Paepalanthus spp: antimycobacterial activity of extracts, methoxylated flavonoids and naphthopyranone fractions

Paepalanthus spp: antimycobacterial activity of extracts, methoxylated flavonoids and naphthopyranone fractions

Raquel R. D. Moreira^I; Gilmarcio Z. Martins^I; Rosemeire C. L. R. Pietro^I; Daisy N. Sato^I; Fernando R. Pavan^I; Sergio R.A. Leite^II; Wagner Vilegas^II; Clarice Q. F. Leite^I

^IFaculdade de Ciências Farmacêuticas, Universidade do Estado de São Paulo, Brazil

^IIInstituto de Química, Universidade do estado de São Paulo, Brazil

^{Correspondence}

ABSTRACT

Paepalanthus spp., Eriocaulaceae, are native plants from Brazil known as "sempre-vivas" (everlasting flowers). In this work, we evaluated the potential anti-mycobacterial activity of two methoxylated flavonoids (flavonoid 7-methylquercetagetin and 7-methylquercetagetin-4'-O-β-D-glucopyranoside) isolated and identified from P. latipes and the naphthopyranone fractions from P. bromeliodes ethanolic extracts. The MIC value of 500 µg/mL was verified for all compounds tested against M. tuberculosis H37Rv. For M. avium, the MIC value ranged from 1000-2000 µg/mL excepting to naphthopyranone fractions with MIC of 500 µg/mL. This is the first report of activity determination of Paepalanthus spp. flavonoids activity against Mycobacterium tuberculosis and M. avium.

Keywords: flavonoids, Mycobacterium, naphthopyranone, Paepalanthus spp

Introduction

Tuberculosis still remains an important public health problem worldwide, accounting for eight million new cases per year (WHO, 2007). The agent of tuberculosis, Mycobacterium tuberculosis kills nearly three million people a year throughout the world. Despite improvements in chemotherapy, tuberculosis is severely affected by the development of multi-resistant strains to drugs that are commonly used to treat the disease (WHO, 2007). The treatment of infections caused by other opportunistic environmental Mycobacterium species as M. avium is difficult, due theirs intrinsic resistance to a wide range of medications (Nuermberger & Grosset, 2004). Most Mycobacterium species are resistant to isonicotinic acid hydrazide (isoniazid), one of the most used therapeutic agents for the treatment of tuberculosis (Chung et al., 2006; Zhang et al., 1992). These challenges have motivated the research of novel antimycobacterial agents to reduce the global burden of tuberculosis and have been discussed in the current biomedical literature (Wilkinson et al., 2007; Pavan et al., 2009; Higuchi et al., 2011, Ma et al., 2010).

Natural products and/or their semi-synthetic derivatives can lead to novel antimycobacterial drugs and may have important role in the chemotherapy of tuberculosis. The literature reports the antimycobacterial activity of many classes of natural products such as alkanes, phenolics, acetogenic quinones, flavonoids, and triterpenes (Pauli et al., 2005; Copp & Pearce, 2007).

Eriocaulaceae is a pantropical, predominantly herbaceous monocotyledonous family, comprising around 1400 species in ten genera (Giulietti et al., 2012). Plants of this family are known as "everlasting flower" ("sempre-vivas" in Portuguese, since they maintain their appearance and color after drying) and they can be found in "Campos Rupestres" vegetation in Brazil. These plants are exported to Europe, Japan and North America as an ornamental flower, representing an important source of income to the population of Minas Gerais State, Brazil (Giulietti et al., 2012).

Taxonomic studies to delimit the genus, about which there is still some confusion, and the biological investigation into the isolated molecules of Eriocaulaceae, are of great importance, because several molecules possess antioxidant actions (Santos et al., 2003), citotoxic and mutagenic activities (Moreira et al., 2002), macrophage activation (Moreira et al., 2001).

This study aimed to evaluate the antimycobacterial activity against M. tuberculosis and M. avium of the two flavonoids (6-hydroxy-7-methoxyquercetin (1) and 6-hydroxy-7-methoxyquercetin-4'-O-β-D-glucopyranoside (2) from 96% crude ethanolic extract from P. latipes and the naphthopyranone fractions obtained from P. bromeliodes.

