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Profile of antimicrobial potential of fifteen Hypericum species from Portugal
Industrial Crops and Products 47 (2013) 126–131
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Profile of antimicrobial potential of fifteen Hypericum species from Portugal Teresa Nogueira a,b,∗ , M. Augusta Medeiros a , M. João Marcelo-Curto a,b , B.E. García-Pérez c , J. Luna-Herrera c , M. Céu Costa a,b,d a
Laboratório Nacional de Energia e Geologia, I.P., Estrada do Pac¸o do Lumiar, 22, 1649-038 Lisboa, Portugal Sociedade Portuguesa de Fitoquímica e Fitoterapia, Rua da Sociedade Farmacêutica, 18, 1169-075 Lisboa, Portugal c Laboratorio de Inmunoquímica II, Departamento de Inmunología, Escuela Nacional de Ciências Biológicas, Casco de S.Tomás 11340, Mexico d Faculdade de Ciências e Tecnologias da Saúde, Grupo Lusófona, Campo Grande, 376, 1749-024 Lisboa, Portugal b
a r t i c l e
i n f o
Article history: Received 10 December 2012 Received in revised form 26 February 2013 Accepted 5 March 2013 Keywords: Antibacterial activity Antimycobacterial activity Hypericaceae Hypericum species Agrobacterium tumefasciens Multidrug-resistant Mycobacterium tuberculosis Mycobacterium species Hyperforin Hypericin Pseudohypericin Staphylococcus aureus
a b s t r a c t The aim of the present study was the search for the industrial exploitation potential of 15 Hypericum species crops from Portugal. Although Hypericum perforatum is well known worldwide, scarce studies have been published of Hypericum spp. identified in Portugal. Extracts from 15 Hypericum species were screened for its antimicrobial activities against 2 Gram− and 2 Gram+ bacteria, 4 non-tuberculous Mycobacterium species, a reference strain H37 Rv and 4 drug-resistant strains of Mycobacterium tuberculosis, as well as 4 drug-resistant clinical isolates. In terms of Gram – standards, H. humifusum and H. elodes were the most active against Agrobacterium tumefasciens, with MIC of 2.5 g/mL. H. elodes and H. hircinum subsp. majus extracts were the most active against MDR-TB strains and isolates, with MIC of 25–100 g/mL and both exhibited significant effect against MDR-TB clinical isolates. With the exception of H. androsaemum and H. linarifolium all Hypericum species were active against Staphylococcus aureus, the H. perfoliatum and H. elodes at the level of 6 and 12 g/mL, respectively, and none showed activity on E. coli. Reference compounds isolated from H. perforatum were also tested and might contribute to the activities observed. The profile of the 15 Hypericum spp. as effective antimicrobial therapy against multidrug resistant pathogens is now available, providing scientific validation on a few available ethnopharmacological data resources. This study may be a starting point for the research on the role of various Hypericum species in integrative medicine for infection control of S. aureus and MDR-MTB. Hypericum species may also constitute a source of new leads towards the discovery of either new candidates and biologically active compounds for pharmaceutical interest, for the treatment of multidrug resistant diseases. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Continuing search for effective antimicrobials is required to improve disease management against multidrug resistant pathogens. Gram-positive cocci have reemerged as predominant pathogens of human hosts within the past decade as an increasing cause of hospital-acquired infections. Staphylococcus aureus is a virulent and invasive pathogen that produces a variety of pyrogenic toxins and superantigens which contribute to its overall virulence. In the past several years Methicillin-resistant S. aureus were also
∗ Corresponding author at: Laboratório Nacional de Energia e Geologia, I.P., Estrada do Pac¸o do Lumiar, 22, 1649-038 Lisboa, Portugal. Tel.: +35 1210924736; fax: +35 1217166966. E-mail address: [email protected] (T. Nogueira). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.03.005
spreading clonally into the community leading to an increased use of vancomycin therapy. Vancomycin-resistant enterococci became a major hospital-acquired pathogen (Okuma et al., 2002). Tuberculosis is still the leading killer infectious disease in the world, with one-third of the world’s population infected with Mycobacterium tuberculosis. The World Health Organization estimates that 2 billion people have latent tuberculosis and 2 million people die each year worldwide because of this infection (WHO, 2008). Although a vaccine (BCG) and effective chemotherapy against tuberculosis were available 50 years ago, the increase in tuberculosis with the AIDS epidemic has resulted in the emergence of multidrug-resistant isolates of Mycobacterium tuberculosis. In spite of enormous efforts to find good leads, pharmacophore elucidation is still distant from a solution against the mechanism by which mutations induce drug resistance (Figueiredo et al., 2012). This fact demands the search for alternative antimycobacterial
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drugs (Jimenez-Arellanes et al., 2003) by rational drug design and/or by continuous screening of ethnopharmacological data resources. The use of active extracts, proficiently prepared, may constitute one way of overcoming the long time taken from the bioactivity evaluation to the identification of the responsible molecule. Furthermore, the extracts may be more active than the isolated components due to synergistic effects (Luna-Herrera et al., 2007; Paulo et al., 2008). The pharmacological activity of species of the genus Hypericum L. (Hypericaceae) is known from both folk medicine and the interest in therapeutical uses of these plants has been increasing in recent years. The most studied species of this genus, Hypericum perforatum L., is included in the monographs of the German Commission E, ESCOP and in some Pharmacopoeias and presently is the subject of many clinical trials concerning their antidepressant, antiviral (antiretroviral, including anti-HIV effects) and antibacterial activities (Barnes et al., 2001). Antibacterial activities have also been reported for other Hypericum species (Gibbons, 2008; Gibbons et al., 2002; Rabanal et al., 2002; Pistelli et al., 2000). Screenings of antimycobacterial activity of Hypericum have been performed against Mycobacterium tuberculosis (less virulent strain – H37 Ra) and a positive activity was observed for H. triquetrifolium Turra (Tosun et al., 2004) and for H. calycinum (Gottshall et al., 1949). McCutcheon et al. (1997) found that H. perforatum was active against M. tuberculosis and M. avium. H. drummondii (Grev. & Hook.) Torrey & Grey inhibited M. smegmatis (Jayasuriya et al., 1991). The genus Hypericum is a source of potential antibacterial leads (Gibbons, 2008). Plants of this genus have yielded families of compounds such as phloroglucinols, anthraquinones, xanthones and filicilic acid derivatives which are reported to be of pharmacological interest. In particular, hypercalin B and hyperenone A from H. acmosepalum showed significant antibacterial activity against Staphylococcus aureus, Mycobacterium tuberculosis H37Rv and M. bovis BCG bacteria (Osman et al., 2012). Also an acylphloroglucinol showed an interesting antibacterial activity (Gibbons et al., 2005) and drummonidins (filicilic acid derivatives) were described to possess a potent antimycobacterial activity (Jayasuriya et al., 1991). The wide variety of plants affected by the soil plant pathogenic bacterium Agrobacterium makes it still of great concern to the agriculture industry in spite of its unique mediating role on plant transformation for the introduction of foreign genes into plant cells and the subsequent regeneration of transgenic plants (Young et al., 2001). In mainland Portugal some Hypericum species have long been used for traditional medicinal purposes mainly H. androsaemum, H. perforatum and also H. undulatum Schousb. ex Willd. for its hepatoprotective, diuretic, anti-inflammatory and antiseptic activities (Costa, 1994; Valentão et al., 2004). With the aim of contributing to new insights for the development of research on the role of several Hypericum species in antimicrobial therapy against multidrug resistant pathogens we report here an antimycobacterial and antibacterial activity screening of ethanol extracts.
2. Materials and methods 2.1. Plant material Aerial parts of Hypericum species populations were collected during the flowering period of the plant on different places of mainland Portugal, Madeira and Azores Islands and were authenticated by Teresa Nogueira. A voucher specimen of each population has been deposited in the Herbarium of Instituto Superior de Agronomia de Lisboa – LISI and Jardim Botânico da Madeira – MADJ (Table 1).
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2.2. Preparation of extracts The aerial parts of the Hypericum species were dried in a ventilated oven at 35 ◦ C and kept at −20 ◦ C under vacuum. The ethanol extracts were obtained by direct maceration of dried and powdered plant material (500 mg) for 24 h at room temperature in the darkness, followed by sonication during 30 min. Extracts were filtered, evaporated under low pressure, blown down to dryness under nitrogen and stored at −20 ◦ C prior to the assays. 3. Reference compounds Reference compounds hypericin, pseudohypericin and hyperforin, isolated from H. perforatum were purchased from Chromadex (Inc. USA). 3.1. Mycobacterium species and clinical isolates Some Mycobacterium species used in these tests were obtained from the American Type Culture Collection (ATCC, Rockville, MD); Mycobacterium tuberculosis H37 Rv (ATCC 27294), H37 Rv isoniazidresistant (ATCC 35822), H37 Rv rifampin-resistant (ATCC 35838), H37 Rv ethambutol-resistant (ATCC 35837), M. fortuitum, M. smegmatis (ATCC 35798), M. avium (ATCC 35717), M. chelonae. Four drug-resistant pulmonar isolates of M. tuberculosis were obtained from patients at different hospitals in Mexico. These drug resistant isolates were selected based on their drug sensitivity pattern to the antimycobacterial drugs isoniazid, rifampin, rifabutin, ethambutol, ofloxacin, clarithromycin, streptomycin and ethionamide, previously determined. 3.2. Inoculum preparation for microplate Alamar Blue Assay All the strains were cultured at 37 ◦ C in Middlebrook 7H9 broth, supplemented with 0.2% glycerol and 10% OADC enrichment (oleic acid, albumin, dextrose, catalase; DIFCO) until log phase growth was achieved. The inoculum for the microcolorimetric assay for screening was prepared by diluting log phase growth cultures with sterile Middlebrook 7H9 to MacFarland No. 1 turbidity standard and then further diluted 1:10. This suspension was prepared just before inoculation of the microplate. For the non tuberculous mycobacteria, a 1:50 dilution was prepared. The working suspension was prepared just prior to inoculation of the microplate. 3.3. Antimycobacterial screening by microplate Alamar Blue Assay The mycobacterial effect was determined by a microcolorimetric assay with Alamar Blue dye, according to Collins and Franzblau (1997) with some modifications (Jimenez-Arellanes et al., 2003). The test was performed in 96-well sterile microplates (Nunc). Six different extract solutions with concentrations in the range 200–6.25 g/mL were tested in each plate in duplicate, reference compounds were tested at concentrations of 50, 25 and 12.5 g/mL. Stock solutions of extracts and reference compounds were prepared in dimethylsulfoxide at a concentration of 20 mg/mL in sterile conditions and stored at −70 ◦ C until use. Working standard solutions were prepared by diluting stock solutions to 800 g/mL in 7H9. Then 200 L of sterile water was added to the outer-perimeter wells. Then 100 L of 7H9 broth, 100 L of each extract or standard solution and 100 L of each solution of tuberculous and non tuberculous mycobacterial suspensions were added to the other wells and to the drug-free control wells. Simultaneously a 1:10 diluted control was prepared from the bacterial suspension representing the growth of 10% of the bacteria population tested. The plates were incubated at 37 ◦ C. After 5 days of incubation for M. tuberculosis
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Table 1 Collection sites of Hypericum species growing on mainland Portugal, Azores and Madeira. Hypericum species
Collection site
Voucher number
H. calycinum L. H. androsaemum L. H. foliosum Aiton H. hircinum subsp. majus (Aiton) N.Robson H. grandifolium Choisy H. perforatum L. H. undulatum Schoub ex Willd. H. perfoliatum L. H. humifusum L. H. linarifolium Vahl. H. canariense L. H. tomentosum L. H. pubescens Boiss. H. glandulosum Aiton H. elodes L.
