Indian Journal of Traditional Knowledge Vol.12 (4), October 2013, pp 619-622
Evaluation of Antimicrobial and Modulatory activity of the extract of Richardia brasiliensis Gomes Edson C Morais¹, Lavouisier F B Nogueira¹, Jéssica L A Leite, Larissa S M Alencar Bruno F F Lucena¹, Fernando G Figueredo¹, Glaucia M M Guedes², Saulo R Tintino², Celestina E S de Souza², Maria F B M Braga², Micheline A Lima3, Fábio H T Souza3, Celidarque da Silva Dias3 & *Henrique D M Coutinho² ¹Faculdade de Ciências Aplicadas Doutor Leão Sampaio, Juazeiro do Norte-CE, Brasil; ²Laboratório de Microbiologia e Biologia Molecular, Universidade Regional do Cariri, Crato-CE, Brasil; 3Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, João Pessoa-PB, Brasil E-mail: *
[email protected] Received 24.06.13, revised 06.08.2013 The emergence of resistant microorganisms and also the toxicity associated with antimicrobial drugs increase the need of research for new active principles. Richardia brasiliensis, a weed used popularly as an expectorant, antiemetic, and diaphoretic. The extracts have coumarins, flavonoids, steroids, triterpenoids, alkaloids and resin, as secondary metabolites. The present study aimed to test the potential antimicrobial and modulator of the ethanolic and hexanic extracts of R. brasiliensis. The ethanolic and hexanic extracts were tested for their antimicrobial effect and in combination with aminoglycosides and antifungal against standard and multi-resistant microorganisms by the broth microdilution method with culture medium Brain Heart Infusion (BHI). It was observed that the association between antibiotics and ethanolic and hexanic extracts showed clinically relevant results on the tests with multi-resistant bacteria. The natural products from R. brasiliensis demonstrated a modulating action against the microorganisms used. These results can represent a new effort to combat antibiotic resistant bacteria. Keywords: Richardia brasiliensis, Antimicrobial activity, Modulatory effect, Multi-resistant microorganisms. IPC Int. Cl.8: A61K 36/00, C12N, C12M, H03K 7/00
Medicinal plants have been used by man since antiquity as a means to alleviate or even cure diseases, this knowledge was passed from generation to generation, especially after the creation of writing. With the development of new technologies could further study of these plants, isolating substances responsible for its pharmacological effect and thus creating drugs with the active principle purified and improved1. Richardia brasiliensis is a plant belonging to the Rubiaceae family which includes about 637 genera and nearly 10,700 species. It is one of the largest families between the dicotyledons, being abundant in subtropical regions worldwide2. Popularly is known as the white poaia, poaia-of-the-field or poaia. It is an annual plant, herbaceous, prostate, branched, stem densely hirsute, measuring 20-50 cm in length. It has marked presence in the agricultural regions of the Midwest, South and Southeast of Brazil3-5. R. brasiliensis is popularly used in Brazil as —————— *Corresponding author
infusion or decoction of the root as an expectorant, antiemetic, and diaphoretic while plants of the same family are also used as anti-inflammatory and to treat hemorrhoids, coughs, bronchitis, and headache6. In the phytochemical prospection of the aerial parts (stalk fragments, petiole and leaf), was observed the presence of coumarins, flavonoids, steroids, terpenoids, alkaloids and resins. In the underground part were found the same substances from the top except for flavonoids and alkaloids7. Pinto et al.8 isolated phytocompounds from R. brasiliensis as Isorhamnetin-3-O-rutinoside, Oleanolic acid, acid p-Hydroxy-benzoic, m-Methoxy-p-hydroxy-benzoic acid and Scopoletin. All classes of phytocompounds indicated by the literature presented several biological activities, demonstrating the necessity of more pharmacognostic and ethnobiological studies. The aim of this study was to evaluate the potential antimicrobial and modulator of ethanolic and hexanic extracts of the leaves of R. brasiliensis against pathogenic microorganisms.
