South African Journal of Botany 131 (2020) 195 199

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Chemical Composition and antibacterial activity of hexane extract of

Lycoperdon Pyriforme
Fariba Asgharpoura, Ali Akbar Moghadamniab, Yasaman Alizadehc, Sohrab Kazemib,*
a
Dental Materials Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran
b
Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran
c
Students of Islamic Azad University Tehran Medical Branch, Tehran, Iran

A R T I C L E I N F O A B S T R A C T

Article History: Mushrooms contain active ingredients with their own antibacterial propFerties, so they can be used for pre-
Received 6 June 2019 vention or treatment of high-risk diseases as well as chronic infections through extracting and optimizing
Revised 11 January 2020 these ingredients as biological and antibacterial substances. In this research, the aerial parts of the fungus,
Accepted 30 January 2020
Lycoperdon pyriforme (L. pyriforme) species, was extracted via hexane solvent and then analyzed using GC/
Available online xxx
MS. The antibacterial test was also performed employing DD, MIC, and MBC methods on Staphylococcus
Edited by K Dole

zal aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis strains. Following the analysis of L. pyri-
forme; Ergosta-5,7-dien-3-ol and 7,22-Ergostadienone compounds were obtained with values of 26.6% and
Keywords:
9.8%; respectively. This extract was only effective on S. aureus in a concentration of 125g/ml. The results of
Antibacterial
this study demonstrated that the hexane extract of L. pyriforme could have antibacterial properties and also
GC/MS
used as a natural antibiotic source for disinfection and treatment of infectious diseases.
Lycoperdon pyriforme
Staphylococcus aureus © 2020 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction tumor, anti-inflammatory, cardiovascular diseases, as well as immu-

nomodulating and antimicrobial actions against both gram-positive
Bacteria are considered as extremely diverse microscopic single- and gram-negative bacteria (Cui et al., 2018b; Ajith and Janardhanan,
cell organisms, playing a significant role in human life (Hlila et al., 2007). Mushrooms are also endowed with significant nutritional val-
2017). However, in addition to the properties and functions of these ues, since they are rich sources of bioactive metabolites of high
creatures in various foods, pharmaceuticals, and other industries; medicinal value such as lectins, polysaccharides, phenolics and poly-
they can be respected as the source of many dangerous diseases as phenolics, terpenoids, ergosterols, and volatile organic compounds
well as chronic infections. Antimicrobial agents have been further (Elsayed et al., 2014). Moreover, mushrooms can be regarded as a
identified as the most important clinical nutrients since the second good source of many different nutraceuticals and used directly in
half of the 20th century and they have consequently saved numerous human diet to improve human health status due to the synergistic
people from life-threatening bacterial infections (Lee et al., 2013). effects of their bioactive compounds (Valverde et al., 2015). In this
Antibiotic resistant microorganisms can also survive due to their abil- domain, L. pyriforme is considered as a species of puffball fungus in
ity to resist after exposure to antibiotic drugs and thereby increase the Agaricaceae family. This species of mushroom is found in most
mortality (Marchese et al., 2016). Therefore, new alternative agents parts of the world. It is also pear-shaped and is 1.5-4.5 cm wide and
from natural sources such as plants that are effective against antibi- 2-4.5 cm high and grows mostly in areas covered with pine trees. In
otic-resistant bacteria have been constantly examined (Diastuti et al., addition, L. pyriforme species is endowed with enzymatic activities in
2014; Nagaraj et al., 2014). a specific pH and temperature, which can be used in large-scale
Furthermore, pharmaceutical studies have demonstrated that industries and factories as a medicinal plant, different bioactive com-
many plants have compounds with medicinal properties (Sofowora pounds such as polysaccharides, alkaloids, terpenoids, tannins and
et al., 2013). Among these plants, mushrooms as macro-fungi have flavonoids were released by Mushroom species. The mycelium and
been utilized in traditional medicine for the purpose of treating vari- fruiting bodies of mushrooms have a valuable effect on health with
ous types of diseases such as antioxidant, anti-viral, anti-cancer, anti- immunostimulatory, antibacterial, and antioxidative properties of
them (Cui et al., 2018a; Ramesh and Pattar, 2010; Yıldırım Akatın
et al., 2013). Nowadays; considering that researchers have conducted
* Corresponding author: Cellular and Molecular Biology Research Center, Health
studies regarding the antibacterial and antioxidant properties of dif-
Research Institute, Babol University of Medical Sciences, Babol, Iran. Tel: +98-
1132199592; Fax: +98-1132190181. ferent species of mushrooms, and since no investigation has been
E-mail address: kazemi.msm@gmail.com (S. Kazemi). conducted on this species so far, it was decided to shed light on the