Material and Methods

Plant material

Paepaplanthus spp., Eriocaulaceae, were collected at Serra do Cipó, Minas Gerais State, Brazil, and authenticated by Prof. Paulo Takeo Sano. Voucher specimens of Paepalanthus latipes Silveira (SPF/CFSC 13846) and P. bromelioides Silveira (SPF/CFSC 13856), were deposited at the Herbarium of the Institute of Biosciences, University of São Paulo, Brazil.

Preparation and fractionation of extracts and isolation of compounds

The aerial parts of the P. latipes and P. bromeliodes were dried in an oven with air circulation, at 60 ºC for seven days, and then powdered. The dry powders were macerated (528 g of P. latipes and 500 g of P. bromeliodes) for one week, at room temperature, with 96% ethanol. The macerates were filtered and evaporated at 60 ºC under reduced pressure, resulting in 11 g of the dry extract of P. latipes and 8 g of P. bromelioides. The extracts were fractioned by gel permeation column Sephadex LH-20 (Pharmacia) and 96% methanol as eluent, resulting in 110 fractions for P. latipes and 150 fractions for P. bromelioides. Naphthiopyranone fractions were identified by thin layer chromatography (mobile phase: CHCl₃:MeOH:H₂O, 43:37:20 v/v/v, developer: NP/PEG and viewing under UV) (Vilegas et al., 1990). The compounds 6-hydroxy-7-methoxyquercetin (1) and 6-hydroxy-7-methoxyquercetin-4'-O-β-D-glucopyranoside (2) were identified by spectroscopic methods (IR, UV, ES-MS, NMR 1D and 2D) (Nehme, 1997).

Mycobacterial strains

Mycobacterium tuberculosis H37Rv ATCC 27294 and Mycobacterium avium ATCC 15769 were obtained from the American Type Culture Collection (Manassas, VA, USA). The mycobacteria were cultured in Middlebrook 7H9 supplemented with 10% OADC and additional 0.05% Tween 80 (to avoid clumps), at 37 ºC for 7-10 or 3-5 days, respectively for M. tuberculosis and M. avium.

Antimycobacterial activity-Determination of in vitro Minimum Inhibitory Concentration (MIC)

The antimycobacterial activity was assessed using the Alamar Blue^TM (MABA) method (Collins & Franzblau, 1997). Microplates of 96 wells were used. The wells were filled with different concentrations of extracts and compounds in dimethyl sulfoxide (DMSO), which varied from 4000 to 250 µg/mL. Isoniazid (Difco, Detroit, MI, USA) was the antimycobacterial drug standard, at concentrations of 0.015-0.50 µg/mL. The tests were performed in triplicate. According to this technique, the MIC of a compound is the concentration that inhibit 90% of the growth of M. tuberculosis H₃₇Rv, using Alamar Blue^TM (resazurin) as a vital fluorescent dye. The bacterial suspensions were prepared at a concentration equivalent to the turbidity standard of McFarland nº 1 and diluted in the ratio 1:25 in Middlebrook 7H9 Broth. The inoculum was supplemented with 10% OADC. Cultures were incubated for seven days at 37 ºC and then added Alamar Blue for reading. A negative control was performed without mycobacteria to confirm the absence of reaction of the tested compounds with the resazurin. The dye fluorescence (indicative of bacterial growth) was measured in a microfluorimeter (Tecan SPECTRAfluor Plus microplate reader), with excitation at 530 nm and emission at 590 nm (Collins & Franzblau, 1997).

Results

The fractionation and rechromatographed of crude ethanolic extracts in silica gel CC resulted in isolation of two methoxylated flavonoids previously isolated from P. latipes (Villegas et al., 1999) and naphthopyranone fraction from P. bromeliodes.

The NMR studies showed that both flavonoids have a methoxy group at position 7, and a hydroxyl group at position 6 which does not occur in quercetin, luteolin and 3-methylquercetin. Besides, this the flavonoid 7-methylquercetagetin (1) presents two free hydroxyl at C3' and C4', analogously to the three flavonoids mentioned. The 7-methylquercetagetin (1) was identified in fractions 90-102 (9.0 mg) and 7-methylquercetagetin-4'-O-β-D-glucopyranoside (2) was identified in fractions 69-78 (10.0 mg), as well as in the crude ethanolic extract from P. latipes. The results obtained from the ES-MS (70 V, positive mode) confirmed the identification of the flavonoids 1 and 2, by comparison with the results of Nehme (1997), which also support our NMR data. The naphthopyranones (paepalantine-9-O-β-D-glycopyranosyl-(1-6)-β-D-glycopyranoside (3), paepalantine-9-O-β-D-allopyranosyl-(1-6)-β-D-glycopyranoside (4)) were identified in fractions 25-35 (6.5 mg) of the P. bromeliodes crude ethanolic extract by thin layer chromatography (R_f 0.40 and 0.45) and the results were confirmed using benchmarks according Vilegas et al (1990).

