Anticancer, antioxidant and antibiotic activities of

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May 15, 2013 - The medicinal use of mushrooms has a very long tradition in the. Asian countries due to their bioactivities such as anticancer, antiox- idant and ...
Food and Chemical Toxicology 58 (2013) 375–380

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Anticancer, antioxidant and antibiotic activities of mushroom Ramaria flava Kun Liu a,b, Junli Wang a,⇑, Le Zhao a, Qian Wang a a b

College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, People’s Republic of China College of Biology Science and Engineering, Hebei University of Economics and Business, Shijiazhuang, Hebei 050061, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 20 February 2013 Accepted 1 May 2013 Available online 15 May 2013 Keywords: Ramaria flava Anticancer activity Antioxidant Antibiotic activity Total phenolic compounds

a b s t r a c t Ramaria flava is a species of edible mushroom with some bioactivity. The anticancer, antioxidant and antibiotic activities and chemical composition of R. flava ethanol extract (EE) were evaluated. The present study exhibited that the EE displayed the strongest inhibitory activity against tumor cell MDA-MB-231 with an IC50 value of 66.54 lg/mL in three tested tumor cell lines, and the inhibition percent was 71.66% at the concentration of 200 lg/mL (MTT assay). The total phenolic compounds varied among four fractions of the EE from 6.66 to 61.01 mg gallic acid equivalent (GAE) per g dry weight. Water fraction exhibited high DPPH and OH radical-scavenging activities with low IC50 values of 5.86 and 18.08 lg/ mL, respectively. Meanwhile, three phenolic compounds from water fraction were also identified by HPLC. The antibiotic activities of the EE were evaluated against three microorganisms and three fungi strains by means of the agar well diffusion method and the poisoned medium technique, respectively. The EE also showed moderate antibiotic activities. These results suggest that R. flava could hold a good potential source for human health. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Ramaria flava, commonly known as broom fungus, usually lives in deciduous forest, on acidic soil, often in larger groups of several specimens, from July to October. It is edible and of high quality. It can be fried, prepared in form of a salad, pickled, and it can be prepared in many other ways as well. As edible mushroom, R. flava has some bioactivities, such as antitumor and antioxidant activities (Dai and Yang, 2008). The ethanol extract EE from R. flava is known to possess DPPH free-radical-scavenging, and anti-lipid peroxidation qualities attributed to flavonoids and phenolic compounds (Gezer et al., 2006). In the case of total phenolic and flavonoid assays, the methanol extract from R. flava found to have the high phenolic content, with a value of 10.51 lg GAE/mg extract (GAE, gallic acid equivalent). On the other hand, total flavonoid content of R. flava was 0.50 ± 0.01 lg QE/mg extract (QE, quercetin equivalent). At the concentration of 20 mg/mL, the strongest reducer was determined as R. flava with a value of 1.915, and chelating effect was 96.75% for the methanol extract of R. flava at a concentration of 2.0 mg/mL (Gursoy et al., 2010). In recently, there are few reports about the research of chemical composition of R. flava. For instance, some ⇑ Corresponding author. Address: College of Life and Environmental Sciences, Minzu University of China, Zhongguancun South Street 27, Beijing 100081, People’s Republic of China. Tel./fax: +86 10 68932633. E-mail address: [email protected] (J. Wang). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.05.001

polyacetylenes were isolated from the culture of R. flava. (Hearn et al., 1973) and eleven compounds were isolated from the fruit body of R. flava in our previous work (Liu et al., 2012b). Many species of the genus Ramaria possess some bioactivities, for instance, the ethyl acetate fractions of Ramaria formosa has antitumor and antioxidant activities (Ramesh and Pattar, 2010; Yoo et al., 1982). The medicinal use of mushrooms has a very long tradition in the Asian countries due to their bioactivities such as anticancer, antioxidant and antimicrobial activities in animals and humans (Liu et al., 2010; Zaidman et al., 2005). The aim of the present study was to evaluate the antitumor, antioxidant and antibiotics activities of the EE from R. flava. The amount of total phenolic compounds in the EE and four fractions was also determined. Meanwhile, three phenolic compounds from water fraction were also identified by HPLC compared with authentic standards.

