Anti-mycobacterial and anti-inflammatory activity of restinga plants: a dual approach in searching for new drugs to treat severe tuberculosis

Abstract

Tuberculosis (TB) still constitutes a threat to public health in various regions of the world. The existing treatment is long and has many side effects. The need to identify new anti-TB compounds and also adjuvants to control exacerbated inflammation in severe TB cases is relevant. Therefore, the objective of this study was to evaluate the anti-mycobacterial activity of extracts and fractions in vitro from plant species collected in the Restinga of Jurubatiba, in Rio de Janeiro state, Brazil. In addition, to verify their immunomodulatory action and cytotoxicity on macrophages. The dichloromethane fraction of Kielmeyera membranacea and Eremanthus crotonoides showed the lowest MIC₅₀ against Mycobacterium bovis BCG (0.95 ± 1.08 and 2.17 ± 1.11 μg/mL, respectively) and M. tuberculosis H₃₇Rv (4.38 ± 1.19 and 15.28 ± 1.21 μg/mL, respectively). They were also able to inhibit the NO and TNF-α production in LPS-stimulated macrophages, without being toxic to cells. Using gas chromatography analysis coupled with mass spectrometer it was possible to suggest the presence of fatty acids and terpenes in the most promising fractions. Those compounds have been described for their anti-mycobacterial activity. These results have enabled identifying Kielmeyera membranacea and Eremanthus crotonoides as the most promising studied species in searching for new anti-TB compounds with dual activity.

Key words:
anti-mycobacterial; immunomodulatory; restinga ; terpenes; tuberculosis

Resumo

A tuberculose (TB) ainda representa um problema de Saúde Pública em várias regiões do mundo. O tratamento existente é longo e apresenta diversos efeitos adversos. Neste contexto, é relevante a necessidade de identificar novas substancias anti-TB e complementares ao controle do processo inflamatório exacerbado em quadros severos da doença pulmonar. O objetivo deste trabalho foi avaliar, in vitro, extratos e frações de espécies vegetais coletadas na Restinga de Jurubatiba, quanto a sua atividade antimicobacteriana, assim como verificar a ação imunomoduladora e citotóxica em macrófagos. As frações em diclorometano de Kielmeyera membranacea e Eremanthus crotonoides apresentaram os menores CIM₅₀ contra Mycobacterium bovis BCG (0,95 ± 1,08 e 2,17 ± 1,11 μg/mL; respectivamente) e M. tuberculosis H₃₇Rv (4,38 ± 1,19 e 15,28 ± 1,21 μg/mL; respectivamente). Essas também foram capazes de inibir a produção de NO e TNF-α em macrófagos estimulados por LPS, sem serem tóxicas para as células. Através de análise por cromatografia em fase gasosa acoplada ao espectrômetro de massas foi possível sugerir a presença de ácidos graxos e terpenos nas frações mais promissoras, substancias estas descritas por apresentarem atividade antimicobacteriana. Estes resultados permitiram a identificação de Kielmeyera membranacea e Eremanthus crotonoides como as espécies mais promissoras desse estudo, tendo em vista a busca de novos fármacos anti-TB com dupla atividade.

Palavras-chave:
anti-micobacteriano; imunomodulatório; restinga; terpenos; tuberculose

Introduction

Tuberculosis (TB) is an infectious disease responsible for 1.2 million deaths in 2018 and an additional 0.25 million deaths resulting from TB among human immunodeficiency virus (HIV)-positive people (WHO 2019). The World Health Organization estimated 10 million new cases, with 484,000 being rifampicin or multidrug-resistant TB (MDR-TB) (WHO 2019), where approximately one in five tuberculosis isolates worldwide are resistant to at least one major first-line drug (Dheda et al. 2017).

Furthermore, in the cases of severe forms of TB such as military TB or tuberculous meningitis, additional anti-inflammatory therapy is required to prevent excessive inflammation (Pai et al. 2016; Zhang et al. 2017). Macrophages are the main host cells for mycobacteria which play an important role and are a potential target for modulating improper or excessive production of inflammatory mediators, which contributes to the pathogenesis of tuberculosis (Koul et al. 2004; Garlanda et al. 2007).

Thus, there is an important need to explore new treatment strategies and natural products are an important source of antibacterial compounds (Harvey et al. 2010). Different plant species and isolated compounds have shown significant in vitro anti-mycobacterial activity (Okunade et al. 2004; Coop & Pearce 2007; Salomon & Schmidt 2012). Potential dual anti-mycobacterial and antiinflammatory activity has also been studied as an alternative and shown promising in vitro and in vivo results (Machado et al. 2014; Ventura et al. 2015a,b,c).

