Biological activities and DNA interactions of Amanita ovoidea

Abstract

Context: Amanita ovoidea (Bull.) Link (Amanitaceae) is a well-known species due to its pleasant aroma and flavor since ancient times in the worldwide. This species is also known in Turkey and people consume it extensively.

Objective: To evaluate medicinal importance of A. ovoidea for human health, to explain the effect of mushroom extracts on bacterial DNA, and to find preventive role on bacterial disease.

Materials and methods: Chloroform, acetone, and methanol extracts of A. ovoidea were tested for the antimicrobial activities against four Gram-positive bacteria, five Gram-negative bacteria, and yeast using a micro-dilution method. In addition, DNA binding, DNA cleavage activity, and restriction enzyme digestion of the methanol extract of A. ovoidea were examined at different concentrations (40.000–78.125 µg/mL).

Results: The highest minimum inhibitory concentration (MIC) value observed against the test micro-organisms was with the chloroform extract (MIC 19.5 µg/mL concentration) against Candida albicans. Other highest antimicrobial effects observed against the test micro-organisms were with the methanol extracts against Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes, Candida albicans, Klebsiella pneumoniae, Proteus vulgaris, and Salmonella enteritidis (MICs, 78 µg/mL concentrations). All concentrations reduced the mobility of plasmid DNA. BamHI and HindIII targeted specially to supercoils and cut them. Amanita ovoidea extract prevented cutting with HindIII by binding especially to the AA region in open circular DNA.

Discussion and conclusion: Present results demonstrated that A. ovoidea has excellent antimicrobial and antifungal activities by its DNA interaction activity on pBR322.

• Antimicrobial activity
• DNA binding
• DNA cleavage
• micro-dilution

Introduction

Mushrooms contain some special components which are therapeutic value and they have been used from the ancient times as remedy for human disease. They are natural sources of antimicrobial, antioxidant, or antiviral agents primarily because of the large biodiversity of such micro-organisms and relatively large quantity of metabolites that can be extracted from these mushrooms. Some mushrooms also have special bioactive compounds namely β-d-glucans, β-d-glucans with heterosaccharide chains of xylose, mannose, galactose or uronic acid, or β-d-glucan–protein complexes (Smith et al., 2002). These compounds are very effective against pathogen micro-organisms mainly bacteria or viruses. Some mushroom species that have these bioactive compounds are Ganoderma lucidum (Curtis) P. Karst. (Ganodermataceae), Flammulina velutipes (Curtis) Singer (Physalacriaceae), Cordyceps spp. (Cordicipitaceae), Lentinula edodes (Berk.) Pegler (Omphalotaceae), Grifola frondosa (Dicks.) Gray (Meripilaceae), Pholiota nameko (T. Itô) S. Ito & S. Imai (Strophariaceae), Sparassis crispa (Wulfen) Fr. (Sparadissidaceae), etc. These species have been used for both traditional medicine and medical industry.

DNA interaction, cleavage, and binding studies of a mushroom extract have not been seen. But DNA properties (binding and cleavage) of the metal complex or compounds are the subject of intense investigation in the fields of chemistry, biology, and medicine in recent years (Dede et al., 2009; McMillin & McNett, 1998; Rao et al., 2007). Such studies are becoming increasingly important in the development of pharmacological properties such as cancer chemotherapeutic agents, molecule probes, and potential artificial gene regulators (Dede et al., 2009; Halli & Patil, 2011; Li et al., 2005; Neelakantan et al., 2008).

Although Amanita ovoidea (Bull.) Link (Amanitaceae) is an important food source in Turkey, there are no conclusive reports on the antimicrobial effects about this mushroom. The main objectives of this study are (i) to reveal the antimicrobial effects of chloroform, acetone, and methanol extracts of A. ovoidea against Gram-positive and Gram-negative bacteria and yeast, and (ii) to observe the interaction mechanism with plasmid DNA.

Materials and methods

Collection of A. ovoidea samples

The samples of A. ovoidea were collected in the field in mixed Pinus brutia, Quercus sp. and Arbutus sp. forest in 2009. The collection site was Antalya-Gazipaşa, Çampınarı, Göce Mah., in mixed Pinus brutia, Quercus sp. and Arbutus sp. forest in needle litter, 36°22′10N–32°21′56E, 420 m, 11 November 2009, HD5054.

