Abstract: Lactarius vellereus is a well known macro fungus used for food in Turkey. Although it has been consumed as food, there is no information on the biological properties. Therefore we aimed to reveal phenolic substances, antioxidant capacity and antimicrobial effect of L. vellereus. Seven phenolics were analysed by High Performance Liquid Chromatography (HPLC) in L. vellereus and four of them were determined to be catechin, ferulic acid, p-coumaric acid and cinnamic acid. The scavenging effect of L. vellereus on 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH) was measured as 86% at 1 mg mL-1 concentration and its reducing power was 0.205 at 0.4 mg mL-1 concentration. The IC50 values were determined as 0.68 mg mL-1 of L. vellereus, 0.43 mg mL-1 of BHA and 0.72 mg mL-1 of BHT. Chloroform, acetone and methanol extracts of L. vellereus were tested for its antimicrobial activities using a micro dilution method against four gram-positive and five gram-negative bacteria. The Minimum Inhibitory Concentration (MIC) was observed with the acetone extract (MIC, 19 μg mL-1) against K. pneumoniae. All extracts of the fungus was usually found to be antimicrobial activity against the test microorganisms. The results showed that L. vellereus can be recommended as a source of natural antioxidants and antimicrobials.
INTRODUCTION
Some fungal species have been used as tea or nutritious food source for many years in Eastern cultures because of their unique scents and soft structures.
Some species of edible, inedible and poisonous mushrooms are known to have important medicinal properties. Some studies have demonstrated that fungi have high amounts of antioxidants and antimicrobial effects (Mau et al., 2002; Yang et al., 2002; Cui et al., 2005; Barros et al., 2007a; Nakajima et al., 2007; Jua et al., 2010). Antioxidants, or the molecules that have a scavenging effect on DPPH radicals, are known as potentially protective substances (Ramirez-Anguiano et al., 2007). This protective effect is mainly attributed to well-known antioxidants such as ascorbic acid, tocopherols and β carotenes, but plant phenols also play an important role. According to Barros et al. (2007b), mushrooms can be regarded as rich sources for molecules with antioxidant, antitumor and antimicrobial properties.
L. vellereus is used as a food in Turkey. Local people consume this mushroom and also sell it at the open markets. It grows in coniferous or mixed coniferous and deciduous forests. Although, L. vellereus is an important food source, there are no conclusive reports concerning its phenolics, antioxidant activities or antimicrobial effects. The main objectives of the current study are as follows: (1) To reveal the phenolics, (2) To study the antioxidant activity of L. vellereus methanolic extract and (3) To find the antimicrobial effect of L. vellereus.
MATERIALS AND METHODS
Collection of L. vellereus samples: The samples were collected in the mixed Pinus sylvestris and Quercus sp. forest from Ordu in 2007. The fungarium number of the species is HD3958. The species identification was performed as described in the literature (Basso, 1999). A stock sample of the species was also deposited at the Fungarium of the Mushroom Application and Research Centre, Selçuk University, Konya, Turkey.
Antioxidant activities
Determination of the phenolics: The total phenolic content of the methanol
crude extract was determined by the Folin-Ciocalteu method with some modifications,
according to Singleton and Rossi (1965).
A Shimadzu 1100 series HPLC equipped with a SIL-10AD vp autosampler and LC-10Advp pump system, Diode Array Detector (DAD) and an Inertsil Agilent Eclipse XDB column (240x4.60 mm, 5 μm particle size) was used to analyse phenolic compounds. The mobile phase consisted of (A) 100% methanol and (B) 3% (v/v) aqueous acetic acid. HPLC separation was performed as described by Maltas and Yildiz (2011). Gallic acid, catechin, caffeic acid, p-coumaric acid, ferulic acid, cinnamic acid and quercetin (Sigma-Aldrich) were analysed as standard phenolic structures. The samples were run in triplicate.
Preparation of the methanol extracts for testing antioxidant activities: Briefly, 100 g of the dried and powdered sample was extracted with stirring in methanol at 60°C for 6 h in a Soxhlet apparatus. The extract was then filtered through Whatman No. 4 filter paper and concentrated under a vacuum at 45°C using a rotary evaporator. The extracts were then lyophilised and stored in the dark at 4°C.