The abundance of Brazilian flora provides great possibilities for finding novel anti-TB compounds. The restinga vegetation has great biodiversity among Brazilian ecosystems and covers around 79% of the coastal sandy plains. This biodiversity is likely due to a combination of physical and chemical factors such as high temperature, soil salinity, extensive deposition of salt and high exposure to light (Cogliatti-Carvalho et al. 2001). These conditions favor a great diversity of habitats which are colonized by a wide variety of plant, animal and microorganism communities. In turn, this leads to differentiated production of secondary metabolites with the aim of environmental adaptation (Amaral et al. 2013). In the present study, the search for active compounds was based on random selection from Restinga of Jurubatiba National Park flora, located in the Northeast of the state of Rio de Janeiro (22°23’S, 41°45’W), Brazil. The distribution of these species in the studied area was taken into account in this process, and only abundant species were selected.

The first objective of the present work was to determine the activity of Tapirira guianensis Aubl., Mandevilla moricandiana (A.DC.) Woodson, Eremanthus crotonoides (DC.) Sch.Bip. (= Vernonia crotonoides), Kielmeyera membranacea Casar., Stachytarpheta schottiana Schauer, Vitex polygama Cham. and Tocoyena bullata (Vell.) Mart. from the restinga of Jurubatiba against avirulent Mycobacterium bovis bacille Calmette-Guérin (BCG) in order to evaluate the usefulness of the non-virulent vaccine strain as a model for detecting activity against the virulent Mycobacterium tuberculosis H₃₇Rv strain. This non-virulent M. bovis is an attenuated strain, but closely related to M. tuberculosis (Mahairas et al. 1996). Thus, M. bovis BCG could be used to provide useful information for screening studies.

Secondly, the modulation of pro-inflammatory mediators such as nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) and the cytotoxicity effect in the macrophages were evaluated. These mediators can indicate an anti-inflammatory profile for the samples, and together with anti-mycobacterial activity data can enable discovering compounds with dual activity.

In a third component, infected macrophages were treated with samples to confirm the role of these mediators and anti-mycobacterial activity together, and the production of NO, TNF-α was measured, as well as the cytotoxic effect. In screening studies, this could reinforce the rationale on the role of mediators during mycobacterium infection, helping to identify natural products with both anti-mycobacterial and anti-inflammatory activity. While some work has been done in this regard, further data are required to support this reasoning.

Materials and Methods

Reagents

Cell culture reagents were purchased from Gibco/Invitrogen (Grand Island, NY, USA). Mycobacteria-specific Middlebrook 7H9 and 7H10 media were obtained from Difco (Detroit, MI, USA); albumin dextrose catalase (ADC) and oleic albumin dextrose catalase (OADC) supplements were from BD Biosciences (BD, Sparks, MD, USA).

Murine recombinant TNF-α from Biosource®. Lipopolysaccharide (LPS) from serotype 0111:B4 Escherichia coli, N^G-monomethyl-L-arginine acetate salt (L-NMMA - 98% purity) (cod. M7033), rifampicin (cod. R7382) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The samples and rifampicin were dissolved in dimethyl sulfoxide (DMSO, Sigma Aldrich); other reagents indicated for cell treatment were dissolved in sterile phosphate buffered saline (PBS) and sterilized by passage through 0.22 μm nylon filters (Corning Inc., Wilkes-Barre, PA, USA).

Plant materials and preparation of extracts and fractions

Leaves of the 7 plant species were collected: BRASIL. RIO DE JANEIRO: Quissamã, Restinga de Jurubatiba National Park, 22°11’53.8”S, 41°27’48.2”W, 10 m above sea level, 20.II.2011, fl., T.U.P. Konno (RFA), (Herbarium of the Federal University of Rio de Janeiro). The voucher specimen codes and relevant information for each species are described in Table 1. This study complied with all relevant federal guidelines and institutional policies related to the botanical material for research purposes, Sisbio/ICMBio number: 62422-11 and SisGen number: AAA989F.

Table 1
Relevant information of the studied species and evaluation of inhibition of Mycobacterium bovis BCG growth when treated with extracts and fractions from restinga species.