Serra de Sintra (mainland Portugal) Serra do Gerês (mainland Portugal) Furna de Enxofre, Terceira (Azores) Serra de Sintra (mainland Portugal) Levada Faja Nogueira (Madeira) Serra de Sintra (mainland Portugal) Aljezur (mainland Portugal) Alter do Chão (mainland Portugal) Serra de Sintra (mainland Portugal) Braganc¸a (mainland Portugal) Quinta Grande (Madeira) Serra de Sintra (mainland Portugal) Serpa (mainland Portugal) Curral das Freiras (Madeira) Alcácer do Sal (mainland Portugal)
LISI:193/2003 LISI:186/2003 LISI:371/2009 LISI:131/2003 MADJ: 10193 LISI:174/2003 LISI:168/2003 LISI:109/2003 LISI:158/2003 LISI:149/2003 MADJ: 10188 LISI:120/2003 LISI:118/2003 MADJ: 10187 LISI:127/2003
and after 2 days for the non tuberculous mycobacteria, one control was developed with 20 L of Alamar blue solution (Trek Diagnostics, Westlake, OH) and 12 L of sterile 10% Tween 80. The plates were reincubated at 37 ◦ C for 24 h. After this incubation, if the well turned pink, all the wells received Alamar Blue and Tween solutions in the same way and were incubated for an additional 24 h. Wells with a well-defined pink colour or a shade that was deeper than the 10% growth well were scored positive for growth. The minimal inhibitory concentration (MIC) was defined as the lowest extract concentration that prevented a colour change to pink. Extracts were considered active if they gave a MIC ≤ 200 g/mL, standards were considered active if they presented a MIC ≤ 50 g/mL. 3.4. Bacteria The following strains of bacteria were used: Agrobacterium tumefasciens 2, Escherichia coli CCMI 270, Staphylococcus aureus CCMI 335 and Streptococcus faecium CCMI 338. The bacteria are from the Culture Collection of Industrial Microbiology LMI (INETI, Lisbon). Brain heart infusion (Merck, Darmstadt, Germany) was used to grow bacteria. 3.5. Antibacterial assays The MICs of the active extracts and compounds were determined by the broth dilution method (Muroi and Kubo, 1996). A 105 mL−1 cfu suspension was obtained by measurement of the cell concentration in a spectrophotometer (UNICAM 8700), at 620 nm wavelength and successive dilutions in saline solution. The following concentrations (g/mL) were tested: 200, 100, 50, 25 and 12.5. After incubation for 24 h at 30–37 ◦ C the microbial growth was examined and the MIC was defined as lowest concentration of the test compound yielding no visible growth. The MIC of each extract and compound was determined in duplicate. Samples with no visible growth were inoculated into the agar culture medium in order to detect bactericidal/bacteriostatic activity. Rifampicin (Sigma) was used as a positive control. 4. Results and discussion The ethanol extracts from 15 Hypericum species and three reference compounds were evaluated for their antimycobacterial and other antibacterial activities (Table 2). The most active extracts against M. tuberculosis H37 Rv were those obtained from H. elodes and H. hircinum subsp. majus, with MICs ranging from 25 to 50 g/mL. The screening of those active extracts against drug-resistant variants of the H37 Rv was also performed and a potent activity
was observed from H. foliosum, H. hircinum subsp. majus, H. grandifolium, H. humifusum and H. elodes with MICs ranging from 25 to 50 g/mL. H. elodes and H. hircinum subsp. majus were also active against drug resistant clinical isolates with MICs ranging from 12.5 to 50 g/mL. In spite of the weak activity of H. humifusum, H. foliosum and H. grandifolium against the reference strain H37 Rv, with MICs of 200 g/mL, they presented higher activities against the drug-resistant reference strains and clinical isolates. H. hircinum subsp. majus showed the higher activity only against M. fortuitum (MICs 50 g/mL), a non tuberculous Mycobacterium species. The lack of activity of H. perforatum observed in this work is contrary to that described by McCutcheon et al. (1997) using the disc diffusion method. This could be attributed to the differences in the screening methods and to the extremely high concentration used in the McCutcheon study. The activity observed for the H. calycinum extract is in accordance with the qualitative preliminary results of Gottshall et al. (1949). The highest level of Gram-negative antibacterial activity (MICs 2.5–200 g/mL) was found towards Agrobacterium tumefaciens [Rhizobium radiobacter] (UniProt; Young et al., 2001), the causal agent of crown gall disease (the formation of tumours) in over 140 species of dicotyledons, A. tumefaciens is still a serious pathogen of walnuts, grape vines, stone fruits, nut trees, sugar beets, horse radish and rhubarb. The chemical isolation to establish the phytochemical profiling and to identify the active constituents may open new perspectives for lead compounds. Staphylococcus aureus was the more susceptible Gram-positive bacteria (MICs 6–50 g/mL). H. perfoliatum, H. elodes, H. glandulosum, H. canariense, H. grandifolium, H. foliosum, H. hircinum subsp. majus, H. humifusum and H. calycinum strongly inhibited S. aureus (MICs 6–25 g/mL). Gibbons et al. (2002) and Gottshall et al. (1949) have reported no activity for the extract of H. calycinum. The positive results obtained for H. foliosum and H. hircinum subsp. majus against this microorganism are in accordance with those found by Gibbons et al. (2002) and Pistelli et al. (2000). The remarkable activity of H. glandulosum, H. grandifolium and H. canariense is in accordance with results formerly reported by Rabanal et al. (2002). Worthy of note is the potential for these active Hypericum spp. to be investigated for synergistic effects in treating staphylococcal infections, since there are currently being performed studies for combination therapies (Smirnova et al., 2011). Five Hypericum extracts, namely H. androsaemum, H. foliosum, H. grandifolium, H. canariense and H. glandulosum, also showed significant activity against Streptococcus faecium (MICs 12.5–50 g/mL). In Table 3 the composition data is gathered on the quantification of biologically active phenolic compounds, such as anthraquinones, flavonoids, flavonoid glycosides and phenolic
Table 2 Antimycobacterial and antibacterial activity of ethanol crude extracts of Hypericum spp. and pure compounds. Hypericum spp.