The species identification was performed by Hasan Huseyin Doğan as described in the literature (Galli, 2001). A stock sample of the species was also deposited at the Fungarium of the Mushroom Application and Research Centre, Selcuk University, Konya, Turkey.

Chemicals used for the studies

Chloroform, acetone, methanol, EDTA (ethylenedinitrilotetraacetic acid), DMF, and glacial acetic acid were supplied by Merck (Darmstadt, Germany). Ampicillin, Amphotericin B, Brain Heart Infusion Broth (BHIB), dimethyl sulfoxide (DMSO), Malt Extract Broth (MEB), and phosphate buffered saline (PBS) were purchased from Oxoid Limited, Hampshire, UK. Tris base and agarose were supplied by Sigma-Aldrich (St. Louis, MO). pBR322 plasmid DNA, BamHI, and HindIII were obtained from Fermentas (Thermo Fisher Scientific Inc., Waltham, MA).

Antimicrobial activity

Preparation of the extracts

The fruiting bodies of mushroom sample were dried and powdered. Chloroform, acetone, and methanol were used as solvents. Each powdered fungus sample (30 g) was suspended with 250 mL of chloroform in a Soxhlet apparatus (Isolab, Istanbul, Turkey) for 8 h. The resultant extract was concentrated by means of a rotary evaporator (Staufen, Germany) at 40 °C and at low pressure, the desired phase was separated from the crude extract with chloroform. Later, the residue was extracted with acetone and methanol. 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 concentration and filtered through a sterile filter (0.45 µg) and stored at 4 °C.

Test micro-organisms

All the micro-organisms were obtained from the Department of Biology, Faculty of Science, Selcuk University, Turkey.

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

BHIB was used to cultivate the bacteria and MEB 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), and the final concentration of each bacterial culture was adjusted to 10⁵ Cfu/mL. 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.

Determination of antimicrobial activity by micro-dilution method

The MIC values were evaluated in accordance with NCCLS (2008). Amanita ovoidea extract in the stock solutions was prepared at a 20.000 µg/mL concentration in PBS:DMSO (1:1). MHB (100 µL) was dispensed into each well of a flat-bottom, 96-well microtiter plate. Serial dilutions of 100 µL of A. ovoidea chloroform, acetone, or methanol extract were separately prepared. Finally, a dilution series of each extract from 20.000 to 0.305 µg/mL was obtained. After the dilution series of each extract was prepared, 100 µL of each bacterial suspension was added separately into each well containing the MHB and the mushroom extract mixture. The current procedure was also repeated for the yeast in different plate wells.

The absorbance of each well was measured by employing an ELISA reader, ELx800 at 630 nm (Biotek Instruments Inc, Winooski, VT). The lowest concentration that produced an inhibitory effect was recorded as the minimum inhibitory concentration (MIC) for each extract (as described by Devienne & Raddi, 2002 with some modifications). Ampicillin (100 μg/mL concentration) for bacteria and Amphotericin B (50 μg/mL concentration) for yeast were utilized as positive controls. Each experiment was conducted in triplicate.

Extract–DNA interaction

Interactions between mushroom extract and pBR322 plasmid DNA were studied by agarose gel electrophoresis (Nyxtechnik, San Diego, CA). The methanol extract of A. ovoidea in dimetilformamid (DMF) was diluted. Then 65 μL aliquots of decreasing concentrations of mushroom extract ranging from 40.000 to 78.125 μg/mL were added to 1 μL of plasmid DNA (conc. 0.5 μg/mL) in a buffer solution containing TE (10 mM Tris–HCl, 0.1 mM EDTA, pH 7.4). The mixtures were left in an incubator (Nüve, Ankara, Turkey) at 37 °C for overnight in the dark. Then 15 μL aliquots of extract/DNA mixtures were loaded onto the 1.5% agarose gel with loading buffer (0.1% bromophenol blue and 0.1% xylene cyanol). Electrophoresis was carried out in TAE buffer (0.05 M Tris base, 0.05 M glacial acetic acid, 1 mM EDTA, pH 8.0) for 3 h at 60 V (Asmafiliz et al., 2009; Gumus et al., 2009; Ozturk et al., 2012). After electrophoresis, the gel was visualized under UV light using a transilluminator (DNA image system, Vilber Lourmat, Eberhardzell, Germany).

