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Document heading doi: 10.1016/S1995-7645(14)60236-4

Antimicrobial constituents from three endophytic fungi

Hidayat Hussain1,2*, Christine Kliche-Spory

1, Ahmed Al-Harrasi

2, Ahmed Al-Rawahi

2, Ghulam Abbas

2,3, Ivan Robert

Green4, Barbara Schulz

5, Karsten Krohn

1, Afzal Shah

6

1 Department of Chemistry, University of Paderborn, Warburger Strasse 100, 33098 Paderborn, Germany

2UoN Chair of Oman’ s Medicinal Plants and Marine Natural Products, University of Nizwa, P.O Box 33, Postal Code 616, Birkat Al Mauz, Nizwa,

Sultanate of Oman

3 Department of Chemistry, CIIT, Abbottabad Campus, Abbottabad-22060, Pakistan

4 Department of Chemistry and Polymer Science, University of Stellenbosch, P/Bag X1 Matieland 7602, South Africa

5

 Institute of Microbiology, University of Braunschweig, Spielmannstraße 7, 38106 Braunschweig, Germany6  Department of Chemistry, Quaid-i-Azam University, 45320 Islamabad, Pakistan

Asian Pac J Trop Med 2014; 7(Suppl 1): S224-S227

Asian Pacific Journal of Tropical Medicine

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

  *Corresponding author: Hidayat Hussain, Department of Chemistry, University of

Paderborn, Warburger Strasse 100, 33098 Paderborn, Germany.

  Tel: +495251602172;

Fax: +495251603245

  E-mail: Hidayat110@gmail.com

  Foundation Project: Supported by BASF AG (Chemical Company) and the BMBF (The

Federal Ministry of Education and Research, Germany (Project no. 03F0360A).

 

1. Introduction

  There are a large number of bioactive compounds that

have been isolated from endophytic fungi and these

bioactive natural products have demonstrated a broad

range of biological activities. Endophytic fungi derived

natural products showed a number of interesting biological

activities viz., antioxidant, anticancer, immunomodulatory,

antiviral, antitubercular, antiparasitic and insecticidal

activities. In addition, these bioactive natural products

could possibly be used as starting materials for

pharmaceuticals or agrochemical products [1] . These

bioactive natural products provided by endophytic fungi

originate from different biosynthetic pathways and belong

ARTICLE INFO ABSTRACT

 Keywords:Natural productEndophytic fungiAntimicrobial activity

Objective:  To evaluate the antimicrobial potential of extracts of the endophytic fungi Plectophomella sp.,  Physalospora sp., and Crataegus monogyna  (C. monogyna) and study thetentative identification of their active constituents.Methods: Crude extracts and isolated compounds were screened for antimicrobial activityusing the agar well diffusion method. Four compounds were purified from three endophytic fungiusing column chromatography and their structures have been assigned based on their

1H and

13C 

nuclear magnetic resonance spectra.Results:  Plectophomella sp.,  Physalospora sp., and C. monogyna extracts showed promisingantifungal, antibacterial and herbicidal properties. (-)-Mycorrhizin A was isolated from

 Plectophomella  sp. while cytochalasins E and K were isolated from  Physalospora sp. Similarlyradicinin was purified from the endophytic fungus C. monogyna. The ethyl acetate extract of

 Plectophomella sp. showed significant antifungal activity towards Ustilago violacea (U. violacea)and Eurotium repens ( E. repens) and significant antibacterial activity against Bacillus megaterium.Interestingly, the ethyl acetate extracts of  Physalospora sp. and C. monogyna showed strongherbicidal and antifungal activities towards Chlorella fusca, U. violacea, E. repens, Mycotyphamicrospora (M. microspora), Fusarium oxysporum, Escherichia coli, and Bacillus megaterium.(-)-Mycorrhizin A  showed significant antifungal activity towards U. violacea and E. repens.Cytochalasins E and K showed strong antifungal activity against  E. repens and  M. microspora especially towards fungal Mycotypha microspora. Similarly cytochalasins E and K showed goodherbicidal activity towards Chlorella fusca. Radicinin showed strong antifungal activity against E.repens and M. microspora.Conclusions: Antimicrobial activities demonstrated by the extracts of the endophytic fungi

 Plectophomella sp.,  Physalospora sp., and C. monogyna and four isolated compounds clearlydemonstrate that these fungi extracts and active compounds present a great potential use in the

food, cosmetic and pharmaceutical industries.

