Comparative Antitumor Activity of Jelly Ear Culinary-Medicinal Mushroom, Auricularia auricula-judae (Bull.) J. Schrot. (Higher Basidiomycetes) Extracts Against Tumor Cells In Vitro Md. Ahsanur Reza,1 Woo-Sik Jo,2 & Seung-Chun Park1,*
1Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Republic of Korea; 2Department of Agricultural Environment, Gyeongbuk Agricultural Technology Administration, Daegu 702-701, Republic of Korea
*Address all correspondence to: Seung-Chun Park, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Republic of Korea; [email protected].
ABSTRACT: The present study compares the antitumor activity of extracts from Auricularia auricula-judae, Phellinus gilvus, Ganoderma lucidum, and 100 Korean wild plants in the P388D1 macrophage cell line. The antitumor activity of A. auricula-judae extract (44.21%) did not differ significantly (P < 0.05) from those of Ph. gilvus (39.46%) and G. lucidum (36.64%) at 1 mg/mL of concentration. Among 100 wild plants, Morus bombycis f. kase, Draba nemorosa var. hebecarpa, Sedum oryzifolium, Lotus corniculatus var. japonicus, and Auricularia auricula-judae 70% ethanol extracts inhibited the viability of tumor cells by 41.85%, 37.31%, 30.29%, 31.98%, and 25.40% at 3 mg/mL of concentration, while inhibition concentration (IC50) values were 1.81, 1.49, 1.05, 1.10, and 0.72 mg/mL, respectively. In Sarcoma 180, NCI H358, and SNU 1 cell lines, the inhibitory activities of A. auricula-judae extract were 65.71%, 69.76%, and 68.01%, respectively. Taken together, the results obtained from the present study indicated that four plant extracts (4% of tested wild plants) and A. auricula-judae extract with similar levels of Ph. gilvus and G. lucidum extracts may be new potential antitumor agents.
ABBREVIATIONS: AAE: ethanol extract of Auricularia auricula-judae; AAW: water extract of Auricularia auric-ula-judae; DMSO: dimethyl sulfoxide; FBS: fetal bovine serum; GLE: Ganoderma lucidum extract; IC50: the half maximal inhibitory concentration; i.u.: international unit; KRIBB: Korea Research Institute of Bioscience and Bio-technology; MTT: 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; NK: natural killer; PBS: phosphate buffer saline; PGE: Phellinus gilvus extract; TB: trypan blue
I. INTRODUCTIONIn the development of antitumor agents, plants and mushrooms are significant sources in terms of safe-ty. They have been used as food or medicine since ancient times. Over the last few decades, anticancer agents such as vinblastine, vincristine, camptoth-ecin derivatives, topotecan, irinotecan, etoposide, and paclitaxel have been investigated in clinical tri-als against cancer.1 On the other hand, various kinds of medicinal mushrooms have been used to prevent various cancers and to improve human life for a long time. Recently, many medical researchers have be-come very interested in studying mushrooms for their biological properties and applications. The higher
Basidiomycetes mushrooms contain highly potent polysaccharides and protein complexes that act as antitumor, immunomodulating, cardiovascular and hypercholesterolemia, antiviral, antibacterial, and an-tiparasitic elements.2 Therefore, the importance and utilization of medicinal mushrooms are inevitable for pharmaceutical sciences. As a result, researchers are studying natural substances with antitumor activ-ity from plants, bacteria, and mushrooms, in order to contribute to solving the problems of cancer therapy.3 The higher Basidiomycetes mushrooms have been extensively investigated for use in cancer therapies and have been introduced as basic remedies since the 1960s.4,5 The species of genera Agaricus, Pleu-
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Biotechnology (KRIBB), Daejeon, Korea.
C. Cell CultureThe P388D1 macrophage, Sarcoma 180, human NSCLC NCI H358 (bronchioalveolar), and SNU1 (gastric carcinoma) cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in a RPMI-1640 medium sup-plemented with 10% FBS, L-glutamine (2 mM), penicillin (100 i.u./mL), and streptomycin (10 mg/mL). The cultured cells were incubated at 37°C in a humidified atmosphere of 5% CO2.
D. Determination of Cell Viability by MTT AssayThe mitochondria-dependent reduction of MTT to formozan was determined by rapid colorimetric assay that measured cell respiration, as an indica-tor of cell viability. The procedure was previously described by Mosmann.7 For the determination of cell viability, P388D1 cells (1 × 105 cells/well) in flat-bottomed 96-well plates were cultured and treated at various concentrations (1 mg/mL, 0.3 mg/mL, 0.1 mg/mL, 0.03 mg/mL, and 0.01 mg/mL) of A. auricula-judae and 100 plant extracts for 72 h at 37°C with 5% CO2, while doxorubicin at different concentrations (10 µg/mL, 1 µg/mL, and 0.1 µg/mL) was used as a positive control.
