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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 59  |  Page : 495-498  

Characterization of the secondary metabolites from endophytic fungi Nodulisporium sp. isolated from the medicinal plant Mikania laevigata (Asteraceae) by reversed-phase high-performance liquid chromatography coupled with mass spectrometric multistage


1 Department of Health, State University of Feira de Santana, Feira de Santana, Brazil
2 Department of Biology, State University of Feira de Santana, Feira de Santana, Brazil
3 Department of Biological Sciences, State University of Santa Cruz, Ilheus, BA, Brazil

Date of Submission22-Dec-2017
Date of Decision28-Feb-2018
Date of Web Publication17-Jan-2019

Correspondence Address:
Alexsandro Branco
Departamento de Saúde, Universidade Estadual de Feira de Santana, Campus Universitário, Av. Transnordestina, s/nº, Bairro Novo Horizonte, 44.036-900 Feira de Santana, BA
Brazil
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_616_17

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   Abstract 


Background: The endophytic fungi are an excellent source of secondary metabolites as a natural product with antimicrobial, insecticidal, cytotoxic, antioxidant, and anticancer activities. No studies on the chemical compounds of the endophytes of Mikania laevigata have been described in the literature. Objective: The objective of the study is to investigate by reversed phase high-performance liquid chromatography coupled with mass spectrometric multistage (RP-HPLC-MS/MS) the secondary metabolites of the endophytic fungi Nodulisporium sp., isolated from the medicinal plant M. laevigata (Asteraceae). Materials and Methods: The mycelia biomass strains of Nodulisporium sp. were accumulated and separated from the aqueous medium by filtration; the filtrate was subjected to a liquid–liquid partition with ethyl acetate (EtOAc) resulting in the crude EtOAc extract. This extract was analyzed by RP-HPLC-MS/MS. Results: The total mass chromatogram of the EtOAc crude strain Nodulisporium sp. extract showed compounds eluted between 13 and 20 min. The peak 1 (Rt 13.8 min), peak 2 (Rt. 14.8 min), peak 3 (Rt. 15.7 min) and peak 4 (Rt. 19.6 min) were characterized as dechlorogriseofulvin (C17H18O6), griseofulvin (C17H17O6Cl), cytochalasin D (C30H37NO6) and nodulisporic acid B2 (C43H57NO7), respectively. Conclusion: The present work provides the first scientific report on constituents of endophytic fungi Nodulisporium sp. isolated from the M. laevigata (Asteraceae).
Abbreviations used: EtOAc: Ethyl acetate; RP-HPLC-MS/MS: Reversed-Phase High-Performance Liquid Chromatography coupled with mass spectrometric multistage; CCMB: Culture collection of microorganisms from Bahia; Rt: Retention time.

Keywords: Endophytic fungi, Mikania laevigata, Nodulisporium, reversed-phase high-performance liquid chromatography coupled with mass spectrometric multistage, Xylariaceae


How to cite this article:
A. Reis IM, C. Ribeiro FP, M. Almeida PR, B. Costa LC, Kamida HM, T. Uetanabaro AP, Branco A. Characterization of the secondary metabolites from endophytic fungi Nodulisporium sp. isolated from the medicinal plant Mikania laevigata (Asteraceae) by reversed-phase high-performance liquid chromatography coupled with mass spectrometric multistage. Phcog Mag 2018;14:495-8

How to cite this URL:
A. Reis IM, C. Ribeiro FP, M. Almeida PR, B. Costa LC, Kamida HM, T. Uetanabaro AP, Branco A. Characterization of the secondary metabolites from endophytic fungi Nodulisporium sp. isolated from the medicinal plant Mikania laevigata (Asteraceae) by reversed-phase high-performance liquid chromatography coupled with mass spectrometric multistage. Phcog Mag [serial online] 2018 [cited 2019 Sep 15];14:495-8. Available from: http://www.phcog.com/text.asp?2018/14/59/495/250187





SUMMARY

  • The endophytic fungi Nodulisporium sp. was previous isolated from the medicinal plant Mikania laevigata (Asteraceae).
  • The crude EtOAc extract of Nodulisporium sp. was analyzed by RP-HPLC-MS/MS.
  • The compounds dechlorogriseofulvin, griseofulvin, cytochalasin D and nodulisporic acid B2 were characterized.



