The activity of flavones and oleanolic acid from Lippia lacunosa against susceptible and resistant Mycobacterium tuberculosis strains

The activity of flavones and oleanolic acid from Lippia lacunosa against susceptible and resistant Mycobacterium tuberculosis strains

Aline Castellar^I; Tatiane S. Coelho^II; Pedro E. A. Silva^II; Daniela F. Ramos^II; Maria Cristina S. Lourenço^III; Celso L. S. Lage^I; Lisieux S. Julião^I; Ymira G. Barbosa^IV; Suzana G. Leitão^IV,*

^IPrograma de Biotecnologia Vegetal, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Brazil

^IILaboratório de Micobactérias, Fundação Universidade Federal do Rio Grande, Brazil

^IIILaboratório de Bacteriologia e Bioensaios em Micobactérias, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz, Brazil

^IVDepartamento de Produtos Naturais e Alimentos, Faculdade de Farmácia, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Brazil

ABSTRACT

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the world's number one killer among infectious diseases. The search for new naturalproducts that can act as drugs against TB has received increased attention duringthe last years. In this work we describe the isolation and identification of the active antimycobacterial principles of the dichloromethane extract from Lippia lacunosa Mart. & Schauer, Verbenaceae. Compounds were evaluated for their in vitro activity against Mycobacterium tuberculosis (susceptible and rifampicin resistantstrain) using a redox bioassay. From the dichloromethane extract of L. lacunosa leaves, seven methoxy-flavones named cirsimaritin (1), eupatilin (2), eupatorin (3), salvigenin (4), 3'-O-methyl-eupatorin (5), 3',7-dimethoxy-5,6,4'- trihydroxyflavone (6), and 7'-O-methylapigenin (7), and one triterpene, named oleanolic acid (8), were isolated. All compounds were found to display antimycobacterial activity against susceptible strain, with MIC ranging from 25 to 200 µg/mL. None of them was active against rifampicin resistant strain. This is the first report in the antimycobacterial activity of 6-substituted flavones, as well as the first report of the occurrence of these substances in L. lacunosa.

Keywords:Lippia lacunosa, Verbenaceae, flavonoids, antimycobacterial activity, Mycobacterium tuberculosis, methoxyflavones, oleanolic acid.

Introduction

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the world's number one killer among infectious diseases (Lin et al., 2002). Each year, about 400,000 people develop TB with isoniazid- and rifampicin-resistant strains (MDR) (Silva et al., 2008). The emergence of MDR strains with human immunodeficiency virus (HIV) co-infection, as well as the decline of socioeconomic conditions in several places around the world, has amplified the difficulty in controlling this disease (Lin et al., 2002; Lechner et al., 2008).

The search for new drugs against TB has received increased attention during the last couple of years. Several candidates are being investigated and an interesting array of compounds constitutes the current portfolio of promising drugs. These drugs should be active against both susceptible and resistant strains (Silva & Ainsa, 2007).

In the last couple of years, several reports and review articles have appeared in the literature about medicinal plants and natural products with antimycobacterium activity (Okunade et al., 2004; Copp, 2003; Newton et al., 2002; Cantrell et al., 2001). Over 350 natural products, mainly from plant species, have been assessed for their antimycobacterial activities (Newton et al., 2002). A number have demonstrated significant in vitro activity and active plant-derived compounds belonging to various chemical classes have been isolated (Newton et al., 2002).

The genus Lippia (Verbenaceae) includes approximately 200 species and is found in Central and South America, as well as in some areas of Tropical Africa (Salimena-Pires, 1991). One of the main diversity centers of this genus is located in the central region of Brazil, mainly in "cerrado" biome, at the "Cadeia do Espinhaço" Mountains, in the State of Minas Gerais (Leitão et al., 2008). Some species, such as Lippia alba (Mill.) N. E. Brown, Lippia origanoides H. B. K., Lippia sidoides Cham., and Lippia graveolens Kunth, are widely used for different purposes such as medicinal and culinary use (Pascual et al., 2001).

The interest in this genus is based on the presence of flavonoids which is a class of low molecular weight phenolic compounds that are widely distributed in the plant kingdom. They exhibit different biological functions that allow interactions between plants and their environment: they are involved in the plant pathogen, plant-plant interactions and plantinsect interactions (Treutter, 2005). Recent studies have also indicated that flavonoids can act as cellular modulators by interaction with enzymes, receptors and transcription factors of intracellular signaling. These interactions could explain their potential anticancer and neurodegeneration-inhibiting activities (Williams et al., 2004).