2. Materials and methods 2.1. Chemicals The chemicals used in this study were of analytical reagent grade that include: 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), RPMI1640 complete medium, 2,2-diphenyl-1-picrylhydrazyl (DPPH), quercetin, chrysin and pinocembrin (Sigma Chemical Co., St. Louis, MO, USA); paclitaxel (Shenyang Tianfeng Biological Pharmaceutical Co., Ltd., Shenyang, China), clotrimazole (Wuhu Sanyi Xincheng Pharmaceutical Co., Ltd., Wuhu, China) and penicillin sodium (CSPC Pharmaceutical Group Limited, Shijiazhuang, China). Ascorbic acid, hydrogen peroxide

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K. Liu et al. / Food and Chemical Toxicology 58 (2013) 375–380

(H2O2), dimethyl sulfoxide (DMSO) and other reagents were of standard analytical grade and purchased from Beijing Chemical Works (Beijing, China). All solutions were freshly prepared in distilled water.

2.2. Macrofungi materials R. flava was collected from the Aershan Region of Inner Mongolia (Inner Mongolia, China) from July 2010 to September 2010. After the identification of the species, a voucher specimen (NoF121718) was deposited at the College of Life and Environmental Science of Minzu University of China for future reference.

2.3. Extraction The air-dried and powdered fruit body of R. flava (1.0 kg) was extracted four times with 10 L of 95% ethanol each time (7 days) at room temperature. The extract was decanted, filtered and concentrated in a rotary evaporator.

The total phenolic content was determined according to the Folin–Ciocalteau method (Cao et al., 2008; Gursoy et al., 2009) with minor modification. Briefly, 1 mL of properly diluted sample was mixed with 2.0 mL of Folin–Ciocalteu reagent (diluted 1 to 1 with distilled water). One minute later, 2.0 mL of NaCO3 (7.5%, w/v) were added and the mixture was incubated at 50 °C for 15 min. Then, the absorbance was measured at 765 nm. A standard solution of gallic acid was used to prepare a calibration curve. The total phenolic content (TPC) was calculated and expressed as mg gallic acid/g of dry extract.

2.8. DPPH radical-scavenging ability assay Radical scavenging activities of the different fractions were determined using the DPPH radical as a reagent, according to the methods of Liu et al. (2012a). 100 lL of a DPPH radical solution in ethanol (60 lM) was mixed with 100 lL of sample solution in ethanol (different concentrations, w/v). The mixture was incubated for 30 min in the dark at room temperature and the absorbance was measured at 517 nm. Ascorbic acid was used as a standard. The DPPH scavenging activity of each sample was calculated using the following equation:

2.4. Preparation of the different fractions of the ethanol extracts

Scavenging effect of DPPH ð%Þ ¼ ½ðAc  At Þ=Ac   100 The EE (10 g) was suspended in 50 mL of distilled water and sequentially partitioned into petroleum ether (3  150 mL), ethyl acetate (3  150 mL) and then nbutanol (3  150 mL). The resulting four solutions were concentrated in vacuum to dryness, to give petroleum ether (1.10 g), ethyl acetate (0.42 g), n-butanol (7.87 g) and water (0.43 g) soluble fractions. These fractions were kept at 4 °C, in the dark until further analysis.

where Ac is the absorbance of the control reaction (100 lL of ethanol with 100 lL of the DPPH solution), and At is the absorbance of the test sample. The antioxidant ability of the sample was expressed as the IC50, which is defined as the concentration in lg of dry material per mL that inhibits the formation of the DPPH radical by 50%.

