Phytosterols isolated from endophytic fungus Colletotrichum gloeosporioides (Melanconiaceae)

Fotoesteróis isolados do fungo endofítico Colletotrichum gloeosporioides (Melanconiaceae)

ABSTRACT

Endophytic fungi are fungi that colonize internal tissues of plants. There are few studies of compounds isolated from endophytic fungi of Amazon plants. Thus, the aim this study was the isolation and structural identification of sitosterol (1), stigmasterol (2), sitostenone (3), squalene (4), ergosterol (5) and ergosterol peroxide (6) from fungus Colletotrichum gloeosporioidesisolated as endophytic from Virola michelli, a typical Amazon plant, used in folk medicine against skin infection. Compounds were isolated by chromatography column on silica and identified by ¹H and ¹³C NMR and MS. The presence of phytosterols in fungi is rare and this is the first report of the isolation of the phytosterols sitosterol, stigmasterol and sitostenone from the genus Colletotrichum.

KEYWORDS:
Sterols; secondary metabolites; Amazon fungi

RESUMO

Fungos endofíticos são fungos que colonizam os tecidos internos das plantas. Existem poucos estudos de compostos isolados de fungos endofíticos de plantas da Amazônia. Assim, o objetivo deste estudo foi o isolamento e identificação estrutural de sitosterol (1), estigmasterol (2), sitostenona (3), esqualeno (4), ergosterol (5) e peroxido de ergosterol (6) do fungo Colletotrichum gloeosporioidesisolado como endofítico de Virola michelli, uma planta típica da Amazônia, usada na medicina popular no combate a infecções de pele. Os compostos foram isolados por cromatografia em coluna de sílica e identificados por RMN ¹H e ¹³C e EM. A presença de fitoesteróis em fungos é rara e este é o primeiro relato do isolamento dos fitoesteróides sitosterol, estigmasterol e sitostenona do gêreno Colletotrichum.

PALAVRAS-CHAVE:
Esteróides; metabolitos secundários; fungos da Amazônia

INTRODUCTION

Endophytic fungi are those colonizing the internal tissue of plants without causing apparent damage (Petrini et al. 1992). The mechanism of interaction between the endophytic fungi and their host plants has still not been completely understood, mainly from the stand point of chemistry, since it has been known that they live in a neutral or even mutualistic interaction with host plants. Fungi are good producers of secondary metabolites, many with the same biological activity (Rosa et al. 2010; Chandra 2012; Pinheiro et al. 2013), where some compounds may help the host plant with combating infestation by other fungi, viruses and bacteria (Faeth and Saari 2012; Kusari et al.2013).

There are also reports of a possible "learning" known as "horizontal gene transfer" between fungus and plant, which would be the ability of organisms associated with producing the same class of compounds (Cook et al. 2014).

The phytosterols are broadly found in plants. The steroids sitosterol and stigmasterol are present in almost all plants; hence they are so called phytosterols. On the other hand, the steroids ergosterol and ergosterol peroxide are commonly found in fungi, occurrence of phytosterols in fungi is relatively unusual (Fontaine et al. 2004). The phytosterols, mainly sitosterol, have the capacity of reducing the blood cholesterol (Awad and Fink 2000), as well as phytosterols can be used to fight of cardiovascular diseases, cancer and inflammatory process (Fernandes and Cabral 2007; Woyengo et al. 2009; Poli and Marangoni 2010).

Brazil has about 20% of the world's biodiversity, especially in the Amazon forest, largest tropical and wet tropical forest, and invaluable source of raw materials in various sectors. Despite the huge Amazonian biodiversity, species that compose it and their relationships are not well known, much less its organisms and their interactions with other beings (Souza et al. 2004). The vegetal specie Virola michelli (Myristicaceae) is a typical Amazon plant known as "ucuúba" for local population and used in folk medicine as a plaster for irritation relief caused by fungi and to treatment of skin infections. Phytochemical and pharmacological studies have showed the anti-inflammatory potential of the crude extracts and pure compounds isolated from V. michelli (Santos et al. 1996; Santos et al. 2007). Thus, according Cook et al.(2014), there is a big probability to find compounds with useful biological activities produced by microorganisms.

In this context, we intend to contribute to the knowledge of diversity and biotechnological potential of Amazonian microorganisms; especially of endophytic fungi isolated from plant Virola michelli. Then, we describe the isolation and structural identification of sitosterol (1), stigmasterol (2), sitostenone (3), squalene (4), ergosterol (5) and ergosterol peroxide (6) from the endophytic fungus Colletotrichum gloeosporioides (Figure 1). The presence of phytosterols in fungi is rare and this is the first report of the isolation of phytosterols sitosterol, stigmasterol and sitostenone from the genus Colletotrichum.

