmed by a high correla- tion obtained between the results (rTPC/ABTS = 0.966 and rTPC/FRAP = 0.954). Correlation coefficients (r) of all evaluated bioactive and cytotoxic parameters are displayed in Table 2. According to the obtained results, the content of soluble polysaccharides correlated well only with the cytotoxic effect exerted in the SKLU-1 cell line (rSKLU/polysaccharides = 0.659), fol- lowed by a low correlation in the astrocytoma cell line (rastroMTT/polysaccharides = 0.415). A higher correla- tion was observed between the polyphenolic com- pounds, antioxidant capacity, and cytotoxic effects, thus linking the potential cytotoxic effects to poly- phenolic compounds, rather than polysaccharides. TFC and antioxidant potential of examined extracts showed good correlation to the H69V cell line (rH69V/TPC = 0.616, rH69V/ABTS = 0.718, and rH69V/FRAP = 0.568). A high correlation coefficient was obtained in the case of total flavonoid and polyphenol com- pounds and astrocytoma cells (rastroMTT/TPC, rastroMTT/TFC = 0.805andrastroNR/TPC = 0.766). Medium correlation was obtained between SKLU-1 and CaCo2 cells (NR cytotoxicity test; rSKLU-1/CaCo2 = 0.571) as well as CaCo2 and H69V (MTT assay and NR cytotoxic- ity test, respectively: rCaCo2/H69V = 0.571 and rCaCo2/H69V = 0.683). Human colon cancer cells were resistant to the majority of investigated extracts at the recom- mended daily doses. The higher concentrations of Agarikon.1 and Agarikon Plus (10× and 100×) significantly decreased cell survival. A slight cyto- toxic effect at the therapeutic dose was observed after treatment with Agarikon and Agarikon.1. Su- per Polyporin decreased cell survival in a dose-re- sponse manner, with observable cytotoxic effects at the therapeutic dose (Figure 1). Agarikon influenced membrane permeability TABLE 1. Composition and Antioxidant Activity of Blended Mushroom Extracts Extract Dry matter (%) Soluble polysaccharides (mg/g dry matter) Total phenols (mg GAE/L) Total flavonoids (mg GAE/L) Antioxidant capacity ABTS (mM Trolox) FRAP (mM Fe(II)) Agarikon 4.02 579.35 839.17 455.83 6.29 7.28 Agarikon Plus 4.60 490.42 908.33 542.08 7.56 10.33 Lentifom 1.71 402.61 339.58 174.17 3.53 2.97 Superpolyporin 3.61 467.09 788.75 305.83 6.53 8.08 Agarikon.1 93.00 1053.88 448.75 240.83 3.53 3.14 Mykoprotect.1 92.94 935.35 635.42 362.92 4.65 6.81
- 6. International Journal of Medicinal Mushrooms Durgo et al.440 TABLE2.CorrelationCoefficients(r)betweentheResultsofMeasurements Polysac- charidesTPCTFCABTSFRAPSKLU-1NR SKLU-1MTT H69VNR H69VMTT Caco2NR Caco2MTT CCTG- 1NR CCTG- 1MTT Polysaccha- rides 1−0.288−0.145−0.487−0.3440.6590.205−0.644−0.8500.4000.0030.2630.415 TPC10.9040.9660.954−0.638−0.7380.2240.616−0.327−0.1550.4750.661 TFC10.8520.884−0.415−0.8280.3720.3530.0930.1240.7660.805 ABTS10.960−0.669−0.7890.2990.718−0.400−0.1760.3420.471 FRAP1−0.481−0.8540.3000.568−0.2620.0120.3820.560 SKLU-1NR 10.105−0.355−0.9190.5710.366−0.100−0.139 SKLU-1MTT 1−0.223−0.222−0.025−0.091−0.458−0.483 H69VNR 10.4450.3570.6830.3030.039 H69VMTT 1−0.654−0.254−0.138−0.081 Caco2NR 10.7640.5800.295 Caco2MTT 10.2850.122 CCTG-1NR 10.872 CCTG-1MTT 1
- 7. Volume 15, Number 5, 2013 Cytotoxic Effects of Medicinal Mushroom Extracts on Human Cancer Cells 441 at the therapeutic dose after treatment of human adenocarcinoma cells. No effect on mitochondri- al dehydrogenase activity was observed. On the contrary, Agarikon Plus inhibited mitochondrial activity at the therapeutic dose and caused a loss of membrane selectivity to some extent (Figure 2). Super Polyporin decreased membrane stability af- ter treatment of SKLU-1 cells with the therapeutic dose. Agarikon Plus decreased mitochondrial de- hydrogenase activity of small cell lung carcinoma cells in a dose-response manner, while it slightly destabilized cell membranes. Agarikon.1 and Mykoprotect.1 strongly decreased cell survival at the therapeutic dose, affecting both mitochon- drial activity and membrane selectivity. In addi- tion, Super Polyporin caused cytotoxic effects in dose-dependent manner. Cytotoxicity was detect- ed on both levels, with decreased membrane and mitochondrial activity (Figure 3). Lentifom and Super Polyporin had strong cytotoxic effects on human astrocytoma cells (Figure 4). All investi- gated extracts showed significant cytotoxic effects at concentrations 10 or 100 times higher than the FIGURE 1. Comparison of human colon cancer cell (Caco-2) survival measured by the MTT assay (A) and the NR method (B).