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Methodology Microorganisms
The microorganisms used in testing were obtained from National Institute for Quality Control in Health (INCQS) of the Oswaldo Cruz Foundation, Ministry of Health, were used three standard strains of bacteria, Escherichia coli ATCC10536; Staphylococcus aureus ATCC25923; Pseudomonas aeruginosa ATCC15442. To evaluate the modulatory activity of the natural products were used the following bacterial isolates multi-resistant: Pseudomonas aeruginosa 22, Escherichia coli 27 and Staphylococcus aureus 358 with resistance profiles tested. The fungal strains used were: the standard strain Candida tropicalis ATCC 40042 and Candida krusei ATCC 2538. Both for antimicrobial evaluation is to evaluate the modulator effect. All strains were maintained in culture medium Heart Infusion Agar – HIA (Difco Ltda) and before testing the cells were cultured for 24 hrs at 35°C using media Brain Heart Infusion Agar – BHI (Difco laboratories Ltda). Plant material
Leaves of R. brasiliensis were collected in the city of Joao Pessoa, Paraiba, Brazil. The plant material was identified by Dr. Maria de Fátima Agra and one voucher specimen was deposited in the herbarium Prof. Lauro Pires Xavier with a voucher number #Agra et al 3195#. Preparation of ethanol extract and fractions of the Richardia brasiliensis leaves
Fresh leaves of R. brasiliensis (275 gm) were ground and macerated and the en gave into extraction with ethanol and hexane solvents for a period 72 hrs. The solvent was distillated in a rotary evaporator at 80°C under reduced pressure. The extracts obtained were weighed (10.2 and 11.4 gm, respectively) and stored at room temperature. Drugs utilized
The antibiotics used in the tests were the aminoglycosides: amikacin, gentamicin and neomycin (Sigma Co., St. Louis, USA). Antifungal drugs used were: Amphotericin B, Nystatin, Mebendazole and Benzoilmetronidazole. All solutions were prepared using sterile water following the manufacturer's recommendations. Minimal Inhibitory Concentration (MIC)
The method used was the broth microdilution. The ethanolic and hexanic extracts of R. brasiliensis were
dissolved using Dimethylsulfoxide (DMSO) and diluted to a concentration of 1024 µg/mL using sterile water. The bacteria inoculates were diluted using BHI 10% to a final concentration of 105 UFC/mL. It was used 100 µL of the inoculum into each well of a 96-well microdilution plate and, after that, submitted to a dilution of 1:2 in series using 100 µL of the extract, varying concentrations between 512 a 8 µg/mL. The microdilution plates were incubated for 24 hrs at 35°C9. The reading of MIC was made using the resazurin indicator, while the fungal tests reading was verified absence or presence of turbidity. The MIC was determined as the lowest concentration where no growth of the microorganism was observed, according to NCCLS10. Modulation tests with antimicrobials
To verify if the natural product could alter the action of antimicrobial drugs against the strains tested, was used the method proposed by Coutinho et al.11. The ethanolic and hexanic extracts of R. brasiliensis were tested in sub inhibitory concentration (MIC/8=128µg/mL). Were distributed 100 µL of the solution containing BHI with the inoculum and the extract in each well. After this, 100 µL of the antimicrobial was mixed with the solution on the first well, after dilution of 1:2, varying the concentrations of the aminoglycosides and antifungal between 2500 to 2.44 µg/mL and 512 to 2 µg/mL, respectively. Results After testing the minimum inhibitory concentration with bacteria and with fungi were found results ≥ 1024 µg/mL for both ethanol extract and hexane extract. However, both ethanol extract and hexane extract showed modulation levels clinically relevant against multi-resistant bacterial strains S. aureus 358 and E. coli 27. In the study of the modulatory activity, both the ethanol extract and the hexane extract against multi-resistant bacterial strains was observed a 9.8 µg/mL of MIC when the natural product was associated with Neomycin and 2.44 µg/mL associated with Gentamicin against E. coli 27. Against S. aureus 358 was observed 19.53 µg/mL of MIC in combination with Amikacin and 2.44 µg/mL with Gentamicin. It was not observed modulating effect clinically relevant against P. aeruginosa (Table 1).
