Antimycobacterial activity of Indigofera suffruticosa with activation potential of the innate immune system

Abstract

Mycobacterium tuberculosis is responsible for over 8 million cases of tuberculosis (TB) annually. Natural products may play important roles in the chemotherapy of TB. The antimycobacterial activity and the innate immune response of methanol (METH) and dichloromethane (DCM) extracts of Indigofera suffruticosa Miller (Fabaceae) were evaluated. We observed that the minimum inhibitory concentrations (MICs) for METH and DCM extracts were 125 and 1000 µg/mL, respectively. However, they were able to induce the innate immune response through the production of high levels of NO and TNF-α (p < 0.001) by peritoneal exudate cells (PECs). These results suggest that I. suffruticosa extracts may have an important immunological role in the control of TB once macrophage activity is induced by them.

Keywords::

• Antimycobacterial activity
• Indigofera suffruticosa
• innate immune response

Introduction

Mycobacterium tuberculosis is a serious threat to humankind, with over 8 million cases of tuberculosis (TB) annually, killing almost 3 million of people per year around the world (WHO, 2008). Moreover, side effects from first-line anti-TB drugs can cause significant morbidity, and compromise treatment regimens for TB (Yee et al., 2003). Most healthy individuals are able to control TB infection with a vigorous immune response, halting the progression of the disease, but not necessarily eradicating the microorganism (McKinney, 2000). The bacterium resides within macrophages, allowing them to resist the antimicrobial effector mechanisms of the host (Raupach & Kaufmann 2001). Macrophages constitute one of the main phagocytic cells of the immunological system, and they are the first cells involved in an immunological response. Part of their effectiveness is due to the production of nitric oxide (NO), hydrogen peroxide (H₂O₂), and other reactive nitrogen intermediates (RNIs), as well as phagocytosis of alien particles (Keil et al., 1999; Carlos et al., 2004; Allavena et al., 2008). Hydrogen peroxide generated by macrophages in a reaction catalyzed by an NADPH (reduced nicotinamide adenine dinucleotide phosphate) oxidase was the first identified effector molecule that mediated mycobactericidal effects of mononuclear phagocytes (Walker & Lowrie, 1981; Lopes et al., 2005). NO, formed by the action of the inducible form of nitric oxide synthase (iNOS), reacts with oxygen radical-forming RNIs. NO and related RNIs have been reported to possess antimycobacterial activity (Kwon, 1997; Yang et al., 2009). Tumor necrosis factor-α (TNF-α), a cytokine that plays multiple roles in immune and pathologic responses in tuberculosis, is also required for acute infection control (Flynn et al., 1995; Palladino et al., 2003; Babbar & Casero, 2006).

Indigofera suffruticosa Miller (Fabaceae) is found in tropical and subtropical areas and is well adapted to growth in semi-arid regions and soils of low fertility (Paiva et al., 1987). A chemical investigation of extracts of leaves of I. suffruticosa in Natural Products Alert (NAPRALERT, 2003) and Chemical Abstracts databases has revealed the presence of alkaloids, flavonoids, steroids, proteins, carbohydrates, and indigo. Some recent reports have demonstrated the in vitro bioassay activity of plant-derived terpenoids against M. tuberculosis (Cantrell et al., 2001; Hastings, 1990; Higuchi et al., 2008a). The literature also reports the antimycobacterial activity of many classes of natural products such as alkanes, phenolics, acetogenic quinines, flavonoids, triterpenes, flavonones, and chalcones (Copp, 2003; Higuchi et al., 2008b; Leite et al., 2006; Pavan et al., 2009).

At present, there is no new drug generation able to eliminate the bacillus. Thus, we investigated the antimycobacterial and immunological activity of methanol (METH) and dichloromethane (DCM) extracts of I. suffruticosa.

Materials and methods

Plant material and samples

Aerial parts of I. suffruticosa were collected in Rubião Junior, Botucatu City, São Paulo State, Brazil, and identified by Prof. Dr. Jorge Yoshio Tamashiro. Immunological assays were performed as soon as the plant was collected. A voucher specimen (HUEC 129598) was deposited at the Herbarium of the Universidade Estadual de Campinas (Unicamp), Campinas, SP, Brazil. The aerial parts of I. suffruticosa (1.1 kg) were dried (40°C), powdered, and extracted exhaustively at room temperature with dichloromethane and methanol, successively. Solvents were evaporated at 40°C under reduced pressure to afford the DCM (15.2 g) and METH (30.0 g) extracts.

Each extract was first solubilized in dimethyl sulfoxide (DMSO) and then diluted in an appropriate culture medium, RPMI-1640 for immunological assays and Middlebrook 7H9 for the determination of antimycobacterial activity (62.5–4000 μg/mL).

