Antimycobacterial and anti-inflammatory activities of fractions and substances from <i>Erythrina verna</i> Vell focusing on dual severe TB treatment approach

SIMÃO, THATIANA L.B.V.; AGUIAR, GABRIELA V.; RAMOS, AMARO C.; SILVA, GLAUBER P. DA; MUZITANO, MICHELLE F.; LASSOUNSKAIA, ELENA; OLIVEIRA, RODRIGO R. DE

doi:10.1590/0001-3765202220211032

person THATIANA L.B.V. SIMÃO

schoolUniversidade Estadual do Norte Fluminense Darcy Ribeiro, Laboratório de Biologia do Reconhecer, Centro de Biociências e Biotecnologia, Av. Alberto Lamego, 2000, Parque Califórnia, 28013-602 Campos dos Goytacazes, RJ, BrazilschoolUniversidade Federal do Rio de Janeiro, Laboratório de Produtos Bioativos, Campus Macaé, Av. Aluizio da Silva Gomes, 50, Novo Cavaleiros, 27930-560 Macaé, RJ, Brazil

0000-0001-7563-1833

person GABRIELA V. AGUIAR

schoolUniversidade Estadual do Norte Fluminense Darcy Ribeiro, Laboratório de Ciências Químicas, Centro de Ciência e Tecnologia, Av. Alberto Lamego, 2000, Parque Califórnia, 28013-602 Campos dos Goytacazes, RJ, Brazil

0000-0002-8542-0172

person AMARO C. RAMOS

schoolUniversidade Estadual do Norte Fluminense Darcy Ribeiro, Laboratório de Ciências Químicas, Centro de Ciência e Tecnologia, Av. Alberto Lamego, 2000, Parque Califórnia, 28013-602 Campos dos Goytacazes, RJ, Brazil

0000-0001-9850-2065

person GLAUBER P. DA SILVA

schoolUniversidade Estadual do Norte Fluminense Darcy Ribeiro, Laboratório de Ciências Químicas, Centro de Ciência e Tecnologia, Av. Alberto Lamego, 2000, Parque Califórnia, 28013-602 Campos dos Goytacazes, RJ, Brazil

0000-0001-93889440

person MICHELLE F. MUZITANO

schoolUniversidade Federal do Rio de Janeiro, Laboratório de Produtos Bioativos, Campus Macaé, Av. Aluizio da Silva Gomes, 50, Novo Cavaleiros, 27930-560 Macaé, RJ, Brazil

0000-0003-0155-3240

person ELENA LASSOUNSKAIA

schoolUniversidade Estadual do Norte Fluminense Darcy Ribeiro, Laboratório de Biologia do Reconhecer, Centro de Biociências e Biotecnologia, Av. Alberto Lamego, 2000, Parque Califórnia, 28013-602 Campos dos Goytacazes, RJ, Brazil

0000-0002-6726-273X

person RODRIGO R. DE OLIVEIRA

schoolUniversidade Estadual do Norte Fluminense Darcy Ribeiro, Laboratório de Ciências Químicas, Centro de Ciência e Tecnologia, Av. Alberto Lamego, 2000, Parque Califórnia, 28013-602 Campos dos Goytacazes, RJ, Brazil

0000-0002-3145-4844

Correspondence to: Rodrigo Rodrigues de Oliveira E-mail:
roroliveira@uenf.br

AUTHOR CONTRIBUTIONS

Thatiana Lopes Biá Ventura Simão: Conceptualization, Methodology, Validation, Investigation, Analysis and/or interpretation of biological data and writing (original draft and review & editing). Gabriela Vigneron Aguiar: Conceptualization, Methodology and Investigation of the Phytochemistry study. Amaro Chaves Ramos: Advising of the counter-current chromatography analysis and interpretation of NMR data. Glauber Pereira da Silva: Methodology and Investigation of the Phytochemistry study. Michelle Frazão Muzitano: Conceptualization, Methodology, Investigation, Analysis and/or interpretation of biological data and writing. Elena Lassounskaia: Conceptualization, Methodology, Investigation, Supervision of the biological study and Resources and funding acquisition. Rodrigo Rodrigues de Oliveira: Conceptualization, Methodology, Investigation, Supervision of the Phytochemistry study, Resources and funding acquisition and writing (original draft and review & editing).