During the course of our search for Brazilian plant extracts that are active against Mycobacterium tuberculosis, the dichloromethane extract from the leaves of Lippia lacunosa Mart. & Schauer showed moderate anti-mycobacterial activity (minimal inhibitory concentration, MIC of 25 µg/mL) (Leitão et al., 2006). In this study, we report the fractionation of this extract, which resulted in the isolation of the seven flavonoids and one triterpene, besides the evaluation of their antimycobacterial activity against Mycobacterium tuberculosis by the same method.

Materials and Methods

Plant material and morphological analysis

Fresh leaves of Lippia lacunosa Mart. & Schauer, Verbenaceae, cultivated from clones originally from Diamantina, MG, Brazil (Viccini et al., 2004), were collected in October 2006, at the campus of the Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil, (22º46'48.6"S, 43º22'24.5"W). The plant was authenticated by Dr. Fatima Regina Gonçalves Salimena, and a voucher of specimen is deposited at the Herbarium of the Departamento de Botânica, Universidade Federal de Juiz de Fora (CESJ 41.691).

Plant extracts

Leaves (800 g) of L. lacunosa were air-dried, powdered, and extracted by maceration exhaustively with ethanol at room temperature. After filtration and concentration under reduced pressure, the aqueous residue was sequentially extracted with organic solvents of increasing polarities, to afford the new extracts: n-hexane (LLFH, 12.2 g), dichloromethane (LLFD, 6.1 g), ethyl acetate (LLFA, 15.4 g) and n-butanol (LLFB, 46.5 g) extracts, in this order, as well as the remaining water-soluble extract (LLFW).

Isolation and identification of substances from the extracts

Part of the dichloromethane extract (LLFD, 3.0 g) was chromatographed on silica gel, and eluted with dichloromethane containing increasing amounts of ethyl acetate. Then it was eluted with ethyl acetate containing increasing amounts of methanol, affording thirty fractions (I-XXX). Fractions V and VI (375 mg), eluted with dichloromethane-ethyl acetate (9:1), were pooled (monitored by TLC) and re-chromatographed on Sephadex LH-20 (methanol), affording oleanolic acid (8, 66 mg), (Mahato & Kundu, 1994), and a mixture of flavonoids. The flavonoid mixture was further separated by preparative layer chromatography on silica gel plates using dichloromethane-ethyl acetate (9:1) as the solvent system. Using this procedure, salvigenin (4, 1.5 mg) (Skaltsa & Shammas, 1988); 3'-O-methyl-eupatorin (5, 2 mg) (Valant-Vetshera & Wollemweber, 1988), and cirsimaritin (1, 9 mg) (Valant-Vetshera & Wollemweber, 1988) were isolated. Fraction XII (637 mg), eluted with dichloromethane-ethyl acetate (8:2), was rerun on silica gel CC, affording 27 fractions. Fraction XII-8, eluted with dichloromethane-ethyl acetate (9:1), afforded additional 3'-O-methyl eupatorin, (5, 1.2 mg); fraction XII-15 yielded eupatilin (2, 2.3 mg) (Valant-Vetshera & Wollemweber, 1988), and fraction XII-20 afforded eupatorin (3, 1.8 mg) (Valant-Vetshera & Wollemweber, 1988). Fractions XIII-XV (466 mg), eluted with dichloromethane-ethyl acetate (7:3) on the original column, were re-submitted to purification on silica gel CC and eluted with dichloromethane-ethyl acetate (9:1 to 8:2), to afford 35 fractions. From fraction XIII-XV-23, 3',7-dimethoxy-5,6,4'-trihydroxyflavone (6, 10.7 mg) (Christensen & Lam, 1991) was isolated, and from fraction XIII-XV-30, 7'-O-methylapigenin (7, 12 mg, also known as genkwanin) (Wollemweber, 1986) was isolated.

The identity of the isolated compounds was confirmed by 1D and 2D-NMR techniques (¹H, HMBC and HSQC) on a Bruker Avance DRX400 (Karlsruhe, Germany) at 25 ºC, operating at 400.13 MHz for ¹H and 100.61 MHz for ¹³C. NMR spectra were recorded in CDCl₃ (flavonoids) or deuterated pyridine (oleanolic acid), using TMS as an internal standard. The data obtained was in accordance with that found in the literature. Additionally, UV data of the flavonoids in methanol, followed by the use of AcONa as a shift reagent was used for identification purposes (Mabry et al., 1970).

