Chemical Compounds and Antibacterial Activity of Tephrosia Toxicaria Pers.

Introduction

Tephrosia toxicaria Pers. (Fabaceae), also referred as T. sinapou (Buc´hoz), is a shrub popularly known as “timbó de caiena” in Ceará state (Northeast of Brazil) where it is used as pesticide and fishing poison^1-2. The phytochemical studies of Tephrosia genus revealed compounds with anticinoceptive, larvicidal and antiinflammatory activities^3,4. Previous investigations of T. toxicaria led to the identification of flavonoids, mainly rotenoids^1-2,5-7. In the present work, we report the isolation of eight known compounds, including the chromone, 6,7-dimethoxy-chromone, 1 described for the first time for this genus, the flavonoid, villosinol, 2 which has been previously reported without its ¹³C-NMR data and sumatrol, 3 which has its ¹³C-NMR data corrected. In addition, as a support of use of alternative source in the treatment of bacterial infections, we describe the antibacterial activity of ethanolic extract from its roots and of some its compounds.

Material and Methods

Plant Material

Pods and roots of T. toxicaria Pers. were collected in Guaraciaba do Norte (Ceará state, Brazil). A voucher specimen (#32139) is kept at the Herbarium Prisco Bezerra, Universidade Federal do Ceará – Brazil.

Methods

The powdered pods, air-dried (180.0 g), were extracted with 95% EtOH at and provided the extract TTVE (26.0 g). An aliquot (8.7g) of TTVE was fractionated by Si gel column (Merck 60-120 60 Mesh) using hexane, CH₂Cl₂ and EtOAc as solvents. The fraction eluted with CH₂Cl₂ (69.0 mg) furnished, after further Si gel column, 6.7 mg of 6a,12a-desydro-α-toxicarol⁸, 4. The fraction eluted with EtOAc (800.0 mg) was purified by Si gel column, furnished 19.9 mg of luteolin⁹, 5. The powdered roots (500.0 g) were extracted with ethanol to afford  TTRE (14.2 g). TTRE was chromatographed on a Si gel column with gradient mixture of hexane/CH₂Cl₂. Fractions eluted with hexane/CH₂Cl₂ (1:1) yielded 13.2 mg of obovatin¹⁰, 6, and the fractions eluted with CH₂Cl₂ furnished 9.9 mg of deguelin⁸, 7. Another portion of roots (649.0 g) was extracted in a Soxhlet system with water. After liophilization, the material (5.4 g) was extracted with ethyl acetate and yielded 2.1 g of organic fraction. This fraction was chromatographed on Sephadex LH-20 using EtOAc:MeOH (1:1). The fractions obtained were submitted to HPLC RP-18 (MeOH/formic acid 0.1% 4:1) and provided pure compounds villosinol¹¹ (2, 5.0 mg), 12a-hydroxy-α-toxicarol⁸ (8, 32.8 mg), sumatrol¹² (3, 3.6 mg), α–toxicarol⁸ (9, 13.0 mg), 12a-hydroxyrotenone¹³ (10, 27.8 mg), tephrosin¹⁴ (11, 62.0 mg), and 6,7-dimethoxy-chromone¹⁵ (1, 2.0 mg).

All compounds were identified by ¹H-NMR, ¹³C-NMR and 2D NMR analysis and comparison with those reported in the literature.

Antibacterial Activity

Microorganisms

The following strains used in this study were provided by the Oswaldo Cruz Foundation – FIOCRUZ: K. pneumoniae ATCC 10031; P. aeruginosa ATCC 15442; S. mutans ATCC 0046; S. aureus ATCC 6538. Strains isolated from clinical material of Escherichia coli Ec 27 and Staphylococcus aureus Sa 358 were also used. The bacteria were activated in Brain Heart Infusion (BHI, Himedia laboratories PVT. LTD., Mumbai, INDIA) for 24 h at 35°C.