 Article history:Received 4 Feb 2014Received in revised form 16 May 2014Accepted 12 Jun 2014Available online 4 Aug 2014

 Contents lists available at ScienceDirect

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to diverse structural groups,  viz., isocoumarins, phenols,

steroids, terpenoids, xanthones, quinones, benzopyranones,

tetralones, cytochalasins and enniatins[1].

Continuing our important work on the characterization

of structurally novel and/or biologically active metabolites

from fungal endophyte cultures[2-17], we found that the

ethyl ethanoate extract of the cultures of the endophytic

fungi  Plectophomella sp., Physalospora sp., and Crataegus

monogyna (C. monogyna) showed significant antifungal and

good antibacterial and herbicidal activities. Fractionation

of the ethyl acetate extracts of these three fungi led to the

isolation and structural determination of four compounds,

namely, (-)-mycorrhizin A, cytochalasins E and K and

radicinin. Details of the isolation and biological activity of

these compounds are reported in the present study.

2. Materials and methods

2.1. General experimental procedures

  Column chromatography was performed using commercial

silica gel (Merck, 0.040–0.063 mm )  and analytical and

preparative thin-layer chromatography (TLC) was performed

with pre-coated silica gel plates Merck G60 F-254 or G50 

UV-254. Infrared radiation spectra were recorded on a

Nicolet-510P spectrophotometer.1H and

13C nuclear magnetic

resonance spectra were recorded on a Bruker Avance 500 

(500 MHz for1H and 125 MHz for

13C) spectrometer. Mass

spectrum and high resolution mass spectrum were recordedin the electron ionization mode on a MAT 8200 and Micromass

LCT mass spectrometer. Microbiological methods and culture

conditions are as described previously[18,19].

2.2. Culture, extraction, and isolation

  The culture filtrate from  Plec tophomel la   sp. was

exhaustively extracted with ethyl ethanoate and concentrated

in vacuo to afford 7.0 g of extract which was separated into

six fractions by column chromatography on silica gel, using

gradients of dichloromethane/ethyl acetate (85:15, 50:50,0:100). (-)-Mycorrhizin A  (9.5 mg) was isolated from fraction

1 by further chromatography on silica gel 60  (Merck) and in

this case using dichloromethane-MeOH  (1%-10% MeOH) as

eluent.

The endophytic fungus  Physalospora sp. was extracted

with ethyl acetate to afford 4.2  g of a residue which was

separated into three fractions by column chromatography on

silica gel, using gradients of dichloromethane/ethyl acetate

(85:15, 50:50, 0:100). The three fractions were further purified

by silica gel column chromatography and preparative

TLC  with CH2

Cl2

 /MeOH  (9:1) as eluent to provide pure

cytochalasins E (9.5 mg) and K (10 mg).

The endophytic fungus C. monogyna was extracted with

ethyl acetate to afford 4.5 g of a residue which was separated

into four fractions by column chromatography on silica gel,

using gradients of dichloromethane/ethyl acetate (85:15,

50:50, 0:100)  as eluent. Fraction 2 was further purified by

silica gel column chromatography and preparative TLC using

CH2Cl2 /MeOH (9:1) as eluent to give pure radicinin (9.1 mg).

2.3. Agar diffusion test for biological activity

  Tests for antifungal, antibacterial, and herbicidal activities

were performed as previously described[19]. Extracts

and compounds isolated were dissolved in acetone at a

concentration of 1 mg/mL. Fifty microliters of the solutions

(50 µg) was pipetted onto a sterile filter disk (Schleicher

and Schuell, 9 mm), which was placed onto an appropriate

agar growth medium for the respective test organism

and subsequently sprayed with a suspension of the testorganism [19]. The test organisms were Chlorella fusca (C.

 fusca), Ustilago violacea (U. violacea),  Eurotium repens ( E.

repens), Fusarium oxysporum (F. oxysporum),  Mycotypha

microspora ( M. microspora),  Escherichia coli  ( E. coli) and

 Bacillus megaterium ( B. megaterium).