The antimycobacterial activities of the extracts, fractions and flavonoids from Paepalanthus spp. and P. latipes against M. tuberculosis and M. avium, using the Alamar Blue assay (MABA), are showed in Table 1. The ethanol extracts and isolated compounds tested against M. tuberculosis H₃₇Rv showed low activity, with MIC value of 500 µg/mL. M. avium was even more resistant to the ethanol extracts and compounds (MIC ranging on 1000-2000 µg/mL), excepting to naphthopyranone fraction, whose MIC was 500 µg/mL.

Discussion

Many natural products, including essential oils and phenolic compounds have been extensively tested for antibacterial activity (Friedman et al., 2002; 2003; 2004; 2006).

In this study, the ethanol extract and methoxylated flavonoids (flavonoid 7-methylquercetagetin and 7-methylquercetagetin-4'-O-β-D-glucopyranoside) from P. latipes, and naphthopyranone fractions isolated from P. bromeliodes ethanolic extract, tested against M. tuberculosis and M. avium, exhibited no significant antimycobacterial activity, despite their characteristic of polyphenolic compounds. The MIC values ranged from 500 µg/mL against M. tuberculosis to 1000-2000 µg/mL to M. avium, except for naphthopyranone, that presented the same 500 µg/mL MIC against both bacteria.

Tosun et al. (2004) considered inactive the plant extracts that could not prevent growth of M. tuberculosis up to concentration of 200 µg/mL. According to Gu et al. (2004), a MIC<128 mg/mL defines a substance as active against M. tuberculosis (Gu et al., 2004). For Cantrell et al. (2001), isolated compounds that exhibit a MIC of 64 µg/mL or lower are considered promising (Cantrell et al., 2001).

Thus, the high MIC value of 500 µg/mL, disqualifies the isolated flavonoids compounds or purified naphthopyranone fractions as promising candidates against M. tuberculosis and M. avium. The antimycobacterial activity of flavonoids has already been reported by many authors. Lin et al. (2002) described the antimycobacterial activity of 142 synthetic chalcones, flavonoids (including some methoxyflavones) and chalconeslike compounds. Murillo et al. (2003) analyzed fourteen flavonoids from Derris indica, including rotenoids and prenylated isoflavones (Murillo et al., 2003). The antimycobacterial methoxyflavones from Kaempferia parviflora was also report by Gu et al. (2004). However, according to Castellar et al (2011), in all researches, no correlation on structure-activity of flavonoids against mycobacteria could be drawn. In the opinion of Silva et al. (2009), high lipophilicity of a substance is an important feature for its antimycobacterial activity.

Due to the fact that the cell wall of mycobacteria contain high amount of lipids, such as mycolic acid, lipophilic substances are likely to penetrate more easily into the cell (Palomino et al., 2002). It has been demonstrated for a series of terpenes that the activity improves with the lipophilicity of a given substance, when compared to their more polar analogues (Cantrell et al., 2001). Pavan et al. (2009), from 37 plant extracts analyzed, found sixteen with promising activity, twelve of which were chloroform extracts and only four methanol extracts. The chloroform as extractor agent, could extracts more apolar compounds and the lipophilic nature of those compounds allows them to more easily penetrate the mycobacterial lipid rich cell wall. This probably favors their antimycobacterial activity.

In previous reports, we have described the isolation and structure determination of paepalantine, a naphthopyran-1-one isolated from the chloroform extracts of the capitulae of Paepalanthus bromelioides, as well as from Paepalanthus vellozioides, with potent antibiotic effect against S. aureus, S. epidermidis, Enterococcus faecalis and Escherichia coli (Coelho et al., 2000; Kitagawa et al., 2003; Devienne et al., 2005) and also antiviral activities (Cotterill et al., 2003).

In another study, we verified that ethanol extracts of P. bromeliodes and P. latipes were able to induce innate immune response through the production of high levels of H₂O₂ by mouse peritoneal macrophages (Moreira et al., 2001). Since M. tuberculosis can survive inside macrophages, their activation is essential for tuberculosis control. Thus, it is possible that the antimicrobial activity of the tested products, poorly in vitro, may be higher in vivo depending on macrophage activation.