Tested material

Cirsimaritin (1), eupatilin (2), eupatorin (3), salvigenin (4), 3'-O-methyl-eupatorin (5), 3',7-dimethoxy 5,6,4'-trihydroxyflavone (6), 7'-O-methylapigenin (genkwanin) (7) and oleanolic acid (8), obtained as described above.

Bacterial strains

M. tuberculosis strain H37Rv (ATCC27294) was used for all experiments in both of the microbiology laboratories. Additional assays with a rifampicin-resistant strain (ATCC-35838, His-526-Tir) were performed at the FURG laboratories.

The isolates were maintained in Ogawa-Kudoh medium for ca. fourteen days. The bacterial suspensions were prepared in sterile water containing 3-mm glass beads. The suspensions were homogenized by vortex agitation and turbidity was adjusted in agreement with tube one of the McFarland scale (3.2 x 10⁶ colonyforming units/mL). The inoculums were prepared by diluting the bacterial suspension 1:25 in Middlebrook 7H9 OADC medium (4.7 g Middlebrook 7H9 base; Difco, Becton Dickinson) enriched with 10% (v/v) oleic acid-dextrose-albumin-catalase (BBL).

In vitro evaluation of anti-tuberculosis activity and Minimum Inhibitory Concentration (MIC)

Samples were screened against Mycobaterium tuberculosis strain H37Rv (ATCC-27294) using microtiter assays containing a redox indicator (Franzblau et al., 1998; Palomino et al., 2002). The final concentration of the substances and fractions was either 100 or 200 µg/mL. Media plus bacteria, with and without rifampicin, were used as controls. MIC determination was carried out as described in literature (Franzblau et al., 1998; Palomino et al., 2002). In brief, redox assays were performed in 96-well microplates using resazurin, Alamar Blue or MTT as the indicator of cellular viability, and the extracts, flavonoids or oleanolic acid, dissolved in DMSO. The minimal inhibitory concentration (MIC) was carried out by two fold serial dilutions of the drugs, starting from 200 µg/ mL.

Results and Discussion

In a previous study from our group (Leitão et al., 2006) the dichloromethane extract from leaves of L. lacunosa showed antimycobacterial activity (MIC 25 µg/mL) against M. tuberculosis H37Rv. In this work, seven methoxyflavones (1-7) and one triterpene (8) were isolated from this extract and evaluated against M. tuberculosis H37Rv and rifampicin resistant strains (Table 1). These compounds were identified by the combination of 1D and 2D NMR techniques, as well as UV data of flavonoids.

All tested substances showed activity against M. tuberculosis H37Rv. The most active compound was 3'-O-methyl-eupatorin (5, MIC 25 µg/mL), followed by cirsimaritin (1), eupatilin (2) and eupatorin (3, MIC 50 µg/mL). Salvigenin (4), and 3',7-dimethoxy5,6,4'-trihydroxyflavone (6) were the least active substances tested, affording MIC of 100 and 200 µg/ mL, respectively. Even though there are many reports on the antimycobacterial activity of the triterpene oleanolic acid (MIC of 50-69 µg/mL) in the literature (Jiménez-Arellanes, 2007; Copp, 2003; Cantrell et al., 2001; Caldwell et al., 2000; Koysomboon et al., 2006; Tanachatchairatana et al., 2008) it was re-evaluated against H37Rv as well as against the rifampicin resistant strain. Our results for H37Rv are compatible with those previously reported. However, oleanolic acid was inactive against this rifampicin-resistant strain at the maximum assayed concentration of 200 µg/mL. Due to the isolation of very small amounts of the flavonoids 2-7, they were not assayed against the rifampicin-resistant strain.

The antimycobacterial activity of flavonoids has already been reported by many authors. Lin and coworkers (2002) have described the antimycobacterial activity of 142 synthetic chalcones, flavonoids (including some methoxyflavones) and chalconeslike compounds against M. tuberculosis H37Rv, and concluded that flavones showed decreased activity with respect to the corresponding chalcones. Another paper on antimycobacterial activity of flavonoids describes the results of the susceptibility of M. tuberculosis H37Ra, an avirulent strain, against fourteen flavonoids, including rotenoids and prenylated isoflavones from Derris indica (Murillo et al., 2003). In this paper, the authors regret that even with the concomitant isolation of fourteen different compounds from the same class (flavonoids), the careful inspection of the comparison of their molecular structures and their activities did not reveal any useful or consistent trends. Three other publications on antimycobacterial flavonoids report the activity against M. tuberculosis H37Rv of methoxyflavones obtained from Haplopappus sonorensis (Yenjai et al., 2004), Kaempferia parviflora (Gu et al., 2004), and Valeriana laxiflora (Haermers et al., 1990). Again, no conclusion on structure-activity could be drawn.