Scavenging effect on DPPH radicals: A 5 mL of the methanol extract in a range of concentrations (0.5-5 mg mL-1) was added to 1 mL of a DPPH radical solution in methanol (the final concentration of DPPH was 0.2 mM). The mixture was shaken vigorously and left to stand for 30 min in the dark and the absorbance was then measured at 517 nm against a blank using the Hitachi U-2001 spectrophotometer (Shimada et al., 1992). BHT and BHA were used as standard controls. The inhibition of DPPH free radicals was calculated in percent (I%) using the following equation:
where, Ablank is the absorbance of the control reaction and Asample is the absorbance of the test compound. The extract concentration providing 50% inhibition (IC50) was calculated from a graph plotting inhibition percentage against extract concentration. Tests were performed in triplicate.
β-carotene-linoleic acid assay: One milliliter of β-carotene solution in chloroform (3.34 mg mL-1) was pipetted into a flask containing 40 mg linoleic acid and 400 mg Tween 20. The chloroform was then removed using a rotary evaporator at 40°C for 5 min. To the resulting residue, 100 mL of oxygen passed through distilled water was added slowly with vigorous agitation to form an emulsion. A 5 mL aliquot of this emulsion was added to a tube containing 0.2 mL of the 200 mg mL-1 antioxidant solution and the absorbance was measured immediately at 470 nm against a blank, which consisted of the emulsion without β-carotene. The tubes were then placed in a water bath at 40°C and the absorbance was measured again at 15 min intervals.
Reducing power: Each extract (0.04-0.4 mg mL-1) in methanol (2.5 mL) was mixed with 2.5 mL of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide and the mixture was incubated at 50°C for 20 min. Next, 2.5 mL of 10% trichloroacetic acid (w/v) was added and the mixture was centrifuged at 200 g for 10 min. The upper layer (5 mL) was mixed with 5 mL of deionised water and 1 mL of 0.1% ferric chloride. Finally, the absorbance was measured at 700 nm against a blank in a Hitachi U-2001 spectrophotometer.
Determination to antimicrobial effects
Preparation of the extracts: Powdered fungus sample (30 g) was extracted
with 250 mL of chloroform in a glass beaker for 8 h at 55°C. The resultant
extract was concentrated using a rotary evaporator at 40°C and low pressure
and the desired phase was separated from the crude extract with chloroform.
Later, the residue was extracted with acetone and methanol, respectively. After
extraction, all the semi-solid extracts were dried by a freeze-dryer to yield
powders. The powdered extracts were dissolved in DMSO:PBS (1:1) at a 100.000
μg mL-1 concentration. These extracts were filtered through
a sterile filter (0.45 μm) and stored at 4°C.
Test microorganisms: All the microorganisms were obtained from the Department of Biology, Faculty of Science, Selçuk University. Four Gram-positive bacteria (Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6633, Listeria monocytogenes type 2 NCTC 5348 and Streptococcus pyogenes ATCC 19615) and five gram-negative bacteria (Escherichia coli ATCC 35218, Klebsiella pneumoniae ATCC 10031, Pseudomonas aeruginosa ATCC 15442, Proteus vulgaris ATCC 7829 and Salmonella enteritidis RSHMB) were chosen as the test bacteria. Candida albicans ATCC 1023 was chosen as the test yeast.
Antimicrobial assay: Brain Heart Infusion Broth (BHIB, Oxoid) was used to cultivate the bacteria and Malt Extract Broth (MEB, Difco) was used for the yeast. Each bacterial species obtained from stock cultures were added into 4-5 mL BHIB and incubated at 35°C for 24 h. The bacterial cultures were prepared in the same medium at a density adjusted to 0.5 McFarland turbidity standards (108 CFU mL-1) and the final concentration of each bacterial culture was adjusted to 105 CFU mL-1. The yeast strain obtained from the stock culture was added into 4-5 mL MEB and incubated at 25°C for 48 h. After incubation, the final concentration of the yeast was adjusted to 104 CFU mL-1.