Figure 1
Compounds isolated from Colletotrichum gloeosporioides. Sitosterol (1), stigmasterol (2), sitostenone (3), squalene (4), ergosterol (5) and ergosterol peroxide (6)

MATERIAL AND METHODS

General procedures

The ¹H and ¹³C NMR experiments were recorded on a NMR spectrometer (Mercury 300, Varian, Oxford, Oxfordshire, UK) with CDCl₃ as solvent and standard. The MS spectra were carried out in the mass spectrometer using ESI (+) ion mode (Acquity TQD, Waters, Milford, MA, USA).

Microorganism

The fungus C. gloeosporioides (Arx, 1957; Melanconiaceae) was obtained from a collection of the "Laboratorio de Bioensaios e Química de Micro-organismos - LaBQuiM / UFPA". This collection contains isolates from V. michelli. The fungus was inoculated into a Petri dish containing PDA culture medium (Potato, Dextrose, and Agar) and incubated at BOD 25 °C for eight days to reactivation. One strain is deposited as a code CGGVM05.

Cultivation of C. gloeosporioides CGGVM05 in rice and isolation of the compounds

Twenty Erlenmeyer flasks (1000 mL) containing 200 g of rice ("Uncle's Been(r)") and 75 mL of water per flasks were autoclaved for 45 min at 121 °C. Small pieces of PDA containing mycelium of C. gloeosporioides were added to 18 Erlenmeyer flasks under sterile conditions, then the Erlenmeyer flasks were incubated at 25 °C for 30 days for colony growth, two Erlenmeyer flaks were used as control. Biomass was macerated with hexane, ethyl acetate and methanol, in which the hexane (5.44 g), ethyl acetate (27.09 g) and MeOH (26.70 g) extracts were obtained after evaporation in rotary evaporator of resulting solutions. The hexane extract was fractionated on silica column using a mixture of hexane, ethyl acetate and methanol, in order increasing polarity, as eluent. The resulting fractions were successively chromatographed on silica gel CC by using hexane, ethyl acetate and methanol as mobile phase in a gradient of polarity and monitored by TLC. Following chemical constituents were isolated sitosterol + stigmasterol (1+2, 18.3 mg), sitostenone (3, 5.9 mg), squalene (4, 23.9 mg), ergosterol (5, 462.2 mg) and ergosterol peroxide (6, 113.9 mg).

RESULTS

The chemical study of the fungus C. gloeosporioides CGGMV05 lead the isolation of sitosterol (1), stigmasterol (2), sitostenone (3), squalene (4), ergosterol (5) and ergosterol peroxide (6). This is the first report of the isolation of the phytosterols sitosterol, stigmasterol and sitostenone from the genus Colletotrichum.

DISCUSSION

The phytosterols are widely spread in the plant kingdom. Normally, they are obtained in mixture of hard separation, because of showing similarity in physical proprieties. The most common are sitosterol and stigmasterol. Mixture containing stigmasterol are identified in ¹H NMR spectrum by signals referents to the vinyl hydrogens (H-22 and H-23) that show up as two doublets between δ_H 5.00 and δ_H 5.20. In all cases H-6 show up as a broad doublet in the ¹H NMR spectrum at δ_H 5.35 and H-3 hydrogen show as multiple around δ_H 3.50. The phytosterols can be free, glycosylated or esterified.

The mixture of compounds 1 + 2 was analyzed by mass spectrometry ESI (+) and peaks m/z 415 [M+H]⁺ and m/z 413 [M+H]⁺ were observed, allowing propose the molecular formulas C₂₉H₅₀O to 1 and C₂₉H₄₈O to 2. Analysis of the ¹H NMR spectrum of the fraction studied showed that it had a mixture of two compounds, coded 1 and 2. The spectrum indicated signals characteristic to sterols, such as olefinic hydrogens δ_H 5.35 (d, J = 6.0 Hz, H-6) and carbinolic hydrogen δ_H3.50 (m, H-3), including the accumulation of signals in the region between δ_H 0.60 and δ_H 2.40 referent to many groups of hydrogens such as methyl, methylene and methine, which characterize the sitosterol (1). The double doubet at δ_H 5.13 (J = 15.5 and 8.0 Hz, H-22) and δ_H 5.01 (J = 15.5 and 8.0 Hz, H-23) are referent to the olefinic hydrogens of double bond with stereochemistry trans in the side chain of a stigmastane. These double doublet combined to others signals discussed to sitoterol characterize the compound stigmasterol (2). Through the strength of the signals relating to the olefinic protons H-22, H-23 and H-6 was also observed that sitosterol is in a higher proportion in the mixture. The ¹³C NMR spectrum of the mixture of 1 and 2 showed characteristic signs of sitosterol and stigmasterol δ_C 121.7 (C-6); δ_C 140.6 (C-5); δ_C129.3 (C-23) and δ_C 138.3 (C-22) confirming identification (Goulart et al. 1993).

