Saudi Pharmaceutical Journal 27 (2019) 401–405

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Original article

Evaluation of antimicrobial activity of secondary metabolites of fungi

isolated from Sultanate Oman soil
Manal M. Alkhulaifi a, Amani S. Awaad b,⇑, Hind A. AL-Mudhayyif a, Monerah R. Alothman a,
Saleh I. Alqasoumi c, Sarah M. Zain d
a
Botany and Microbiology Department, College of Science, King Saud University, Riyadh, Saudi Arabia
b
Director Of Gateway to United Kingdom Education Ltd., Bradford, UK
c
Pharmacognosy Department, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
d
School of Live Sciences, M-Pharm Department, University of Bradford, Bradford, UK

a r t i c l e i n f o a b s t r a c t

Article history: Seven fungal species were isolated from soil samples collected from the University of Sultan Qaboos,
Received 2 October 2018 Muscat, Sultanate of Oman. The fungal isolates were identified as Aspergillus athecius, A. terreus var. afri-
Accepted 31 December 2018 cans, A. flavus, A. terreus, A. foetidus, Fusarium chlamydosporum and F. nygamai. Phytochemical and chro-
Available online 3 January 2019
matographic investigation showed variety of secondary metabolites in all of the fugal extracts (extra and
intra cellular). The antimicrobial activity of internal and external extracts of the isolated fungal species
Keywords: were screened against Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, Pseudomonas aeruginosa,
Aspergillus flavus
Lactobacillus acidophilus, Streptococcus gordonii, S. mutans. The antimicrobial activity of external sec-
Phytochemical contents
Intra cellular
ondary metabolites was generally better than the internal metabolites. The highest antimicrobial activity
Extra cellular (32 mm, 30 mm and 29 mm) was obtained from external secondary metabolites of Aspergillus flavus
Fusarium chlamydosporum against Candida tropicals, Candida parapsilosis and Candida albicans, respectively.
Candida albicans Ó 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an
Antimicrobial open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Soil microbes
Aspergillus
Fusarium

1. Introduction The necessity for new antibiotics increasing daily due to the
emergence of drug-resistant pathogens. Despite the huge potential
Antibiotic is a natural substance of biological origin used to sources of antibiotics including medicinal plants, soil still the most
treat diseases caused by microbes to eukaryotic organisms includ- important reservoir for novel antibiotics with pharmaceutical and
ing human being (Pannapa and Pattra, 2017; Hacioglu and Dulger, biological activity (Rajaperumal et al., 2013). There are more than
2011; Nikodinovic et al., 2003). According to their mode of action, 500 antibiotics are discovered every year, however, almost 60% are
antibiotics are known as wide- or narrow-spectrum. These include obtained from the soil (Molinari, 2009). Interestingly, a few grams
cell wall, protein and nucleic acid inhibition (Tortora et al., 2007; of soil contain numerous microorganisms (Makut and Owolewa,
Brooks et al., 2001). 2011).
Numerous species of fungi including Penicillium, Aspergillus,
Fusarium, Cladosporium and Yeasts are able to produce enzymes
and secondary metabolites including antibiotics (Hyde, 2001).
⇑ Corresponding author at: Pharmacognosy and Director, Of gateway to United Approximately 20% of antibiotics have been obtained from fungi
Kingdom Education Ltd., Bradford, UK. isolated from soil (Berdy, 2005). Those antibiotics, produced by
E-mail address: [email protected] (A.S. Awaad). fungi, including fusidic acid (Akpotu et al., 2017), cephalosporin
Peer review under responsibility of King Saud University. and penicillin are widely used for treatment of many diseases
(Sethi et al., 2013) and they are the most important source of
potentially significant pharmaceutical drugs (Wasser, 2002). The
objective of this study was to isolate and identify soil fungi and
Production and hosting by Elsevier to determine their ability to inhibit the growth of microorganisms.

https://doi.org/10.1016/j.jsps.2018.12.009
1319-0164/Ó 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
402 M.M. Alkhulaifi et al. / Saudi Pharmaceutical Journal 27 (2019) 401–405