Determination of antimicrobial activity by the micro dilution method: L. vellereus extract in the stock solutions was prepared at 20000 μg mL-1 concentration in PBS: DMSO (1:1). MHB (100 μL) was dispensed into each well of a flat-bottom, 96-well microtiter plate. To prepare serial dilutions, 100 μL of L. vellereus chloroform, acetone or methanol extract was separately dispensed into MHB-containing microplate wells (A1, A2, A3, etc.) and mixed well. Next, 100 μL of this mixture was pipetted from the first well into a second well, generating a ½ dilution. Finally, this process was repeated to generate a dilution series of each extract from 20000 to 0.305 μg mL-1. After the dilution series of each extract was prepared, 100 μL of each bacterial suspension were separately added into each well containing the MHB and the mushroom extract mixture. This procedure was also repeated for the yeast in different plate wells.
The absorbance of each well was measured using an ELISA reader at 630 nm (EL x 800). After the first reading was finished, all the plates were covered and incubated at 37°C for 24 h. Afterwards, the absorbance was measured again. The first absorbance was subtracted from the second absorbance and the difference was used to calculate the MIC values. The lowest concentration that produced an inhibitory effect was recorded as the Minimum Inhibitory Concentration (MIC) for each extract (as described by Devienne and Raddi (2002), with some modifications). Ampicillin (100 μg mL-1 concentration) for bacteria and Amphotericin B (50 μg mL-1 concentration) for yeast was used as positive controls. Each experiment was conducted in triplicate. Due to a lack of correlation and statistical significance between each bacterial species, statistical analysis was not performed.
RESULTS AND DISCUSSION
Phenolic contents of L. vellereus: The amount of phenolic compounds in the methanol extract of L. vellereus was determined to be 0.17 mg mL-1 gallic acid equivalents. The phenolic composition of L. vellereus was reported here for the first time (Table 1). The amount of total phenolics in wild mushrooms varies in different ranges in the literature. The phenolics were found to be 23.28 mg g-1 of Coriolus versicolor (L.) Quél., 47.25 mg g-1 of Ganoderma lucidum (Curtis) P. Karst. and 51.28 mg g-1 of G. tsugae Murril (Mau et al., 2002). Wong and Chye (2009) observed the total phenolics of Pleurotus porrigens (Pers.) P. Kumm., Xerula furfuracea (Peck) Redhead, Ginns and Shoemaker, Schizophyllum commune Fr., Polyporus tenuiculus (P. Beauv.) Fr., Hygrocybe conica (Schaeff.) P. Kumm. and Pleurotus florida singer with petroleum ether and methanolic extract. According to their results, the amount of the total phenolics in wild mushrooms varied from 0.98 to 28.23 mg GAE g-1 of PE extract. Among the PE and methanolic extracts, H. conica had the highest phenolic content of 28.23 and 42.21 mg GAE g-1, respectively while other species varied between 0.83-27.15 mg GAE g-1 for both PE and methanolic extract. Barros et al. (2007b) studied the phenolics of three mushrooms and they found that Leucopaxillus giganteus (Quél.) Singer extracts showed the highest phenolic content (6.29 mg g-1) whereas Agaricus arvensis Schaeff. extracts (2.83) was slightly lower than in Sarcodon imbricatus (L.) P. Karst. (3.76 mg g-1). The total phenolic content of Pleurotus ostreatus (Jacq.) P. Kumm. was found to be 5.49 g/100 g (Jayakumar et al., 2009). Compared to relevant literature, the total phenolic of L. vellereus is relatively lower. The yield of extracts do not associated with the total phenolics content of the fungal species but might be influenced by the types of phenolic compounds present along with the extractability of the solvent used in the preparation. These results indicate that phenolic quantities in fungal species can vary dependent on the methods applied or the solvents.
Seven phenolics from the methanolic extract of L. vellereus were analysed and four of them were identified as catechin, ferulic acid, p-coumaric acid and cinnamic acid as illustrated in Table 1, there are significant qualitative and quantitative differences in the components of the extracts. Catechin is the predominant phenolic acid with a value of 20 mg g-1, followed by ferulic acid at 18 mg g-1, p-coumaric acid at 12 mg g-1 and cinnamic acid at 8 mg g-1. However, the extract did not contain any flavonoids, such as quercetin, gallic acid and caffeic acid. Antioxidant, antimicrobial, antiallergy and anticancer effects of catechin were reported in some previous studies (Kondo et al., 2000; Shimamura et al., 2007). Catechin was found in R. delica at 5.33 mg g-1 by Yaltirak et al. (2009). In the present study, it was found at 20 mg g-1 and this ratio is quite high. Antimicrobial activities were already appearing in high-impact in the present study and this can be related with high catechin level.