Antibacterial Test (MIC) and Resistance Modulation Bacterial

MIC (minimal inhibitory concentration) was determined in a microdilution assay¹⁶ utilizing an inoculum of 100 µL of each strain, suspended in BHI broth up to a final concentration of 10⁵ CFU/mL in 96-well microtiter plates, using serial dilutions (1:1). Each well received 100 µL of each (roots ethanolic extract (TTRE) and of the compounds 6, 7, 8, 10 and 11). The concentrations of the extract and organic compounds varied 512 – 8 µg/mL. MICs were recorded as the lowest concentrations required to inhibit growth.

The minimum inhibitory concentration for antibiotics was determined in BHI by the microdilution test, using suspensions of 10⁵ CFU/mL and a drug concentration ranging from 2,500 to 2,4 μg/mL (1:1 serial dilutions). MIC was defined as a lower concentration at which growth was observed. For evaluation of the absence and presence of deguelin (7) and 12a-hydroxy-α-toxicarol (8) in P. aeruginosa and Staphylococcus aureus modulators of antibiotic resistance, a MIC of the antibiotics was determined in the presence or absence of subunits and as plates were incubated for 24 h at 37ºC. Each antibacterial test for MIC determination was performed in triplicate.

Results and Discussions

It is worth to mention that villosinol, 2 has its ¹³C-NMR data being reported for the first time, and that the ¹³C-NMR assignment for sumatrol, 3 was corrected through bidimensional NMR analysis.

Villosinol (2). C₂₃H₂₂O₈, ¹H-NMR (500 MHz, CDCl₃) δ_H: 1.74 (s, 3H, CH₃-8’), 2.84 (dd, J = 15.0 and 7.5 Hz, 1H, H-4’a), 3.20 (dd, J = 15.0 and 9.5 Hz, 1H, H-4’b), 3.76 (s, 3H, 2-OCH₃), 3.83 (s, 3H, 3-OCH₃), 4.18 (s, 1H, 12a-OH), 4.47 (d, J = 12.0 Hz, 1H, H_eq-6), 4.55 (sl, 1H, H-6a), 4.58 (dd, J = 12.0 and 2.5 Hz, 1H, H_ax-6), 4.93 (s, 1H, H-7’), 5.05 (s, 1H, H-7’), 5.20 (t, J = 8.5 Hz, 1H, H-5’), 6.04 (s, 1H, H-10), 6.49 (s, 1H, H-4), 6.71 (s, 1H, H-1), 11.83 (s, 1H, 11-OH). ¹³C-NMR (125 MHz, CDCl₃) δ_C: 194.4 (C-12), 170.4 (C-9), 166.1 (C-11), 156.1 (C-7a), 151.6 (C-3), 148.7 (C-4a), 144.4 (C-2), 143.1 (C-6’), 113.2 (C-7’), 109.7 (C-1), 109.0 (C-1a), 105.0 (C-8), 101.4 (C-4), 100.2 (C-11a), 92.5 (C-10), 88.7 (C-5’), 75.8 (C-6a), 67.1 (C-12a), 63.9 (C-6), 56.7 (2-OCH₃), 56.2 (3-OCH₃), 30.8 (C-4’), 17.3 (C-8’).