The compound 3 was analyzed by mass spectrometry ESI (+) and peak m/z 413 [M+H]⁺ was observed, allowing propose the molecular formula C₂₉H₄₈O to 3. The ¹H NMR spectrum of compound 3 showed the same pattern signals for 1 and 2 with the exception of the signal at δ_H 3.50 referring to the carbinolic hydrogen H-3. In the ¹³C NMR spectrum was observed an additional signal at δ_C199.3 (C-3) referent to carbonilic carbon, thus the compound 3 was identified as sitostenone (Prachayasittikul et al.2009).

The compound 4 was isolated from the nonpolar fractions of extract in the form of oil. In its ¹H NMR spectrum verified the presence of olefinic hydrogens in the region between δ_H 5.07 and δ_H 5.14. Signals assigned to methylene hydrogens neighboring double bonds were observed at δ_H 2.00. In δ_H 1.60 and δ_H 1.68 are signs commonly attributed to hydrogens of methyl attached to carbon sp2. In the ¹³C NMR spectrum showed six signs related to olefinic carbons (δ_C 135.1; 134.9; 131.2; 124.4; 124.3; 124.2) signals for six methylene carbons (δ_C39.7 2x ; 29.7; 28.3; 26.7 and 26.6) and four signals related to methyl carbons (δ_C 25.7; 17.7; 16.0; 15.9). A comparison of the NMR data of the compound 4 with the literature enabled us to identify as squalene (Barreto et al.2013).

The compounds 5 and 6 are commonly isolated from fungi and were identified through analysis of ¹H and ¹³C NMR spectra and compared to the literature data and presented total similarity to ergosterol and ergosterol peroxide (Marinho et al. 2009).

To confirm that the phytosterols were produced by the fungus C. gloeosporioides the control extract (only the rice culture medium) was analyzed by direct-infusion electrospray mass spectrometry in positive ion mode ESI(+), where the spectrum obtained showed the total absence of peaks for phytosterols.

Phytosterols have presented important biological activities are broadly found in plants. Although isolation of phytosterols in fungi to be relatively unusual, some studies have demonstrated the isolation of these compounds from some fungi species (Ling et al.2007; Yan et al. 2010; Tarawneh et al. 2013) which opens up new possibilities to the isolation and obtaining this important class of compounds. This is the first report of the isolation of phytosterols sitosterol, stigmasterol and sitostenone from the genus Colletotrichum. The isolation of phytosterols from the fungus C. gloeosporioidescorroborates with the theory of transfer of skills between endophytic fungus and host plant for the production of secondary metabolites, also contribute to knowledge secondary metabolites from Amazon fungi.

ACKNOWLEDGEMENTS

The authors thank the Fundação Amazônia de Amparo a Estudos e Pesquisas do Pará (FAPESPA), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Vale S/A and Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) for the financial support.