MORAIS et al: ANTIMICROBIAL AND MODULATORY ACTIVITY OF RICHARDIA BRASILIENSIS GOMES
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Houghton et al. (MIC ≥ 1024 µg/mL)17. Results are divergent from Figueiredo et al.18. These divergent results probably occur due the fact of plants were been reaped in different regions and also the use of different methodologies, as disk diffusion. The Discussion maximum concentration of the antimicrobial agent In the phytochemical prospection of the aerial parts used in this study was 512 µg/mL, this concentration of R. brasiliensis were found flavonoids, steroids, was a bit more higher of the lowest MIC that was triterpenoids, alkaloids and resin. Studies show that found by Figueiredo et al.18. According to Greger & secondary metabolites like flavonoids and terpenes Hadacek19 the methodology used in the study cited have an antimicrobial12,13. In species, Coussarea above is not well accepted because it’s not possible to platyphylla Mull. Arg also shows the presence of precise the quantity of natural product that spreads terpenes and alkaloids14, as was shown the presence through the agar. of triterpenes and Phytosterol in Guettarda 15 The resistance of the microorganisms occurs by grazielae . Steroids have also been isolated from the 16 several mechanisms like efflux pump, production of specie R. grandiflora . enzymes that inactivate the drug and change the target The ethanol and hexanic extracts showed no of action of the antibiotic. The resistance mechanisms antimicrobial activity clinically relevant before the are transmitted through exchange of genetic material tested strains of fungi and bacteria according between microorganisms of the same species20. Table 1—Test result for modulation of bacterial Many studies have shown that extracts and resistance (µg/mL) essential oils from plants can modify the activity of EERB + HERB + Antibiotic antimicrobial and thereby improve its performance by Escherichia coli 27 antibiotic antibiotic alone decreasing the concentration necessary for there to be Amikacin 39.1 39.1 156.2 growth inhibition. Fact demonstrated with the plants Neomycin 9.8 9.8 78.1 Turnera ulmifolia, Momordica charantia, Mentha Gentamicin 2.4 2.4 9.8 arvensis, Cordia verbenaceae21,22. The results indicate Staphylococcus aureus 358 the potential of this and other natural products for Amikacin 19.5 19.5 78.1 future clinical and in-vivo assays. These assays must Neomycin 19.5 9.8 19.5 be directed using information from the traditional Gentamicin 2.4 2.4 19.5 knowledge and after an appropriate study about the Pseudomonas aeruginosa 22 toxicity and chemical prospection. Amikacin 156.2 321.5 312.5 The tests of modulation showed with the bacteria Neomycin 156.2 156.2 312.5 modulating effect clinically relevant against S. aureus Gentamicin 19.5 19.5 39.1 (associated with gentamicin) and E. coli (associated EERB = Ethanol extract of Richardia brasiliensis. HERB = with neomycin and gentamicin). This effect is Hexane extract of Richardia brasiliensis. probably coming from the secondary metabolites Table 2- Test result for modulation of fungal resistance (µg/mL). present in the plant under study. The capacity that Candida tropicallis EERB + HERB + Antibiotic natural products have to modify the action of ATCC40042 antibiotic antibiotic alone antimicrobials can be seen from studies that show that Mebendazole ≥ 1024 ≥ 1024 ≥ 1024 association of synthetic drugs and plant extracts can Amphotericin B ≥ 1024 ≥ 1024 ≥ 1024 act reversing microbial resistance eliminating Nystatin ≥ 1024 ≥ 1024 ≥ 1024 plasmids and inhibiting the efflux pump22,23. Benzoilmetronidazol ≥ 1024 ≥ 1024 ≥ 1024 Bioprospecting, a vital step in the pharmaceutical Candida krusei production process, is also one of the most ATCC2538 controversial and socially complex aspects in the Mebendazole ≥ 1024 ≥ 1024 ≥ 1024 pharmaceutical industry. The use of the traditional Amphotericin B ≥ 1024 ≥ 1024 ≥ 1024 knowledge for this process have the aim to alleviate Nystatin ≥ 1024 ≥ 1024 ≥ 1024 social inequalities by providing access to end Benzoilmetronidazol ≥ 1024 ≥ 1024 ≥ 1024 24 EERB = Ethanol extract of Richardia brasiliensis. HERB = Hexane products, such as new drugs or biotechnologies . So, extract of Richardia brasiliensis. countries rich in biodiversity can develop these The modulation tests with fungi Candida tropicalis and Candida krusei showed MIC ≥1024 µg/mL in all of the tests (Table 2).
