(4S)-4,8-dihydroxy-1-tetralone and other chemical constituents from Pestalotiopsis sp. EJC07, endophytic fromBauhinia guianensis

Abstracts

The present work reports the isolation of eight compounds fromPestalotiopsis sp. EJC07 isolated as endophytic fromBauhinia guianensis, a tipical plant of the Amazon. The compounds (4S)-4,8-dihydroxy-1-tetralone (1), uracil (2), uridin (3), p-hydroxybenzoic acid (4), ergosterol (5), ergosterol peroxide (6), cerevisterol (7) and ducitol (8) were isolated by chromatographic procedures and identified by spectral methods of 1D and 2D NMR and MS. The compound 1 is being reported for the first time in the genusPestalotiopsis.

B. guianensis; endophityc fungus; Pestalotiopsis; tetralone

O presente trabalho reporta o isolamento de oito compostos dePestalotiopsis sp. EJC07 isolado como endofítico deBauhinia guianensis, uma planta típica da Amazônia. Os compostos (4S)-4,8-diidroxi-1-tetralona (1), uracila (2), uridina (3), ácido p-hidroxibenzoico (4), ergosterol (5), peróxido de ergosterol (6), cerevisterol (7) e ducitol (8) foram isolados por procedimentos cromatográficos e identificados por métodos espectrais de RMN 1D e 2D e EM. O composto 1 está sendo reportado pela primeira vez no gêneroPestalotiopsis.

B. guianensis; fungo endofítico; Pestalo tiopsis; tetralona

INTRODUCTION

The endophytic microorganisms live in the internal tissues of plants without causing apparent diseases to their hosts (Petrini et al. 1992). In the tropical and temperate forests, covering only 1.44% of the terrestrial surface, there are approximately 300,000 (three hundred thousand) plant species so far analyzed, home to more than 60% of the world biodiversity (Strobel et al. 2004). As a result, the opportunity to find new microorganisms and producers of bioactive compounds for agriculture, industry and medicine has increased the bioprospection of this microbiota of great importance in biotechnology (Strobel 2002).

Fungi generally are characterized by rapid growth of their colonies, adapting to various culture media, mostly in cereals such as rice and corn, where their metabolism is potentiated and, still producing a wide variety of secondary metabolites with the most diverse activities (Marinho et al. 2005).

Fungi of the Pestalotiopsis genus are broadly distributed, occurring in soil, seeds, fruits and leafs, can be parasite, endophytic or saprobes (Jeewon et al. 2004). Frequently endophytic association has been described to Pestalotiopsis generally found in subtropical and tropical regions (Wei and Xu 2004, Strobel and Daisy 2003). There are many reports of the metabolites secondary produced by Pestalotiopsis with biological activity and biotechnological applicability (Strobel and Long 1998, Li et al. 2005).

Thus, we decided study the biomass produced by endophytic fungusPestalotiopsis sp. EJC07 isolated from Bauhinia guianensis and this work led to the isolation of the eight known compounds biologically active (Figure 1). The (4S)-4,8-dihydroxy-1-tetralone is being reported the first time in the Pestalotiopsis genus.

Figure 1
- Compounds isolated from Pestalotiopsis sp. EJC07.

MATERIALS AND METHODS

General Procedures

ESIMS data were acquired in positive and negative mode using a Waters Acquity TQD instrument. 1D and 2D NMR spectra were recorded on a Varian Mercury 300, using solvent signal as reference. The chemical shifts are given in delta (δ) values and the coupling constants (J) in Hertz (Hz).

Plant Material

Bauhinia guianensis was collected in the city of Belém-PA and a voucher specimen (number 177.179) was deposited at the Herbarium of Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA).

Microorganism

Pestalotiopsis sp. was obtained from a collection of the Laboratório de Bioensaios e Química de Micro-organismos (LaBQuiM), Faculdade de Química - Universidade Federal do Pará. This collection contains isolates fromBauhinia guianensis. One strain is deposited in the LaBQuiM with the code EJC07.

Culture of Pestalotiopsis sp. in Rice and Chemical Constituents Isolation

Twenty-two Erlenmeyer flasks (1,000 mL) containing 200 g rice (Tio João(r)) and 125 mL distilled water per flask were autoclaved for 45 min at 121°C. Small cubes of PDA medium containing mycelium of Pestalotiopsis sp. EJC07 were added in 20 Erlenmeyer flasks under sterile condition. Two flasks were used as control. After 30 days of growth at 25°C the biomass obtained was macerated with hexane, ethyl acetate and methanol. The ethyl acetate was evaporated under reduced pressure, producing a yellowish residue (8.9 g). After successive fractionations the ethyl acetate extract on silica gel chromatography column eluted with hexane, ethyl acetate and methanol in polarity gradient were obtained the compounds 1-8.