Family and botanical name	Common name#	Voucher number	Part used and solvent extractor	Samples	Sample code	Mycobacterium bovis BCG
						MIC50±SD (μg/mL)	Maximum inhibition (%)
Anacardiaceae	Micumi	RFA38757	Leaves	Crude Extract Fractions	TgCE	>100	22.33±0.0
Tapirira guianensis Aubl.			Methanol	Hexane Dichloromethane Ethyl acetate Butanol Aqueous	Tg HEX Tg DCM Tg EtOAc Tg BuOH Tg Aq	86.53±1.29 22.47±1.12 49.16±1.38 80.62±1.30 >100	56.39±0.90 77.83±1.32 79.06±0.00 54.17±1.62 43.21±1.97
Apocynaceae	Chifre-de-veado	RFA38748	Leaves	Crude Extract Fractions	Mm CE	>100	18.74±0.00
Mandevilla moricandiana (A. DC.) Woodson			Ethanol/water (7:3, v/v)	Hexane Dichloromethane* Ethyl acetate Butanol Aqueous	Mm HEX Mm DCM Mm EtOAc Mm BuOH Mm Aq	>100 11.97±1.23 18.44±1.16 >100 >100	26.65±4.89 93.8±0.12 83.25±1.02 16.67±3.37 43.86±4.02
Asteraceae	Papel sanitário das indias	RFA38749	Leaves	Crude Extract* Fractions	Vc CE	4.42±1.38	94.64±1.26
Eremanthus crotonoides (DC.) Sch.Bip.			Ethanol/water (8:2, v/v)	Hexane Dichloromethane* Ethyl acetate Butanol Aqueous	Ec HEX Ec DCM Ec EtOAc Ec BuOH Ec Aq	20.14±1.21 2.17±1.11 21.67±1.49 84.42±1.69 90.26±2.01	80.6±2.88 93.1±0.07 70.98±3.72 50.70±0.0 56.62±2.82
Calophyllaceae	Pequiá-branco	RFA38752	Leaves	Crude Extract Fractions	Km CE	29.92±1.14	74.60±2.34
Kielmeyera membranacea Casar.			Ethanol/water (7:3, v/v)	Hexane Dichloromethane* Ethyl acetate Butanol Aqueous	Km HEX Km DCM Km EtOAc Km BuOH Km Aq	>100 0.95±1.08 >100 30.58±1.20 41.10±1.25	46.3±2.42 96.83±0.14 10.27±5.29 77.14±1.38 59.88±2.04
Lamiaceae	Carabuçu	RFA38750	Leaves	Crude Extract Fractions	Vp CE	22.64±1.28	81.55±1.54
Vitex polygama Cham.			Ethanol/water (8:2, v/v)	Hexane Dichloromethane* Ethyl acetate Butanol Aqueous	Vp HEX Vp DCM Vp EtOAc Vp BuOH Vp Aq	13.43±1.43 8.90±1.26 >100 3.40±1.90 >100	89.09±0.84 91.65±0.54 50.55±7.68 83.25±3.30 38.85±2.28
Verbenaceae	Gervão-mirim	RFA38759	Leaves	Crude Extract Fractions	Ss CE	78.77±1.32	54.09±1.26
Stachytarpheta schottiana Schau			Ethanol/water (8:2, v/v)	Hexane* Dichloromethane Ethyl acetate Butanol Aqueous	Ss HEX Ss DCM Ss EtOAc Ss BuOH Ss Aq	1.52±1.30 44.52±1.13 29.36±1.23 32.41±1.30 >100	97.42±0.00 75.26±5.36 76.68±0.72 71.03±4.02 37.32±4.20
Rubiaceae	Jenipapo-da-praia	RFA38974	Leaves and Flowers	Crude Extract Fractions	Tb CE	>100	27.01±2.20
Tocoyena bullata (Vell.) Mart.			Ethanol	Hexane Dichloromethane Ethyl acetate Butanol Aqueous	Tb HEX Tb DCM Tb EtOAc Tb BuOH Tb Aq	>100 42.06±1.13 58.24±1.50 >100 66.38±1.32	36.08±3.89 73.99±1.04 79.76±4.01 3.75±2.46 57.25±2.64

• Antibiotic rifampicin was used at concentrations of 0.001, 0.003, 0.01 and 0.03 μg/mL, demonstrating 16.78±4.88%, 71.58±2.13%, 98.85±1.06% and 99.63±0.32% growth inhibition, respectively; # = et al.; * = The most promising activities; Maximum inhibition (%) = the highest percentage of inhibition obtained at the concentrations tested; MIC₅₀ = lowest concentration at which 50% of the growth was inhibited.

The leaves were air-dried at room temperature, triturated and extracted by maceration for 24 h at room temperature with the described solvents (Tab. 1). The solvents were renewed five times, completing the extraction after 120 h. After extraction, the materials were concentrated under vacuum in a rotary evaporator at temperatures below 45 °C and the solvent was eliminated to leave the crude extract. The crude extract was lyophilized, dissolved in water:methanol (H₂O:MeOH) (1:9) and fractions were obtained by liquid-liquid partition, using solvents with increasing polarity: firstly, hexane (Hex - 1/3 of the total volume by 3 times), then MeOH phase was concentrated, dissolved in H₂O and partitioned with dichloromethane (DCM - 1/3 of the total volume by 3 times), aqueous phase was partitioned with ethyl acetate (EtOAc - 1/3 of the total volume by 3 times), at long last aqueous phase was partitioned butanol (BuOH - 1/3 of the total volume by 3 times) and the last residual aqueous phase (Aq) was concentrated and lyophilized. This procedure was repeated for each plant species and the respective fraction codes are shown (Tab. 1).