REFERENCES

• Awad, A. B.; Fink, C. S. 2000. Phytosterols as anticancer dietary components: evidence and mechanism of action. The Journal of Nutrition, 130: 2127-2130.
• Barreto, M.B.; Gomes, C.L.; Freitas, J.V.B.; Pinto, F.C.L.; Silveira, E.R.; Gramosa, N.V.; Torres, D.S.C. 2013. Flavonoides e terpenoides de Croton muscicarpa (Euphorbiaceae). Química Nova, 36: 675-679.
• Chandra, S. 2012. Endophytic fungi: novel sources of anticancer lead molecules. Applied Microbiology and Biotechnology, 95: 47-59.
• Cook, D.; Gardner, D.R.; Pfister, J.A.; Grum, D. 2014. Biosynthesis of natural products in plants by fungal endophytes with an emphasis on Swainsonine. In: Jetter, R. (Ed.). Phytochemicals - biosynthesis, function and application,recent advances in phytochemistry. v.44. Springer International Publishing, USA, p. 23-41.
• Faeth, S.H.; Saari, S. 2012. Fungal grass endophytes and arthropod communities: lessons from plant defence theory and multitrophic interactions. Fungal Ecology, 5: 364-371.
• Fernandes, F; Cabral, J.M.S. 2007. Phytosterols: Applications and recovery methods. Bioresource Technology, 98: 2335-2350.
• Fontaine, J.; Grandmougin-Ferjani, A.; Glorian, V.; Durand, R. 2004. 24-Methyl/methylene sterols increase in monoxenic roots after colonization by arbuscular mycorrhizal fungi. New Phytologist, 163: 159-167.
• Goulart, M.O.F.; Sant`Ana, A.E.G.; Lima, R.A.; Cavalcante, S.H.; Carvalho, M.G.; Braz-Filho, R. 1993. Fitoconstituintes químicos isolados de Jatropha elliptica Atribuição dos deslocamentos químicos dos átomos de carbono e hidrogênio dos diterpenos jatrofolonas A e B. Química Nova, 16: 95-100.
• Kusari, P.; Kusari, S.; Spiteller, M.; Kayser, O. 2013. Endophytic fungi harbored in Cannabis sativa L.: diversity and potential as biocontrol agents against host plant-specific phytopathogens. Fungal Diversity, 60: 137-151.
• Ling, Q.; Bogang, L.; Jiafa, G.; Guolin, Z. 2007. Chemical study on Aspergillus sp 136. Journal of Applied and Environmental Biology, 13: 66-68.
• Marinho, A.M.R.; Marinho, P.S.B.; Rodrigues-Filho, E. 2009. Esteroides produzidos por Penicillium herquei, um fungo endofítico isolado dos frutos de Melia azedarach (Meliaceae). Quimica Nova, 32: 1710-1712.
• Petrini, O.; Sieber, T.N.; Toti, L.; Viret, O. 1992. Ecology, metabolite production, and substrate utilization in endophytic fungi. Natural Toxis, 1: 185-196.
• Pinheiro, E.A.A.; Carvalho, J.M.; Santos, D.C.P.; Feitosa, A.O.; Marinho, P.S.B.; Guilhon, G.M.S.P.; Santos, L.S.; Souza, A.L.D.; Marinho, A.M.R. 2013. Chemical constituents of Aspergillus sp EJC08 isolated as endophyte from Bauhinia guianensis and their antimicrobial activity. Anais da Academia Brasileira de Ciências, 85: 1247-1252.
• Poli, A; Marangoni, F. 2010. Pythosterols and cardiovascular health. Pharmacological Research, 61: 193-199.
• Prachayasittikul, S.; Suphapong, S.; Worachartcheewan, A.; Lawung, R.; Ruchirawat, S.; Prachayasittikul, V. 2009. Bioactive metabolites from Spilanthes acmella Murr. Molecules, 14: 850-867.
• Rosa, L.H.; Gonçalves, V.N.; Caligiorne, R.B.; Alves, T.M.A.; Rabello, A.; Sales, P.A.; Romanha, A.J.; Sobral, M.E.G.; Rosa, C.A.; Zani, C.L. 2010. Leishmanicidal, trypanocidal, and cytotoxic activities of endophytic fungi associated with bioactive plants in Brazil. Brazilian Journal of Microbiology, 41: 420-430.
• Santos, L.S.; Andrade, M.A.; Corrêa, M.J.C.; Campos, L.M.O. 1996. Constituents from the leaves of Virola michellii Fitoterapia, 67: 55- 556.
• Santos, L.S.; Borges, F.C.; Oliveira, M.N.; Souza-Filho, A.P.S.; Guilhon, G.M.S.P.; Arruda, M.S.P.; Muller, A.H.; Santos, A.S.; Arruda, A.C. 2007. Allelochemicals isolated from the leaves of Virola michelli Heckel. Allelopathy Journal 20: 235-243.
• Souza, A.Q.L.; Souza, A.D.L.; Astolfi-Filho, S.; Pinheiro, M.L.B.; Sarquis, M.I.M.; Pereira, J.O. 2004. Atividade antimicrobiana de fungos endofíticos isolados de plantas tóxicas da Amazônia: Palicourea longiflora (aubl.) rich e Strychnos cogens bentham Acta Amazonica, 34: 185-195.
• Tarawneh, A.H.; León, F.; Radwan, M.M.; Rosa, L.H.; Cutler, S.J. 2013. Secondary metabolites from the fungus Emericella nidulansNatural Products Communication, 8: 1285-1288.
• Woyengo, T.A.; Ramprasath, V.R.; Jones, P.J.H. 2009. Anticancer effects of phytosterols. European Journal of Clinical Nutrition, 63: 813-820
• Yan, H.; Gao, S.; Li, C.; Li, X.; Wang, B. 2010. Chemical constituents of a marine-derived endophytic fungus Penicillium commune G2M. Molecules, 15: 3270-3275.

Publication Dates

• Publication in this collection
  Mar 2016

History

• Received
  11 Jan 2015
• Accepted
  22 June 2015