Thereafter, 50 µL of MTT solution (2 mg/mL) was added into each well of flat-bottomed 96-well plates and incubated again at 37°C with 5% CO2 for 4 h. Then, the supernatant was aspirated and the insoluble formozan product dissolved in 200 µL of DMSO (Sigma-Aldrich). The optical densi-ties (OD) of the culture wells were measured us-ing the VERSA max microplate reader (Associates of Cape Cod, Inc., East Falmouth MA, USA) at 540 nm, yielding absorbance, which measured the activity of mitochondrial dehydrogenase enzymes to reduce the purple formazan from yellow MTT [3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide, a tetrazole] that directly corre-lates to the number of metabolically active cells in the wells. The formula of inhibition of tumor cell proliferation was as follows:
Viability of cells (%) = (OD value of sample/OD value of control) × 100 (I)
Inhibition rate (%) = [1- (OD value of sample/OD value of control)] × 100 (II)
rotus, Lentinus, Ganoderma, Grifola, Volvariella, Auricularia, and Tremella are the most popular in the mushroom industry for their medicinal and nu-tritional values.6 Therefore, in the present study, we conducted experiments on comparative antitumor activities of 100 wild plants and Auricularia auricu-la-judae, Phellinus gilvus, and Ganoderma lucidum extracts in the P388D1 macrophage tumor cell line.
II. MATERIALS AND METHODSA. Chemicals/ReagentsRPMI 1640 medium, fetal bovine serum (FBS), L-glutamine (2 mM), penicillin (100 i.u./mL) and streptomycin (10 mg/mL), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-y l)-2,5-dip henyltetrazolium bromide (MTT), trypan blue (TB), and doxorubicin were purchased from Sig-ma-Aldrich Chemical Co. (St. Louis, MO, USA). All analytical grade chemicals were used in this experiment.
B. Preparation of Mushroom and Plant Extracts
Before the start of the experiment, dried Au-ricularia auricula-judae, Phellinus gilvus, and Ganoderma lucidum were ground into powder (particle diameter: 0.2–0.5 mm). Samples of 5 g were soaked with 70% ethanol solvent (solid to liquid ratio of 1/50 w/v) and extracted at 100°C for 6 h. Immediately after heat extraction, the superna-tant was collected by filtration (70 mm, Advantec, Toyo Roshi Kaisha Ltd., Tokyo, Japan).
The extract was put in a boiling bottle and placed in a water bath (Buchi Water Bath B-480, Buchi Labortechnik AG, Flawil, Switzerland) for vacuum concentration of 70% ethanol extracts at 70°C temperature using a Buchi Rotavapor R-114 at 10 rpm and Eyela CCA-1111 (Tokyo Rikakikai Co., Ltd., Shanghai, China). To make powders with the extract concentrations, they were dried in a vacuum concentrator (BioTron Inc., Puchon, Ko-rea) at a controlled temperature (<50°C).
In the experiments, they were dissolved in sterile PBS and filtered by 0.2 µm filter. The fil-trates were aliquoted in small tubes and stored in a freezer as stock solutions. In order to compare antitumor activity between A. auricula-judae and Korean wild plants, 100 plant extracts (methanol extraction) were provided by Plant Resource Cen-ter, Korea Research Institute of Bioscience and
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E. Statistical AnalysisAll values were expressed as the mean ± S.D. Sta-tistical analysis was done by one-way analysis of variance (ANOVA) using the SAS program. Non-linear regression analysis (GraphPad Prism 5 pro-gram, GraphPad Software, Inc., USA) was used to obtain IC50 values. P-values less than 0.05 were considered significant.