RESULTS AND DISCUSSION

Constituents Isolated

The extract was submitted the chromatographic column on silica gel eluted with hexene, EtOAc and MeOH given (4S)-4,8-dihydroxy-1-tetralone (1), uracil (2), uridin (3),p-hydroxybenzoic acid (4), ergosterol (5), ergosterol peroxide (6), cerevisterol (7) and ducitol (8). All the compounds were identified by 1D and 2D NMR methods and comparison with literature data.

Chemical Constituents Identification

The mass spectrum ESI(+) to 1 showed m/z = 179 [M+H]⁺, that together with NMR data allowed propose the molecular formula C₁₀H₁₀O₃. The ¹H NMR spectrum to 1 showed signals characteristic to the aromatic ring at d 7.49 (t, 8.0 Hz, H-6), d 7.02 (dt, 7.8, 0.9 e 0.9 Hz, H-5) and d 6.92 (dd, 8.1 and 1.0 Hz, H-7), a singlet signal at d 12.41 typical of the chelated hydroxyl and a double duplet signal at d 4.92 with coupling constants of 7.4 and 4.0 Hz attributed to the hydrogen H-4. Also was observed in the ¹H NMR spectrum two signalsddd attributed to the diastereotopics hydrogens H-2 at d 3.00 (17.8, 8.2 and 4.7 Hz, H-2β) and d 2.65 (17.8, 8.2 and 4.7 Hz, H-2α), it was also observed two multiplets signals at d 2.35 and d 2.19 attributed to the hydrogens H-3β e H-3α, respectively.

In the ¹³C NMR observed 10 signals to car bons, being six signals referents to the aromatic carbons (d 162.7, d 145.8, d 137.0, d 117.8, d 117.3 and d 115.2), one signal at d 204.2 attributed to the carbonyl carbon (C-1), the signal at d 67.7 was attributed to the carbinolic carbon (C-4) and the signals at d 34.6 and d 31.2 were attributed to the sp³ carbons. The analysis of the specific rotation showed [α]^D+12,6 (CH₂Cl₂, 0,00116) to the compound 1. The signals were attributed in the base the HMBC, HSQC and COSY correlations. The spectral data were compared with the data described in the literature to (4S)-4,8-di-hydroxi-α-tetralone and showed total similarity (Zhu et al. 2008). According Inácio et al. (2006) the (4S)-4,8-di-hydroxi-1-tetralone showed antifungal activity by bioautography test against Cladosporium cladosporioides and C. sphaerospermum comparable to the standard nystatin and interesting allelopathic activity (Li et al. 2012).

The compounds 2, 3 and 4 showed as principal characteristic two signals double doublet in the hydrogens aromatic region (d 6.50 - d 8.00) in their ¹H NMR spectra. Then, the¹H and ¹³C NMR data were compared with the literature allowing identify them as uracil (2), uridin (3) andp-hydroxybenzoic acid (4) (Marinho et al. 2007). The compounds 2 and 3 are nitrogenous bases and show antiviral activity (Pinto et al. 2002).

The compounds 5, 6 and 7 showed in their¹H NMR spectra characteristics to steroidal compounds as the signals singlet and double duplet in the methyl region at 0.60 to d 1.20; methylene hydrogens signals at d 1.20 to d 2.00; signals to carbinolic hydrogens at d 3.63 (m) to 5, d 3.95 (m) to6 and d 3.61 (m) and d 4.07 (m) to 7; further the signals to olefinics hydrogens at d 5.15 to d 5.20. These data together with ¹³C NMR data to the compounds allowed identify them as the steroids ergosterol (5), ergosterol peroxide (6) and cerevisterol (7) (Marinho et al. 2009). The steroid ergosterol and ergosterol peroxide have significant anticancer activity (Kawagishi et al. 1988). Already the compound 8showed only signals to carbinolic hydrogens in their ¹H NMR spectrum. The comparison the ¹H and ¹³C data of the 8with the literature allowed identify as ducitol (Marinho et al. 2007).

The chemical study of the endophytic fungus Pestalotiopsis sp. EJC07 led to the isolation of eight biologically active compounds and the (4S)-4,8-di-hydroxi-1-tetralone is being reported for the first time in the genus Pestalotiopsis. This compound have been isolated of other fungi such as Sclerotinia sclerotiorum (Morita and Aoki 1974), Cytospora eucalypticola (Kokubun et al. 2003), Caryospora callicarpa (Zhu et al. 2008),Aspergillus fumigatus (Li et al. 2012) andParaconiothyrium variabile (Prado et al. 2013) and from plants belonging to the family Juglandaceae (Li et al. 2014, Talapatra et al. 1988). (4S)-4,8-dihydroxy-1-tetralone, a dihydronaphthalenone, has a particular interest as building blocks for more complex natural compounds featuring a spirobisnaphthalene structure (Prado et al. 2013).