Figures (4)
Tables (2)

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Figure 1 Chemical structures of compounds 1 (erythratidinone), 2 (alpinumisoflavone) and 3 (erysenegalensein M).

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Figure 2 Effect of isolated substances from Erythrina verna on production of proinflammatory mediators (NO and TNF-α) by LPS-stimulated RAW 264.7 macrophages. Macrophages RAW 264.7 were plated and stimulated with 1.0 µg/mL de LPS for 24 h at 37 ºC with or without isolated substances from E. verna (0.8, 4, 20 and 100 µg/mL). (a) Nitrite was determined by the method of Griess reaction. Macrophages not treated and not stimulated with LPS were used as negative control; and macrophages stimulated with 1 μg/mL LPS (56.1 ± 1.5 µg/mL) and not treated as positive control. L-NMMA was also used as positive control at 20 μg/mL inhibiting 51.4 ± 2.4 % NO production. (b) In the TNF-α assay, TNF-α values determined in untreated and only LPS-treated cell cultures were 1.1 ± 0.1 pg/mL and 1428.6 ± 6.6 pg/mL, respectively. The results presented are mean values obtained over three experiments, each done in triplicate. *** p < 0.001, ** p < 0.01 and * p < 0.05 compared to untreated group (0 μg/mL) determined by Tukey test.

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Figure 3 Effect of isolated substances from Erythrina verna on M. tuberculosis growth in bacterial culture. Bacterial suspensions (1 × 10⁶ CFU/well) of M. tuberculosis strain H37Rv (a) and clinical isolate M299 (b) were treated or untreated with substances (0.8, 4, 20 and 100 µg/mL) or rifampicin (0.00032, 0.0016, 0.008, 1 µg/mL for the strain H37Rv and 0.008, 0.04, 0.2 and 10 µg/mL for the strain M299) for 5 days. Bacterial growth in the resulted cultures was quantified by MTT test. Data are presented as a percentage of bacterial growth of each treated culture compared to the growth of corresponding untreated culture (100%). The bars for each substance refer to concentrations tested in ascending order. The results presented are mean values obtained over three experiments, each done in triplicate. *** p < 0.001 compared to untreated bacterial suspension (0 μg/mL) determined by Tukey test. Bacterial suspensions treated with antibiotic rifampicin were used as a positive control.

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Figure 4 Effect of isolated substances and most active fractions from Erythrina verna on M. tuberculosis growth in macrophages. RAW 264.7 macrophages were infected with M. tuberculosis strain H37Rv at the infection ratio of 1:1(macrophage:mycobacteria) and treated with samples for 4 days. Bacterial intracellular viability after the treatment was determined by bacterial CFU counts. Statistical analyses were calculated and compared to untreated group (*** p < 0.001, ** p < 0.01 and * p < 0.05). Minimum inhibitory concentrations of samples on M. tuberculosis growth in macrophages were calculated and values in the same column with different superscript letters (a-c) are significantly different (p < 0.05 or p < 0.001; determined by Tukey test). Values are reported as mean value ± SD (n = 3).

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Table I Inhibitory effects of Erythrina verna fractions on production of NO and TNF-α by LPS-stimulated RAW 264.7 macrophages, on growth of M. bovis BCG, M. tuberculosis H37Rv and clinical isolate M. tuberculosis M299 in culture and evaluation of cytotoxicity by MTT test.

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Table II Inhibitory effects of isolated substances from Erythrina verna on production of NO and TNF-α by LPS-stimulated RAW 264.7 macrophages, evaluation of cytotoxicity and on growth of the laboratory M. tuberculosis strain H37Rv and clinical isolate M. tuberculosis M299.