- 8. International Journal of Medicinal Mushrooms Durgo et al.442 therapeutic concentration. The cytotoxic effect of medicinal mushroom blends was stronger than that of single mushroom species extracts. IV. DISCUSSION The use of edible and inedible mushroom prepara- tions in medicinal purposes as a supportive therapy is well known and documented.15 The compounds in the majority of mushrooms with biological ac- tivity against infections (both viral and microbial, high blood pressure, cancer development, oxida- tive stress–related diseases, consequences of ion- izing radiation) are similar for both edible and in- 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 polyphenolic antioxidants are an important group of secondary metabolites because of their contribu- tion to human health and their multiple biological effects, such as antioxidant activity, antimutagenic and/or anticancerogenic activities, and anti-inflam- matory action. However, there is a lack of data re- FIGURE 2. Comparison of human lung adenocarcinoma cells (SKLU-1) survival measured by the MTT assay (A) and the NR method (B).
- 9. Volume 15, Number 5, 2013 Cytotoxic Effects of Medicinal Mushroom Extracts on Human Cancer Cells 443 garding their content in mushrooms, especially in blended mushroom extracts. This work determined the content of soluble polysaccharides, total polyphenols, and flavo- noids and the antioxidant capacity of six blended mushroom extracts, four liquid extracts (Agarikon, Agarikon Plus, Lentifom, and Super Polyporin) and two tablet products (Agarikon.1 and Myko- protect.1). Compared with single mushroom species, the blended mushroom preparations analyzed in this study contained a higher content of soluble poly- saccharides (e.g., 6.8% in L. edodes versus 16.0% in T. fuciformis16 ) (Table 1), confirming the effec- tive polysaccharide extraction procedure devel- oped by the producer. According to the findings of Song and van Griensven,21 the polysaccharide con- tent of three mushroom mixtures ranges from 1.2 to 15.0 mg glucose equivalent/mL (mg GE/mL), which is higher than the mushroom blends evaluat- ed in our study.21 The same authors also evaluated the TPC of several medicinal mushroom species and their mixtures. Their results showed high vari- ability of polyphenolic content in the same mush- FIGURE 3. Comparison of human small cell lung carcinoma cell (H69V) survival measured by the MTT assay (A) and the NR method (B).
- 10. International Journal of Medicinal Mushrooms Durgo et al.444 room species derived from different geographical origins; the TPC of their three mushroom mixtures ranged from 213.8 to 647.8 µg GAE/mL, which is in accordance with the results obtained in this study. However, the results indicate that both phe- nolic content and polysaccharides depend primar- ily on the composition of mushroom blends.21 The results displayed in Table 1 also reveal a higher content of total phenols in liquid mushroom blends, rather than in the tablets. This may be due to the use of other substances during tablet form- ing process, which may interfere in the analytical determination of polyphenols. The difference be- tween the liquid and tablet preparations can also be observed in the content of soluble polysaccharides, since the tablet preparations exhibit almost double the content compared to the liquid ones. It is well known that one of the main characteristics of poly- phenols is their propensity to form complexes with proteins, polysaccharides, and alkaloids (e.g., caf- feine).22–24 Therefore, in tablet preparations with higher polysaccharide content, the formation of polyphenol-polysaccharide complexes may have occurred, leading to underestimation of total phe- nols in these mushroom blends. However, this could also be attributed to the mushroom composi- FIGURE 4. Comparison of human astrocytoma cell (CCTG-1) survival measured by the MTT assay (A) and the NR method (B).