MIC (g/mL) Antimycobacterial activity
Antibacterial activity
Drug-resistant strains
Mycobacterium spp.
H37Rv
H37Rv INH-R
H37Rv ETAM-R
H37Rv SM-R
H37Rv RIF-R
Is.cl. SIN-3
Is.cl. SIN-4
Is.cl. HG8
Is.cl. MMDO
M. fortuitum
M. smeg.
M. avium
M. chelonae
E. coli
S. aureus
S. faecium
100 >200 200 25 200 >200 >200 >200 200 >200 >200 >200 >200 100 50
100 >100 50 25 50 >100 >100 >100 100 >100 100 >100 >100 100 50
>100 >100 50 100 50 >100 >100 >100 50 >100 100 >100 >100 100 50
100 >100 100 50 100 >100 >100 >100 50 >100 >100 >100 >100 100 25
100 >100 100 25 100 >100 >100 >100 50 >100 >100 >100 >100 100 50
100 n.t. 100 12.5 100 >100 >100 >100 >100 >100 >100 >100 >100 >100 50
>100 >100 100 100 100 >100 >100 >100 >100 >100 >100 >100 >100 >100 50
100 n.t. 100 50 100 n.t. n.t. n.t. 100 n.t. n.t. n.t. n.t. >100 50
100 >100 100 50 100 >100 >100 >100 >100 >100 >100 >100 >100 >100 50
>100 >100 >100 50 >100 >100 >100 >100 100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 100
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
>200 >200 n.t. >200 n.t. >200 >200 >200 >200 >200 n.t. >200 >200 n.t. >200
20 >200 12.5a 25 12.5 50 50 6 25 >200 12.5 >200 50 12.5 12
>200 50a 12.5a >200 50a >200 200 >200 200a >200 25a >200 >200 12.5a >200
>200 >200 25 ≤0.06
>50 >50 25 ≤0.06
>50 >50 25 ≤0.06
>50 >50 25 ≤0.06
>50 >50 25 >2
n.t. n.t. n.t. >2
>50 >50 25 >2
n.t. n.t. n.t. n.t.
>50 >50 25 0.125
>50 >50 >50 n.t.
50 >50 >50 n.t.
>50 >50 >50 n.t.
>50 >50 50 n.t.
n.t. n.t. n.t. 1
100a >100 50a 0.001
25a 100a 50a 0.5a
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H. calycinum H. androsaemum H. foliosum H. hircinum ssp. majus H. grandifolium H. perforatum H. undulatum H. perfoliatum H. humifusum H. linarifolium H. canariense H. tomentosum H. pubescens H. glandulosum H. elodes Compounds Hypericin Pseudohypericin Hyperforin Rifampicin
Drug-resistant clinical isolates
n.t.: not tested. Antimycobacterial activity: H37Rv – reference strain; drug resistant strains: H37Rv INH-R, H37Rv ETAM-R, H37Rv SM-R, H37Rv RIF-R – strains resistant to isoniazid, ethambutol, streptomycin and rifampin, respectively. Drug resistant clinical isolates: Is.cl.SIN-3 – resistant to streptomycin, isoniazid, rifampin, rifabutin, ethambutol, clarithromycin and ofloxacin, Is.cl.SIN-4 – resistant to streptomycin, isoniazid, rifampin, rifabutin, ethambutol, ethionamide and ofloxacin, Is.cl.HG8 – resistant to ethambutol, clarithromycin and ethionamide, Is.cl.MMDO – resistant to isoniazid and ethambutol. Mycobacteria species: M. fortuitum, M. smegmatis, M. avium, M. chelonae. Antibacterial activity: Gram− (Escherichia coli) and Gram+ (Staphylococcus aureus, Streptococcus faecium); rifampicin – positive control. a Bacteriostatic.
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−b −b n.c. 0.3g 0.012h 0.02g n.c. n.c. +e n.c. +c − − −; 0.12f 0.07 0.04 0.04 0.24 0.07 0.04 0.02 −; +c 0.002 n.c. 0.004 0.004 0.01 0.001 0.002 0.005 0.004 0.004 0.002 0.07 0.002 − 0.13 0.11 0.03 0.06 0.19 0.0009 0.0007 0.007
5. Conclusion Industrial development of medicinal plants play an important role in the discovery of new drugs. Natural products are directly or indirectly responsible for almost 40% of the drugs used in modern therapeutics. Considering that the development of new synthetic drugs demand for high costs the ethnopharmacology may constitute a precious guide for the selection of plant extracts and compounds (Grando et al., 2008). On the other hand the increasing incidence of multi-drug resistant strains worldwide determines the urgent need for new anti-multidrug resistant bacterial drugs. The assays performed in the present study describe the antimicrobial activity of fifteen Hypericum extracts, some of them herein investigated for the first time, with MICs ranging from 2.5 to 50 g/mL. In the assays with reference compounds the results obtained suggest that they might contribute to the antimicrobial activities observed in the extracts. From this study, new insights and evidence are open on the potential of Hypericum species as a source of new leads towards the discovery of either new candidates for the treatment of multidrug resistant tuberculosis and as potent antimicrobials for drug design.