High lipophilicity of substances has been reported as an important feature for antimycobacterial activity (Silva et al., 2007). Due to the fact that the cell wall of mycobacteria contain lipophilic substances such as mycolic acid, more 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). To date, no flavonoid glycosyl derivative has been shown to be active against M. tuberculosis, and compounds such as quercetin have demonstrated only weak activity. Thus, if polarity is considered as an important factor of the antimycobacterial activity of flavonoids, it would be expected that in a series of methoxyflavones, that the higher the degree of methyl substitution the higher the activity of the derivatives. Analysis of the MIC of methoxyflavones 1-7 in Table 1, shows, as expected, that the tetramethoxy flavone derivative 5 (3'-O-methyleupatorin) is the one displaying the best activity (MIC 25 µg/mL). However, this hypothesis does not hold when we compare the MIC of the dimethoxy flavone 6 (200 µg/mL) with that of monomethoxy flavone 7 (50 µg/mL). Furthermore, a critical analysis of the structures of the trimethyl derivatives 2 and 3, as well as the dimethyl derivatives 1 and 6 does not reveal any structure-activity relationship.

Recently, the mechanisms of resistance of mycobacteria have garnered a lot of attention. Besides the classic resistance mechanisms, other mechanisms related to intrinsic and acquired resistance, such as efflux pump mechanisms, have been described. Drugs that can decrease resistance by inhibition of the efflux pump have been an important goal in the research of new drugs. For instance, INH resistance has been associated with the activation or induction of an energy-dependent efflux pump (Lechner et al., 2008). In this work, two of the isolated compounds, cirsimaritin (1), and oleanolic acid (8), were obtained in large enough quantities to be assayed against the rifampicin- resistant Mycobacterium tuberculosis strain 35838. One interesting feature about oleanolic acid (8), is its inhibitory activity on the multidrug resistance protein ABCC1 (MRP1) (Braga et al., 2007). Furthermore, a number of flavonoids have been described as MRP1 inhibitors (Felipe et al., 2008) to date. However, even at 200 µg/mL, compounds 1 and 8 failed to show any activity against this rifampicin-resistant strain. The antimycobacterial activity of oleanolic acid against a rifampicin-resistant strain has already been reported in literature (Jimenez-Arellanes, 2007) with a MIC of 50 µg/mL. However, as we don't know the molecular basis of rifampicin resistance of the strain assayed by Jimenez-Arellanes (2007) it is very difficult to establish some comparative analysis between these different results, since we can't infer that they are genetically identical.

From the dichloromethane extract of Lippia lacunosa seven flavones, including six 6-substituted flavones, were isolated. Our results of the antimycobacterial activity of 6-substituted flavones from Lippia lacunosa show the importance of investigating the phytochemistry area to discover new drugs against TB.

This is the first report concerning the nonvolatile chemistry of this plant, as well as the first report of the antimycobacterial activity of 6-substituted methoxyflavones.

Acknowledgements

The authors wish to thank CNPq (Edital MCT-CNPq/MS-SCTIE-DECIT 25/2006, Process no. 410475/2006-8) and FAPERJ (E-26/111.614/2008) for financial support. We are also indebted to Centro Nacional de Ressonância Magnética Nuclear Jiri Jones, UFRJ, Rio de Janeiro, for the use of NMR equipment, and to Prof. Lyderson Viccini and Prof. Fatima G. Salimena, from Universidade Federal de Juiz de Fora, for supplying us with plant material. Some of us are indebted to CAPES and CNPq for fellowships, CDTS/ Plataforma de Bioensaios II, FIOCRUZ, and for UNICEF/UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR). Collaborative work was performed under the auspices of the Iberoamerican Program for Science and Technology (CYTED), Project X.11:PIBATUB.

Received 22 Dec 2010

Accepted 20 Jan 2011

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Publication Dates

• Publication in this collection
  06 May 2011
• Date of issue
  Oct 2011

History

• Received
  22 Dec 2010
• Accepted
  20 Jan 2011