   Introduction Top


Mikania laevigata (Asteraceae), a Brazilian medicinal plant commonly known as “guaco,” is used for the treatment of respiratory disorders, such as asthma, bronchitis, and chronic lung diseases and also for coughing.[1],[2] The antiulcer, anti-inflammatory, analgesic, antispasmodic, and antimicrobial activities have also been investigated for this plant.[3],[4],[5] Chemical studies on M. laevigata leaves report the isolation and identification of coumarin, terpenes, steroids, flavonoid glycosides, o-coumaric acid, cinnamoyl grandifloric acid, cupressenic acid, isopropiloxi-grandifloric acid, isobutiloxi-grandifloric acid, saponins, and tannins.[1],[2],[4],[5]

The symbiotic relationship among endophytic fungi as well as plants gives endophytes the powerful capability to produce new bioactive substances. The endophytic fungi are an excellent source of secondary metabolites as a natural product with antimicrobial, insecticidal, cytotoxic, antioxidant, and anticancer activities. Thus, they have great promising applications in agriculture and medicine.[6],[7]

No studies on the chemical compounds of the endophytes of M. laevigata have been described in the literature. Henceforth, previous studies conducted by our researcher group showed the isolation and identification of the endophytic fungus from M. laevigata and the evaluation of its extracts against four strains of Salmonella spp.[8],[9] All the fungal extracts showed antimicrobial activity, and the microorganisms of genus Nodulisporium, Xylariaceae family, were the dominant group of fungi associated with this plant species. Nodulisporium, a group of common endophytic fungi known to produce bioactive secondary metabolites, contained antiflea, cytotoxic, antiplasmodium, antifungal, human DNA polymerase k inhibitors, and mycotoxin activity.[10]

In this work, the ethyl acetate (EtOAc) crude strain Nodulisporium sp. extract (isolated from M. laevigata) was chemically analyzed by reversed-phase high-performance liquid chromatography coupled with mass spectrometric multistage (RPH-PLC-MS/MS).


   Materials and Methods Top


Collection of plant material and isolation of endophytic fungus

The strains of Nodulisporium sp. were isolated from the leaves of M. laevigata, collected in a floral Garden located in the State University of Santa Cruz, Ilheus, state of Bahia, Brazil, in September 2009. Three plants were selected for the withdrawal of healthy leaves. From each plant, five leaves were collected, totaling a sample of 15 leaves which were immediately subjected to endophytic fungi isolation. Both the isolation and identification of these fungi are described in a previous study of the same research group.[8] These fungi were deposited as Nodulisporium sp. Culture Collection of Microorganisms from Bahia (CCMB) 562 (Genbank n° JNO51356), at the CCMB, in the Department of Biology, State University of Feira de Santana.

Cultivation and preparation of the fungal strain

The endophytic fungi Nodulisporium sp. grown on Malt Extract Agar at 28°C for 5 days was inoculated into 500-ml Erlenmeyer flasks containing 300-ml Malt Extract Broth at room temperature for 4 weeks. The mycelia biomass accumulated in the Erlenmeyer was separated from the aqueous medium by filtration, and the filtrate was subjected to a liquid–liquid partition with EtOAc (3 mL × 200 mL). The EtOAc fraction was evaporated resulting in the crude EtOAc extract.

Reversed-phase high-performance liquid chromatography coupled with mass spectrometric multistage analysis

The crude EtOAc extract of mycelium was analyzed by RP-HLC-MS/MS in positive ion mode using an Esquire 3000-plus mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a CBM-20A controller, LC-20AD pump, SIL-20AC autosampler, and SPD-20A detector. A Phenomenex Luna C-18 (2) column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase consisted of water containing 0.05% phosphoric acid (A) and methanol (B) at a flow rate of 1.0 mL/min using the following gradients: 0–20 min: A (75%–0%) and B (25%–100%), 20–24 min: A/B: 0/100%, 24–25 min: A (0%–75%) and B (100%–25%), and 25–35 min: A/B: 0/100%. The detection was done on a DAD detector set at 340 nm. The mobile phase was prepared daily, filtered through a 0.45-mm membrane filter (Millipore), and sonicated before use.