0.08 0.03 0.02 0.41 0.17 0.03 0.31 0.21 0.12 0.27 0.003 0.05 0.13 0.65 0.97 0.70 0.19 0.88 1.02 0.003 − − 0.04 n.c. 0.005 0.001 0.001 − − 0.004 − 0.08 n.c. − 0.0006 − − 0.0001 − − − 0.0007 0.07 −
Isoquercitrina Rutina Caffeic acida
Hyperosida
0.12 0.0004 0.36 0.14 0.07 0.13 0.07 0.21 1.01 0.70 0.003
0.06 0.03 0.05 0.03 0.14 0.04 0.04 0.08 0.02 0.003 0.03
Amentoflavonea
Pseudohypericin
+d −e 0.14f 0.55g −h 0.03g n.c. n.c. n.c. n.c. +c
acids, including previous studies (Farinha et al., 2002). Significant differences of composition among Hypericum species were observed which might be related with the differences of biological activities verified in the present study. A patent on extracts from some Hypericum species useful for the treatment of persistent tuberculosis was previously registed (Nogueira et al., 2009). Three reference compounds isolated from H. perforatum, hypericin, pseudo-hypericin and hyperforin, were active against all the drug-resistant strains and some of the clinical isolates tested, with MICs from 25 to up to 50 g/mL. However only hyperforin showed activity against the strain H37 Rv at a MIC of 25 g/mL. All of them showed also some activity against non tuberculous Mycobacterium species (MICs ≥ 50 g/mL). The antibacterial activity was also assayed against Gram+ bacteria, Staphylococcus aureus and Streptococcus faecium, with MICs of hypericin and hyperforin ranging from 25 to 50 g/mL. Prior QSAR studies described quinones (Dimov et al., 2001) and biflavonoids (Dias et al., 2004) as active compounds against tuberculosis. Hypericin and pseudohypericin, two anthraquinones, are present in some Hypericum species (Farinha et al., 2002; Mathis and Ourisson, 1964; Kartnig et al., 1996; Umek et al., 1999). However, none of these compounds presented a huge activity against the whole group of Mycobacteria tested.
Acknowledgement Collaborative work was performed under the auspices of the Iberoamerican Program for Science and Technology (CYTED), Project X.11: PIBATUB.
n.c.: not confirmed; −: not present. a Farinha et al. (2002). b Kitanov (2001). c Piovan et al. (2004). d Klingauf et al. (2005). e Rainha et al. (2011). f Sagratini et al. (2008). g Hosni et al. (2010). h Rainha et al. (2012).
H. calycinum H. androsaemum H. hircinum ssp. majus H. perforatum H. undulatum H. perfoliatum H. humifusum H. linarifolium H. tomentosum H. pubescens H. elodes
0.9 1.46 − 0.06 0.19 0.91 4.18 4.81 0.97 0.84 0.006
References Chlorogenic acida
Compounds (mg/100 mg of plant) Species
Table 3 Cumulative data on quantitative HPLC analysis of extracts of 11 portuguese Hypericum species.
Quercitrina
Quercetina
Kaempferola
Hypericina
Hyperforin
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Barnes, J., Andersen, L.A., Phillipson, J.D., 2001. Hypericum perforatum L. A review of its chemistry, pharmacology and clinical properties. J. Pharm. Pharmacol. 53, 583–600. Collins, L.A., Franzblau, S.G., 1997. Microplate Alamar Blue Assay versus BACTEC 460 system for high-throughput screening of compound against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob. Agents Chemother. 41, 1004–1009. Costa, A.F., 1994. Farmacognosia, vol II. Fundac¸ão Calouste Gulbenkian, Lisboa1021–1023. Dias, J.C., Rebelo, M.M., Alves, C.N., 2004. A semi-empirical study of biflavonoid compounds with biological activity against tuberculosis. J. Mol. Struct. (Theochem) 676, 83–87. Dimov, D., Nedyalkova, Z., Haladjova, S., Schüürmann, G., Mekenyan, O., 2001. QSAR Modeling of antimycobacterial activity and activity against other bacteria of 3-formyl rifamycin SV derivatives. Quant. Struct. Act. Relat. 20, 298–318.