Analysis by gas chromatography (GC) coupled to mass spectrometry (MS)

Fractions were submitted to Shimadzu GC-MS/QP2010 equipment using capillary columns of fused silica RTx-5MS (30 m × 0.25 μm) from Restek Corporation Pennsylvania USA). Injector temperature: 270 °C (SPLIT). Mass detector conditions: ionization source, 200 °C; Interface, 230 °C. The column temperature was maintained at 60 °C for 1 min and then subsequently increased to 280 °C at a rate of 15 °C/min and held for 10 min. Helium was used as carrier gas with a flow rate of 1.1 mL/min. The fractions (1 mg) were derivatized using 100 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) for 30 min. After 15 min of standing the vial with the fraction, MSTFA was thoroughly vortexed for 2 minutes and then returned to stand. The derivatized fractions were dissolved in dichloromethane at 1 mg/mL, and 1 μL was injected with an automatic injector (Carneiro et al. 2018).

Anti-mycobacterial activity

Two mycobacterial strains were used in this study: avirulent Mycobacterium bovis BCG, Moreau vaccine strain, and virulent Mycobacterium tuberculosis H₃₇Rv (ATCC 27294). Mycobacterial strains were grown in suspension in Middlebrook 7H9 medium, supplemented with 0.5% glycerol, 0.05% Tween-80 and 10% of ADC at 37 °C under biosecurity level 3 containment conditions. The suspension densities were measured by spectrophotometry at 600 nm and corresponding concentrations were determined for each serial dilution strain with plating on Middlebrook 7H10 agar plates supplemented with 0.5% glycerol and 10% of OADC. During the middle logarithmic growth phase, the bacterial suspensions were plated on a 96-well microplate (1×10⁶ colony forming unit - CFU/well) in the presence of plant samples at concentrations of 100, 20, 4 and 0.8 μg/mL or rifampicin (ranging from 0.001 to 0.03 μg/mL for M. bovis BCG and from 0.008 to 1 μg/mL for M. tubelculosis H₃₇Rv). The plates were sealed and incubated at 37 °C and 5% of carbon dioxide (CO₂) for 7 days for M. bovis BCG or 5 days for M. tuberculosis strains. The mycobacterial cultures were incubated after these periods for 3 h with MTT solution (5 mg/mL) and then treated overnight with lysis buffer (20% w/v Sodium dodecyl sulfate (SDS)/50% dimethylformamide (DMF) in distilled water - pH 4.7) (Gomez-Flores et al. 1995). The resulting optical densities of the samples were measured in microplate reader at 570 nm. A mycobacterial suspension treated with rifampicin (Sigma-Aldrich ≥ 99% purity) was used as a positive control, while untreated mycobacterial suspensions were used as a negative control.

Culture and treatments of LPS-activated RAW 264.7 cells

Murine macrophages RAW 264.7 (ATCC, TIB-71) were cultured in Dulbecco Modified Eagle medium (DMEM-F12) supplemented with 10% Fetal bovine serum (FBS) and Gentamicin (0.2%) in 5% CO₂ atmosphere at 37 °C. Cells were seeded in 96-well microplates (5×10⁴ cells/well) for the experiments and incubated for 24 h. Cells were stimulated after this period with 1 μg/mL LPS (Escherichia coli 0111:B4; Sigma-Aldrich) and treated with plant samples at concentrations of 100, 20, 4 and 0.8 μg/mL for 24 h. Next, culture supernatants were collected for NO and TNF-α assays (Araujo et al. 2017).

Determination of nitric oxide (NO) production

In the nitric oxide experiments, an inhibitor of the inducible isoform of nitric oxide synthase (iNOS), L-7VMMA was used as a positive control at 20 μg/mL. Stimulated and untreated macrophages were used as a negative control. Nitrite, which is a stable NO metabolite, was assessed by the Griess method (Griess 1879). The nitrite concentration was calculated from a NaNO₂ standard curve. The optical density was measured in microplate reader at 540 nm.

Determination of tumor necrosis factor-alpha (TNF-α) production

Tumor necrosis factor-alpha (TNF-α) was measured by an L929 fibroblast (ATCC, CCL-1) bioassay based on the sensitivity of L929 cells to the cytotoxic effect of TNF-α. To do so, the L929 cells were seeded in 96-well microplates (2.5×10⁴ cells/well). The resulting cell monolayers after 24 h of incubation were treated with the macrophage culture supernatants in the presence of actinomycin D (2 μg/mL). After an additional 24 h incubation, the L929 cell viability was assayed by the MTT assay (Mosmann 1983). The cytokine concentrations were determined by using a recombinant mouse cytokine to obtain a standard curve correlating cellular viability and TNF-α concentration. The optical density of each well was measured at 570 nm employing a microplate reader.