Sumatrol (3). C₂₃H₂₂O₇. mp 213.6–215.1 ºC. ¹H-NMR (500 MHz, CDCl3) δ_H: 1.75 (s, 3H, CH₃-8’), 2.86 (dd, J = 15.0 and 8.0 Hz, 1H, H-4’a), 3.24 (dd, J = 15.0 and 9.5 Hz, 1H, H-4’b), 3.79 (s, 3H, 2-OCH₃), 3.82 (s, 3H, 3-OCH₃), 3.85 (d, J = 4.0 Hz, 1H, H-12a), 4.17 (d, J = 12.0 Hz, 1H, Heq-6), 4.59 (dd, J = 12.0 and 3.0 Hz, 1H, Hax-6), 4.88 (t, J = 3.0 Hz, 1H, H-6a), 4.93 (s, 1H, H-7’), 5.06 (s, 1H, H-7’), 5.20 (t, 1H, J = 8.5 Hz, 1H, H-5’), 6.02 (s, 1H, H-10), 6.46 (s, 1H, H-4), 6.87 (s, 1H, H-1), 12.42 (s, 1H, 11-OH). ¹³C-NMR (125 MHz, CDCl3) δ_C: 193.8 (s, C-12), 169.6 (s, C-9), 166.4 (s, C-11), 156.5 (s, C-7a), 150.1 (s, C-3), 147.6 (s, C-4a), 144.4 (s, C-2), 143.2 (s, C-6’), 112.8 (t, C-7’),110.9 (d, C-1), 105.0 (s, C-1a), 104.3 (s, C-8), 101.5 (s, C-11a), 101.4 (d, C-4), 92.1 (d, C-10), 88.4 (d, C-5’), 72.0 (d, C-6a), 66.3 (t, C-6), 56.7 (q, 2-OCH3), 56.2 (q, 3-OCH3), 44,0 (d, C-12a), 30.9 (t, C-4’), 17.3 (q, C-8’).

Figure 1: Compounds isolated from T. toxicariaClick here to View figure

 

In vitro Antibacterial Activity

The MICs of Tephrosia toxicaria extract, TTRE, obovatin, (6), deguelin (7), 12a-hydroxy-α-toxicarol (8), 12a-hydroxy-rotenone (10), and tephrosin (11) against six bacterial strains are presented in Table 1. All tested compounds and extract showed antimicrobial activity against Gram-positive and Gram-negative bacteria, with the best effect of 12a-hydroxy-α-toxicarol against to the grown of Gram-positive S. aureus 358 with MIC 256 µg/mL, while Deguelin is responsible for the best result, the Gram-negative bacteria, P. aeruginosa was inhibited at 64 µg/mL.

Table 2 shows the results concerning the modulation tests of bacterial resistance to aminoglycosides. When 7 and 8 compounds are combined with the antibiotic amikacin, tested against P. aeruginosa and S. aureus strains, the isolated and combined MIC values were the same. Synergism was observed in the combinations of 7 with gentamicin against the two bacterial strains used in the test and in the combination of 7 with neomycin against P. aeruginosa, characterized by reduction of MIC by 50% compared to MIC of the antibiotics tested alone. Only the 8 combination with neomycin against S. aureus showed antagonism, increasing the MIC by 50% compared to the MIC of the neomycin tested alone.

Some natural products of origin plant and phytochemicals are known to have antimicrobial properties, which may be of great importance in treatment against infections. With the increased incidence of antibiotic resistance, alternative natural plant products may be of interest. ¹⁷. Several studies have been conducted in different countries, demonstrating the efficacy of this type of treatment. Many natural products of plants were evaluated not only for direct antimicrobial activity, but also as resistance modifying agents^18,19.

Various chemical compounds (synthetic or natural) have direct antibacterial activity against many species, expanding the activity of an antibiotic, reversing the natural resistance of bacteria to specific antibiotics, causing the inhibiting the active efflux of antibiotics through the plasma membrane and / or elimination of plasmids. Potentiation of antibiotic activity or reversion of resistance to antibiotics allows the classification of these compounds as modifiers of antibiotic activity^20,21.

The mechanism described as “synergistic multi-effect targeting” or “Herbal shotgun” is a possible strategy that explains modulatory effects and refers to the use of plants and drugs in an approach using various-substance combinations, which may not only affect a single target, but several ones, where the different therapeutic components contribute in a synergistic-agonistic effect. This approach is not only for combinations of extracts: Combinations between complex mixtures (extracts and/or oil) and compounds chemical isolated synthetic or naturals or antibiotics are also possible ^22,23.