T. Nogueira et al. / Industrial Crops and Products 47 (2013) 126–131 Farinha, A., Martins, J.M., Nogueira, T., Tavares, R., Duarte, F.A., 2002. HPLC analysis of Hypericum L. species from Portugal. In: Nat. Prod. in the New Millennium. Kluwer Acad. Pub.125–134. Figueiredo, R., Ramos, D.F., Moiteiro, C., Medeiros, M.A., Marcelo Curto, M.J., Menezes, J.C., Pando, R.H., Silva, P.E.A., Costa, M.C., 2012. Pharmacophore insights into rpoB gene mutations in Mycobacterium tuberculosis rifampicin resistant isolates. Eur. J. Med. Chem. 47, 186–193. Gibbons, S., Ohlendorf, B., Johnsen, I., 2002. The Genus Hypericum – a valuable resource of anti-Staphylococcal leads. Fitoterapia 73, 300–304. Gibbons, S., Moser, E., Hausmman, S., Stavri, M., Smith, E., Clennett, C., 2005. An antistaphylococcal acylphloroglucinol from Hypericum foliosum. Phytochemistry 66, 1472–1475. Gibbons, S., 2008. Phytochemicals for bacterial resistance – strengths, weaknesses and opportunities. Planta Med. 74, 594–602. Gottshall, R.Y., Lucas, E.H., Lickfeldt, A., Roberts, J.M., 1949. The occurrence of antibacterial substances active against Mycobacterium tuberculosis in seed plants. J. Clin. Invest. 28, 920–921. Grando, R., António, M.A., Araújo, C.E.P., Soares, C., Medeiros, M.A., Carvalho, J.E., Lourenc¸o, A.M., Lopes, L.C., 2008. Antineoplastic 31-Norcycloartanones from Solanum cernuum Vell. Z. Naturforsch. 63c, 507–514. Hosni, K., Msaâda, K., Taârit, M.B., Hammami, M., Marzouk, B., 2010. Bioactive components of three Hypericum species from Tunisia: a comparative study. Ind. Crops Prod. 31, 158L 163. Jayasuriya, H., Clark, A.M., McChesney, J.D., 1991. New antimicrobial filicinic acid derivatives from Hypericum drummondii. J. Nat. Prod. 54, 1314–1320. Jimenez-Arellanes, A., Meckes, M., Ramirez, R., Torres, J., Luna-Herrera, J., 2003. Activity against multidrug-resistant Mycobacterium tuberculosis in Mexican Plants used to treat respiratory diseases. Phytother. Res. 17, 903–908. Kartnig, T., Göbel, I., Heydel, B., 1996. Production of hypericin, pseudohypericin and flavonoids in cell cultures of various Hypericum species and their chemotypes. Planta Med. 62, 51–53. Kitanov, G.M., 2001. Hypericin and pseudohypericin in some Hypericum species. Biochem. Syst. Ecol. 29, 171–178. Klingauf, P., Beuerle, T., Mellenthin, A., El-Moghazy, S.A.M., Boubakir, Z., Beerhues, L., 2005. Biosynthesis of the hyperforin skeleton in Hypericum calycinum cell cultures. Phytochemistry 66, 139–145. Luna-Herrera, J., Costa, M.C., González, H.G., Rodrigues, A.I., Castilho, P.C., 2007. Synergistic antimycobacterial activities of sesquiterpene lactones from Laurus spp. J. Antimicrob. Chemother. 59 (3), 548–552. McCutcheon, A.R., Stokes, R.W., Thorson, L.M., Ellis, S.M., Hancock, R.E.W., Towers, G.H.N., 1997. Anti-mycobacterial screening of British Columbian medicinal plants. Int. J. Pharm. 35, 77–83. Mathis, C., Ourisson, G., 1964. Étude chimio-taxonomic du genre Hypericum II. Identification de constituent de diverses Huilles Essentielles D’ Hypericum. Phytochemistry 3, 115–131. Muroi, H., Kubo, I., 1996. Antibacterial activity of anacardic acid and totarol alone and in combination with methicillin, against methicillin-resistant Staphylococcus aureus. J. Appl. Bacteriol. 80, 387–394. Nogueira, T., Costa, M.C., Moiteiro, C.M., Medeiros, M.A., Curto, M.J, Luna-Herrera, J., Rodriguez, E., 2009. Extracts from Hypericum L. species useful for the treatment of persistent tuberculosis. European Patent EP 1803463 B1.
131
Okuma, K., Iwakawa, K., Turnidge, J.D., Grubb, W.B., Bell, J.M., O‘Brien, F.G., Coombs, G.W., Pearman, J.W., Tenover, F.C., Kapi, M., Tiensasitorn, C., Ito, T., Hiramatsu, K., 2002. Dissemination of new methicillin-resistant Staphylococcus aureus clones in the community. J. Clin. Microbiol. 40, 4289–4294. Osman, K., Evangelopoulos, D., Basavannacharya, C., Gupta, A., McHugh, T.D., Bhakta, S., Gibbons, S., 2012. An antibacterial from Hypericum acmosepalum inhibits ATP-dependent MurE ligase from Mycobacterium tuberculosis. Int. J. Antimicrob. Agents 39, 124–129. Piovan, A., Filippini, R., Caniato, R., Borsarini, A., Maleci, L.B., Cappelletti, E.M., 2004. Detection of hypericins in the red glands of Hypericum elodes by ESI-MS/MS. Phytochemistry 65, 411–414. Pistelli, L., Bertoli, A., Zucconelli, S., Morelli, I., Panizzi, L., Menichini, F., 2000. Antimicrobial activity of crude extracts and pure compounds of Hypericum hircinum. Fitoterapia 71, S138–S140. Paulo, A., Martins, S., Branco, P., Dias, T., Borges, C., Rodrigues, A.I., Costa, M.C., Teixeira, A., Mota-Filipe, H., 2008. The opposing effects of the flavonoids isoquercitrin and Sissotrin, isolated from Pterospartum tridentatum, on oral glucose tolerance in rats. Phytother. Res. 22, 539–543. Rabanal, R.M., Arias, A., Prado, B., Hernández-Pérez, M., Sánchez-Mateo, C.C., 2002. Antimycrobial studies on three species of Hypericum from the Canary Island. J. Ethnopharmacol. 81, 287–292. Rainha, N., Lima, E., Baptista, J., 2011. Comparison of the endemic Azorean Hypericum foliosum with other Hypericum species: antioxidant activity and phenolic profile. Nat. Prod. Res. 25 (2), 123–135. Rainha, N., Lima, E., Baptista, J., Fernandes-Ferreira, M., 2012. Content of hypericins from plants and in vitro shoots of Hypericum undulatum Schousb. ex Willd. Nat. Prod. Res., pp. 1–11, iFirst. Sagratini, G., Ricciutelli, M., Vittori, S., Öztürk, N., Öztürk, Y., Maggi, F., 2008. Phytochemical and antioxidant analysis of eight Hypericum taxa from Central Italy. Fitoterapia 79, 210–213. Smirnova, M.V., Strukova, E.N., Portnoy, Y.A., Dovzhenko, S.A., Kobrin, M.B., Zinner, S.H., Firsov, A.A., 2011. The antistaphylococcal pharmacodynamics of linezolid alone and in combination with doxycycline in an in vitro dynamic model. J. Chemother. 