ACKNOWLEDGMENTS

The authors thank the Fundação Amazônia de Amparo a Estudos e Pesquisas do Pará (FAPESPA), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) for the financial support, Secretaria de Estado de Educação do Pará (SEDUC) for the research scholarship.

• Inácio ML,Silva GH, Teles HL, Trevisan HC, Cavalheiro AJ, Bolzani VS, Young Mcm, Pfenning LH and Araújo AR. 2006. Antifungal metabolites fromColletotrichum gloeosporioides, an endophytic fungus in Cryptocarya mandioccana Nees (Lauraceae). Biochem Syst Ecol 34: 822-824.
• Jeewon R,Liew Ecy and Hyde KD. 2004. Phylogenetic evaluation of species nomenclature of Pestalotiopsis in relation to host association.Fungal Divers 17: 39-55.
• Kawagishi H,Katsumi R, Sazawa T, Mizuno T, Hagiwara T and Nakamura T. 1988. Cytotoxic Steroids from the Mushroom Agaricus blazei. Phytochemistry27: 2777-2779.
• Kokubun T,Veitch NC, Bridge PD and Simmonds Msj. 2003. Dihydroisocoumarins and a tetralone from Cytospora eucalypticola. Phytochemistry 62: 779-782.
• Li H,Qing C, Zhang Y and Zaho Z. 2005. ScreenIng for endophytic fungi with antitumour and antifungal activities from Chinese medicinal plants. World J Microbiol Biotechnol 21:1515-1519.
• Li XJ,Zhang Q, Zhang AL and Gao JM. 2012. Metabolites from Aspergillus fumigatus, an endophytic fungus associated with Melia azedarach, and their antifugal, antifeedant, and toxic activities. J Agric Food Chem 60: 3424-3431.
• Li XX,Yu MF, Ruan X, Zhang YZ and Wang Q. 2014. Phytotoxicity of 4,8-dihydroxy-1-tetralone isolated from Carya cathayensis Sarg. to various plant species. Molecules 19: 15452-15467.
• Marinho Amr,Rodrigues-Filho E, Moitinho Mlr and Santos LS. 2005. Biologically active polyketides produced by Penicilium janthinellum isolated as an endophytic fungus fruit of Melia azedarach. J Braz Chem Soc 16: 280-283.
• Marinho Amr,Marinho Psb and Rodrigues Filho E. 2007. Constituintes Químicos de Penicillium sp, um Fungo Endofítico Isolado de Murraya paniculata (Rutaceae). RECEN 9: 189-199.
• Marinho Amr,Marinho Psb and Rodrigues Filho E. 2009. Esteroides produzidos por Penicillium herquei, um fungo endofítico isolado dos frutos de Melia azedarach (Meliaceae). Quim Nova32: 1710-1712.
• Morita T andAoki H. 1974. Isosclerone, a new metabolite of Sclerotinia sclerotiorum (LIB.) De Bary. Agric Biol Chem 50: 997-1001.
• Prado S,Buisson D, Ndoye I, Vallet M and Nay B. 2013. One-step enantioselective synthesis of (4S)-isosclerone through biotranformation of juglone by an endophytic fungus. Tetrahedron Lett54: 1189-1191.
• Petrini O,Sieber TN, Toti L and Viret O. 1992. Ecology, metabolite production, and substrate utilization in endophytic fungi. Natural Toxins 1: 185-196.
• Pinto AC,Silva Dhs, Bolzani VS, Lopes NP and Epifanio RA. 2002. Produtos naturais: atualidade, desafios e perspectivas. Quim Nova25: 45-61.
• Strobel Gand Daisy B. 2003. Bioprospecting for microbial endophytes and their natural products. Microbiol Mol Biol Rev 67: 491-502.
• Strobel GA. 2002. Rainforest endophytes and bioactive products. Crit Rev Biotechnol 22: 315-333.
• Strobel GA,Daisy B, Castillo U and Harper J. 2004. Natural Products from Endophytic Microorganisms. J Nat Prod 67:257-268.
• Strobel GAand Long DM. 1998. Endophytic microbes embody pharmaceutical potential. ASM News 64: 263-268.
• Talapatra SK, Karmacharya B,De SC and Talapatra B. 1988. (-)-regiolone, an α-tetralone from Juglans regia: structure, stereochemistry and conformation.Phytochemistry 27: 3929-3932.
• Wei JG andXu T. 2004. Pestalotiopsis kunmingensis, sp. nov., an endophyte from Podocarpus macrophyllus. Fungal Divers 15: 247-254.
• Zhu Y,Dong J, Wang L, Zhou W, LiL, He H, Liu H and Zhang K. 2008. Screening and isolation of antinematodal metabolites against Bursaphelenchus xylophilus prodiced by fungi. Ann Microbiol 58: 375-380.

Publication Dates

• Publication in this collection
  05 Feb 2016
• Date of issue
  Mar 2016

History

• Received
  07 July 2014
• Accepted
  13 July 2015