- 11. Volume 15, Number 5, 2013 Cytotoxic Effects of Medicinal Mushroom Extracts on Human Cancer Cells 445 tion (species) in each evaluated mushroom blend. In addition, there were no proteins or peptides in the examined extracts because these compounds were degraded during mushroom extract prepara- tion (unpublished data). Therefore, it can be con- cluded that polyphenols, flavonoids, and polysac- charides play a crucial role in biological activity of the examined extracts. High correlation coefficients observed be- tween the TPC and antioxidant capacities of mush- room blends indicate that antioxidant activity of the examined mushroom preparations is attributed to the polyphenolic compounds, rather than poly- saccharides. Although Song and van Griensven21 determined a high correlation among the polyphe- nols and polysaccharides of different medicinal mushroom extracts (R=0.82), our study revealed no connection between these compounds (Table 2). According to Lu and Ding,25 naturally derived an- tioxidant compounds of Coprinus comatus include total phenols, flavonoids, tocopherols, and poly- saccharides. The results of our study revealed no correlation between the content of polysaccharides and antioxidant capacity of mushroom blends. Tablets contain the highest content of polysac- charides, while Agarikon and Agarikon Plus pos- sess the highest content of polyphenols and flavo- noids. The comparison of cancer cell line survival after treatment with the six blends and single com- pounds reveals a strong cell type–specific cytotox- ic effect. Agarikon was the most effective against human lung adenocarcinoma cells (SKLU-1) at the therapeutic dose. This effect was detected through changes in membrane active transport. This com- pound caused decreased mitochondrial activity in colon cancer cells (Caco2) and small cell lung carcinoma cells (H69V) at the therapeutic dose to some extent. Agarikon Plus inhibited mitochondrial dehy- drogenase activity in human lung adenocarcinoma cells and colon cancer cells, while astrocytoma cells were susceptible to Lentifom at the therapeu- tic dose through mitochondrial activity depriva- tion. Super Polyporin inhibited both mitochondrial and membrane activity of small cell lung carcino- ma cells and colon cancer cells in a dose-depen- dent manner, whereas it caused a slight decrease in cell survival in other cell lines. Polysaccharide- rich extracts, Agarikon.1 and Mykoprotect.1, were the most effective through both mitochondrial and membrane activity against small cell lung carcinoma cells and human lung adenocarcinoma cells. Agarikon.1 decreased cell survival of small cell lung carcinoma and human lung adenocarci- noma cells by 80%. Super Polyporin and Myko- protect.1 also inhibited growth of small cell lung carcinoma and human lung adenocarcinoma cells at 50% and 30%, respectively. Single pharmaceuti- cal compound PSP decreased cell survival at 20% (Figure 3), pointing out that mushroom blends have stronger cytotoxic effects compared with the single compound. A possible explanation of observed cytotoxic effects lies in the fact that polysaccharide extracts of various mushrooms are highly active in reactive oxygen species (ROS) generation; however, there is evidence that some 1,4 glucans cannot induce ROS.12,21 ROS mediate in signal transduction and regulation of diverse processes, such as phagocyte activation, cell proliferation, and migration, and fi- nally apoptosis. Molecular damage caused by ROS in normal cells, induces repair mechanisms, while in tumor cells ROS activate cell death processes through apoptosis.22 Shnyreva et al.5 found that ROS-dependent activation of apoptotic cell death is crucial to antitumor activity. In this work, we showed that Agarikon and Agarikon Plus, which have high polyphenol and flavonoid content, decrease cell survival of hu- man lung adenocarcinoma cells by 70% and 30%, respectively, at the daily recommended dose (Figure 2). Astrocytoma cells were most sensitive to Super Polyporin and Lentifom, which inhibited astrocytoma cell growth by 30% (Figure 4). These blends contain the lowest levels of polysaccha- rides, polyphenols, and flavonoids. It is possible that the synergistic effect of all three major groups of compounds decreased astrocytoma cell survival. In addition, Lentifom is rich in lentinan polysac- charide, which has been proven to inhibit cancer cell growth. Lentinan causes release of cytokines,