23 (3), 140–144. Tosun, F., Kizilay, C¸.A., Sener, B., Vural, M., Palittapongarnpim, P., 2004. Antimicobacterial screening of some Turkish plants. J. Ethnopharmacol. 95, 273–275. Umek, A., Kreft, S., Katnig, T., Heydel, B., 1999. Quantitative phytochemical analyses of six Hypericum species growing in Slovenia. Planta Med. 65, 388–390. UniProt Taxonomy Rhizobium radiobacter (Agrobacterium tumefaciens) (Agrobacterium radiobacter). http://pir.uniprot.org/taxonomy/358 Valentão, P., Carvalho, M., Fernandes, E., Carvalho, F., Andrade, P.B., Seabra, R.M., Bastos, M.L., 2004. Protective activity of Hypericum androsaemum infusion against tert-butyl hydroperoxide-induced oxidative damage in isolated rat hepatocytes. J. Ethnopharmacol. 92, 79–84. World Health Organization Global, 2008. Tuberculosis Control Surveillance, Planning, Financing. WHO/HTM/TB/2008, 393. Young, J.M., Kuykendall, L.D., Martínez-Romero, E., Kerr, A., Sawada, H., 2001. A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al., 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis. Int. J. Syst. Evol. Microbiol. 51, 89–103.
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Profile of antimicrobial potential of fifteen Hypericum species from Portugal Teresa Nogueira a,b,∗ , M. Augusta Medeiros a , M. João Marcelo-Curto a,b , B.E. García-Pérez c , J. Luna-Herrera c , M. Céu Costa a,b,d a
Laboratório Nacional de Energia e Geologia, I.P., Estrada do Pac¸o do Lumiar, 22, 1649-038 Lisboa, Portugal Sociedade Portuguesa de Fitoquímica e Fitoterapia, Rua da Sociedade Farmacêutica, 18, 1169-075 Lisboa, Portugal c Laboratorio de Inmunoquímica II, Departamento de Inmunología, Escuela Nacional de Ciências Biológicas, Casco de S.Tomás 11340, Mexico d Faculdade de Ciências e Tecnologias da Saúde, Grupo Lusófona, Campo Grande, 376, 1749-024 Lisboa, Portugal b
a r t i c l e
i n f o
Article history: Received 10 December 2012 Received in revised form 26 February 2013 Accepted 5 March 2013 Keywords: Antibacterial activity Antimycobacterial activity Hypericaceae Hypericum species Agrobacterium tumefasciens Multidrug-resistant Mycobacterium tuberculosis Mycobacterium species Hyperforin Hypericin Pseudohypericin Staphylococcus aureus
a b s t r a c t The aim of the present study was the search for the industrial exploitation potential of 15 Hypericum species crops from Portugal. Although Hypericum perforatum is well known worldwide, scarce studies have been published of Hypericum spp. identified in Portugal. Extracts from 15 Hypericum species were screened for its antimicrobial activities against 2 Gram− and 2 Gram+ bacteria, 4 non-tuberculous Mycobacterium species, a reference strain H37 Rv and 4 drug-resistant strains of Mycobacterium tuberculosis, as well as 4 drug-resistant clinical isolates. In terms of Gram – standards, H. humifusum and H. elodes were the most active against Agrobacterium tumefasciens, with MIC of 2.5 g/mL. H. elodes and H. hircinum subsp. majus extracts were the most active against MDR-TB strains and isolates, with MIC of 25–100 g/mL and both exhibited significant effect against MDR-TB clinical isolates. With the exception of H. androsaemum and H. linarifolium all Hypericum species were active against Staphylococcus aureus, the H. perfoliatum and H. elodes at the level of 6 and 12 g/mL, respectively, and none showed activity on E. coli. Reference compounds isolated from H. perforatum were also tested and might contribute to the activities observed. The profile of the 15 Hypericum spp. as effective antimicrobial therapy against multidrug resistant pathogens is now available, providing scientific validation on a few available ethnopharmacological data resources. This study may be a starting point for the research on the role of various Hypericum species in integrative medicine for infection control of S. aureus and MDR-MTB. Hypericum species may also constitute a source of new leads towards the discovery of either new candidates and biologically active compounds for pharmaceutical interest, for the treatment of multidrug resistant diseases. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Continuing search for effective antimicrobials is required to improve disease management against multidrug resistant pathogens. Gram-positive cocci have reemerged as predominant pathogens of human hosts within the past decade as an increasing cause of hospital-acquired infections. Staphylococcus aureus is a virulent and invasive pathogen that produces a variety of pyrogenic toxins and superantigens which contribute to its overall virulence. In the past several years Methicillin-resistant S. aureus were also
∗ Corresponding author at: Laboratório Nacional de Energia e Geologia, I.P., Estrada do Pac¸o do Lumiar, 22, 1649-038 Lisboa, Portugal. Tel.: +35 1210924736; fax: +35 1217166966. E-mail address: [email protected] (T. Nogueira). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.03.005