III. RESULTS AND DISCUSSIONAuricularia auricula-judae is an culinary-medic-inal mushroom, and its fruiting bodies have been used in Chinese food and medicine since ancient times.8,9 The nutritional content of dried fungus in-cludes 293.1 Kcal per 100 g, 64.82% carbohydrate, 9.69% proteins, 1.22% fat, and 7.87% ash.10 Re-cently, the many biological activities of A. auricu-la-judae extracts have become an interest in medi-cal therapies. Hypoglycemic,11 prevention of liver damage,12 anticoagulant,13 anti-complementary,14 in vivo hypolipidemic,15 antioxidant,16,17 and pre-vention of ischemia18 effects have been reported. Previously, the antitumor activity of A. auricula-judae aqueous extract was also determined in ani-mal models.19 In mushrooms and wild plants, there has been much research on antitumor activity, but there is no research on comparative antitumor ac-tivity. For this reason, the degree of antitumor ac-
tivity of mushrooms is still unknown. In the present study, therefore, we compared antitumor activity of a non-toxic edible mushroom, A. auricula-judae 70% ethanol extract, and 100 Korean wild-plant extracts, and then measured the antitumor effica-cy by comparing the activity of medicinal mush-rooms, Phellinus gilvus and Ganoderma lucidum 70% extracts, together with A. auricula-judae 70% extract in the same concentrations. On the basis of preliminary screening, the antitumor activities of A. auricula-judae 70% ethanol and 100 indig-enous plant extracts were measured by MTT as-say in P388D1 macrophage in vitro. The antitumor activity of A. auricula-judae 70% ethanol and 100 plant extracts is presented in Table 1. As shown in Table 1 and Fig. 1, the mean antitumor activity (42.21%) of A. auricula-judae extract significantly differed from that of selected plant extracts (Morus bombycis f. kase, Draba nemorosa var. hebecarpa, and Lotus corniculatus var. japonicas except Se-dum oryzifolium). The results indicated that A. au-ricula-judae extract has a stronger cytotoxic effect than the selected plant extracts. Furthermore, we investigated the differences of antitumor efficacy between water and 70% ethanol extracts from A. auricula-judae and compared the findings to those of organic extracts of Ph. gilvus and G. lucidum. The comparative antitumor activity of water and
TABLE 1. Antitumor Activity Screening of 100 Korean Wild Plants and Auricularia auricular-juadae extracts on P388D1 Macrophage Tumor Cells (1 mg/mL) Sample Name Antitumor activity (%) Sample Name Antitumor activity (%)Ardisia japonica 24.26 ± 1.30 Potentilla discolor 14.20 ± 1.20Astilbe koreana 16.84 ± 1.20 Potentilla nivea 15.24 ± 1.03Centaurea cyanus 14.23 ± 1.01 Prunus salicina var. co-
Philadelphus schrenckii 17.65 ± 1.05 Doxorubicin* 36.35 ± 1.89Phlomis umbrosa 22.30 ± 0.96Note. Data are expressed as mean ± SD. Values with different superscripts differed significantly among selected plant and mushroom extracts at P < 0.05.
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70% ethanol extracts of A. auricula-judae and or-ganic extracts of Ph. gilvus and G. lucidum are pre-sented in Fig. 2. The 70% ethanol extracts of A. au-ricula-judae, Ph. gilvus, and G. lucidum exhibited
4.5, 4.0, and 3.5 times greater antitumor activities, respectively, than A. auricula-judae hot water ex-tract, while the efficacy of 70% ethanol extracts of A. auricula-judae, Ph. gilvus, and G. lucidum did not differ significantly (P < 0.05). After dose-de-pendent antitumor activities were determined, IC50 values of selected plant extracts and A. auricula-judae 70% ethanol extract were calculated (Fig. 3).
The IC50 values of Morus bombycis f. kase, Draba nemorosa var. hebecarpa, Sedum oryzifo-lium, and Lotus corniculatus var. japonicas were 2.5, 2.0, 1.45, and 1.53 times greater than Auricu-laria auricula-judae extracts, respectively. The cytotoxicities on tumor cells of extracts were also significant (P < 0.05) in a dose-dependent decreas-ing manner. Also, the potent cytotoxic activities of A. auricula-judae 70% ethanol extract on Sarcoma 180, human NSCLC NCI H358 (broncho-alveo-lar), and SNU 1 (gastric carcinoma) cell lines in vitro (Fig. 4) were determined and had about 1.5 times greater activities than P388D1 cells. The cy-totoxic activities of 70% ethanol extract from A.
FIGURE 1. Cytotoxicity of P388D1 macrophage tumor cells by administration of plant and mushroom extracts (1 mg/mL) and doxorubicin (1 µg/mL) for 48 h. (A) control, (B) doxorubicin, (C) Morus bombycis f. kase, (D) Draba nemorosa, (E) Sedum oryzifolium, (F) Lotus corniculatus var. japonicus, (G) Auricularia auricula-judae.