Peritoneal macrophages

Peritoneal macrophages, thioglycollate-elicited peritoneal exudate cells (PECs), were harvested from Swiss mice using 5.0 mL of sterile phosphate-buffered saline (PBS, pH 7.4). The cells were washed twice by centrifugation at 200 g for 5 min at 4°C and re-suspended in RPMI-1640 medium (Sigma). The adherent cells were obtained by incubation for 1 h at 37°C in an atmosphere of air/CO₂ (95:5, v/v) (Forma Scientific), and incubated with lipopolysaccharide (LPS) or RPMI-1640 medium. This protocol was in agreement with the regulations of the Research Ethics Committee (# 01/2005).

MTT assay for cell viability

PECs (5 × 10⁶ cells/mL) was re-suspended in RPMI-1640 medium. The suspension (100 μL) and the extracts (100 μL) were added to each well of a 96-well tissue culture plate and they were incubated for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric assay was performed as described by Mosmann (1983). Cells only and culture medium (RPMI-1640) were used as a control that corresponded to 100% of macrophage viability.

Measurement of H2O2 production

Hydrogen peroxide production of adherent PECs (2 × 10⁶ cells/mL) was measured using the horseradish peroxidase-dependent phenol red oxidation microassay (Pick & Mizel, 1981). Phorbol myristate acetate (PMA; Sigma, St. Louis, MO) was used as a positive control.

Measurement of NO production

NO production of adherent PECs (5 × 10⁶ cells/mL) was measured using Griess reagent (Green et al., 1982). Escherichia coli O111B lipopolysaccharide (LPS 1 µg/mL) solution was used as a positive control.

Measurement of TNF-α production

Determination of TNF-α in the supernatant was based in its property to destroy the L929 tumoral cell line (mouse tumor fibroblast) (Carlos et al., 1994). LPS (1 µg/mL) was used as a positive control.

Determination of antimycobacterial activity by MABA

The minimum inhibitory concentration (MIC) of METH and DCM extracts was determined against M. tuberculosis H₃₇Rv (American Type Culture Collection 27294) in Middlebrook 7H9 medium using the microplate Alamar blue assay (MABA) (Collins & Franzblau, 1997). For the standard test, the MIC value of isoniazid (Sigma) was determined each time. The acceptable MIC of isoniazid ranged from 0.015 to 0.05 µg/mL.

Statistical analysis

The results are expressed as mean ± SD of at least four experiments. One-way analysis of variance (ANOVA) with Dunnett’s post-test was performed using GraphPad InStat software (San Diego, CA, USA) with the level of significance set at p < 0.05.

Results and discussion

TB multiple drug resistance has become a major threat worldwide, and thus calls for an urgent search for new and effective treatments for this deadly disease. Naturally occurring compounds obtained as extracts from plants indicate that inhibitory activity against M. tuberculosis is widespread in nature (Okunade et al., 2004).

The cytotoxic effect of the extracts was evaluated by the determination of MTT (a tetrazolium salt: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) (Mosmann, 1983). The half maximal inhibitory concentration (IC₅₀) was found to be 200 µg/mL (Table 1).

Table 1.  Effect of methanol and dichloromethane extracts of Indigofera suffruticosa on the viability of peritoneal macrophages.

I. suffruticosa extract	Viability (%)
METH
600 µg/mL	15.73 ± 1.90
400 µg/mL	30.29 ± 4.32
200 µg/mL	55.29 ± 1.36
100 µg/mL	71.57 ± 1.82
DCM
600 µg/mL	11.54 ± 0.85
400 µg/mL	26.49 ± 2.95
200 µg/mL	51.35 ± 3.71
100 µg/mL	63.91 ± 4.11

Note. Negative control corresponds to 100% viability because it contains only RPMI-1640 and cells. Each value represents the mean ± SD for at least four independent experiments carried out in triplicate.

This study evaluated the antimycobacterial activity of extracts of I. suffruticosa and their action in the innate immune system. The antimycobacterial activities of METH and DCM are presented in Table 2. Gu et al. (2004) considered plant extracts with an MIC value ≤ 128 µg/mL to be active. Thus, we considered the result of MIC 125 µg/mL found for the METH crude extract to be promising. The extracts presented a high production of nitric oxide with statistically significant values compared to the negative control (p < 0.001). The amount of NO produced by the METH extract (105.99 µmol/5 × 10⁵ cells) was larger than the production of the DCM extract (58.9 µmol/5 × 10⁵ cells) (Figure 1). The results regarding TNF-α confirmed a significant production of this cytokine, at levels near that of the positive control (252.7 and 234.6 units/mL, METH and DCM extracts, respectively), confirming a correlation between the synthesis of TNF-α and NO (Figure 2) (Bogdan et al., 1991; Carli et al., 2009). I. suffruticosa did not produce significant amounts of H₂O₂ when compared with the negative control (p > 0.05): METH, 0.59 nmol/2 × 10⁵cells and DCM, 3.3 nmol/2 × 10⁵cells) (Figure 3). This fact can be justified by the presence of tannins, such as gallic acid, in extracts of I. suffruticosa. This class of substances has been shown to have antioxidant potential, being responsible for the scavenging of free radicals such as hydrogen peroxide (Akira et al., 2002). Thus, this screening suggests that both extracts of I. suffruticosa promoted the activation of macrophages. The significant production of studied mediators (NO and TNF-α) by activated macrophages in the presence of I. suffruticosa is very important, since macrophages produce several effector molecules that can enhance or restore the ability of the innate immune system to fight against TB infection.