   Results and Discussion Top


[Figure 1] shows the total mass chromatogram of the EtOAc crude strain Nodulisporium sp. extract. This chromatogram showed the natural compounds eluted between 13 and 20 min. The peaks after 22 min were characterized as culture contamination and were not considered in this study. [Figure 2] and [Figure 3] show MS and MS2 spectra of peaks 1, 2, 3, and 4 of EtOAc extract of mycelium from Nodulisporium sp. The peak with retention time (Rt) at 13.8 min was characterized as dechlorogriseofulvin (C17H18O6, molecular ion at 318). This compound showed protonated ion at m/z 319 [M+H]+ and sodium adducts at m/z 341 [M+Na]+ and 659 [2M+Na]+. The MS2 of the protonated ion showed the following fragments: 287 [M-OCH3+H]+ (1a), 251 [M-C4H3O+H]+ (1b), 181 [M-C8H9O2+H]+ (1c), and 165 [M-C8H9O3+H]+ (1d). This fragmentation pathway was in accordance with literature for the same compound [Figure 4].[11]
Figure 1: Total mass chromatogram of EtOAc extract of mycelium of Nodulisporium sp.: (1) dechlorogriseofulvin, (2) griseofulvin, (3) cytochalasin D, and (4) nodulisporic acid B2

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Figure 2: MS spectra of peaks 1, 2, 3, and 4 of ethyl acetate extract of mycelium from Nodulisporium sp.

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Figure 3: (1) MS2 analysis of MS 319 (peak 1); (2) MS2 analysis of MS 353 (peak 2); (3) MS2 analysis of MS 508 (peak 3); and MS2 analysis of MS 700 (peak 4)

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Figure 4: Dechlorogriseofulvin (1a-1d), griseofulvin (2a-2d), cytochalasin D (3a-3b), and nodulisporic acid B2 (4a) fragments

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The compound 2 (Rt. 14.8 min) showed protonated ion at m/z 353 [M+H]+ and sodium adducts at m/z 375 [M+Na]+ and 727 [2M+Na]+. The MS2 analysis showed fragmentation profile at m/z 321 [M-OCH3+H]+ (2a), 285 [M-C4H3O+H]+ (2b), 215 [M-C8H9O2+H]+ (2c), and 165 [M-C8H9O3 Cl+H]+ (2d) [Figure 4]. Thus, the compound 2 was characterized as griseofulvin (C17H17O6 Cl).[12] Griseofulvin is a fungistatic agent used for the treatment of dermatophytes. It is a known metabolic product of several species of fungus. It is deposited in keratin precursor cells, which become resistant to the invasion of dermatophytes. Dechlorogriseofulvin is formed by the degradation of griseofulvin.[13]

The compound 3 (Rt. 15.7 min) was characterized as cytochalasin D (C30H37 NO6). This compound showed protonated ion at m/z 508 [M+H]+ and sodium adducts at m/z 530 [M+Na]+. The MS2 fragmentation profile of protonated ion showed loss of 18 Da (H2O) and 60 Da (C2H2O and H2O) correspondent to m/z 490 (3a) and 448 (3b), respectively [Figure 4].[14],[15] The cytochalasin member is well known as mycotoxins widely distributed in various fungi. Its potent inhibition of the actin polymerization affects a wide range of cellular events. Thus, cytochalasins are important biochemical tools for studying fundamental cellular processes.[14],[15]

The compound 4 (Rt. 19.6 min) showed protonated ion at m/z 700 [M+H]+. A search for fungus metabolite with the same mass of compound 4 suggests its characterization as nodulisporic acid B2. The MS2 of protonated ion showed losses of 18 (H2O) and 45 (COOH); ions m/z 682 (4a) and 637 (4b), respectively [Figure 4]. Nodulisporic acids are indole diterpenes that exhibit potent insecticidal properties.[16]


   Conclusion Top


This study demonstrated that RP-HPLC-MS/MS is a suitable tool for characterization of different metabolites compounds for the endophytic fungi, it was possible to characterize four metabolites: dechlorogriseofulvin, griseofulvin, cytochalasin D, and nodulisporic acid B2. The present work provides the first scientific report on phytoconstituents of endophytic fungi Nodulisporium sp. isolated from the the M. laevigata (Asteraceae) and these fungi proved to be a source of bioactive metabolites.

Acknowledgements

The authors are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the grants and fellowship.