BamHI and HindIII restriction enzyme digestion

After mushroom extract–DNA mixtures were incubated for overnight, it was restricted with digestion by enzymes BamHI or HindIII (1 Unit) for 1 h at 37 °C. The restricted DNA was run in 1% agarose gel electrophoresis for 2 h at 60 V in TAE buffer (Sambrook et al., 1989). The gel was viewed with a transilluminator in the image system.

Results and discussion

Antimicrobial results

According to Doğan and Akbaş (2013) and Doğan et al. (2013), in order to evaluate antimicrobial activity, MIC values ought to be measured from the fourth through the 16th dilutions and the values should be in the ranges given below:

1. MIC values are lower than 100 µg/mL = antimicrobial activity is high.

2. MIC values are between 100 and 500 µg/mL =antimicrobial activity is moderate.

3. MIC values are between 500 and 1000 µg/mL =antimicrobial activity is weak.

4. MIC values are more than 1000 µg/mL = no antimicrobial effect.

In accordance with these ranges, the antimicrobial results are provided in Table 1. Amanita ovoidea exhibited different antimicrobial effects in various concentrations against each test micro-organism. The maximum inhibitory effect on the test micro-organisms was observed with chloroform extract (MIC value, 19.5 µg/mL at 10th dilution) against C. albicans. Overall, the methanol extract was observed to display the maximum antimicrobial effect with values generally lower than 100 µg/mL, placing it in the high-activity category. The micro-organisms inhibited at this level were Bacillus subtilis, S. aureus, L. monocytogenes, S. pyogenes, C. albicans, K. pneumoniae, P. vulgaris, and S. enteritidis (MIC values, 78 µg/mL at the eighth dilution). The effects of the methanol extract E. coli and P. aeruginosa were moderate and higher than 100 µg/mL (MIC values, 156 and 312.5 µg/mL at the seventh and sixth dilutions, respectively). Second effective solvent was acetone. The range of antimicrobial effect was between 78 and 312.5 µg/mL. For the chloroform extract, the effective result was revealed on C. albicans (MIC value, 19.5 µg/mL at the 10th dilution) and P. aeruginosa (MIC value, 39 µg/mL at the ninth dilution), and other results were between 156 and 312.5 µg/mL at seventh and sixth dilutions. The lowest antimicrobial effect was noted on S. pyogenes (MIC value 625 µg/mL at the fifth dilution). In general, methanol and acetone extracts of A. ovoidea were more effective than chloroform extracts against bacteria and yeast. The most effective MIC concentrations of the extracts were typically measured between the sixth and ninth dilutions (MIC values, 312.5–19 µg/mL), and these results demonstrated that the inhibition values were lower than 100 µg/mL or between 500 and 1000 µg/mL, except for chloroform extract against to S. pyogenes.

Table 1. MIC values of A. ovoidea extracts (μg/mL) are presented, and the highest MIC value was underlined*.

Micro-organisms	Chloroform	Acetone	Methanol
B. subtilis	312.5 (100–500)	312.5 (100–500)	78 (<100)
S. aureus	312.5 (100–500)	312.5 (100–500)	78 (<100)
L. monocytogenes	156 (100–500)	312.5 (100–500)	78 (<100)
S. pyogenes	625 (500–1000)	312.5 (100–500)	78 (<100)
C. albicans	19.5 ≤100	312.5 (100–500)	78 (<100)
E. coli	156 (100–500)	78 (<100)	156 (100–500)
K. pneumoniae	156 (100–500)	78 (<100)	78 (<100)
P. aeruginosa	39 (< 100)	78 (<100)	312.5 (100–500)
P. vulgaris	156 (100–500)	312.5 (100–500)	78 (<100)
S. enteritidis	156 (100–500)	156 (100–500)	78 (<100)

*<100 μg/mL = high, 100–500 μg/mL = moderate, 500–1000 μg/mL = weak.