Conclusion

This is the first report of antimycobacterial activity of Paepalanthus spp. flavonoids. The results showed that the ethanol extracts, methoxylated flavonoids and naphthopyranone fractions exhibited no significant activity against M. tuberculosis and M. avium. Further, biological studies are needed to verify whether the antituberculosis activity in vivo is more intense, since the alcoholic extracts of the plant species studied were shown to be capable of producing macrophage activation.

Acknowledgements

We are thankful to Mr. Nivaldo from the Nuclear Magnetic Resonance Laboratory of the Institute of Chemistry, UNESP, Araraquara-SP, Brazil, for helping in RMN analysis. Fundação de Amparo à Pesquisa do Estado de São Paulo, ref. Processes: 2008/10390-2 and 2011/11593-7 and CNPq for financial support.

Received 5 Oct 2012

Accepted 13 Oct 2012

• Cantrell CL, Franzblau SG, Fischer NH 2001. Antimycobacterial plant terpenoids. Planta Med 67: 685-694.
• Castellar A, Coelho TS, Silva PEA, Ramos DF, Lourenço MCS, Lage CLS, Julião LS, Barbosa YG, Leitão SG 2011. The activity of flavones and oleanolic acid from Lippia lacunosa against susceptible and resistant Mycobacterium tuberculosis strains. Rev Bras Farmacogn 21: 835-840.
• Chung MJ, Lee KS, Koh WJ, Kim TS, Kang EY, Kim SM, Kwon OJ, Kim S 2006. Drug-sensitive tuberculosis, multidrug-resistant tuberculosis and nontuberculous mycobacterial pulmonary disease in non AIDS adults: comparisons of thin-section CT findings. Eur Radiol 16: 1934-1931.
• Coelho RG, Vilegas W, Devienne KF, Raddi MS. 2000. A new cytotoxic naphthopyrone dimer from Paepalanthus bromelioides. Fitoterapia 71: 497-500.
• Collins LA, Franzblau SG 1997. Microplate Alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Ch 41: 1004-1009.
• Copp BR, Pearce NA 2007. Natural product growth inhibitors of Mycobacterium tuberculosis. Nat Prod Rep 24: 278-297.
• Cotterill AS, Donner CD, Gill M, White JM 2003. Pigments of fungi. Part 70. Total synthesis of (R)-semixanthomegnin and the X-ray crystal structure of (+/-)-7-chloro-10-methoxy-3-methyl-3, 4-dihydro-1H-naphtho 2,3-c pyran-1,6,9-trione. Aust J Chem 56: 49-57.
• Devienne KF, Raddi G, Coelho RG, Vilegas W. 2005. Structure-antimicrobial activity of some natural isocoumarins and their analogues. Phytomedicine 12: 378-381.
• Friedman M, Buick R, Elliott CT 2004. Antibacterial activities of naturally occurring compounds against antibiotic-resistant Bacillus cereus vegetative cells and spores, Escherichia coli, and Staphylococcus aureus. J Food Protect 67: 1774-1778.
• Friedman M, Henika PR, Levin CE, Mandrell RE, Kozukue N. 2006. Antimicrobial activities of tea catechins and theaflavins and tea extracts against Bacillus cereus. J Food Protect 69: 354-361.
• Friedman M, Henika PR, Mandrell RE 2002. Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, and Salmonella enterica. J Food Protect 65: 1545-1560.
• Friedman M, Henika PR, Mandrell RE. 2003. Antibacterial activities of phenolic benzaldehydes and benzoic acids against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, and Salmonella enterica. J Food Protect 66: 1811-1821.
• Giulietti AM, Andrade MJG, Scatena VL, Trovó M, Coan A I, Sano PT, Santos F AR, Borges R LB, van den Berg C 2012. Molecular phylogeny, morphology and their implications for the taxonomy of Eriocaulaceae. Filogenia molecular, morfologia e suas implicações para a taxonomia de Eriocaulaceae. Rodriguésia 63: 1-19.