2.9. Hydroxyl radical-scavenging activity assay 2.5. HPLC analysis of phenolic compounds Water fraction of the EE was analyzed using an analytical HPLC unit (Gilson), with a Shim-pack VP-ODS C18 column (5 lm particle size, 150  4.6 mm i.d. Shimadzu, Japan), material and thermostated at 30 °C. The solvent system used was a mixture of 0.5% formic acid in deionized water (A) and 0.5% formic acid in acetonitrile: methanol (50:50 V/V) (B). Elution was performed at a flow rate of 1.0 mL/min and the gradient was as follows: 15–40% B in 20 min, 40–50% B in 10 min, 50–100% B in 10 min followed by 10 min at 100% B. Detection was achieved with a Gilson diode array detector (DAD) and chromatograms were recorded at 330, 310, 290, 270 and 250 nm. The compounds in water fraction were identified by their UV spectra recorded with the diode array detector and by chromatographic comparisons (retention times) with authentic standards. Phenolic compounds quantification was achieved by the absorbance recorded in the chromatograms relative to external standards, at 270 nm for quercetin, chrysin and pinocembrin.

2.6. Anticancer assay Test sample was subjected to in vitro cell growth inhibitory activity against the human cancer cell lines (BGC-803, NCI-H520 and MDA-MB-231) by MTT assay (Khan et al., 2007). For the assay, the cells were grown in tissue culture flasks in RPMI 1640 medium at 37 °C in an atmosphere of 5% CO2 and 100% relative humidity in a CO2 incubator. Analiquot of 100 lL of cells (105 cells/mL) was transferred to a well of 96-well tissue culture plate. The cells were allowed to grow for 12 h and then treated with the sample. 100 lL test samples (200, 100, 50, 25, 12.5, and 6.25 lg/mL) were added to the wells and cells were further incubated for another 48 h at 37 °C in an atmosphere of 5% CO2. 20 lL MTT (5 mg/mL in phosphate-buffered saline) was then added to each well and cells were further cultured for 4 h. After removal of the medium, 100 lL dimethyl sulfoxide (DMSO) was added to each well. The absorbance was measured on a microplate reader (Thermo LabSystems, Grand Rapids, OH, USA) at the wavelength of 570 nm (Wang et al., 2012). Suitable blanks and positive control were also included, and paclitaxel was used as positive control. The inhibition percentage was calculated using the following formula:

Inhibition activity ð%Þ ¼ ½ðAcontrol  Asample Þ=Acontrol   100 where Acontrol is the absorbance of the control reaction and Asample is the absorbance in the presence of the sample. Each test was done in triplicate and the concentration required for a 50% inhibition of viability (IC50) was determined. 2.7. Determination of total phenolic content The Folin–Ciocalteu reagent was freshly prepared according to the method of the reports (Cao et al., 2008) by refluxing a mixture of 5.0 g sodium molybdate, 20.0 g sodium tungstate, 10 mL phosphoric acid (85%) and 20 mL concentrated hydro-chloric acid for 2 h followed successively by reacting it with 3 g lithium sulfate and 15 mL oxydol by a few drops of bromine. The total volume of the resulting solution was adjusted to 250 mL with distilled water. The solution was filtered and stored at 4 °C in the dark.

The OH radical-scavenging activity assay was conducted according to the Fenton method (Grymonpré et al., 2001; Wu and Hansen, 2008) with some modifications. Briefly, an aliquot of each sample (100 mL, different concentrations, w/v) was incubated with a mixture of 20 mL of FeSO47H2O (9 mM), 20 mL of hydroxybenzoic acid–ethanol solution (9 mM) and 20 mL of H2O2 (8.8 mM) in a 37 °C water bath for 30 min (Liu et al., 2012a). Scavenging capacity was read spectrophotometrically by monitoring the decrease of the absorbance at 510 nm. The percent OH radical-scavenging effect of each sample was calculated using the following equation:

OH scavenging activity ð%Þ ¼ ½ðAc  AtÞ=Ac   100 where Ac is the absorbance of the control reaction, and the sample is replaced by 100 mL ethanol. Tests were performed in triplicate.

2.10. Antibiotic (antibacterial and antifungal) assay 2.10.1. Microbial strains The bacterial strains, Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 25922), and Bacillus subtilis (ATCC 6633) were used for the studies. And the fungi strains were Fusarium graminearum, Gibberella zeae and Cercosporella albo-maculans.