Ferulic acid, likes many phenols, is an antioxidant ‘in vitro’ in the sense that it is reactive towards free radicals, such as Reactive Oxygen Species (ROS).
| Table 1: | Percentage values of Identified Phenolic substances of L. vellereus |
| ND: Not deleted | |
ROS and free radicals are implicated in DNA damage, cancer and accelerated cell aging. Ferulic acid may be effective at preventing cancer induced by exposure to carcinogenic compounds (Kampa et al., 2004).
Coumarin derivatives are important substances for human health. They have anti-thrombotic, anti-inflammatory and vasodilatory effects as well as antiviral and antimicrobial activities (Cowan, 1999).
Cinnamic acids are common representatives of a wide group of phenylpropane-derived compounds that are in the highest oxidation state. Cinnamic acids are effective against viruses, bacteria and fungi (Cowan, 1999).
As a result, high and varied phenolic compounds in L. vellereus increase its medical importance.
Antioxidant activity results
The scavenging effects on DPPH radicals: The DPPH radical scavenging effects
of L. vellereus, as well as those of BHA and BHT controls increased
with the increasing concentration from 0.5 to 5 mg mL-1 (Fig.
1). The scavenging values at 0.5 mg mL-1 concentration was 0.046
mg mL-1 for L. vellereus, 0.047 mg mL-1
for BHT and 0.038 mg mL-1 for BHA, while the scavenging values at
5 mg mL-1 was 0.035 mg mL-1 for L. vellereus,
0.035 mg mL-1 for BHT and 0.028 mg mL-1 for BHA. As illustrated
in Fig. 1, the scavenging effect of L. vellereus
extract on DPPH radicals rose with the increasing concentrations.
The IC50 value of L. vellereus extract was higher than BHA and lower than BHT. The IC50 values was 0.68 mg mL-1 for L. vellereus, 0.43 mg mL-1 for BHA and 0.72 mg mL-1 for BHT (Fig. 2). A lower IC50 value indicates higher antioxidant activity (Pourmorad et al., 2006). The IC50 of L. vellereus was lower than the standard of BHT and it has an effective antioxidant activity. The inhibition values of L. vellereus extract and the standards on the DPPH radicals at 5 mg mL-1 were 88% for L. vellereus, 90% for BHA and 88% for BHT (Fig. 3).
In previous studies, the scavenging effects of mushroom extracts ranged from 36-96% at the concentrations of 5 to 10 mg mL-1. Because different methods and concentrations were used in the literature, there are no standard levels for measuring scavenging effects. Some studies can be summarised as follows. The methanolic extract of Dictyophora indusiata (Vent.) Desv. was 92% at 6.4 mg mL-1 (Mau et al., 2004). Yang et al. (2002) investigated mushrooms of some commercial importance (Flammulina velutipes (Curtis) Singer, Lentinula edodes (Berk.) Pegler, Pleurotus ostreatus (Jacq.) P. Kumm. and P. cystidiosus O.K. Mill.) and they found that the scavenging effects of these mushrooms varied between 42.9 and 81.8% at 6.4 mg mL-1.