https://doi.org/10.1016/j.sajb.2020.01.044
0254-6299/© 2020 SAAB. Published by Elsevier B.V. All rights reserved.
196 F. Asgharpour et al. / South African Journal of Botany 131 (2020) 195 199

antibacterial effect of L. pyriforme, through hexane extraction, culti- 2.4. Antibacterial test
vated in the northern regions of Iran.
The testing microbes used were two Gram-positive bacteria
(Staphylococcus aureus ATCC 25923) and Bacillus subtilis ATCC 6051)
2. Materials and methods
and two Gram-negative bacteria (Escherichia coli PTCC 1533 and
Pseudomonas aeruginosa PTCC 1707). To prepare microbial culture,
2.1. Chemicals and reagents
bacterial strains were cultivated in Brain-Heart Infusion Broth at 37 °
C for 24 h.
Hexane (Daejung, Korea), media and standard antibiotics (Merck,
Germany), were obtained and the other chemicals were purchased
from local commercial sources. 2.4.1. Disc diffusion method
The agar disc diffusion method was employed for the determina-
tion of antimicrobial activity of the extracts. Suspensions containing
2.2. Preparation of extract 108 CFU/ml of bacteria, in exponential growth phase, were dispersed
in Mueller-Hinton agar medium. Filter paper disks (6 mm of diame-
Lycoperdon Pyriform (L. Pyriform, when young and fresh) were har- ter) were impregnated with 20 l of each extract, and the concentra-
vested from Northern region of Iran and identified by a botanist in Agri- tions of the extracts ranged from 125 to 1000 g/ml. They were then
cultural and Natural Resources Research Center of Mazandaran. incubated at 40 °C and after drying, they were placed on the inocu-
(Figure 1). All aerial organs of the plant were separated and dried under lated Petri dishes. Negative control was performed using related sol-
laboratory fume hood in the shade for ten days. After drying, these vent employed to dissolve extract and gentamicin (10 g) and
organs of the plant were extracted with hexane for 72 hours in the chloramphenicol (30 g) were used as positive controls. Then petri
incubator shaker. The hexane extracts were filtered and evaporated dishes were incubated for 24 h at 37 °C for bacterial strains. Antimi-
from the solvent. The extracts were collected and kept in refrigerator. crobial activity was measured by estimating the diameter of inhibi-
tion zone (mm). The work was repeated three times and the values
were the average of the three replicates (Jane
s et al., 2007).
2.3. GC-MS analysis

Analysis of the hexane extract of L. Pyriform was performed using a 2.4.2. Determination of MIC and MBC
GC-MS equipment (Agilent technology, 7890B-5977B MSD). Experi- Broth dilution assay was used to determine the MIC of the n-hex-
mental conditions were as follows: DB-5 MS capillary column, dimen- ane extract of L. Pyriform against bacterial strains. This test was per-
sion: 30 M, ID: 0.25 mm, Film thickness: 0.25 m, the carrier gas was formed in sterile microplate Elisa, which were loaded with 100 l of
Helium (99.999%) with flow rate of 1 ml/min and sample volume of 1 l each extracted dilution (125 1000 g/ml) into each well. Bacterial
was injected with auto sampler in split ratio of 10:1. The injector tem- suspensions (100 l) containing 5 £ 105 CFU were added to each well.
perature was set at 250 °C and the oven temperature was programmed Positive control (without extract) and negative control (no inocu-
from 50 °C (storage time of 1min), increased by rate 8 °C/min to 120 °C lums) were also added to each well. Microplate was incubated for
(storage time of 1 min), increased by rate 6 °C/min to 250 °C, ending 24 h at 37 °C. After that growth of bacteria, its apparent turbidity was
with a 15 min isothermal at 250 °C. The solvent delay was 0 3 min, evaluated. MIC was determined as the lowest concentration of the
and the total GC MS running time was 47 min. Mass-spectrum extract that had no macroscopically visible growth. Minimum bacte-
GC MS was interpreted using the database of National Institute of ricidal concentration (MBC) was determined by sub-culturing of clear
Standards and Technology (NIST 11 Variant) and the name, molecular wells on Mueller Hinton agar. Then it was incubated for 24 h. The
weight, and structure of the components of the extract were identified lowest concentration of samples with no bacterial growth (99% inhi-
(Zhou et al., 2015). bition) was reported as MBC (Jane
s et al., 2007).