3. Results

3.1. Structures of isolated compounds

  (-)-Mycorrhizin A was isolated from  Plectophomella sp.

while cytochalasins E and K were isolated from Physalospora 

sp. Similarly radicinin were purified from endophytic fungus

C. monogyna  (Figure 1). The structures of (-)-mycorrhizin

A[20], cytochalasins E[21], and K[21], and radicinin[22], were

identified by comparison with published spectral data.

CI O

O

O

OO

O

O

O

HH

H

HNOH

O

O

O O

O

O O

O

O

OAc

HH

HHN

1

3 4

2

OH

HO

Figure 1.  Structures of compounds isolated from  Ple ctophomel la  sp.,

 Physalospora sp., and C. monogyna.

1: (-)-Mycorrhizin A; 2: cytochalasins E; 3: cytochalasins K; 4: radicinin.

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3.2. Biological activity

  The antibacterial, fungicidal, and herbicidal properties of

the three extracts of the endophytic fungi viz., Plectophomella 

sp.,  Physalospora   sp., and C. monogyna  and four pure

compounds  viz., (-)-mycorrhizin A, cytochalasins E  and

K and radicinin are compiled in Table 1. The extracts and

isolated compounds were tested in an agar diffusion assay

for their antifungal, antibacterial, and herbicidal properties

towards C. fusca, U. violacea, E. repens, M. microspora, F.

oxysporum, E. coli, and  B. megaterium. The ethyl acetate

extract of Plectophomella sp. showed significant antifungal

activity towards U. violacea and  E. repens and significant

antibacterial activity against  B. megaterium. On the other

hand the extract from  Plectophomella sp. showed moderate

antifungal activity towards M. microspora and F. oxysporum 

while it demonstrated week herbicidal activity. Interestingly,

the ethyl acetate extracts of  Physalospora   sp. and C.

monogyna showed strong herbicidal and antifungal activitiestowards C. fusca, U. violacea,  E. repens,  M. microspora, F.

oxysporum,  E. coli, and  B. megaterium. (-)-Mycorrhizin A 

showed significant antifungal activity towards U. violacea 

and  E. repens and moderate activity against  M. microspora 

and F. oxysporum.

Table 1

Biological activities of extracts and pure metabolites against microbial test

organisms in agar diffusion assaya.

Extract/Compound Radius of zone of inhibition (mm)

Herbicidal Antifungal Antibacterial

Chl Ust Eur  Mm Fo Ec Bm

 Plectophomella sp. extract 4 12 12 7 7 1 13

 Physalospora sp. extract 30 24 22 20 17 13 16

C. monogyna extract 35 25 30 22 n.t 15 15

(-)-Mycorrhizin A 3 14 14 10 n.t 3 8

Cytochalasins E  10 6 22 14 n.t n.t n.t

Cytochalasins K 9 5 21 13 n.t n.t n.t

Radicinin 2 3 15 13 0 2 3a10 mg/mL of extracts and compounds were tested for inhibitions of C. fusca 

(Chl), U. violacea (Ust), E. repens (Eur ), M. microspora (Mm), F. oxysporum (Fo),

 E. coli (Ec) and B. megaterium (Bm); n.t: not tested.

  In our investigation cytochalasins E and K  showed

strong antifungal activity against  E. repens   and  M.

microspora  especially towards fungal  M. microspora .Similarly cytochalasins E and K  showed good herbicidal

activity towards C. fusca. In our investigation radicinin

showed strong antifungal activity against  E. repens and M.

microspora while week antibacterial and herbicidal activity

were observed.

4. Discussion

  Fungal derived natural products have primarily served

as lead structures for the development of antibacterial,

anticancer and antifungal agents. In the present study,

antimicrobial activity is observed for three extracts

and four pure compounds. The ethyl acetate extracts of

endophytic fungi Plectophomella sp., Physalospora sp., and

C. monogyna showed significant antibacterial, antifungal,

and herbicidal activities. The halogenated natural product

(-)-mycorrhizin A which was isolated from  Plectophomella 

sp. demonstrated significant antifungal activity towards U.

 violacea and  E. repens. Previously (-)-mycorrhizin A has

also been reported from the fungi  viz.,  Pezicula carpinea,

 Pe zi cu la li vida ,  Mono tropa hypopi ty s and  Lachnum

 papyraceum[23].