| Fig. 1: | The DPPH radical scavenging effects of L. vellereus, BHA and BHT |
| Fig. 2: | IC50 values of L. vellereus, BHA and BHT |
| Fig. 3: | Percentage inhibition values of L. vellereus and standards on the DPPH radicals |
The scavenging effect of Volvariella volvacea (Bull.) Singer was also found as 57.8% at 9 mg mL-1 (Cheung et al., 2003). Mau et al. (2004) used 10 mg mL-1 concentration in their studies, which reported scavenging effects of 78.8%, 79.9% and 94.1% for Termitomyces albuminosus (Berk.) R. Heim, Grifola frondosa (Dicks.) Gray and Morchella esculenta (L.) Pers., respectively. Additionally, Lee et al. (2008) studied the fruiting body and mycelia of Hypsizygus marmoreus (Peck) H.E. Bigelow with hot water and methanol extraction. According to their results, the scavenging effects of the fruiting body and mycelia ethanol extracts, at 5 mg mL-1 concentration, were both 75.5%, while the scavenging effects of the fruiting body and mycelia hot water extracts were 36.8 and 55.5%, respectively, also at 5 mg mL-1. The scavenging effect of Russula delica extract at 10 mg mL-1 was found as 26% (Yaltirak et al., 2009). Compared with the inhibition values reported in those studies, L. vellereus is effective at lower concentration. In the present study, inhibition levels of L. vellereus reached 88% at 5 mg mL-1.
β-carotene-linoleic acid assay: The rate of absorbance change was calculated from T0 to 120th min and used to calculate the coefficient of oxidation prevention as a percent (%). The bottom absorbance curve in Fig. 4 is the control sample. The absorbance at the 120th min was measured as 1.643 mg mL-1 for L. vellereus, 0.73 mg mL-1 for control, 1.74 mg mL-1 for BHT and 1.89 mg mL-1 for BHA. Antioxidant activity was assayed as the ability to inhibit the peroxidation of linoleic acid. The inhibition values of L. vellereus were determined to be higher than Trolox, but lower than BHT and BHA. L. vellereus, BHA, BHT and Trolox exhibited 64.6, 80.8, 73.7 and 50.51% inhibition, respectively (Fig. 5).
According to literature, the inhibition values of mushroom extracts vary from 50 to 96% at the different concentrations. The antioxidant activity of methanolic extracts of young and mature Agaricus brasiliensis Fr. specimens were evaluated with β-carotene and were found to inhibit oxidation 92% at 0.2 mg mL-1 (Soares et al., 2009). Gursoy et al. (2009) studied six Morel species (Morchella rotunda (Pers.) Boud., M. esculenta (L.) Pers. var. umbrina (Boud.) S. Imai, M. deliciosa Fr., M. elata Fr., M. conica Krombh. and M. angusticeps Peck) and found M. esculenta var. umbrina and M. angusticeps to be the most active species (96.89 and 96.88%, respectively, at 4.5 mg mL-1 concentration). Sarikurkcu et al. (2010) investigated Amanita caesarea (Scop.) Pers., Clitocybe geotropa (Bull.) Quél. and Leucoagaricus pudicus (Bull.) Bon and they discovered that L. pudicus possessed the highest oxidation level (81.8% at 25.5 mg mL-1), followed by A. caesarea (70.1%) and C. geotropa (61.3%). In comparison to previous studies, L. vellereus has a high oxidation level even at a low concentration (64.6% at 2.284 mg mL-1 concentration).
Reducing power: The reducing power of L. vellereus showed a parallelism with the increasing concentration (Fig. 6). The highest reducing power, 0.04 to 0.4 mg mL-1, was observed with BHA (1.895 mg mL-1) followed by BHT (1.654 mg mL-1) and L. vellereus (0.205 mg mL-1).
| Fig. 4: | Oxidation of linoleic acid depented on the time |
| Fig. 5: | Percentage of inhibition of linoleic acid oxidation |
| Fig. 6: | Reducing powers of L. vellereus and synthetic antioxidants |
According to Elmastas et al. (2007), the reducing power of R. delica and Verpa conica (O.F. Müll.) Sw extracts were 1.32 and 1.22 μg mL-1, respectively, at 200 μg mL-1. At 20 mg mL-1, the reducing powers of A. caesarea, C. geotropa and L. pudicus were 1.5, 1.2 and 1.3 mg mL-1, respectively (Sarikurkcu et al., 2010). In the present study, the reducing power of L. vellereus was 0.205 at 0.4 mg mL-1 concentration. In comparison to the relevant studies, L. vellereus possesses a high reducing power at a low concentration. Antimicrobial results: According to Craig (1998), to evaluate antimicrobial activity, MIC values should be measured from the 4th through the 16th dilutions. The antimicrobial effects of L. vellereus against bacteria and yeast were measured in accordance with the following ranges (Gulay, 2002; Morales et al., 2008):
| • | MIC values are lower than 100 μg mL-1 = antimicrobial activity is high |
| • | MIC values are between 100 and 500 μg mL-1 = antimicrobial activity is moderate |
| • | MIC values are between 500 and 1000 μg mL-1 = antimicrobial activity is weak |
| • | MIC values are more than 1000 μg mL-1 = no antimicrobial effect |
In accordance with these ranges, the antimicrobial results are given in Table 2. The Minimum inhibitory effect on the test microorganisms was observed with acetone extract (MIC, 19 μg mL-1) against K. pneumoniae. Overall, all extracts exhibited good antimicrobial effect with values generally lower than 100 μg mL-1 (MIC, 39 μg mL-1) placing it in the high activity category, while the MIC of chloroform extract was much lower (MIC, 78 μg mL-1) against K. pneumoniae.