2.10.2. Inhibitory zone assay The antimicrobial activity of the sample was determined using the agar well diffusion method (Wang et al., 2012) with some modifications. Briefly, 15 mL of the culture medium (beef-protein medium, pH 7.2) were poured in each 90 mm diameter sterilized Petri plate, and then plates were kept for 5 min for drying the surface of agar. 0.1 mL of the given microorganism (1  106–1  107 cells/mL) was added onto the plate containing cooled culture medium as mentioned before. 6 mm diameter wells were punched in the agar, and 50 lL of the sample (100 mg/mL) to be tested was poured into each well. Plates were incubated at 37 °C for 24 h. Control wells, containing methanol (negative control) and penicillin sodium (positive control) for the tested bacteria, were also run parallel in the same plate. Antimicrobial activity was assessed by measuring the diameter of the zone of inhibition for the respective drug. Each test was done in triplicate and the values reported herein are mean values of three experiments.

2.10.3. Minimum inhibitory concentration The minimum inhibitory concentration (MIC) of the EE was evaluated by two fold serial dilution method (Siddiqi et al., 2011). The sample was dissolved in methanol (filtered, 0.22 lm), and then diluted to obtain 200 mg/mL stock solutions. 0.5 mL of stock solution was incorporated into 0.5 mL of sterilized nutrient broth for bacteria and serially diluted to achieve 100, 50, 25, 12.5 and 6.25 mg/mL respectively. Analiquot of 100 lL standardized suspension of the test bacteria (105 CFU/ mL) was transferred to a well of 96-well tissue culture plate. Then, another 100 lL diluted samples were also added to each well and the inoculated 96-well tissue culture plates were incubated at 37 °C for 24 h. The MIC was defined as the lowest concentration of samples which inhibited the visible growth of tested microorganisms.

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K. Liu et al. / Food and Chemical Toxicology 58 (2013) 375–380 2.10.4. Antifungal assays Three fungal strains, namely F. graminearum, G. zeae and C. albo-maculans, were obtained from College of Life and Environmental Sciences, Minzu University of China. The antifungal activity of the EE was evaluated by means of the poisoned medium technique used in the previous study (Kanwal et al., 2010). Potato Dextrose Agar (PDA) medium was prepared by autoclaving at 121 °C for 30 min. Weighed 200 mg of the EE were dissolved in 1 mL of methanol, and added to flasks containing 99 mL of sterilized PDA medium, when still molten, to obtain 2 mg/mL final concentrations. The control received the same amount of methanol. 15 mL of the medium were poured in per sterilized Petri plate of 90 mm diameter and the medium was allowed to solidify. Mycelial disks of 6 mm diameter were prepared from the tips of 2–4 day-old culture of the three test fungal species with the help of a sterilized cork borer and placed in the centre of each plate. Each treatment was replicated three times. Plates were incubated in an incubator at 28 °C for 2–4 days. Fungal growth was measured by averaging the three diameters, taken at right angles, for each colony. The standard antifungal agent clotrimazole was used as a positive control and each test was repeated three times. The antifungal index was calculated using the following equation:

100 90

Inhibition activity (%)

80 70 60 50 40 30 20 10 0 6.25

12.5

where Da is the diameter of growth zone in the experimental dish (cm), Db is the diameter of growth zone in the control dish (cm), and Dc is the diameter of the mycelial disks placed in the centre of each petri plate (cm). 2.11. Statistical analysis Values are given as the mean values ± standard deviation of three measurements. The significance of difference was calculated by Student’s t-test, and values P < 0.05 were considered to be significant. Regression analysis was carried out using the Statistical Package for the Social Sciences (SPSS) 17.0 software (SPSS Inc., Chicago, IL).