Fractions E.verna	IC₅₀ (µg/mL)	CC₅₀ (µg/mL)		MIC₅₀ (µg/mL)
Fractions E.verna	NO	TNF-α	MTT	Mbv BCG	Mtb H37Rv	Mtb M299
1	12.8 ± 2.3^a,^b	>100	>100	> 100	> 100	XX
2	5.1 ± 0.4^c,^d	4.7 ± 1.6 a	18.2 ± 1.3	67.8 ± 1.1	82.6 ± 1.2^a	XX
3	4.8 ± 1.6^c,^d	>100	91.1 ± 0.8^a	54.3 ± 0.6	78.9 ± 1.1^a	XX
4	9.2 ± 1.9^b	24.7 ± 1.2	>100	14.6 ± 2.3	15.8 ± 1.8^b	XX
5	2.5 ± 0.2^d	13.4 ± 1.0	>100	31.7 ± 1.9	48.6 ± 0.9	XX
6	3.4 ± 1.2^c,^d	89.7 ± 2.3	4.3 ± 0.2	5.4 ± 0.2 a	20.1 ± 2.3^b	XX
7	3.5 ± 1.0^c,^d	>100	91.3 ± 1.2^a	72.9 ± 1.1	> 100	XX
8	4.1 ± 0.9^c,^d	>100	>100	21.5 ± 0.5	> 100	XX
9	2.4 ± 0.1^d	>100	>100	6.1 ± 0.1^a	10.1 ± 2.1^c	XX
4.1	18.1 ± 0.3	>100	>100	85.2 ± 1.3	>100	>100
4.2	12.4 ± 1.3^a,^b	17.7 ± 0.3	>100	90.1 ± 2.2	>100	>100
4.3	>100	>100	>100	> 100	>100	>100
4.4	>100	>100	>100	> 100	>100	>100
4.5	>100	>100	>100	> 100	>100	>100
4.6	>100	>100	>100	99.1 ± 2.4	>100	>100
4.7	37.2 ± 1.9	>100	>100	2.5 ± 0.4^a,^b	16.8 ± 0.1^b	20.5 ± 0.5
4.9	6.8 ± 1.5^d	5.3 ± 0.7^a	42.8 ± 1.2	2.6 ± 1.8^a,^b	2.7 ± 1.7^d	2.8 ± 1.7^a
L-NMMA¹	27.1 ± 2.6	XX	XX	XX	XX	XX
Rifampicin²	XX	XX	XX	0.01 ± 0.03^b	0.2 ± 0.01^d	3.3 ± 0.2^a

Substances	IC₅₀ (µg/mL)		CC₅₀ (µg/mL)	MIC₅₀ (µg/mL)
Substances	NO	TNF-α	MTT	Mtb H37Rv	Mtb M299
Erythratidinone	5.2 ± 1.1^a	5.9 ± 1.2^a	>100	3.4 ± 1.2	2.6 ± 1.2^a
Alpinumisoflavone	24.2 ± 1.0	17.7 ± 1.1	95.9 ± 1.1	9.7 ± 1.1	16.8 ± 1.1^b
Erisenegalensein M	4.6 ± 1.0^a	5.9 ± 1.0^a	11.3 ± 1.2	6.5 ± 1.0	18.3 ± 1.1^b
L-NMMA¹	27.1 ± 2.6	XX	XX	XX	XX
Rifampicin²	XX	XX	XX	0.2 ± 0.01	1.0 ± 0.2^a

[tfnI] ¹Standard nitric oxide inhibitor.²Standard antimycobacterial drug. XX—not defined. Values in the same column with different superscript letters (a–d) are significantly different (p < 0.05, p < 0.01or p < 0.001); determined in Tukey test. Values are reported as mean value ± SD (n = 3).

Antimycobacterial and anti-inflammatory activities of fractions and substances from Erythrina verna Vell focusing on dual severe TB treatment approach