3. Results

3.1. Bioactive compounds present in the hexane extract

The GC-MS chromatogram analysis of the hexane extract of L. pyr-

iforme peaks indicated the presence of nine phytochemical constitu-
ents (Figure 2). Comparing the mass spectra of the constituents with
the NIST library, phytocompounds were characterized and then rec-
ognized (Table 1). The mass spectra of two major components of the
phytochemicals identified in the whole plant were also illustrated in
Figures 3 and 4. Of the nine identified compounds, the most predomi-
nant compounds were Ergosta-5, 7-dien-3-ol (26.64%) and 7, 22-
Ergostadienone (9.83%) as unsaturated steroid compounds.

3.2. Antimicrobial activity

The antibacterial activity of the hexane extract of L. pyriform was

determined via disk diffusion and MIC methods against five patho-
genic isolates (Table 2). The hexane extract of L. pyriform (500 g/ml)
also showed the largest zone of inhibition (12 mm) against S. aureus
(ATCC 25923) (Figure 5). Moreover, the MIC of the hexane extract of
L. pyriform varied from 125 to >1000 g/ml for gram-positive isolates
and MIC value from 1000 to >1000 g/ml for gram-negative ones
Figure 1. Lycoperdon pyriforme collected from the northern region of Iran. (Table 2).
 F. Asgharpour et al. / South African Journal of Botany 131 (2020) 195 199 197

Figure 2. Chromatogram obtained from the GC/MS analysis of hexane extract of Lycoperdon Pyriforme.