Table 2.  Minimal inhibitory concentration (MIC) against M. tuber-culosis of I. suffruticosa methanol and dichloromethane extracts.

	MIC (µg/mL)
Methanol extract	125
Dichloromethane extract	1000
Isoniazid^a	0.05

Note. The fluorescence of dye was measured in a SPECTRAfluor Plus (Tecan®). MIC value ≤ 125 μg/mL is defined as active against M. tuberculosis. ^aStandard drug.

Figure 1.  Induction of nitric oxide (NO) production by methanolic and dichloromethanic extracts of I. suffruticosa from peritoneal macrophages. Cells incubated with lipopolysaccharide (1 µg/mL) were used as a positive control (C+) and cells in culture medium (RPMI-1640) were used as a negative control (C–). Data are reported as mean ± SD for at least four independent experiments carried out in triplicate. One-way ANOVA with Dunnett’s post-test was performed; ***p < 0.001 vs. C–.

Figure 2.  Induction of tumor necrosis factor-α (TNF-α) production by methanolic and dichloromethanic extracts of I. suffruticosa from peritoneal macrophages. Cells incubated with lipopolysaccharide (1 µg/mL) were used as a positive control (C+) and cells in culture medium (RPMI-1640) were used as a negative control (C–). Data are reported as mean ± SD for at least four independent experiments carried out in triplicate. One-way ANOVA with Dunnett’s post-test was performed; ***p < 0.001 vs. C–.

Figure 3.  Induction of hydrogen peroxide (H2O2) production by methanolic and dichloromethanic extracts of I. suffruticosa from peritoneal macrophages. Cells incubated with phorbol myristate acetate (0.2 mM) were used as a positive control (C+) and cells in potassium phosphate buffer as a negative control (C–). Data are reported as mean ± SD for at least four independent experiments carried out in triplicate.

Nitric oxide (NO), formed by the action of the inducible form of nitric oxide synthase (iNOS), reacts with oxygen radical-forming RNIs. NO and related RNIs have been reported to possess antimycobacterial activity (Kwon, 1997). Phagocytes kill intracellular organisms during an initial oxidative phase dependent on NADPH oxidase, followed by a prolonged nitrosative phase, during which bacterial growth is inhibited by iNOS (Nathan & Shiloh, 2000). There are several potential mechanisms that can explain how NO may affect the microbial life-cycle. NO and other RNIs can modify bacterial DNA, protein, and lipids at both the microbial surface and intracellularly. They can alter cytokine production and induce or prevent apoptosis of host cells by controlling caspase activity (Raupach & Kaufmann, 2001).

M. tuberculosis strongly induces the release of several cytokines during infection. TNF-α is a cytokine that plays multiple roles in immune and pathologic responses in tuberculosis, and is also required for acute infection control (Flynn et al., 1995). It plays a major role in the recruitment of inflammatory cells to the site of infection and in the formation and maintenance of granulomas (Gaemperli et al., 2006). This cytokine is necessary for optimal co-ordination of both the differentiation of specific T cells to secrete the appropriate T helper 1 cytokines and the development of granulomas in which activated macrophages restrict mycobacterial growth (Ehlers, 2003). TNF-α is required for control of latent TB, and it is also a key element for activating macrophages to produce iNOS and thus in maintaining the pathway for generating NO and preventing reactivation of the disease (Adams et al., 1995).

We suggest that the extracts may be an important bactericidal source against M. tuberculosis of natural origin and do not present the toxic effects provoked by the drugs currently used in the treatment of tuberculosis. Moreover, possible association with traditional drugs can be suggested, considering that most standard drugs do not present the same simultaneous microbiological and immunological effect of the extracts tested here. The results described herein highlight the importance of conducting an in-depth study of the species of the Brazilian biome, and show the great potential of its biodiversity in the treatment of infectious diseases.

Declaration of interest

The authors thank FAPESP for its financial support.