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resources to combat social and environmental problems. This is one of the most important role of the studies of the folk medicine and the traditional knowledge. Conclusion The extracts of R. brasiliensis demonstrated results with clinical relevance showing moderate modulation effect against S. aureus 358 and E. coli 27. Being this a pioneering study on the modulating effect of R. brasiliensis. Deepest research about its secondary metabolites could help to elucidate which one of these metabolites is responsible for such effects. Conflict of Interest There is no conflict of interest Acknowledgment The authors are grateful to the institutions FALS and URCA, where all the tests were performed, and to the Brazilian research support agencies CNPq and FUNCAP References 1 Schenkel EP, Gosmann G & Petrovick PR, Produtos de origem vegetal e o desenvolvimento de medicamentos, In: Simões CMO, Schenkel EP, Gosmann G, Mello JCP, Mentz LA & Petrovick PR. (org) Farmacognosia: da planta ao medicamento, 3 edn, (Editora da Universidade UFRGS/ Editora da UFSC, Porto Alegre/Florianópolis), 2001. 2 Pereira MS & Barbosa MRV, A família Rubiaceae na Reserva Biológica Guaribas, Paraíba, Brasil. Subfamílias Antirheoideae, Cinchonoideae e Ixoroideae, Acta Bot Bras, 18 (2004) 305-318. 3 Agra MF, Freitas PF & Barbosa-Filho JM, Synopsis of the plants know as medicinal and poisonous in Northeast of Brazil, Rev Bras Farmacogn, 17 (2007) 114-140. 4 Lorenzi H, Plantas daninhas do Brasil: terrestres, aquáticas, parasitas, tóxicas e medicinais. (Ed. Plantarum LTDA, Curitiba), 1991. 5 Pedrinho Júnior AFF, Bianco S & Pitelli RA, Acúmulo de massa seca e macronutrientes por plantas de Glycine max e Richardia brasiliensis, Planta Daninha, 22 (2004) 53-61. 6 Grandi TSM, Trindade JÁ, Pinto MJF, Ferreira LL & Catella AC. Plantas medicinais de Minas Gerais, Brasil, Acta Bot Bras, 3 (1989) 185-224. 7 Figueiredo ADL, Estudo Farmacognóstico e Avaliação Antimicrobiana de Richardia brasiliensis Gomez (Rubiaceae), (MSc Thesis, Universidade Federal de Goiás), 2005. 8 Pinto DS, Tomaz ACA, Tavares JF, Tenório-Souza FH, Dias CS, Braz-Filho R & da-Cunha EVL, Secondary metabolites isolated from Richardia brasiliensis Gomes (Rubiaceae), Rev Bras Farmacogn, 18 (2008) 367-372. 9 Javadpour MM, Juban MM, Lo W.C, Bishop SM, Alberty JB & Cowell SM, De novo antimicrobial peptides with low mammalian cell toxicity, J Med Chem, 39 (1996) 3107-3113.
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