Cytochalasins E and K, isolated from  Physalospora  sp.,

showed strong antifungal activity and good herbicidal

activity. Cytochalasins are a group of fungal metabolites

with complex and diverse molecular structure and

possessing various biological activities. Cytochalasins

have a rigid bicyclic isoindolinone core which is fused to

a macro cycle. Cytochalasins showed profound effects oncytoskeletal proteins, resulting in pronounced morphogenic

changes in animals and plants. Despite the slight structural

differences in their macrocycles, cytochalasins E bearing

an β, γ-unsaturated ketone moiety, was found to be slightly

more active than cytochalasins K, with an α,β-unsaturated

lactone moiety, suggesting the importance in the position of

the double bond for an increase in antimicrobial activity of

these cytochalasins.

Radicinin which was isolated from endophytic fungus

C. monogyna showed strong antifungal activity and week

antibacterial and herbicidal activities. Radicinin is aphytotoxic and antibiotic metabolite produced by some

phytopathogenic fungi. It was intially reported from

Stemphylium radicinum  in the 1950s, and since then has

been reported to be produced by several fungal species viz.,

Cochliobolus lunatus,  Alternaria chrysanthemi,  Alternaria

helianthi,  Phoma andina , Curvularia sp.,  Alternaria

radicina and Alternaria petroselini[22]. It has been reported

that radicinin demonstrated phytotoxic activity and also

produced necrotic lesions in Coix lachryma-jobi as well as

exhibiting root growth in carrot seedlings[22].

  The lower antimicrobial activity against some organismsby the extracts may be attributed to the lower concentration

of the antimicrobial compounds. All the tested compounds

have good antifungal, antibacterial, and algicidal properties.

Assuming that the metabolites produced in the culture are

also synthesized in the plant, they could, for example, play

a role in inhibiting competitive microorganisms within the

endophyte’s natural habitat. Additionally, due to the fact

that a broad range of microorganisms is inhibited, it would

be of interest to discover whether compounds isolated in

the present study are generally cytotoxic.

  The antimicrobial properties of three extracts of the

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endophytic fungi  viz.,   Plectophomella sp.,  Physalospora 

sp., and C. monogyna and four pure compounds  viz.,

(-)-mycorrhizin A  , cytochalasins E  and K and radicinin

confirm their potential use in the food and pharmaceutical

industries. Similarly, the antimicrobial activity of the extracts

and compounds isolated also confirms their potential use in

the preservation of foodstuffs against bacteria and fungi and

that these compounds may also be valuable for extending

the shelf life of foodstuffs.

 

Conflict of interest statement

  We declare that we have no conflict of interest.

Acknowledgements

  We thank BASF AG and the BMBF  (Bundesministerium f ürBildung und Forschung, project No. 03F0360A).

References

[1] Kaul S, Gupta S, Ahmed M, Dhar MK. Endophytic fungi from

medicinal plants: a treasure hunt for bioactive metabolites.

 Phytochem Rev 2012; 11: 487-505.

[2] Hussain H, Krohn K, Ahmed I, Draeger S, Schulz B, Pietro SD, et

al. Phomopsinones A-D: four new pyrenocines from Endophytic

fungus,  Phomopsis sp. European J Org Chem 2012; doi: 10.1002 /

ejoc.201101788.

[3] Hussain H, Krohn K, Floerke U, Schulz B, Draeger S, Pescitelli

G, et al. Absolute configurations of globosuxanthone A and

secondary metabolites from Microdiplodia sp.– a novel solid-

state CD /TDDFT approach. European J Org Chem 2007; 2: 292-

295.

[4] Hussain H, Krohn K, Floerke U, Schulz B, Draeger S, Pescitelli

G, et al. Absolute configuration of hypothemycin and new 5’-O-

methylhypothemycin from Phoma sp.-a novel solid state CD /

TDDFT approach. Tetrahedron Asymmetry 2007; 18: 925-930.

[5] Hussain H, Krohn K, Ullah Z, Draeger S, Schulz B. Bioactivechemical constituents of two endophytic fungi. Biochem Syst

 Ecol 2007; 35: 898-900.

[6] Hussain H, Krohn K, Schulz B, Draeger S. Exochromone:

structurally unique chromone sdimer with antifungal and

algicidal agent from Exophiala sp. Heterocycles 2007; 74: 331-337.