- 12. International Journal of Medicinal Mushrooms Durgo et al.446 nitric oxide, and other cell signal messengers, po- tentiating immune cells.4 Human colon cancer cells seem most resis- tant to cytotoxic effects of the examined blends and single mushroom extracts. Super Polypo- rin decreased cell survival by 40% at the thera- peutic dose, while isolated compounds, PSP and β-glucan, decreased cell survival by 30% and 40%, respectively (Figure 1). Lentifom also decreased colon cancer cell survival, but this was only note- worthy at concentrations higher than the daily rec- ommended dose. There are various events responsible for the biological effects of different mushrooms and their selected components. Postemsky et al.26 detected that Grifola gargal extract inhibits the mutagenic effect of standard promutagen DMBA in the fruit fly (Drosophila melanogaster). The extract’s bio- active compounds modify detoxification in vivo, preventing the promutagen from metabolizing into active intermediates that can damage cellular mac- romolecules (including DNA). The study excluded analysis of the responsible compound(s).26 Joseph et al.18 isolated a polysaccharide–protein complex from Phellinus rimosus that prevents gamma radia- tion–induced oxidative stress. The mushroom also contains important immunomodulatory polysac- charides as well as a protein-bound polysaccharide that inhibits cell matrix-degrading enzyme produc- tion, tumor cell-induced platelet aggregation and cell mobility, and angiogenesis by modulating cy- tokine production, which explain its antimetastatic activity.27,28 β-glucans from Agaricus brasiliensis (=A. blazei) showed anti-inflammatory, antialler- gic, and antiasthmatic properties in mouse mod- els.29 Contrary to those findings, Goncalves et al.30 proved that extracts from A. brasiliensis caused induction of CD4+ T cells, natural killer T cells, phagocytes, monocytes, and proinflammatory cy- tokines, excreting both local and systemic inflam- mation in mice. One explanation given by the au- thors was that the proinflammatory action could be a consequence of applying of whole fruit body ex- tract instead of concentrated selected components of A. brasiliensis, for which an anti-inflammatory effect was proven. Some higher Basidiomycetes glucans showed a direct antioxidant effect in vitro.4 In in vivo conditions, β-glucans rapidly enter the small intestine, where they are partially degraded to smaller units and transferred to the bone marrow and endothelial reticular system. These units are released from macrophages and are consequently taken up by circulating granulocytes, monocytes, and dendritic cells, inducing humoral and cell- mediated immunity that eliminates tumor cells and pathogens.4 Smina et al.31 isolated triterpenes from Gano- derma lucidum that cause its strong antioxida- tive activity with insignificant toxicity. Grifolin, a secondary metabolite from Albatrellus confluens, causes deprivation of kinases and G1 arrest.32 V. CONCLUSIONS At the recommended doses, the most effective blended extracts were as follows: Super Polyporin was effective against colon cancer cells (Caco2), human lung adenocarcinoma cells (SKLU-1), small cell lung carcinoma cells (H69 V) and hu- man astrocytoma cells (CCTG-1). Agarikon and Agarikon Plus were most effective against human lung adenocarcinoma cells (SKLU-1). Lentifom was effective against human lung adenocarci- noma cells (SKLU-1), small cell lung carcino- ma cells (H69 V), and human astrocytoma cells (CCTG-1). Agarikon.1 and Mykoprotect.1 were effective against small cell lung carcinoma cells (H69 V). In this work, it was determined that the blends of mushroom extracts consistently elicit greater cytotoxic effects than single species extracts. Anti- oxidant activity is not correlated with the polysac- charide content of the blends, but is strongly cor- related with their TPC. The concentration of any single compound cannot predict the overall cyto- toxic effect of the mushroom (or the mushroom blend); the ratio of all components as well as the tumor cell type determine the effectiveness of cy- totoxicity of the mushroom extract blend. Many companies worldwide produce dietary supplements from medicinal mushrooms. Most are made from a single mushroom species and usually