Macrophage cytotoxicity assay

The cytotoxic effects of plant samples on RAW 264.7 cell viability in cultures stimulated with LPS were determined using the lactate dehydrogenase (LDH) release assay as previously described (Muzitano et al. 2006). First, the cells (5×10⁴ cells/well) were seeded in 96-well microplates and then treated with plant samples at 100, 20, 4 and 0.8 μg/mL for 24 h. Cytoplasmic LDH enzyme release into cell culture supernatants was detected using a commercial LDH kit (Doles, GO, Brazil). Cell lysates obtained through the treatment with 1% Triton X-100 were used as a positive control. DMSO was used as solvent for the sample dilutions, and was tested in parallel as control. Stimulated and untreated cells supernatants were used as a negative control.

Infection of macrophage and quantification of intracellular growth

RAW 264.7 macrophages were plated (1×10⁵ cells/well) in antibiotic-free DMEM-F12 medium supplemented with 10% FBS and incubated for 24 h. Mycobacterial suspensions were sonicated prior to infection for 1 minute to disperse clumps and optical densities were adjusted to 0.1. The macrophage cultures were infected at a multiplicity of infection (MOI) of 1:1 (macrophage:mycobacterium). Phagocytosis was allowed to progress for 3 h. Extracellular mycobacteria were removed after 3 h by washing with PBS 1X and the infected cell monolayers were treated for 4 days with plant samples or rifampicin. Macrophage viability was monitored by LDH assay and was over 80% in all experiments. Cells were lysed on day 4 after infection with 1% saponin to release intracellular bacteria. Lysate aliquots were diluted 10-fold in PBS, plated in triplicate on 7H10 agar plates and incubated at 37 °C. Total CFU were determined after 21 days (Lasunskaia et al. 2010).

Statistical analysis

Statistical analysis was performed using oneway analysis of variance (ANOVA) and the Tukey test for multiple range tests, employing GraphPad Prism 5 software to assess statistical significance between data groups. The results were considered statistically significant for p < 0.05. The half maximal inhibitory concentration (IC₅₀), lowest concentration at which 50% of the growth was inhibited (MIC₅₀) and 50% cytotoxic concentration (CC₅₀) values were calculated by non-linear regression analysis of log[concentration]/inhibition curves by GraphPad Prism 5 Software, applying a sigmoidal dose-response variable slope curve fitting using the different percentages obtained for each corresponding concentration in triplicate and were expressed as means with a corresponding 95% confidence interval.

Results and Discussion

Crude extracts and fractions of Tapirira guianensis, Mandevilla moricandiana, Eremanthus crotonoides (=Vernonia crotonoides), Kielmeyera membranacea, Stachytarpheta schottiana, Vitex polygama and Tocoyena bullata were screened against Mycobacterium bovis BCG and showed mean MIC₅₀ values ranging from 0.95 to greater than 100 μg/mL (Tab. 1). Higher inhibitory activities were observed for crude extract from E. crotonoides (94.64 ± 1.26%), hexane fraction from S. schottiana (97.42 ± 0.01%), dichloromethane fraction from V. polygama, E. crotonoides, M. moricandiana and K. membranacea (91.65 ± 0.54, 93.11 ± 0.07, 93.81 ± 0.12 and 96.83 ± 0.14%, respectively).

The plant species investigated in this study or species of the same genus have traditionally been used and/or reported with antibacterial activity or used for cough and fever and related symptoms for tuberculosis, among other diseases. The compounds isolated from Tapirira guianensis bark have shown activity against Staphylococcus aureus and S. epidermidis (Roumy et al. 2009). A study on the antibacterial activity of northern-peruvian medicinal plants reported Mandevilla trianae Woodson as an effective inhibitor of Escherichia coli (Bussmann et al. 2008). In addition, vine extract from Mandevilla veraguasensis Seem.) Hemsl. was able to boost the antimicrobial activity of human blood against S. aureus (Yaseen et al. 2017).

The extracts and isolated compounds from Vernonia amygdalina Delile flowers showed considerable antibacterial activities against different pathogenic bacteria (Habtamu & Melaku 2018). Vernonia condensata Baker, traditionally used to treat infectious processes and inflammation, also showed promising activity against S. aureus, providing scientific support to ethnopharmacological use (Silva et al. 2018).