2. Materials and methods the obtained fungal isolates was confirmed by the Regional Center
for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt.
2.1. Fungal isolation and identification
2.2. Extraction
2.1.1. Isolation
2.1.1.1. Soil samples collection. The soil samples were collected from 2.2.1. Internal fungal secondary metabolites extraction
different sites at Sultan Qaboos University, Muscat, Sultanate The mycelial mat for each fungus (250 g) was obtained, washed
Oman. The direct inoculation method was used for sampling and with distilled water, extracted by refluxing in 2 liters of boiled
isolation of fungal isolates. Soil samples were separately trans- ethanol for 120 min and re-extracted again for three times (as
ferred onto the surface malt agar media and incubated for 5– mentioned before). The obtained filtrates were then concentrated
7 day at 25 °C. together and dried from ethanol under reduced pressure at low
temperature and kept in the fridge for the investigation.

2.1.1.2. Fungal isolation. For isolation of all fungi four different 2.2.2. External fungal secondary metabolites extraction
media were used (yeast extract sucrose potato dextrose, czapek’s One liter of each fungal growth medium was separately
dox and yeast malt extract agar malt extract) were uses. The tech- extracted using 2 L of n-butanol (saturated with water), and re-
nique used was described by Al-Enazi et al. (2018). extracted again for four repetitive times. The obtained butanol
extract was filtered using Buchner funnel contains anhydrous
sodium sulphate. The collected butanol extracts was dried from
2.1.2. Identification of fungal isolates solvent using rotatory evaporator at temperature not exceeding
For identification of the isolated fungi, each fungal isolate was 35C and the dry extract was then kept in fridge for investigation
microscopically examined be by transferring fungal mycelia onto (Zain et al., 2012).
a glass slide containing a drop of lactophenol cotton blue stain,
and then covered with the cover slip and was viewed under micro- 2.3. Phytochemical screening and chromatographic investigation
scope. Also, the macroscopic observation was determined by com-
paring the fungal isolate characters with the Pictorial atlas of soil 2.3.1. Phytochemical screening were carried out for all both
and seed fungi (Casero et al., 2017). However, identification of extra and intra fungal extracts to investigate their phytochemical

Fig. 1. The microscopic structures of fungal isolates; Aspergillus athecius (a), Aspergillus terreus var. africans (b), Aspergillus flavus (c), Fusarium chlamydosporum (d), Aspergillus
terreus (e), Aspergillus foetidus (f) and Fusarium nygamai (g).
 M.M. Alkhulaifi et al. / Saudi Pharmaceutical Journal 27 (2019) 401–405 403

contents of secondary metabolites, these were done according to aeruginosa, Streptococcus gordonii, and Streptococcus mutans were
the standard published methods (Tiwari et al., 2011). obtained from the Regional Center for Mycology and Biotechnology
All of Each fungal extracts (intra and extra) was tested chro- (RCMB), Al-Azhar University, Cairo, Egypt as test organisms.
matographically using three systems a- c [(a) ethyl acetate: metha-
nol: water 90: 5: 4 v/v/v, (b) chloroform: methanol 95: 5 v/v & (c) 2.4.2. Antimicrobial screening
benzene: ethyl acetate 86: 14 v/v]. Visualization of the spots were The antimicrobial activity of internal and external secondary
carried out under UV before and after spraying with Antimony metabolites of the isolated fungal strains grown on malt extract,
trichloride (SbCl3). yeast extract sucrose, and yeast-malt extract media were deter-
mined using disc-diffusion method (Al-Enazi et al., 2018). Petri
2.4. Antimicrobial activity dishes containing 20 mL of agar medium were seeded with test
organisms. Standard discs (6 mm in diameter) loaded with 50 lL
2.4.1. Test organisms of fungal extract were placed onto the agar, and incubated at
The test organisms used were Candida albicans, C. glabrata, C. 37 °C for 24–48 h. The antimicrobial activity was recorded as the
parapsilosis, C. tropicalis, Lactobacillus acidophilus, Pseudomonas diameter of the inhibition zone formed around the disc.

Table 1
Phytochemical Screening of all fungal extracts (extra and intra cellular).