Table 1: Values of the minimal inhibitory concentration (MIC) (μg/mL) of Tephrosia toxicaria extract and obovatin (6), deguelin (7), 12a-hydroxy-α-toxicarol (8), 12a-hydroxy-rotenone (10), and tephrosin (11)

	E. coli (27)	K. pneumoniae (10031)	P. aeruginosa (15442)	S. mutans (0046)	S. aureus (6538)	S. aureus (358)
TTRE	512	512	≥1024	512	512	≥1024
6	≥1024	≥1024	≥1024	512	512	≥1024
7	≥1024	512	64	≥1024	512	512
8	512	512	≥1024	≥1024	512	256
10	512	512	512	512	512	≥1024
11	512	512	512	≥1024	512	≥1024

 

Table 2: MIC values (μg/mL) of aminoglycosides in the absence and presence of compounds 7 and 8 in P. aeruginosa and Staphylococcus aureus.

Antibiotics	P. aeruginosa (15442)		S. aureus (358)
	MIC alone	MIC combined (7)	MIC alone	MIC combined (8)
Gentamicin	64	32	64	32
Amicacin	32	32	64	64
Neomycin	32	16	32	64

 

Conclusion

In conclusion, the prospective health-promoting effects of plant secondary metabolites have encouraged the research about their chemical constitution and their potential to treat several diseases, among them bacterial infection. The plant substances and their extracts display rarely toxic side effects when compared in treatments with conventional drugs, so these results could be leads for the development of new antibacterial agent, or the compounds from T. toxicaria even may be used associates with standard antibiotics.

Authors’ Contribuitions

FRLS, MVST, JNV, JQL (Master and PhD sudents), IGP (Graduate student) contributed running the laboratory work and drafted the paper; AMCA, JM, MRS, RBF, GMPS did the analysis and interpretation of data of RMN, critical revision of the manuscript. JGMC, EFFM and FFGR contributed with the microbiological tests. All the authors have read the final manuscript and appoved the submission.

Acknowledgement

The authors are grateful to CNPq, CNPq/Pronex and CAPES for financial support and fellowships.