Cancer diseases are one of the main causes of death worldwide. The discovery of new molecules from natural origin is a global trending currently for the less toxicity of natural products (Wang et al., 2012). Mushrooms comprise a vast and yet largely untapped source of powerful new pharmaceutical products. And there are approximately 651 species of higher basidiomycetes that have been found to possess antitumor activities (Fan et al., 2006). For example, the EE of Antrodia cinnamomea (AC) mycelia possessed high antihepatoma activity. The IC50 of EE of AC mycelia fermented for 4 week in a 500 mL fermentor against Hep3B and HepG2 cells were 36.9 and 3.1 lg/mL respectively. And no adverse effects of all samples on normal primary rat hepatocytes were observed (Chen et al., 2008). Fungal anticancer substances can be generally divided into two groups of high- and low-molecular-weight compounds. Most of the high-molecular-weight compounds are polysaccharides or protein-bound polysaccharides (Kidd, 2000). The second group comprises low-molecular-weight secondary compounds that can penetrate the cell membrane and act on specific signal-transduction pathways (Zaidman et al., 2005). These include mainly sesquiterpenes, triterpenes, steroids and sterols, and a few polyketides (Mahajna et al., 2009). To study the growth inhibitory activity of the EE from R. flava in vitro, human cancer cell lines (BGC-803, NCI-H520 and MDAMB-231) were incubated with various concentrations of the EE from R. flava. The potentially toxic effect of the EE was assessed using the MTT assay (Fig. 1 and Table 1). The EE showed inhibition activity on all these three human cancer cells, and the percentage of inhibition was 33.83%, 54.63% and 71.66% respectively, when the concentration was at 200 lg/mL. In particular, the EE displayed the strongest growth inhibitory activity on human breast cancer cell line MDA-MB-231 (IC50 = 66.54 ± 4.27 lg/mL). Ergosterol peroxide (EP) is a major antitumor sterol present in edible and medicinal mushrooms (Lindequist et al., 2005; Russo et al., 2010). Several studies reported anticancer activity of EP in few types of cancer cells such as U266 (multiple myeloma), SCC4

50

100

200

EE-MDA-MB-231

P-MDA-MB-231

EE-NCI-H520

P-NCI-H520

EE-BGC-803

P-BGC-803

Fig. 1. Inhibition activity of the EE on three cancer cell lines. Values are mean ± SD (n = 3). EE, ethanol extract; P, paclitaxel.

Table 1 The in vitro antitumor activity of the ethanolic extracts from R. flava (IC50 (lg/mL). Sample

3. Results and discussion 3.1. Anticancer activity

25

Concentration (µg/mL)

Antifungal index ð%Þ ¼ ðDa  Dc Þ=ðDÞb  DcÞ  100

Ethanol extract (EE) Paclitaxela a

Cancer cell MDA-MB-231

BGC-803

NCI-H520

66.54 ± 4.27 16.52 ± 0.33

743.99 ± 21.09 14.67 ± 0.89

134.44 ± 2.42 19.75 ± 1.00

Paclitaxel as positive control.

(head and neck squamous cell carcinoma), DU145 (prostate cancer), and MDA-MB-231 (breast cancer) cells (Rhee et al., 2012). For instance, EP attenuated cell growth and induced apoptosis in human prostate cancer LNCaP and DU-145 cells (Russo et al., 2010), and EP from the fermentation mycelia of Ganoderma lucidum cultivated in the medium exerted the cytotoxic effect against Hep 3B cells (Chen et al., 2009). In our previous study, six sterols including EP were isolated from R. flava (Liu et al., 2012b). And the EE displayed the strongest growth inhibitory activity on human breast cancer cell line MDAMB-231; this result suggests that EP in R. flava may be one of the reasons for the inhibitory activities of the EE on MDA-MB-231 cells. 3.2. Antioxidant activity 3.2.1. Recovery percent and phenolic content of R. flava fractions The EE from R. flava was fractionated by sequential extraction with solvents of increasing polarity: petroleum ether, ethyl acetate, n-butanol and water. It is well known that phenolic compounds are considered to be major contributors to the antioxidant capacity of plants (Sarikurkcu et al., 2010). Therefore, in the present study, the total phenolic content (TPC) of these fractions was evaluated. Extraction yield of the different solvent fractions obtained from R. flava are given in Table 2. The amount of extractable compounds, expressed as percentage by weight of dried powder, ranged from 0.42% for ethyl acetate fraction to 7.87% for n-butanol fraction. The total phenolic contents of the samples, as obtained from the calibration curve (y = 0.0062x + 0.0647, R2 = 0.9993, x is the absorbance; y is the concentration of gallic acid solution, lg/mL) and expressed as gallic acid equivalents (mg gallic acid/g dried extract), ranged from 6.66 mg GAE/g for n-butanol fraction to 61.01 mg GAE/g for water fraction (Table 2). The data presented in Table 2