Extracts from Kielmeyera variabilis Mart. & Zucc., K. lathrophyton Saddi, K. neglecta Saddi showed antibacterial activity, with the latter species being active against multidrug-resistant bacteria (Pinheiro et al. 2003; Toledo et al. 2011; Sousa et al. 2012). Stachytarpheta indica (L.) Vahl and S. urticifolia (Salisb.) Sims have also had their traditional uses as antimicrobials supported scientifically (Princely et al. 2013; Sreelatha et al. 2014).

Vitex negundo L., traditionally used to treat colds, coughing, and bacterial dysentery was identified as having anti-mycobacterial activity (Gupta et al. 2011). In another work, Vitex species, namely, V. altissima L.f., V diversifolia Kurz ex C.B.Clarke, V. peduncularis Wall. ex Schauer and V. trifolia L., were investigated against bacterial strains, in which V. peduncularis was the most interesting species (Kannathasan et al. 2011). No previous anti-mycobacterial studies have been reported from Tocoyena bullata. However, phytochemical study of this species has been carried out by our group and phenolic compounds showed inhibitory activity in mast cells degranulation (Santos et al. 2019).

The plant species were found to be active against M. bovis BCG in the primary screening, and eleven samples showed more than 80% inhibition and were screened against a virulent strain of Mycobacterium tuberculosis H₃₇Rv. This strain was found to be more sensitive towards three plant species, and the results are compiled in Table 2. The most antimycobacterial activity was observed in the dichloromethane fraction from K. membranacea with 97.40 ± 1.99% of growth inhibition at 100 μg/mL and a MIC₅₀ value of 4.38 ±1.19 μg/mL (Tab. 2).

Table 2
Evaluation of inhibition of Mycobacterium tuberculosis H₃₇Rv growth when treated with extracts and fractions from restinga species.

Botanical name	Samples	Sample code	MIC50±SD(μg/mL)	Maximum inhibition (%)
Mandevilla moricandiana	Dichloromethane	Mm DCM	>100	19.04±2.59
	Ethyl acetate	Mm EtOAc	>100	11.41±4.34
Eremanthus crotonoides	Crude Extract	Vc CE	48.11±1.29	94.38±0.96
	Hexane	Vc HEX	>100	23.66±1.64
	Dichloromethane	Vc DCM	15.28±1.21	94.89±0.60
Kielmeyera membranacea	Dichloromethane	Km DCM	4.38±1.19	97.40±1.99
Stachytarpheta schottiana	Hexane	Ss HEX	>100	34.03±1.26
Vitex polygama	Crude Extract	Vp CE	>100	34.76±1.59
	Hexane	Vp HEX	>100	46.07±3.97
	Dichloromethane	Vp DCM	78.32±1.47	50.47±0.30
	Butanol	Vp BuOH	>100	44.32±5.00

• Antibiotic rifampicin was used at concentrations of 0.008, 0.04, 0.2 and 1 μg/mL, demonstrating 83.92±1.80%, 95.40+0.68%, 98.16±1.49% and 100.13±0.19% growth inhibition, respectively; MIC₅₀ = lowest concentration at which 50% of the growth was inhibited; SD = standard deviation; Maximum inhibition (%) = the highest percentage of inhibition obtained at the concentrations tested.

Antibiotic rifampicin was used at concentrations of 0.008, 0.04, 0.2 and 1 μg/mL, demonstrating 83.92±1.80%, 95.40+0.68%, 98.16±1.49% and 100.13±0.19% growth inhibition, respectively; MIC₅₀ = lowest concentration at which 50% of the growth was inhibited; SD = standard deviation; Maximum inhibition (%) = the highest percentage of inhibition obtained at the concentrations tested.Six samples in our study showed inhibition ≥ 90% at 100 μg/mL against M. bovis BCG and three samples against M. tuberculosis H₃₇Rv. Our findings are in agreement with Tosun et al. (2004), where extracts which do not inhibit M. tuberculosis in at least 200 μg/mL are considered inactive. Active samples should have MIC values ≤ 128 μg/mL (Gu et al. 2004).

The production of pro-inflammatory mediators such as interleukin-1 (IL-1), IL-12, TNF-α and NO by the infected macrophages is generally essential for protection against mycobacteria (Garlanda et al. 2007). However, additional anti-inflammatory therapy is required to prevent excessive inflammation in the cases of severe forms of TB such as military TB or tuberculous meningitis and pericarditis (Zhang et al. 2017). In addition, anti-inflammatory therapy reduces mortality in patients exhibiting hyperinflammatory phenotype which could be determined by host genetic polymorphisms, increased bacterial virulence or specific comorbid states (Critchley et al. 2013).