- 13. Volume 15, Number 5, 2013 Cytotoxic Effects of Medicinal Mushroom Extracts on Human Cancer Cells 447 contain many compounds. Isolated, single com- pound supplements are rare. However, attempts to register mushroom preparations as dietary supple- ments in Western countries have been almost com- pletely denied.33 Although cancer therapies (such as surgery, chemotherapy, and radiotherapy) can inhibit tumor growth and prolong patients’ life span to some ex- tent, they usually compromise the immune system (often already damaged by the cancer), induce free radical production, and damage genetic material of healthy cells. Supportive biotherapy, including the use of effective mushroom extracts, is gaining at- tention since it reduces side effects and helps over- come cancer growth.4 The only registered mushroom drugs come from the Far East; Western medicine remains in- different, and thus medicinal mushrooms are unde- rused. Medicinal mushroom research has discov- ered many promising compounds that are in need of follow-up (e.g., in clinical studies) and develop- ment into medical drugs. Contemporary pharma- ceutical drugs are well-defined single compounds with completely defined physical and chemical properties and understood mechanisms of ac- tion (pharmacokinetics and pharmacodynamics). Mushroom extracts contain a mixture of active compounds that interact with intracellular signal- ing pathways, changing the course of inflamma- tion, cell differentiation, survival, apoptosis, an- giogenesis, and tumor progression.15,16 CONFLICTS OF INTEREST To avoid any doubt in objectivity, the coauthors af- filiated to Dr Myko San–Health from Mushrooms Company were not involved in the laboratory mea- surements and have not influenced the results in any way. ACKNOWLEDGMENTS The authors thank Dr Myko San–Health from Mushrooms Company for the purchase of the hu- man tumor cell lines, growth media, and medici- nal mushroom preparations. The authors also ac- knowledge support from the Republic of Croatia Ministry of Science, Education, and Sports (proj- ects 058-0000000-3470 and 058-0582261-2246). REFERENCES 1. Wasser SP. Medicinal mushroom science: history, current status, future trends, and unsolved problems. Int J Med Mushrooms. 2010;12:1–16. 2. Hobbs C. Medicinal Mushrooms. 2 ed. Santa Cruz, CA: Botanica Press, 1995. 3. Halpern GM. Healing Mushrooms. Garden City Park, NY: Square One Publishers, 2007. 4. De Silva, DD, Rapior S, Fons F, Bahkali AH, Hyde KD. Medicinal mushrooms in supportive cancer therapies: an approach to anti-cancer effects and putative mechanisms of action. Fungal Div. 2012;55:1–35. 5. Shnyreva AV, Song W, Van Griensven LJLD. Extracts of medicinal mushrooms Agaricus bisporus and Phellinus linteus induce proapoptotic effects in the human leukemia cell line K562. Int J Med Mushrooms. 2010;12:167–75. 6. Ivankovic S, Hirsl N, Jakopovic I, Jurin M. The influence of medicinal mushroom preparations on mouse tumors. Int J Med Mushrooms. 2004;6:107–17. 7. Chang ST, Wasser SP. The role of culinary-medicinal mushrooms on human welfare with a pyramid model for human health. Int J Med Mushrooms. 2012;14:95–135. 8. Association of Official Analytical Chemists (AOAC). Official Methods of Analysis. 16 ed. Baltimore, MD: AOAC International, 1995. 9. Lachman J, Hosnedl V, Pivec V, Orsák M. Polyphenols in cereals and their positive and negative role in human and animal nutrition. In: Proceedings of Conference Cereals for Human Health and Preventive Nutrition. Brno, Czech Republic: Mendelova Zemedelska a Lesnicka Univer- zita, 1998, p. 118–25. 10. Benzie IF, Strain JJ. The ferric reducing ability of plasma (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 M, Rice-Evans C. Antioxidant activity applying an im- proved ABTS radical cation decolorisation assay. Free Radical Biol Med. 1999;26:1231–37. 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. 1990;18:129–44. 14. Mickish G, Fajta S, Keilhauer G, Schlick E, Tschada R, Alken P. Chemosensitivity testing of primary human re- nal cell carcinoma by tetrazolium based microculture as- say (MTT). Urol Res. 1990;18:131–36.
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