Test for Aspergillus Aspergillus Aspergillus Fusarium Aspergillus Aspergillus Fusarium

athecius terreus var. flavus chlamydo- terreus toetidus nygama
africans sporum
Intra Extra Intra Extra Intra Extra Intra Extra Intra Extra Intra Extra Intra Extra
Alkaloids – – – – – – – – – – – – – –
Carbohydrates and /or glycosides + + + + + + + + + + + + + +
Flavonoids – – – – – – – – – – – – – –
Saponins ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Tannins ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Unsaturated sterols and/orTriterpens + + + + + + + + + + + + + +
Anthraquinones – – – – – – – – – – – – – –
Proteins and/or amino acids + + + + + + + + + + + + + +
Coumarins and lactones ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Cardenolides – – – – – – – – – – – – – –

(+): present; ( ) absent, (±) trace.

Table 2
TLC results for phytochemical Screening of all extracts (extra and intra cellular).

Rf values Colour Aspergillus Aspergillus Aspergillus Fusarium Aspergillus Aspergillus Fusarium

athecius terreus var. flavus chlamydo- terreus toetidus nygama
africans sporum
a b c UV NH3 SbCl3 Intra Extra Intra Extra Intra Extra Intra Extra Intra Extra Intra Extra Intra Extra
– 0.86 – bl. bl. bl. _ + _ + _ + _ + _ + _ + _ +
– 0.86 – br. br. br. _ + _ + _ + _ + _ + _ + _ +
– – 0.73 bl gr. bl gr. bl gr. _ + _ + _ + _ + _ + _ + _ +
– – 0.64 bl gr. bl gr. bl gr. _ + _ + _ + _ + _ + _ + _ +
– 0.85 0.55 bl gr. bl gr. bl gr. + + + + + + + + + + + + + +
– 0.83 0.49 bl gr. bl gr. bl gr. _ + _ + _ + _ + _ + _ + _ +
– – 0.43 bl gr. bl gr. bl gr. _ + _ + _ + _ + _ + _ + _ +
– – 0.43 br. br. br. _ + _ + _ + _ + _ + _ + _ +
– – 0.35 bl. bl. bl. ± + ± + ± + ± + ± + ± + ± +
– – 0.31 bl. bl. bl. _ ± _ ± _ ± _ ± _ ± _ ± _ ±
– – 0.18 bl. bl. bl. _ ± _ ± _ ± _ ± _ ± _ ± _ ±
– – 0.15 bl. bl. bl. _ ± _ ± _ ± _ ± _ ± _ ± _ ±
– – 0.13 bl gr. bl gr. bl gr. _ + _ + _ + _ + _ + _ + _ +
– 0.51 0.05 bl. bl. bl. _ ± _ ± _ ± _ ± _ ± _ ± _ ±
0.80 0.28 0.02 bl. bl. bl. + + + + + + + + + + + + + +
0.77 0.03 0.18 y. br. br. + + + + + + + + + + + + + +
0.71 0.04 0.02 y. br. br. + + + + + + + + + + + + + +
0.66 0.22 – bl. bl. bl. + _ + _ + _ + _ + _ + _ + _
0.59 0.21 – bl. bl. bl. ± _ ± _ ± _ ± _ ± _ ± _ ± _
0.53 0.14 – bl. bl. vi. _ _ _ _ _ _ _ _ _ _ _ _ _ _
0.40 0.12 – bl. bl. bl. + + + + + + + + + + + + + +
0.37 0.08 – br. br. br. + + + + + + + + + + + + + +
0.22 0.06 – br. br. br. + + + + + + + + + + + + + +
0.16 0.04 – br. br. br. + + + + + + + + + + + + + +
0.13 – – y. y. y. ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.08 – – y. y. y. ± _ ± _ ± _ ± _ ± _ ± _ ± _
0.03 – – br. br. br. + ± + ± + ± + ± + ± + ± + ±
– 0.86 – bl. bl. bl. _ + _ + _ + _ + _ + _ + _ +

( ): absent; (+): present; (±): trace; (a): ethyl acetate: methanol: water 90:5:4 v/v/v; SbCl3: antimony trichloride; (b): chloroform: methanol 95:5 v/v; (c): benzene: ethyl
acetate 86:14 v/v; bl: blue; br: brown; gr: green; y:yellow; NH3: ammonia; ex: extra cellular; In: intra cellular.
404 M.M. Alkhulaifi et al. / Saudi Pharmaceutical Journal 27 (2019) 401–405

