S224
Asian Pac J Trop Med 2014; 7(Suppl 1): S224-S227
Contents lists available at ScienceDirect
Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm
Document heading
doi: 10.1016/S1995-7645(14)60236-4
Antimicrobial constituents 1,2
from three endophytic fungi 1
2
Hidayat Hussain *, Christine Kliche-Spory , Ahmed Al-Harrasi 4 5 1 6 Green , Barbara Schulz , Karsten Krohn , Afzal Shah
, Ahmed Al-Rawahi2, Ghulam Abbas2,3, Ivan Robert
Department of Chemistry, University of Paderborn, Warburger Strasse 100, 33098 Paderborn, Germany
1
UoN 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
2
Department of Chemistry, CIIT, Abbottabad Campus, Abbottabad-22060, Pakistan
3
Department of Chemistry and Polymer Science, University of Stellenbosch, P/Bag X1 Matieland 7602, South Africa
4
Institute of Microbiology, University of Braunschweig, Spielmannstraße 7, 38106 Braunschweig, Germany
5
Department of Chemistry, Quaid-i-Azam University, 45320 Islamabad, Pakistan
6
ARTICLE INFO Article history:
Received 4 Feb 2014 Received in revised form 16 May 2014 Accepted 12 Jun 2014 Available online 4 Aug 2014
Keywords: Natural product Endophytic fungi Antimicrobial activity
ABSTRACT Objective: T o evaluate the antimicrobial potential of extracts of the endophytic fungi Plectophomella sp., Physalospora sp., and Crataegus monogyna (C. monogyna) and study the tentative identification of their active constituents. Methods: Crude extracts and isolated compounds were screened for antimicrobial activity using the agar well diffusion method. Four compounds were purified from three endophytic fungi using 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 promising antifungal, antibacterial and herbicidal properties. (-)-Mycorrhizin A was isolated from Plectophomella sp. while cytochalasins E and K were isolated from Physalospora sp. Similarly radicinin 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 strong herbicidal and antifungal activities towards Chlorella fusca, U. violacea, E. repens, Mycotypha microspora (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 good herbicidal 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 clearly demonstrate that these fungi extracts and active compounds present a great potential use in the food, cosmetic and pharmaceutical industries.
1. Introduction There are a large number of bioactive compounds that have been isolated from endophytic fungi and these *Corresponding author: Hidayat Hussain, Department of Chemistry, University of Paderborn, Warburger Strasse 100, 33098 Paderborn, Germany. Tel: +495251602172; Fax: +495251603245 E-mail: [email protected] Foundation Project: Supported by BASF AG (Chemical Company) and the BMBF (The Federal Ministry of Education and Research, Germany (Project no. 03F0360A).
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] . T hese bioactive natural products provided by endophytic fungi originate from different biosynthetic pathways and belong
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Hidayat Hussain et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S224-S227
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 ( M erck, 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. I nfrared radiation spectra were recorded on a 1 13 Nicolet-510P spectrophotometer. H and C nuclear magnetic resonance spectra were recorded on a Bruker Avance 500 1 13 (500 MHz for H and 125 MHz for C) spectrometer. Mass spectrum and high resolution mass spectrum were recorded in 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
T he culture filtrate from Plectophomella 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 CH 2 C l 2 / M e OH ( 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] . E xtracts 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 test organism[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. O
CI
OH
O
O
O
1
O
H
H
HN 3
H
HN
O
H
O 2
O
O O
OH
O HO
O O O
H
H
OAc
O
O 4
O
Figure 1. S tructures of compounds isolated from Plectophomella 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 activities towards 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
Plectophomella sp. extract Physalospora sp. extract C. monogyna extract (-)-Mycorrhizin A Cytochalasins E Cytochalasins K Radicinin
Radius of zone of inhibition (mm)
Herbicidal Chl
Ust
30
24
4
35
12 25
Antifungal
Eur Mm Fo 12
7
22
20
30
22
3
14
14
10
9
5
21
13
10 2
6 3
22 15
14 13
7
17
n.t n.t n.t n.t 0
Antibacterial Ec 1
13
Bm 13 16
15
15
n.t n.t
n.t n.t
3
2
8
3
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. a
10
I n 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, Pezicula livida, Monotropa hypopitys 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. C ytochalasins have a rigid bicyclic isoindolinone core which is fused to a macro cycle. Cytochalasins showed profound effects on cytoskeletal 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. R adicinin is a phytotoxic and antibiotic metabolite produced by some phytopathogenic fungi. I t 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 organisms by 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
Hidayat Hussain et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S224-S227
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 für Bildung 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: 292295.
[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. Bioactive
chemical constituents of two endophytic fungi. Biochem Syst Ecol 2007; 35: 898-900.
[6] H ussain H , K rohn K , S chulz B , D raeger S . E xochromone:
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.
S227
[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] Q in S , H ussain H , S chulz B , D raeger S , K rohn K . T wo 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] S iddiqui IN , Z ahoor A , H ussain H , A hmed I , A hmad 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] S uzuki M , S akuno E , I shihara A , T amura J , N akajima 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.