FIGURE 2. Cytotoxic effects of water extract (AAW) and 70% ethanol extract (AAE) from Auricularia auricula-judae, Phellinus gilvus (PGE), and Ganoderma lucidum (GLE) extracts (1 mg/mL) and doxorubicin (1 µg/mL) on P388D1 macrophage tumor cells. Mean values ± SD from triplicate, separate experiments are shown. Differ-ent alphabets differed significantly at P < 0.05.
A B C
D E F
G
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auricula-judae varied in various tumor cell lines. There were no remarkable differences between the experimental results of crude A. auricula-judae ethanol extract on Sarcoma 180, lung carcinoma, and gastric carcinoma. These findings indicate that the antitumor effects depend not only on the nature of the extract component, but also on the type of tumor cell line.20
Cancer is a leading cause of death in both men and women throughout the world. Nowadays, hor-monal therapy, radiation, and chemotherapy as well as surgical removal of the tumor are the most com-mon treatments against cancer, and these methods do not ensure a complete cure.21 As a result, the search for anticancer agents from plant sources started in earnest in 1950 to discover and develop anti-cancer drugs, including alkaloids. This led to the identification of the many clinically active che-
motherapeutic agents that exhibit a range of cyto-toxic activities, including taxol and camptothecin.1
Antitumor activities of A. auricula-judae ex-tract varied significantly between water and 70% ethanol extract. The water extract exhibited lower efficacy against tumor cells than the ethanol extract and other selected organic plant extracts, while 70% ethanolic extracts from A. auricula-judae had more potent inhibitory activity than other plant ex-tracts on tumor cells. On the other hand, 70% etha-nol extracts of A. auricula-judae exhibited higher antitumor activity than Ph. gilvus and G. lucidum extracts, though they did not vary significantly (P < 0.05).
The ethanolic extract of G. lucidum inhibited the proliferation of human myeloid leukemia (HL-60) cells by 44.8% and varied from 2.0% to 82.5% inhibition against other tumor cells in vitro,22 while
FIGURE 3. Dose-dependent antitumor effects of plants and mushroom extracts on P388D1 macrophage-like tumor cell line in vitro. A, B, C, D, E, and F stand for Morus bombycis f. kase, Draba nemorosa var. hebecarpa, Sedum oryzifolium, Lotus corniculatus var. japonicus, Auricularia auricula-judae extracts, and doxorubicin, respectively. Concentrations of samples are mg/mL. Mean values ± SD from triplicate, separate experiments are shown and dif-ferent alphabets in the same figure differed significantly at P < 0.05 among different concentrations.
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Ph. gilvus showed dose-dependent antitumor activ-ity against B16F10 melanoma cells both in vitro and in vivo.23 The antitumor activity of mushroom ex-tracts might vary due to chemical modification of antitumor active components during extraction.24
A water-soluble β-glucan from 70% ethano-lic extract of A. auricula-judae exhibited potent inhibitory activity against tumor growth,25 while alkali-insoluble β-glucan did not exhibit antitumor activity, but modified β-glucan exhibited potent antitumor activity.19 It was also reported that the water extract of A. auricula-judae exhibited anti-tumor activity by 42.6% inhibition against solid tumor.26 Therefore, it may be said that fractionated extract or pure extract might have better efficacy than a crude extract as well as other mushroom extracts. Lotus corniculatus extract exhibited an-titumor activity in the first screening test, because it contains kaempferol and quercetin.27 Kaemferol induced apoptosis in glioblastoma cells through oxidative stress,28 while quercetin induced the NK cell–mediated lysis of tumor cell and increased connexin 43, which suppresses the growth of can-cer cells.21,29
IV. CONCLUSIONSMedicinal mushrooms and Korean wild plant ex-tracts contain potential antitumor ingredients that might be effective against the growth of tumor or cancer cells. This study investigated the possibil-ity of A. auricula-judae mushroom as an antitumor candidate, comparing antitumor activity with other mushrooms and 100 Korean plant extracts. The antitumor activities of A. auricula-judae, Ph. gil-vus, and G. lucidum extracts were similar. As com-
pared with Korean plant extracts, the antitumor activity of A. auricula-judae 70% ethanol extract (IC50, 0.72 mg/mL) was higher than studied Korean wild plants. Also, the inhibitory activities of A. auricula-judae extract were 65.71%, 69.76%, and 68.01%, respectively, in Sarcoma 180, NCI H358, and SNU 1 cell lines. From the results obtained in the present study, A. auricula-judae extract may be a new potential antitumor agent.
ACKNOWLEDGMENTSThis research was supported in part by the Tech-nology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, and in part by Kyungpook National University Research Fund, 2012.
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