These current results are also confirmed by previous studies. Quereshi et al. (2010) tested the antimicrobial activity of various solvent extracts of G. lucidum (Curtis) P. Karst (40 mg mL-1 concentration) against six species of bacteria. Acetone extract exhibited maximum antibacterial activity, while the most susceptible bacterium observed was K. pneumoniae. Bala et al. (2011) investigated the antimicrobial effect of forty-seven different specimens from Australia and they found that water and ethanol extracts were more effective against S. aureus than E. coli, whereas a small number of hexane extracts showed better results for their potential antimicrobial effect against E. coli at higher concentration. In generally, a number of macrofungi in the genera Agaricus, Amanita, Boletus, Cantharellus, Fomitopsis, Hohenbuehelia, Lentinus, Ramaria and Strobilomyces showed good inhibition rates. Aqueous and methanolic extracts of Trametes hirsuta (Wulfen) Lloyd were tested against pathogenic fungi and bacteria.
The maximum antibacterial activity of aqueous extract of T. hirsuta was found against S. aureus than that of methanol extract. The significant antifungal activity of aqueous extract was found against Aspergillus flavus than that of methanol extract (Sivaprakasam et al., 2011). The antibacterial effects of three mushrooms extract G. lucidum, Auricularia auricula (L.) Underw., Pleurotus floridanus singer were studied against S. aureus and E. coli.
| Table 2: | Minimal inhibition concentration values of L. vellereus extracts (μg mL-1) against tested microorganisms |
| *-<100 μg mL-1: High, 100-500 μg mL-1: Moderate, 500-1000 μg mL-1: Low | |
A. auricula showed significant antibacterial activity against S. aureus. P. floridanus showed some antibacterial activity while G. lucidum showed no antibacterial activity. None of the extracts showed any activity against E. coli (Iftekhar et al., 2011). Antimicrobial activity of Ganoderma praelongum Murrill, G. resinaceum Boud. and G. lucidum were evaluated against thirty strains of clinical isolates of methicillin resistant and methicillin sensitive S. aureus (MSSA). Maximum activity of crude extracts was exhibited by ethyl acetate. The MIC of Sesquiterpenoids extract of G. praelongum was 0.390-6.25 mg mL-1. Diterpenoids and triterpenoids displayed moderate activity while polysaccharides IIIa and IIIb showed weak activity. All bacterial strains were resistant to polysaccharides I and II (Ameri et al., 2011).
The current results are similar or more effective than those reported in the literature. To the best of our knowledge, there were previously no reports for the antimicrobial effects of L. vellereus and this result was reported here for the first time.
CONCLUSION
The total phenolic, some phenolics, antioxidant activities and antimicrobial effects of L. vellereus were measured. Fungal species can be used a source of healthy foods, contributing high levels of antioxidants and antimicrobial activities.
The present results indicate that economically important and edible mushrooms display significant antioxidant and antimicrobial features. Therefore, these studies should be extended to other economically important and edible mushrooms.
ACKNOWLEDGMENTS
The authors are indebted to the Foundation for TUBİTAK (TBAG/109T584) and Scientific Research Projects (BAP) Coordinating Office (BAP/08201036-BAP10701001) at Selçuk University for their financial support of this work.