Table 1 conditions, etc. (Gil-Ramirez et al., 2018). Studies have revealed that
Compounds identified in hexane extract L. Pyriform using GC/MS analysis. fungal sterols are involved in down-regulated genes of cholesterol
Peak a
R.T. (min) Corr. area Compounds Total (%)
homeostasis in vitro and in vivo. Moreover, b-glucans and other
water-soluble compounds have stimulated transcriptional profiles
1 3.697 284760 Toluene 9.210
similar to simvastatin or ezetimibe (Caz et al., 2015). These results
2 18.919 274473 n-decanoic acid 8.877
3 33.080 167739 Tetradecanoic acid ethyl ester 5.425
have indicated that mushroom extracts have indirectly affected tran-
4 35.461 244727 Di-sec-butyl phthalate 7.915 scriptional and post-transcriptional modulations, leading to hypo-
5 38.451 535318 Dibutyl phthalate 17.314 cholesterolemic effects (Meneses et al., 2016). In this respect, Barros
6 39.503 172167 Methylenebis(2,4,6- 5.568 et al. analyzed phenolic compounds in six species of Portuguese wild
triisopropylphenylphosphine)
mushrooms species through mass spectrometer (HPLC-DAD-ESI/MS).
7 44.484 183838 dimethyl{bis(tridecyloxy)}silane 9.202
8 60.876 386350 Ergosta-5,7-dien-3-ol, 26.648 Flavonoids were not observed, but the most abundant phenolic acid
11 62.980 75414 7,22-Ergostadienone 9.841 compounds included protocatechuic, p-hydroxybenzoic, and p-cou-
a
RT: Retention time (minutes). maric acids, as well as two vanillic acid isomers. The only compound
found in species Cantharellus cibarius, Lycoperdon perlatum, and Mac-
rolepiota procera was cinnamic acid (Barros et al., 2009).
4. Discussion
The use of plant extracts with known antimicrobial activities can
be of utmost importance in therapeutic procedures (Cowan, 1999).
Recently, the use of medicinal plants in the treatment and preven-
Different studies have been also carried out in several areas to show
tion of diseases is attracting the attention of researchers. In between,
the effect of plant extracts in recent years. In this study, the antimi-
mushrooms are rich sources of bioactive metabolites such as lectins,
crobial activity of L. pyriforme was investigated for the first time, and
polysaccharides, phenolics and polyphenolics, terpenoids, ergosterols
no study was found examining the antibacterial properties of this
(Ajith and Janardhanan, 2007). In this study, The GC MS chromato-
mushroom extract. As well; antimicrobial activities of ethanol, meth-
gram analysis of the hexane extract of L. pyriforme indicated two
anol, and water extracts of Lycoperdon perlatum against S. aureus, P.
major components inclusive Ergosta-5, 7-dien-3-ol and 7, 22-Ergo-
aeruginosa, E. coli, B. cereus, Candida albicans, and Candida glabrata
stadienone. Studies have shown that, the most fungal steroids were
confirmed inhibited growth of all the tested pathogenic organisms
ergosterol and its derivatives such as ergost-22-ene-1,3-diol, ergo-
except the aqueous extract of Lycoperdon perlatum against P. aerugi-
sta-5,7-dien-3b-ol, and (22E)-ergosta-1,4,6,22-tetraen-3-one (Gil-
nosa. Moreover, the phytochemical analysis of extracts revealed the
Ramirez et al., 2018). Ergosterol (ergosta-5,7,22-trien-3b-ol)
presence of varying levels of bioactive compounds such as flavonoids,
accounted for about 53 80% of fungal sterols (w/w), considered as
saponins, proteins and carbohydrates, glycosides, as well as alkaloids
the major fungal sterol (Huang et al., 2016). Species such as Chanthar-
and tannins (Akpi, 2017; Cui et al., 2019). In a research by Novakovic
ellus cibarius and Craterellus cornucopioides have also exclusively
et al. on the hexane extract of Lycoperdon perlatum against S. aureus,
shown ergosterol. Certainly, it should be noted that their exact con-
B. subtilis, and E. coli, the hexane extract displayed a modest antibac-
centration in different species of mushrooms depends on environ-
terial activity only against the S. aureus strain with MIC and MBC
mental conditions such as geographic area, growth stage, cultivation
198 F. Asgharpour et al. / South African Journal of Botany 131 (2020) 195 199

Figure 3. Mass Spectrum of Ergosta-5, 7-dien-3-ol.

Figure 4. Mass Spectrum of 7, 22-Ergostadienone.

Table 2
The Disk diffusion and MIC of hexane extract of L. Pyriform.

Isolates DD (mm) MIC (g /ml) MBC (g /ml)

h. extract (500 g) Gentamicin (10 g) h. extract Gentamicin h. extract Gentamicin

Staphylococcus aureus (ATCC 25923) 12 15 125 1.56 250 3.12

Escherichia coli (PTCC 1533) 0 20 1000 3.12 NA 12.5
Pseudomonas aeruginosa (PTCC 1707) 0 17 >1000 200 NA 400
Bacillus subtilis (ATCC 6051) 0 23 >1000 6.25 NA 25

values equal to 3.12 mg/ml and 6.25 mg/ml; respectively (Novakovic 5. Conclusion
et al., 2015). Additionally, Ramesh et al. determined antimicrobial
activity of methanolic extracts of 6 wild edible mushrooms against Phytochemical analysis is regarded as a very important laboratory
standard pathogenic bacteria and fungi in which the highest in-vitro and scientific process. This study was conducted to identify two ste-
antibacterial activity was by Lycoperdon perlatum against E. coli. Fur- roids (Ergosta-5, 7-dien-3-ol and 7, 22-Ergostadienone) in the hexane
thermore, the amount of bioactive compounds in edible mushrooms extract of L. pyriforme. The proposed GC/MS method was also used for
could directly affect their antibacterial capacities (Ramesh and Pattar, identification and determination of Ergosta and other compounds.
2010). Moreover, the hexane extract of L. pyriforme exhibited the antibacterial
 F. Asgharpour et al. / South African Journal of Botany 131 (2020) 195 199 199

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