K. Liu et al. / Food and Chemical Toxicology 58 (2013) 375–380

Table 2 The yield extraction, the total phenolic content, the evaluation of the IC50 values in the DPPH and OH radical-scavenging activity assay of R. flava.

a b

Samples

Yield (%)

Total phenolic contenta (mg GAE/g)b

DPPHa IC50 (lg/ OHa IC50 (lg/ mL) mL)

Ethanol extract (EE) Ascorbic acid Petroleum ether fraction Ethyl acetate fraction n-Butanol fraction Water fraction

10.00 – 1.10

12.95 ± 0.41 – 22.14 ± 0.76

34.41 ± 2.87 5.00 ± 0.31 42.29 ± 1.63

41.46 ± 0.80 20.55 ± 0.25 102.43 ± 2.0

0.42

14.52 ± 0.87

20.37 ± 1.31

32.41 ± 1.51

7.87 0.43

6.66 ± 0.32 61.01 ± 0.98

46.80 ± 1.67 5.86 ± 0.77

116.55 ± 2.11 18.08 ± 1.26

A 100 Scavenging activities (%)

378

80 60 40 20 0 5

Each value represents the mean ± SD of three experiments. (mg GAE/g): mg of gallic acid equivalent per g of dry mushroom ethanol extract.

10

20

50

100

200

Concentration (µg/mL)

indicated that the highest total phenol content of 12.95 mg GAE/g of dried sample was obtained in the EE, which was higher than the values reported for the methanol extracts from R. flava (10.51 mg GAE/g) (Gursoy et al., 2010). 3.2.2. DPPH radical-scavenging activity Free radicals are known to induce oxidative damage in biomolecules and play an important role in aging, cardiovascular diseases, cancer, impaired immune system, and inflammatory diseases (Wang et al., 2012). In this assay, the radical-scavenging activity of the EE and the four fractions was tested using the DPPH freeradical, which has the advantage of being unaffected by certain side reactions, such as metal ion chelation and enzyme inhibition (Amarowicz et al., 2004). The scavenging activity of the extracts tested was compared to those of ascorbic acid, used as positive control, and the DPPH radical-scavenging activity of the samples increased at sample concentrations ranging from 5 to 200 lg/mL (Fig. 2A). The activity can be evaluated by the determination of the IC50 values, which correspond to the amount of extract required to scavenge 50% of the DPPH radicals present in the reaction mixture. High-IC50 values indicate low-antioxidant activity. As shown in Table 2, water fraction shows the highest potent DPPH radical-scavenging activity, followed by ethyl acetate fraction, EE, petroleum ether and n-butanol fractions; and the IC50 values of the samples were ranged from 5.86 ± 0.77 lg/mL (water fraction) to 41.80 ± 1.67 lg/mL (n-butanol fraction). Although the TPC of petroleum ether fraction was significantly higher than that of ethyl acetate fraction, its antioxidant activity was lower. Our results demonstrate that R. flava extracts, particularly ethyl acetate and water fractions, have a better DPPH radical-scavenging activity and therefore exhibited higher antioxidant activities. 3.2.3. Hydroxyl radical-scavenging activity The hydroxyl radical and its subsequent radicals are the most harmful reactive oxygen species; they are mainly responsible for the oxidative injury of biomolecule (Xu et al., 2011). In the assay, the hydroxyl radical was generated through the Fenton reaction in this system, and the hydroxyl radical-scavenging activities of the samples tested were compared with that of ascorbic acid, which was used as a positive control, and the samples also exhibited dose-dependent hydroxyl radical-scavenging activities at concentrations ranging from 5 to 200 lg/mL (Fig. 2B). The scavenging effects of the samples were shown in Table 2, and the IC50 values of the samples were ranged from 18.08 ± 1.26 to 116.65 ± 2.11 lg/ mL. Based on the comparison of the IC50 values, the order of the hydroxyl radical-scavenging activity was found to be water fraction > ethyl acetate fraction > EE > Petroleum ether fraction > nbutanol fraction (Table 2). Water fraction had the highest hydroxyl radical-scavenging activity among all the samples, with the IC50