3. Results and discussion glycosides in addition to traces of coumarins and lactones, sapo-
nins, tannins > no alkaloids, flavonoids, Anthraquinones and Carde-
3.1. Isolation and identification of fungal strains nolides were detected (Table 1).
Thin Layer Chromatography (TLC) investigation of all fungal
Fungal isolates were obtained from the analyses of different soil extracts (extra and intra) showed that all extra cellular extracts
samples collected from the University of Sultan Qaboos, Muscat, of each fungi were similar to each other, and that all intra cellular
and Sultanate of Oman. Seven fungal strains were isolated from extracts of each fungi were similar to each other. However, the
malt extract agar plates incubated at 28 °C for 7 days. All fungal intra cellular extracts contain more compounds than extra cellular
isolates were obtained in pure cultures by single conidial transfer extracts (Table 2).
onto beer agar plates. The fungal isolates were identified as Asper-
gillus athecius, A. terreus var. africans, A. flavus, A. terreus, A. foetidus 3.3. Antimicrobial activity
Fusarium chlamydosporum and F. nygamai (Fig. 1).
Similarly, Raja and others (Raja et al., 2017) isolated and identi- The internal and external secondary metabolites of the obtained
fied different fungal strains from soil samples collected from Loy- fungal species were investigated for their antimicrobial activity
ola College Campus, Chennai, India. Also, it was suggested that against Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis,
the number and frequencies of fungal species isolated from soil Pseudomonas aeruginosa, Lactobacillus acidophilus, Streptococcus
depend on the moisture content and/or level of organic carbon in gordonii, S. mutans. Each fungal strain was grown on Malt Extract
the soil (Salar and Aneja, 2006). (ME), Yeast Extract Sucrose (YES) and Yeast-Malt Extract media
at 28 ± 2 °C for 14 days. Both the intra- and extra-cellular metabo-
3.2. Phytochemical and chromatographic screening lites of the fungal species were screened for their antimicrobial
activity (Tables 3–5). The isolated fungal species exhibited antican-
All extracts of each fungi (extra & intra cellular) were found to didal and antibacterial activity against almost all the investigated
be quite similar to each other, they contain; Proteins and/or amino test organisms. However, activity of secondary metabolites of fungi
acids, unsaturated sterols and/or triterpens, carbohydrates and /or grown on malt extract media was the highest (Table 3). Moreover,

Table 3
Antimicrobial activity of cell extract of fungal isolates grown on malt extract medium.

Test organism Diameter of inhibition zone (mm)

Aspergillus Aspergillus Aspergillus Fusarium Aspergillus Aspergillus Fusarium
athecius terreus var. flavus chlamydo- terreus foetidus nygamai
africans sporum
Int. Ext. Int. Ext. Int. Ext. Int. Ext. Int. Ext. Int. Ext. Int. Ext.
Unicellular fungi
Candida albicans 17.0 19.0 21.0 22.0 16.0 16.0 14.0 14.0 27.0 29.0 17.0 18.0 15.0 16.0
Candida glabrata 15.0 15.0 19.0 19.0 14.0 17.0 13.0 15.0 26.0 25.0 16.0 16.0 14.0 17.0
Candida parapsilosis 17.0 18.0 18.0 17.0 15.0 17.0 13.0 13.0 28.0 30.0 17.0 17.0 16.0 21.0
Candida tropicalis 16.0 19.0 20.0 21.0 17.0 17.0 14.0 15.0 27.0 32.0 17.0 19.0 14.0 19.0
Gram-Negative Bacteria
Pseudomonas aeruginosa 15.0 18.0 16.0 20.0 16.0 18.0 10.0 10.0 22.0 24.0 16.0 18.0 17.0 17.0
Gram-Positive Bacteria
Lactobacillus acidophilus 15.0 15.0 17.0 17.0 19.0 20.0 12.0 15.0 21.0 27.0 19.0 20.0 14.0 15.0
Streptococcus gordonii 16.0 17.0 17.0 19.0 17.0 19.0 13.0 14.0 23.0 26.0 18.0 18.0 17.0 17.0
Streptococcus mutans 14.0 16.0 15.0 16.0 17.0 18.0 14.0 16.0 22.0 25.0 17.0 21.0 15.0 17.0

Int., intracellular metabolites; Ext., extracellular metabolites.