Asian Pac J Trop Med 2014; 7(Suppl 1): S224-S227
Contents lists available at ScienceDirect
Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm
Document heading
doi: 10.1016/S1995-7645(14)60236-4
Antimicrobial constituents 1,2
from three endophytic fungi 1
2
Hidayat Hussain *, Christine Kliche-Spory , Ahmed Al-Harrasi 4 5 1 6 Green , Barbara Schulz , Karsten Krohn , Afzal Shah
, Ahmed Al-Rawahi2, Ghulam Abbas2,3, Ivan Robert
Department of Chemistry, University of Paderborn, Warburger Strasse 100, 33098 Paderborn, Germany
1
UoN 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
2
Department of Chemistry, CIIT, Abbottabad Campus, Abbottabad-22060, Pakistan
3
Department of Chemistry and Polymer Science, University of Stellenbosch, P/Bag X1 Matieland 7602, South Africa
4
Institute of Microbiology, University of Braunschweig, Spielmannstraße 7, 38106 Braunschweig, Germany
5
Department of Chemistry, Quaid-i-Azam University, 45320 Islamabad, Pakistan
6
ARTICLE INFO Article history:
Received 4 Feb 2014 Received in revised form 16 May 2014 Accepted 12 Jun 2014 Available online 4 Aug 2014
Keywords: Natural product Endophytic fungi Antimicrobial activity
ABSTRACT Objective: T o evaluate the antimicrobial potential of extracts of the endophytic fungi Plectophomella sp., Physalospora sp., and Crataegus monogyna (C. monogyna) and study the tentative identification of their active constituents. Methods: Crude extracts and isolated compounds were screened for antimicrobial activity using the agar well diffusion method. Four compounds were purified from three endophytic fungi using 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 promising antifungal, antibacterial and herbicidal properties. (-)-Mycorrhizin A was isolated from Plectophomella sp. while cytochalasins E and K were isolated from Physalospora sp. Similarly radicinin 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 strong herbicidal and antifungal activities towards Chlorella fusca, U. violacea, E. repens, Mycotypha microspora (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 good herbicidal 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 clearly demonstrate that these fungi extracts and active compounds present a great potential use in the food, cosmetic and pharmaceutical industries.
1. Introduction There are a large number of bioactive compounds that have been isolated from endophytic fungi and these *Corresponding author: Hidayat Hussain, Department of Chemistry, University of Paderborn, Warburger Strasse 100, 33098 Paderborn, Germany. Tel: +495251602172; Fax: +495251603245 E-mail: [email protected] Foundation Project: Supported by BASF AG (Chemical Company) and the BMBF (The Federal Ministry of Education and Research, Germany (Project no. 03F0360A).
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] . T hese bioactive natural products provided by endophytic fungi originate from different biosynthetic pathways and belong
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Hidayat Hussain et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S224-S227
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 ( M erck, 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. I nfrared radiation spectra were recorded on a 1 13 Nicolet-510P spectrophotometer. H and C nuclear magnetic resonance spectra were recorded on a Bruker Avance 500 1 13 (500 MHz for H and 125 MHz for C) spectrometer. Mass spectrum and high resolution mass spectrum were recorded in 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
T he culture filtrate from Plectophomella 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 CH 2 C l 2 / M e OH ( 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] . E xtracts 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 test organism[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. O
CI
OH
O
O
O
1
O
H
H
HN 3
H
HN
O
H
O 2
O
O O
OH
O HO
O O O
H
H
OAc
O
O 4
O
Figure 1. S tructures of compounds isolated from Plectophomella sp., Physalospora sp., and C. monogyna. 1: (-)-Mycorrhizin A; 2:
cytochalasins E; 3: cytochalasins K; 4: radicinin.
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Hidayat Hussain et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S224-S227
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 activities towards 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
Plectophomella sp. extract Physalospora sp. extract C. monogyna extract (-)-Mycorrhizin A Cytochalasins E Cytochalasins K Radicinin
Radius of zone of inhibition (mm)
Herbicidal Chl
Ust
30
24
4
35
12 25
Antifungal
Eur Mm Fo 12
7
22
20
30
22
3
14
14
10
9
5
21
13
10 2
6 3
22 15
14 13
7
17
n.t n.t n.t n.t 0
Antibacterial Ec 1
13
Bm 13 16
15
15
n.t n.t
n.t n.t
3
2
8
3
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. a
10
I n 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, Pezicula livida, Monotropa hypopitys 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. C ytochalasins have a rigid bicyclic isoindolinone core which is fused to a macro cycle. Cytochalasins showed profound effects on cytoskeletal 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. R adicinin is a phytotoxic and antibiotic metabolite produced by some phytopathogenic fungi. I t 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 organisms by 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
Hidayat Hussain et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S224-S227
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 für Bildung 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: 292295.
[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. Bioactive
chemical constituents of two endophytic fungi. Biochem Syst Ecol 2007; 35: 898-900.
[6] H ussain H , K rohn K , S chulz B , D raeger S . E xochromone:
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.
S227
[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] Q in S , H ussain H , S chulz B , D raeger S , K rohn K . T wo 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] S iddiqui IN , Z ahoor A , H ussain H , A hmed I , A hmad 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] S uzuki M , S akuno E , I shihara A , T amura J , N akajima 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.