Interaction with pBR322 plasmid DNA

pBR322 plasmid DNA was incubated with 40.000–78.125 µg/mL at different concentrations to determine whether mushroom extract causes conformational difference on DNA helix and to observe the destroyed parts of DNA, the agarose gel electrophoresis was done (Figure 1A). Supercoil DNA (form I) migrated faster than open circular DNA (form II) as shown in Figure 1(A). If the broken occurs in both DNA strands, linear DNA (form III) is formed and it appears between forms I and II in the electrophoresis (Gust et al., 1998; Ozturk et al., 2012; Reddy et al., 2006). It is seen in the form of smear so that plasmid DNA is fragmented in the highest concentration of mushroom extract in the electrophoregram (Figure 1A in line 1). While supercoil DNA (form I) was observed in subsequent three highest concentrations, form II (single nicked open circular DNA) was seen in very small amount. In the descending concentrations of extract (5, 6, 7, 8, 9, and 10 µg/mL), densities of forms I and II are increasingly quite evident. However, this extract decreased the mobility of particularly form I by binding DNA. Even in the lowest concentrations, it quite influenced the electrophoretic mobility in comparison with the control plasmid by binding DNA.

Figure 1. (A) Gel electrophoretic mobility of pBR322 plasmid DNA and different concentrations of the extract. (B) Digestion of the mixtures of pBR322 plasmid DNA and extract by BamHI. (C) Digestion of the mixtures of pBR322 plasmid DNA and extract by HindIII. (P) untreated pBR322 plasmid DNA; (CP) pBR322 plasmid DNA is linearized by BamHI or HindIII.

BamHI and HindIII digestion of mushroom extract–pBR322 plasmid DNA

Both restriction enzymes targeted supercoils and made cutting, but BamHI made partial cutting in open circular DNA (Figure 1B). Therefore, in addition to form III observed, the form II densities are low. The cutting was inhibited partially when connecting to form II of this extract. In the lowest concentration of extract, BamHI enzyme cut both forms I and II and only form III was observed on the gel. In the descending concentrations, cutting with BamHI was better for both supercoil and open circular DNA and it approached cutting mobility of control. It showed that the interest of the extract which is G–G regions of DNA decreased in diminishing concentrations. In the HindIII enzyme activity, uncut density of form II is more (Figure 1C). On the gel, cut supercoils were observed as form III beside form II. Cutting with HindIII of plasmid DNA was inhibited by binding to the A–A region in open circular DNA of extract. Even in the lowest concentrations, cutting did not occur. Mobility of form III was the same with control form III.