• Gu JQ, Wang Y, Franzblau SG, Montenegro G, Yang D, Timmermann BN 2004. Antitubercular constituents of Valeriana laxiflora. Planta Med 70: 509-514.
• Higuchi CT, Sannomiya M, Pavan FR, Leite SRA, Sato DN, Franzblau SG, Sacramento LVS, Vilegas W, Leite CQF 2008. Byrsonima fagifolia Niedenzu apolar compounds with antitubercular activity. eCAM, doi:10.1093/ecam/nen077.
• Kitagawa RR, Raddi MS, Khalil NM, Vilegas W, da Fonseca LM. 2003. Effect of the isocoumarin paepalantine on the luminol and lucigenin amplified chemiluminescence of rat neutrophils. Biol Pharm Bull 26: 905-908.
• Lin YM, Zhou Y, Flavin MT, Zhou LM, Nie W, Chen FC. 2002. Chalcones and flavonoids as anti-tuberculosis agents. Bioorg Med Chem 10: 2795-2802.
• Ma Z, Lienhardt C, McIlleron H, Nunn AJ, Wang X. 2010. Global tuberculosis drug development pipeline: the need and the reality. Lancet 375: 2100-2109.
• Moreira R, Carlos I, Vilegas W 2001. Macrophage activation by Paepalanthus spp. Euriocaulaceae. Rev Bras Farmacogn 9: 37-42.
• Moreira RRD, Santos LE, Varella SD, Varanda EA, Vilegas W 2002. Avaliação da atividade mutagênica do extrato etanólico bruto de Paepalanthus latipes (Eriocaulaceae) e dos compostos flavonoídicos 7-metoxilados relacionados. Rev Bras Farmacogn 12: 11-19.
• Murillo JI, Encarnación-Dimayuga R, Malmstrøm J, Christophersen C, Franzblau SG. 2003. Antimycobacterial flavones from Haplopappus sonorensis. Fitoterapia 74: 226-230.
• Nehme C 1997 Estudo químico de plantas da família Eriocaulaceae-Escapos. P. bromelioides e P. latipes. (Master). Natural Products, São Paulo State University, Faculty of Pharmaceutical Sciences.
• Nuermberger E, Grosset J 2004. Pharmacokinetic and pharmacodynamic issues in the treatment of mycobacterial infections. Eur J Clin Microbiol 23: 243-255.
• Palomino JC, Martin A, Camacho M, Portaels F 2002. Resazurin microtiter assay plate: Simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Ch 46: 2720-2722.
• Pauli GF, Case RJ, Inui T, Wang Y, Cho S, Fischer NH, Franzblau SG 2005. New perspectives on natural products in TB drug research. Life Sci 78: 485-494.
• Pavan FR, Sato DN, Higuchi CT, Santos ACB, Vilegas W, Leite CQE 2009. In vitro anti-Mycobacterium tuberculosis activity of some Brazilian "Cerrado" plants. Rev Bras Farmacogn 19: 204-206.
• Santos LC, Piacente S, Montoro P, Pizza C, Vilegas W 2003. Atividade antioxidante de xantonas isoladas de espécies de Leiothrix (Euriocaulaceae). Rev Bras Farmacogn 13: 67-74.
• Silva ML, Martins CH, Lucarini R, Sato DN, Pavanb FR, Freitas NH, Andrade LN, Pereira AC, Bianco TN, Vinholis AH, Cunha WR, Bastos JK, Silva R, Da Silva Filho AA 2009. Antimycobacterial Activity of Natural and Semi-Synthetic Lignans. Z Naturforsch C 64: 779-784.
• Tosun F, Kizilay CA, Sener B, Vural M, Palittapongarnpim P 2004. Antimycobacterial screening of some Turkish plants. J Ethnopharmacol 95: 273-275.
• Vilegas W, Roque NF, Salatino A, Giesbrecht AM, Davino S 1990. Isocoumarin from Paepalanthus bromelioides. Phytochemistry 29: 2299-2301.
• World Health Organization 2007. Public private mix for TB care and control. Geneva: WHO. (WHO/HTM/TB/2007.378).
• Wilkinson BL, Bornaghi LF, Wright AD, Houston TA, Poulsen SA 2007. Anti-mycobacterial activity of a bis-sulfonamide. Bioorg Med Chem Lett 17: 1355-1357.
• Zhang Y, Heym B, Allem B, Young D, Cole ST 1992. The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature 358: 591-593.

Publication Dates

• Publication in this collection
  22 Nov 2012
• Date of issue
  Apr 2013

History

• Received
  05 Oct 2012
• Accepted
  13 Oct 2012