Table 4
Antimicrobial activity of cell extract of fungal isolates grown on yeast extract sucrose medium.

Test organism Diameter of inhibition zone (mm)

Aspergillus Aspergillus Aspergillus Fusarium Aspergillus Aspergillus Fusarium
athecius terreus var. flavus chlamydo- terreus foetidus nygamai
africans sporum
Int. Ext. Int. Ext. Int. Ext. Int. Ext. Int. Ext. Int. Ext. Int. Ext.
Unicellular fungi
Candida albicans 12.0 12.0 11.0 10.0 10.0 10.0 00.0 00.0 13.0 11.0 09.0 09.0 10.0 09.0
Candida glabrata 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0
Candida parapsilosis 14.0 15.0 12.0 13.0 13.0 13.0 10.0 11.0 15.0 16.0 11.0 12.0 12.0 14.0
Candida tropicalis 10.0 12.0 10.0 10.0 11.0 10.0 07.0 00.0 12.0 12.0 00.0 00.0 10.0 11.0
Gram-Negative Bacteria
Pseudomonas aeruginosa 10.0 10.0 10.0 12.0 11.0 11.0 11.0 11.0 14.0 13.0 12.0 12.0 11.0 10.0
Gram-Positive Bacteria
Lactobacillus acidophilus 11.0 12.0 11.0 12.0 10.0 11.0 00.0 00.0 13.0 12.0 00.0 00.0 13.0 16.0
Streptococcus gordonii 11.0 11.0 12.0 10.0 00.0 00.0 00.0 00.0 12.0 15.0 09.0 10.0 11.0 11.0
Streptococcus mutans 10.0 10.0 10.0 11.0 10.0 11.0 00.0 00.0 11.0 13.0 00.0 00.0 10.0 11.0

Int., intracellular metabolites; Ext., extracellular metabolites.

 M.M. Alkhulaifi et al. / Saudi Pharmaceutical Journal 27 (2019) 401–405 405

Table 5
Antimicrobial activity of cell extract of fungal isolates grown on yeast malt-extract medium.

Test organism Diameter of inhibition zone (mm)

Aspergillus Aspergillus Aspergillus Fusarium Aspergillus Aspergillus Fusarium
athecius terreus var. flavus chlamydo- terreus foetidus nygamai
africans sporum
Int. Ext. Int. Ext. Int. Ext. Int. Ext. Int. Ext. Int. Ext. Int. Ext.
Unicellular fungi
Candida albicans 12.0 13.0 11.0 11.0 10.0 10.0 08.0 10.0 12.0 13.0 00.0 00.0 10.0 11.0
Candida glabrata 11.0 10.0 12.0 12.0 09.0 10.0 07.0 07.0 12.0 12.0 00.0 00.0 10.0 10.0
Candida parapsilosis 12.0 12.0 12.0 11.0 10.0 11.0 00.0 00.0 11.0 11.0 00.0 00.0 12.0 12.0
Candida tropicalis 00.0 00.0 12.0 12.0 11.0 11.0 08.0 09.0 11.0 11.0 00.0 00.0 11.0 11.0
Gram-Negative Bacteria
Pseudomonas aeruginosa 00.0 00.0 10.0 11.0 09.0 09.0 00.0 00.0 10.0 10.0 00.0 00.0 00.0 10.0
Gram-Positive Bacteria
Lactobacillus acidophilus 12.0 12.0 10.0 12.0 10.0 11.0 10.0 09.0 13.0 13.0 00.0 00.0 10.0 11.0
Streptococcus gordonii 13.0 12.0 12.0 12.0 09.0 10.0 00.0 00.0 11.0 11.0 00.0 00.0 12.0 10.0
Streptococcus mutans 13.0 13.0 00.0 09.0 11.0 10.0 00.0 00.0 10.0 11.0 00.0 00.0 12.0 12.0

Int., intracellular metabolites; Ext., extracellular metabolites.

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