References

1. Vasconcelos, J. N.; Lima, J. Q.; Lemos, T. L. G.; Oliveira, M. C. F.; Almeida, M. M. B.; Andrade-Neto, M.; Mafezoli, J.; Arriaga, A. M. C.; Santiago, G. M. P.; Braz-Filho, R. Quim. Nova, 2009, 32, 382-386
   CrossRef
2. Vasconcelos, J. N.; Santiago, G. M. P.; Lima, J. Q.; Mafezoli, J.; Lemos, T. L. G.; Silva, F. R. L.; Lima, M. A. S.; Pimenta, A. T. A.; Braz-Filho, R.; Arriaga, A. M. C.; Cesarin-Sobrinho, D. Quim Nova, 2012, 35, 1097-1100
   CrossRef
3. Chen, Y.; Yan, T.; Gao, C.; Cao, W.; Huang, R. Molecules, 2014, 19, 1432-1458
   CrossRef
4. do Val, D. R.; Bezerra, M. M.; Silva, A. A. R.; Pereira, K. M. A.; Rios, L. C.; Lemos, J. C.; Arriaga, N. C.; Vasconcelos, J. N.; Benevides, N. M. B.; Pinto, V. P. T.; Cristino-Filho, G.; Brito, G. A. C.; Silva, F. R. L.; Santiago, G. M. P.; Arriaga, A. M. C.; Chaves, H. V. European Journal of Pain, 2014,  18, 1280-1289
   CrossRef
5. Pancharoen, O.; Petveroj, S.; Phongpaichit, S. Songklanakarin J. Sci Technol. 2007, 29, 151-156
6. Arriaga, A. M. C.; Malcher, G. T.; Lima, J. Q.; Magalhães, F. E. A.; Gomes, T. M. B. M.; Oliveira, M. C. F.; Andrade-Neto, M.; Mafezoli, J.; Santiago, G. M. P. J. Essent. Oil Res. 2008, 20, 450-451
   CrossRef
7. Arriaga, A. M. C.; Lima, J. Q.; Vasconcelos, J. N.; Oliveira, M. C. F.; Andrade-Neto, M.; Santiago, G. M. P.; Uchoa, D. E. A.; Malcher, G. T.; Mafezoli, J.; Braz-Filho, R. Magnetic Resonance in Chemistry, 2008, 47, 537-540
   CrossRef
8. Andrei, C. C.; Vieira, P. C.; Fernandes, J. B.; da Silva, M. F. G. F.; Rodrigues Filho, E. Phytomchemistry, 1997, 46, 1081-1085
   CrossRef
9. Agrawal, P. K. Carbon-13 NMR of flavonoids; Elsevier, 1989
10. Andrei, C. C.; Ferreira, D. T.; Faccione, M.; de Moraes, L. A. B.; de Carvalho, M. G.; Braz-Filho, R. Phytochemistry, 2000, 55, 799-804
    CrossRef
11. Krupadanam, G. L. D.; Sarma, P. N.; Srimannarayana, G.; Subbaroa, N. V. Tetrahedron Lett., 1977, 24, 2125-2128
    CrossRef
12. Jang, D. S.; Park, E. J.; Kang, Y.; Hawthorne, M. E.; Vigo, J. S.; Graham, J. G.; Cabieses, F.; Fong, H. H. S.; Mehta, R. G.; Pezzuto, J. M.; Kinghorn, A. D.; J. Nat. Prod., 2003, 66, 1166-1170
    CrossRef
13. Phrutivorapongkul, A.; Lipipun, V.; Ruangrunngsi, N.; Watanabe, T.; Ishikawa, T. Chem. Pharm. Bull., 2002, 50, 534-537
    CrossRef
14. Ahmad, V. U.; Ali, Z.; Hussauni, S. R.; Iqbal, F.; Zahid, M.; Abbas, M.; Saba, N. Phytochemical Commun., 1999, 70, 443-445
15. Romussi, G.; Ciarallo, G. J. Heterocyclic Chem., 1976, 13, 211-220
    CrossRef
16. Javadpour, M. M.; Juban, M. M.; Jo, W. C.; Bishop, S. M.; Alberty, J. B.; Cowell, S. M.; Becker, C. L.; McLaughlin, M. L. J. Med. Chem., 1996, 39, 3107-3113
    CrossRef
17. Matias, E. F. F.; Santos, K. K. A.; Almeida, T. S.; Costa, J. G. M.; Coutinho, H. D. M. Rev. Bras. Biocienc. 2010, 8, 294-298
18. Matias, E. F. F.; Alves, E. F.; Santos, B. S.; Souza, C. E. S.; Ferreira, J. V. A.; Lavor, A. K. L. S.; Figueredo, F. G.; Lima, L. F.; Santos, F. A. V.; Peixoto, F. S. N.; Colares, A. V.; Boligon, A. A.; Saraiva, R. A.; Athayde, M. L.; Rocha, J. B. T.; Menezes, I. R. A.; Coutinho, H. D. M.; Costa, J. G. M. J. Evid. Based. Complementary Altern. Med., 2013a, 1-7
19. Matias, E. F. F.; Santos, K. K. A.; Falcão-Silva, V. S.; Siqueira-Júnior, J. P.; Costa, J. G. M.; Coutinho, H. D. M. Indian J. Med. Res., 2013b, 137, 178-182
20. Coutinho, H. D. M.; Costa, J. G. M.; Lima, E. O.; Falção-Silva, V. S.; Siqueira-Júnior, J. P. BMC Complement. Altern. Med. 2009a, 9, 13
    CrossRef
21. Coutinho, H. D. M.; Costa, J. G. M.; Lima, E. O.; Siqueira-Júnior, J. P. Comp. Immunol. Microbiol. Infect. Dis., 2010, 33, 467-471
    CrossRef
22. Hemaiswarya, S. H.; Kruthiventi, A. K.; Doble, M. Phytomedicine, 2008, 15, 639-652
    CrossRef
23. Wagner, H.; Ulrich-Merzenich, G. Phytomedicine, 2009, 16, 97-110
    CrossRef

---

This work is licensed under a Creative Commons Attribution 4.0 International License.