[7] Krohn K, Kouam SF, Kuigoua GM, Hussain H, Cludius-Brand S,

Flörke U, et al. Xanthones and new oxepino[2,3-b]chromones

from three endophytic fungi. Chemistry. 2009; 15: 12121-12132.

[8] Hussain H, Akhtar N, Draeger S, Schulz B, Pescitelli G, Antus S,

et al. New bioactive 2,3-epoxycyclohexenes and isocoumarins

from the endophytic fungus  Phomopsis sp. from Laurus azorica.

 European J Org Chem 2009; 5: 749-756.

[9] Hussain H, Krohn K, Draeger S, Meier K, Schulz B. Bioactive

chemical constituents of a sterile endophytic fungus from

 Meliotus dentatus. Rec Nat Prod 2009; 3: 114-117.

[10] Qin S, Hussain H, Schulz B, Draeger S, Krohn K. Two new

metabolites, epoxydine A and B, from Phoma sp. Helv Chim Acta

2010; 93: 169-174.

[11] Saleem M, Hussain H, Ahmed I, Draeger S, Schulz B, Meier K, et

al. Viburspiran, an antifungal member of the Octadride Class of

maleic anhydride natural products.  European J Org Chem 2011;

4: 808-812.

[12] Siddiqui IN , Zahoor A, Hussain H, Ahmed I, Ahmad VU ,

Padula D, et al. Diversonol and blennolide derivatives from the

endophytic fungus  Microdiplodia sp.: absolute configuration of

diversonol. J Nat Prod 2011; 74: 365-373.

[13] Ahmed I, Hussain H, Schulz B, Draeger S, Padula D, Pescitelli G,

et al. Three new antimicrobial metabolites from the endophytic

fungus Phomopsis sp. European J Org Chem 2011; 15: 2867-2873.

[14] Hussain H, Ahmed I, Schulz B, Draeger S, Floerke U, Pescitelli

G, et al. Solid-state circular dichroism and hydrogen bonding

absolute configuration of massarigenin A  from Microsphaeropsis

sp. Chirality 2011; 23: 617-623.

[15] Krohn K, Hussain H, Ullah Z, Flörke U, Schulz B, Draeger S, et

al. Massarilactones E-G, new metabolites from the endophytic

fungus Coniothyrium sp., associated with the plant  Artimisia

maritime. Chirality 2007; 19: 464-470.

[16] Hussain H, Tchimene MK, Ahmed I, Meier K, Steinert M, Draeger

S, et al. Antimicrobial chemical constituents from the endophytic

fungus from  Phomopsis sp. from  Notobasis syriaca.  Nat Prod

Commun 2011; 6: 1905-1906.

[17] Hussain H, Ahmed I, Schulz B, Draeger S, Krohn K. Pyrenocines

J-M: four new pyrenocines from the endophytic fungus,

 Phomopsis sp. Fitoterapia 2012; 83: 523-526.

[18] Höller U, Wright AD, Matthée GF, König GM, Draeger S, Aust HJ,

et al. Fungi from marine sponges: diversity, biological activity

and secondary metabolites. Mycol Res 2000; 104: 1354-1365.

[19] Schulz B, Sucker J, Aust HJ, Krohn K, Ludewig K, Jones PG, et al.

Biologically active secondary metabolites of endophytic Pezicula

species. Mycol Res 1995; 99: 1007-1015.

[20] Trofast J, Wickberg B. Mycorrhizin a and chloromycorrhizin a,

two antibiotics from a mycorrhizal fungus of Monotropa hypopitys L. Tetrahedron 1977; 33: 875-879.

[21] Steyn PS, van Heerden FR, Rabie CJ. Cytochalasins E  and K,

toxic metabolites from  Aspergillus clavatus. J Chem Soc Perkin

Trans 1 1982; doi; 10.1039 /P19820000541.

[22] Suzuki M, Sakuno E, Ishihara A, Tamura J, Nakajima H.

Conversions of deoxyradicinin to radicinin and of radicinin to

3-epi-radicinin in the phytopathogenic fungus Bipolaris coicis.

 Phytochemistry 2012; 75: 14-20.

[23] Dembitsky VM, Tolstikov GA. Natural nalogenated mononuclear

phenol compounds and their derivatives. Chem Sustain Dev 2003;

11: 567-575.