Scavenging activities (%)

B 100 80 60 40 20 0 5

10

20

50

100

200

Concentration (µg/mL) Fig. 2. The DPPH (A) and hydroxyl (B) radical-scavenging activities of the ethanol extracts and the fractions of R. flava at different concentrations (lg/mL), expressed as percentages of inhibition (%), versus the positive control (ascorbic acid). Data represent the means ± SD of three experiments. A acid, Ascorbic acid; EE, Ethanolic extract; PEF, Petroleum ether fraction; EAF, Ethyl acetate fraction; NBF, n-Butanol fraction; WF, Water fraction.

Table 3 Phenolic compounds content in water fraction of R. flava (mg/kg, dry basis).a

a

Compound

Sample

Quercetin (RT15.24 min) Chrysin (RT 21.15 min) Pinocembrin (RT 21.83 min)

0.81 ± 0.07 0.15 ± 0.03 0.91 ± 0.06

Results are expressed as mean of three determinations.

value of 18.08 lg/mL, which was lower than that of ascorbic acid. Therefore, water fraction produced by R. flava could be suitable for treating diseases in which oxygen-free radical production is involved. 3.2.4. Phenolic compounds Because water fraction had the highest DPPH and hydroxyl radical-scavenging activities among the various solvent-partitioned fractions, it was further purified by HPLC. Data from phenolic compounds in edible mushrooms is scarce (Ribeiro et al., 2007). The analysis by HPLC of water fraction revealed the existence of three phenolic compounds: quercetin, chrysin and pinocembrin; and the quantification of the three compounds were 0.81, 0.15 and 0.91 mg/kg, respectively (Table 3). All these compounds are reported for the first time in R. flava. 3.3. Antibiotic activity 3.3.1. Antibacteria activity The activity of the EE was tested against bacteria while penicillin sodium is used as a standard drug for comparison. The test

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K. Liu et al. / Food and Chemical Toxicology 58 (2013) 375–380 Table 4 Antimicrobial activity of the ethanolic extracts of R. flava. Microorganisms

Strain

MICa (mg/mL)

Inhibition zone (cm) EE

b

Penicillin sodiumc

Negative controld

EE

Penicillin sodium

Gram negative E. coli

ATCC 25922

1.24 ± 0.11e

2.88 ± 0.17

0

100.00

0.32

Gram positive S. aureus B. subtilis

ATCC 6538 ATCC 6633

2.18 ± 0.16 1.68 ± 0.14

3.04 ± 0.26 3.00 ± 0.14

0 0

6.25 25.00

0.16 0.16

a

Minimum inhibitory concentrations (MIC). The ethanol extract of R. flava (5 mg/well). c The concentration of penicillin sodium was 0.5 mg/well to gram negative microorganisms and 0.25 mg/well to gram positive microorganisms. d Methanol (0.005 mL/cell) was used as the negative control. e Each value is expressed as mean ± SD (n = 3). b

Table 5 Antifungi activity of the ethanol extracts of R. flava against three pathogenic fungi. Sample

Ethanol extract (EE) Clotrimazole Negative control a

Antifungal indexa (%) F. graminearum

F. auenaceum

C. albo-maculans

19.99 ± 1.24 69.55 ± 2.46 0.00 ± 0.00

36.64 ± 1.78 77.96 ± 2.23 0.00 ± 0.00

30.03 ± 1.83 70.44 ± 1.76 0.00 ± 0.00

Each value is expressed as mean ± SD (n = 3).