Financial support and sponsorship

This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) of Brazil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Bolina RC, Garcia EF, Duarte MG. Comparative study of the chemical composition of the species Mikania glomerata Sprengel and Mikania laevigata Schultz Bip. ex Baker Rev Bras Farmacognosia 2009;19:294-8.  Back to cited text no. 1
    
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dos Santos SC, Krueger CL, Steil AA, Kreuger MR, Biavatti MW, Wisniewski Junior A, et al. LC characterisation of guaco medicinal extracts, Mikania laevigata and M. glomerata, and their effects on allergic pneumonitis. Planta Med 2006;72:679-84.  Back to cited text no. 2
    
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Suyenaga ES, Reche E, Farias FM, Schapoval EE, Chaves CG, Henriques AT, et al. Antiinflammatory investigation of some species of Mikania. Phytother Res 2002;16:519-23.  Back to cited text no. 3
    
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Yatsuda R, Rosalen PL, Cury JA, Murata RM, Rehder VL, Melo LV. Effects of Mikania genus plants on growth and cell adherence of mutans streptococci. J Ethnopharmacol 2005;97:183-9.  Back to cited text no. 4
    
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Bighetti AE, Antônio MA, Kohn LK, Rehder VL, Foglio MA, Possenti A. Antiulcerogenic activity of a crude hydroalcoholic extract and coumarin isolated from Mikania laevigata schultz bip. Phytomedicine 2005;12:72-7.  Back to cited text no. 5
    
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Stadler M, Ju YM, Rogers JD. Chemotaxonomy of entonaema, rhopalostroma and other xylariaceae. Mycol Res 2004;108:239-56.  Back to cited text no. 6
    
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Jia M, Chen L, Xin HL, Zheng CJ, Rahman K, Han T, et al. Afriendly relationship between endophytic fungi and medicinal plants: A systematic review. Front Microbiol 2016;7:906.  Back to cited text no. 7
    
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Ribeiro FP, Kamida HM, Rodrigues K, Almeida PR, Uetanabaro AP, Costa LC, et al. Isolation and identification of endophytic fungi in the medicinal plant Mikania laevigata (Asteraceae). Phcog J 2014;6:10-5.  Back to cited text no. 8
    
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Ribeiro FP, Fonseca FC, Reis IM, Araújo IS, Kamida HM, Branco A, et al. Xylariaceae endophytic fungi metabolites against salmonella. In: Kumar Y, editor. Salmonella – A Diversified Superbug. Rijeka: InTech; 2012. p. 119-38.  Back to cited text no. 9
    
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Zheng QC, Chen GD, Kong MZ, Li GQ, Cui JY, Li XX, et al. Nodulisporisteriods A and B, the first 3,4-seco-4-methyl-progesteroids from Nodulisporium sp. Steroids 2013;78:896-901.  Back to cited text no. 10
    
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Kahsay G, Adegoke AO, Van Schepdael A, Adams E. Development and validation of a reversed phase liquid chromatographic method for analysis of griseofulvin and impurities. J Pharm Biomed Anal 2013;80:9-17.  Back to cited text no. 11
    
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Mistri HN, Jangid AG, Sanyal M, Shrivastav P. Electrospray ionization LC–MS/MS validated method to quantify griseofulvin in human plasma and its application to bioequivalence study. J Chromatogr B 2007;850:318-26.  Back to cited text no. 12
    
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Develoux M. Griseofulvin. Ann Dermatol Venereol 2001;128:1317-25.  Back to cited text no. 13
    
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Prasain JK, Ueki M, Stefanowicz P, Osada H. Rapid screening and identification of cytochalasins by electrospray tandem mass spectrometry. J Mass Spectrom 2002;37:283-91.  Back to cited text no. 14
    
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Nielsen KF, Smedsgaard J. Fungal metabolite screening: Database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography-UV-mass spectrometry methodology. J Chromatogr A 2003;1002:111-36.  Back to cited text no. 15
    
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Bills GF, González-Menéndez V, Martín J, Platas G, Fournier J, Peršoh D, et al. Hypoxylon pulicicidum sp. nov. (Ascomycota, xylariales), a pantropical insecticide-producing endophyte. PLoS One 2012;7:e46687.  Back to cited text no. 16
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]



 

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