According to these aspects, the eleven samples previously assayed against M. tuberculosis H₃₇Rv were evaluated for inhibition of NO and TNF-α production in LPS-stimulated RAW 264.7 macrophage culture. All the samples were able to inhibit NO production by more than 50%, and six samples were able to inhibit TNF-α production by more than 50%. The K. membranacea and E. crotonoides species showed potential inhibitory capacity, with all IC₅₀ values showing < 0.8 μg/mL for NO and 4.70 ± 1.84, 5.83 ± 1.40, 1.82 ± 1.77 μg/mL for TNF-α from Km DCM, Ec CE and Vc DCM, respectively (Tab. 3). This modulation could present benefits in treating severe tuberculosis.

Table 3
Inhibitory effects of extracts and fractions from studied species on production of NO and TNF-α by LPS-stimulated RAW 264.7 macrophages, and evaluation of cytotoxicity by LDH test.

Botanical name	Samples	Sample code	NO IC50±SD (μg/mL)	TNF-α IC50±SD (μg/mL)	CC50±SD (μg/mL)
Mandevilla moricandiana	Dichloromethane	Mm DCM	33.80±1.89	>100	90.38±1.32
	Ethyl acetate	Mm EtOAc	62.13±1.84	54.66±1.18	>100
Eremanthus crotonoides	Crude Extract	Vc CE	<0.8	5.83±1.40	>100
	Hexane	Vc HEX	<0.8	55.05±1.17	>100
	Dichloromethane	Vc DCM	0.05±1.34	1.82±1.77	>100
Kielmeyera membranacea	Dichloromethane	Km DCM	<0.8	4.70±1.84	>100
Vitex polygama	Crude Extract	Vp CE	78.23±1.97	>100	>100
	Hexane	Vp HEX	48.51±2.02	>100	>100
	Dichloromethane	Vp DCM	12.91±1.61	>100	>100
	Butanol	Vp BuOH	75.07±1.74	>100	>100

• L-NMMA, a inhibitor of the inducible isoform of nitric oxide synthase (iNOS), at 20 μg/mL showed 95.15±7.33 % of inhibition on NO production; NO = nitric oxide; TNF-α = Tumor necrosis factor-alpha; SD = standard deviation; IC₅₀ = the half maximal inhibitory concentration; CC₅₀ = the 50% cytotoxic concentration.

A previous study with methanol stem extract of Kielmeyera rugosa Choisy demonstrated anti-hyperalgesic and anti-inflammatory effects, and these effects were associated with the inhibition of cytokine cascade generated by carrageenan and/or in decreasing the production of inflammatory mediators by significantly reducing the TNF-α and IL-1β (Melo et al. 2014). This suggests that other species of the Kielmeyera genus display anti-inflammatory activity and also inhibit TNF-α. Furthermore, Vernonia cinerea (L.) Less. species also exhibited strong inhibitory effects on nitric oxide production in LPS-stimulated RAW264.7 cells (Kuo et al. 2018)

The cytotoxic effect was evaluated in macrophages and the samples showed no significant cytotoxicity. The selectivity index (SI) was calculated for the three most active species of K. membranacea, E. crotonoides and V. polygama. The SI values calculated for DCM fractions from these species were > 100, 45 and 10 for M. bovis BCG, and > 20, 5 and 1 for M. tuberculosis H₃₇Rv, respectively. The selectivity index > 10 suggests that it will be safer and the sample can then be evaluated further (Orme 2001).

Mycobacterium bovis BCG in our study was sensitive for 28 samples, and four samples among these were active against M. tuberculosis H₃₇Rv, highlighting three active species of K. membranacea, E. crotonoides and V. polygama. These findings are highly significant since there is an impermeability associated with the complex cell wall structure.

Intracellular growth of M. bovis BCG in infected macrophages was also evaluated. The active dichloromethane fractions from K. membranacea and E. crotonoides at 100 μg/mL decreased mycobacterial growth from 7.7×10⁴ to 2.6×10⁴ CFU/mL (66.23 ± 3.67%) and from 11×10⁴ to 8.5×10⁴ CFU/mL (22.73 ± 1.29%), respectively. The rifampicin (positive control) showed concentration-dependent activity and reduced mycobacterial intracellular growth from 9.35×10⁴ to 0.25×10⁴CFU/mL (97.53 ± 2.47%) at 0.8 μg/mL.

These results importantly enabled us to observe that the antimycobacterial activity was maintained, even when evaluated against intracellular growth. Considering that the antimycobacterial activity of extracts and fractions in infected macrophages has still been little discussed, this may be a relevant assay after initial screening.

These studies of infected macrophages may assist in identifying specific samples able to inhibit M. tuberculosis growth intracellularly, thus improving the ability to identify active compounds and excluding possible interferences from the permeability and conversion of the sample to the host cell, as well as the metabolism of the active compound before bacillus elimination (Sorrentino et al. 2015).