The current antimicrobial results were verified by previous studies. According to Yoon et al. (1994), G. lucidum had a good antimicrobial effect against P. vulgaris (MIC, 1.25 mg/mL) and E. coli (MIC, 1.75 mg/mL), and six species of bacteria were had MIC values larger than 5 mg/mL. Within the study, MIC values were generally measured at 78 µg/mL or 312.5 µg/mL and the present results are better than those of Yoon et al. (1994). Gbolagade et al. (2007) studied the antimicrobial effects of certain fungal species by employment of microdilution methods, and they established that the MIC concentration of Marasmius jodocodo Henn. (Marasmiaceae) was 2.75 mg/mL against E. coli, while Tricholoma robustum (Alb. & Schwein.) Ricken (Tricholomataceae) was 15.75 mg/mL against M. bulliardii Quél. Janeš et al. (2007) applied the broth microdilution test for screening of antibacterial activity on extracts of higher and endophytic fungi. Among tested extracts, three significant antibacterial activities were identified on the extracts of Amanita virosa Secr. (Amanitaceae) and Cortinarius praestans (Cordier) Gillet (Cortinariaceae) against P. aeruginosa and S. aureus, respectively, and the extract of endophytic fungus Truncatella hartigii (Tubeuf) Steyaert (Amphisphaeriaceae) against Enterococcus faecalis and S. aureus. Quereshi et al. (2010) tested the antimicrobial activity of various solvent extracts of G. lucidum (40 mg/mL concentration) against six species of bacteria. Acetone extract showed maximum antibacterial activity, whereas the most susceptible bacterium recorded was K. pneumoniae. Bala et al. (2011) investigated the antimicrobial effect of 47 different specimens from Australia, and they ascertained that water and ethanol extracts were more effective against S. aureus than E. coli, whereas an inconsiderable number of hexane extracts showed better results for their potential antimicrobial effect against E. coli at higher concentration. In general, a number of macrofungi from the genera Agaricus, Amanita, Boletus, Cantharellus, Fomitopsis, Hohenbuehelia, Lentinus, Ramaria, and Strobilomyces demonstrated good inhibition rates. Aqueous and methanol extracts of Trametes hirsuta (Wulfen) Lloyd (Polyporaceae) were tested against pathogenic fungi and bacteria. Maximum antibacterial activity of the aqueous extract of T. hirsuta was found against S. aureus compared with that of the methanol extract. Significant antifungal activity of the aqueous extract was found against Aspergillus flavus compared with that of the methanol extract (Sivaprakasam et al., 2011). The antibacterial effects of the extract from three mushrooms G. lucidum, Auricularia auricula (L.) Underw. and Pleurotus floridanus Singer (Pleurotaceae) were studied against S. aureus and E. coli. Auricularia auricularia displayed significant antibacterial activity against S. aureus. Pleurotus florida showed some antibacterial activity, while G. lucidum did not demonstrate any antibacterial activity. None of the extracts exhibited any activity against E. coli (Iftekhar et al., 2011). Antimicrobial activity of Ganoderma praelongum Murrill, G. resinaceum Boud., and G. lucidum was evaluated against 30 strains of clinical isolates of methicillin-resistant and methicillin-sensitive S. aureus (MSSA). The maximum activity of crude extracts was exhibited by ethyl acetate. The MIC of sesquiterpenoid extracts of G. praelongum was 0.390–6.25 mg/mL. Diterpenoids and triterpenoids displayed a moderate activity, while polysaccharides IIIa and IIIb showed weak activity. All bacterial strains were resistant to polysaccharides I and II (Ameri et al., 2011). Doğan and Aktaş (2013) investigated the antimicrobial activity of Amanita caesarea (Scop.) Pers. (Amanitaceae) against Gram-positive and -negative bacteria, and yeast. The MIC observed against the test micro-organisms were with the acetone extract (4.8 µg/mL concentration) against C. albicans. In the other study, T. boudieri, A. brunnescens, and L. vellereus were tested against Gram-positive, -negative bacteria, and yeast using a micro-dilution method. The highest MIC values for all fungal extracts were observed between 78 and 2.4 µg/mL (Doğan et al., 2013). Doğan (2013) studied phenolics, antioxidant activity, and fatty acid composition of A. ovoidea. According to his result, A. ovoidea has a good antioxidant activity, 37 different fatty acids, p-coumaric acid, cinnamic acid, and ferulic acid. In this article, it was also recommend as a good medicinal fungi due to its antioxidant activity, rich fatty acids, and phenolics.

The effect of the extract on mobility of plasmid DNA was observed in all concentrations. The same magnitude of effect was observed between 5th and 10th (2500–78.125 μg/mL) concentrations. Even the effect of low concentrations of the extract on DNA is clearly visible. BamHI and HindIII restriction enzymes prevented cutting DNA by binding G–G and A–A regions of open circular plasmid DNA in all concentrations of extract (Figure 1B and C). These results showed a parallelism with the effects of extract on bacteria.

The antimicrobial effect of the extract on used bacteria can be explained in two ways:

1. Bacteria may become sensitive, since extract can damage plasmid DNA of bacteria by binding or fragmentation.

2. Extract may prevent the growth of bacteria, due to it can also damage to chromosomal DNA with the same way.

The current results have been similar or more effective than those reported in the literature. To the best of our knowledge, there have previously been no reports on the antimicrobial effects of A. ovoidea, and these results are reported here for the first time.

Conclusions

The antimicrobial activity and DNA interactions of A. ovoidea were observed. The present results indicate that the economically important and edible mushroom A. ovoidea demonstrates significant antimicrobial activity and effect to plasmid DNA of bacteria, thus it can be used as a natural antimicrobial agent.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The authors are indebted to the Foundation for TUBITAK (TBAG/109T584) and Scientific Research Projects (BAP) Coordinating Office (BAP/10401054) at Selcuk University for their financial support of the current work.