microorganisms used in the present studies included E. coli (as gram negative bacteria) and S. aureus and B. subtilis (as gram positive bacteria). The agar well diffusion method was used to evaluate the antibacterial activity of the EE (Wang et al., 2012). The EE from R. flava was found to be active against all the tested gram-positive and gram-negative species (Table 4). The zone of inhibition (ZOI) values obtained indicates that among the tested bacteria, the most susceptible bacterium was S. aureus, followed by B. subtilis, E. coli showed the least sensitivity. This agrees with previous reports that, in general, gram-positive bacteria are considered more sensitive than gram-negative bacteria to different microbial compounds because of the differences in the structure of their cell walls (Siddiqi et al., 2011). Further to it, the EE showed low activities as compared to standard drug towards all the organisms. Gezer et al. (2006) reported that the EE of R. flava inhibited the growth of gram-positive bacteria better than gram-negative bacteria and exhibited more significant inhibitory activities against S. aureus than B. subtili. The result was similar to ours. However, the literature also reported that the EE of R. flava showed no antibacterial activity against E. coli (ATCC 35218), and this results is different with ours. It was probably that the isolated method, the E. coli strains adopted and the collected region of the R. flava were different in the two literatures. The MIC values for the EE against B. subtilis, S. aureus and E. coli were 25, 6.25 and 100 mg/mL (Table 4). The values of MIC showed that the EE was found with higher inhibition on S. aureus than on B. subtilis and E. coli. EP in R. flava may be one of the reasons for the inhibitory activities of the EE on microorganisms, as the previous reports showed that steroids like EP, isolated from Ganoderma applanatum, proved to be weakly active against a number of gram-positive and gramnegative microorganisms (Artur Smania et al., 1999). 3.3.2. Antifungal activity The dada demonstrated that the EE from R. flava possess antifungal activities against these three pathogenic fungi (Table 5). The EE was most effective against Fusarium auenaceum, where at the concentration of 2 mg/mL caused a 36.64% reduction in fungal

growth. The EE also suppressed the growth of C. albo-maculans and F. graminearum by 30.03 and 19.99% respectively, at the concentration of 2 mg/mL (Table 5). Further to it, the EE showed low activities as compared to standard drug clotrimazole toward all these three pathogenic fungi. 4. Conclusion In the present study, it was found that the EE from R. flava possessed anticancer, antioxidant, and antibiotic activities. To the best of our knowledge, except the DPPH free-radical-scavenging activity, this is the first report of such activities from this mushroom. Our results revealed that the EE showed anticancer, antioxidant, and antibiotic capacities. Furthermore, three phenolic compounds were also identified from water fraction and they were isolated from R. flava for the first time. These results showed that R. flava could be a potential source of pharmaceuticals. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This work was financially supported by the National Basic Research Program of China (973 Program, 2009CB522300), the ‘‘985’’ Project (MUC98504-14 and MUC98507-08), ‘‘111’’ Project (B08044), and Science Research Program for Colleges and Universities of Hebei Province (2011174). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fct.2013.05.001. References Amarowicz, R., Pegg, R.B., Rahimi-Moghaddam, P., Barl, B., Weil, J.A., 2004. Freeradical scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies. Food Chem. 84, 551–562. Artur Smania, J., Monache, F.D., Smania, E.d.F.A., Cuneo, R.S., 1999. Antibacterial activity of steroidal compounds isolated from Ganoderma applanatum (Pers) Pat. (Aphyllophoromycetideae) fruit body. Int. J. Med. Mushrooms 1, 325–330. Cao, Y.P., Dai, H.Z., Cao, W., Han, Z.P., 2008. Determination of total phenols in Zizyphus jujuba Mill. by Folin-Ciocaileu colorimetry. J. Anhui Agric. Sci. 36 (1299), 1302. Chen, Y.S., Pan, J.H., Chiang, B.H., Lu, F.J., Sheen, L.Y., 2008. Ethanolic extracts of Antrodia cinnamomea mycelia fermented at varied times and scales have differential effects on hepatoma cells and normal primary hepatocytes. J. Food Sci. 73, 179–185.

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