Our findings suggested that bioactivity resides in the dichloromethane fractions of K. membranaceae and E. crotonoides, therefore these two fractions were selected for chemical characterization by gas chromatography coupled with mass spectrometry (CG-MS).

GC-MS chromatogram of the Km DCM showed four peaks, from which one was predominant (Fig. 1) and was identified by comparing with the NIST library. The prevailing compound was a structure of pentacyclic triterpene (89.82%) eluted at 31.82 min (Tab. 4). Ec DCM showed 13 peaks, from which six were predominant (Fig. 2) and were identified using the NIST library (National Institute of Standards Technlogy). The six prevailing compounds were propanoic acid (7.93%) eluted at 4.27 min, hexadecanoic acid (6.22%) eluted at 17.96 min, oleic acid (13.55%) eluted at 19.60 min, octadecanoic acid (7.02%) eluted at 19.82 min, ricinoleic acid (36.10%) eluted at 21.36 min, and most likely a derivative of ricinoleic acid (6.10%) eluted at 23.43 min (Tab. 5).

Figure 1
Chromatographic profile of dichloromethane fraction from Kielmeyera membranacea obtained by gas chromatography coupled with mass spectrometer (GC-MS) after derivatization using N-methyltrimethylsilyltrifluoroacetamide (MSTFA).

Figure 2
Chromatographic profile of dichloromethane fraction from Eremanthus crotonoides obtained by gas chromatography coupled with mass spectrometer (GC-MS) after derivatization using N-methyltrimethylsilyltrifluoroacetamide (MSTFA).

Table 4
Chemical composition of dichloromethane fraction from Kielmeyera membranacea by gas chromatography coupled with mass spectrometer GC-MS.

Peak	tR (min)	Area (% of total)	ID	Similarity (%)
1	24.73	3.26	Squalene	89
2	31.82	89.82	4,4,6a,6b,8a,11,11,14b-octamethyl-1,4,4a,5,6, 6a,6b,7,8,8a, 9,10,11,12,12a, 14,14a,14b-octadecahydro-2H-iceno-3-one	88
3	33.85	3.01	Unidentified	-
4	34.85	3.91	Unidentified	-

Table 5
Chemical composition of dichloromethane fraction from Eremanthus crotonoides by gas chromatography coupled with mass spectrometer GC-MS.

Peak	tR (min)	Area (% of total)	ID	Similarity (%)
1	3.07	6.10	Silanamine, N, 1,1,1-tetramethyl-N-(Trimethylsilyl)	82
2	4.27	7.93	Unidentified	84
3	5.62	3.33	Propanoic acid, 2-[(trimethylsilyl)oxy]	91
4	13.88	2.60	Unidentified	-
5	14.08	1.99	Unidentified	-
6	17.96	6.22	Hexadecanoic acid, trimethylsilyl ester	91
7	19.60	13.55	Oleic acid, trimethylsilyl ester	89
8	19.82	7.02	Octadecanoic acid, trimethylsilyl ester	89
9	19.97	2.71	Unidentified	92
10	21.36	36.10	Ricinoleic acid, trimethylsiloxy, trimethylsilyl ester	96
11	23.43	6.10	Unidentified	85
12	25.11	4.43	Unidentified	-
13	25.62	1.92	Unidentified	-

Thus, the mass spectrometry analyses of dichloromethane fractions indicated the presence of terpenes and fatty acids. These results corroborate with other reports in the literature, which describe anti-mycobacterial activities for the same secondary metabolite classes (Jiménez-Arellanes et al. 2013; Luo et al. 2011; Carballeira 2008).

According to the references of this study, this is the first report with in vitro anti-mycobacterial and anti-inflammatory properties for the studied Restinga species. The obtained results are partial scientific validations for the species upon known ethnopharmacological uses of their genus, highlighting the K. membranaceae and E. crotonoides species.

In summary, anti-mycobacterial activities observed for the species in this study indicate the presence of promising compounds. K. membranaceae and E. crotonoides were the most active species. We have successfully demonstrated that dichloromethane fractions of K. membranaceae and E. crotonoides contain anti-mycobacterial activity which can inhibit both avirulent M. bovis BCG and virulent M. tuberculosis H₃₇Rv strains. They also constitute viable sources for identifying anti-mycobacterial compounds which also present anti-inflammatory properties, and which may be potentially useful for treating severe TB cases.

Acknowledgements

This work was supported by FAPERJ and CNPq research grants.

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Edited by

Area Editor: Dr. Leopoldo Baratto

Publication Dates

• Publication in this collection
  11 June 2021
• Date of issue
  2021

History

• Received
  23 Oct 2019
• Accepted
  20 Apr 2020