spreading clonally into the community leading to an increased use of vancomycin therapy. Vancomycin-resistant enterococci became a major hospital-acquired pathogen (Okuma et al., 2002). Tuberculosis is still the leading killer infectious disease in the world, with one-third of the world’s population infected with Mycobacterium tuberculosis. The World Health Organization estimates that 2 billion people have latent tuberculosis and 2 million people die each year worldwide because of this infection (WHO, 2008). Although a vaccine (BCG) and effective chemotherapy against tuberculosis were available 50 years ago, the increase in tuberculosis with the AIDS epidemic has resulted in the emergence of multidrug-resistant isolates of Mycobacterium tuberculosis. In spite of enormous efforts to find good leads, pharmacophore elucidation is still distant from a solution against the mechanism by which mutations induce drug resistance (Figueiredo et al., 2012). This fact demands the search for alternative antimycobacterial
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drugs (Jimenez-Arellanes et al., 2003) by rational drug design and/or by continuous screening of ethnopharmacological data resources. The use of active extracts, proficiently prepared, may constitute one way of overcoming the long time taken from the bioactivity evaluation to the identification of the responsible molecule. Furthermore, the extracts may be more active than the isolated components due to synergistic effects (Luna-Herrera et al., 2007; Paulo et al., 2008). The pharmacological activity of species of the genus Hypericum L. (Hypericaceae) is known from both folk medicine and the interest in therapeutical uses of these plants has been increasing in recent years. The most studied species of this genus, Hypericum perforatum L., is included in the monographs of the German Commission E, ESCOP and in some Pharmacopoeias and presently is the subject of many clinical trials concerning their antidepressant, antiviral (antiretroviral, including anti-HIV effects) and antibacterial activities (Barnes et al., 2001). Antibacterial activities have also been reported for other Hypericum species (Gibbons, 2008; Gibbons et al., 2002; Rabanal et al., 2002; Pistelli et al., 2000). Screenings of antimycobacterial activity of Hypericum have been performed against Mycobacterium tuberculosis (less virulent strain – H37 Ra) and a positive activity was observed for H. triquetrifolium Turra (Tosun et al., 2004) and for H. calycinum (Gottshall et al., 1949). McCutcheon et al. (1997) found that H. perforatum was active against M. tuberculosis and M. avium. H. drummondii (Grev. & Hook.) Torrey & Grey inhibited M. smegmatis (Jayasuriya et al., 1991). The genus Hypericum is a source of potential antibacterial leads (Gibbons, 2008). Plants of this genus have yielded families of compounds such as phloroglucinols, anthraquinones, xanthones and filicilic acid derivatives which are reported to be of pharmacological interest. In particular, hypercalin B and hyperenone A from H. acmosepalum showed significant antibacterial activity against Staphylococcus aureus, Mycobacterium tuberculosis H37Rv and M. bovis BCG bacteria (Osman et al., 2012). Also an acylphloroglucinol showed an interesting antibacterial activity (Gibbons et al., 2005) and drummonidins (filicilic acid derivatives) were described to possess a potent antimycobacterial activity (Jayasuriya et al., 1991). The wide variety of plants affected by the soil plant pathogenic bacterium Agrobacterium makes it still of great concern to the agriculture industry in spite of its unique mediating role on plant transformation for the introduction of foreign genes into plant cells and the subsequent regeneration of transgenic plants (Young et al., 2001). In mainland Portugal some Hypericum species have long been used for traditional medicinal purposes mainly H. androsaemum, H. perforatum and also H. undulatum Schousb. ex Willd. for its hepatoprotective, diuretic, anti-inflammatory and antiseptic activities (Costa, 1994; Valentão et al., 2004). With the aim of contributing to new insights for the development of research on the role of several Hypericum species in antimicrobial therapy against multidrug resistant pathogens we report here an antimycobacterial and antibacterial activity screening of ethanol extracts.
2. Materials and methods 2.1. Plant material Aerial parts of Hypericum species populations were collected during the flowering period of the plant on different places of mainland Portugal, Madeira and Azores Islands and were authenticated by Teresa Nogueira. A voucher specimen of each population has been deposited in the Herbarium of Instituto Superior de Agronomia de Lisboa – LISI and Jardim Botânico da Madeira – MADJ (Table 1).
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2.2. Preparation of extracts The aerial parts of the Hypericum species were dried in a ventilated oven at 35 ◦ C and kept at −20 ◦ C under vacuum. The ethanol extracts were obtained by direct maceration of dried and powdered plant material (500 mg) for 24 h at room temperature in the darkness, followed by sonication during 30 min. Extracts were filtered, evaporated under low pressure, blown down to dryness under nitrogen and stored at −20 ◦ C prior to the assays. 3. Reference compounds Reference compounds hypericin, pseudohypericin and hyperforin, isolated from H. perforatum were purchased from Chromadex (Inc. USA). 3.1. Mycobacterium species and clinical isolates Some Mycobacterium species used in these tests were obtained from the American Type Culture Collection (ATCC, Rockville, MD); Mycobacterium tuberculosis H37 Rv (ATCC 27294), H37 Rv isoniazidresistant (ATCC 35822), H37 Rv rifampin-resistant (ATCC 35838), H37 Rv ethambutol-resistant (ATCC 35837), M. fortuitum, M. smegmatis (ATCC 35798), M. avium (ATCC 35717), M. chelonae. Four drug-resistant pulmonar isolates of M. tuberculosis were obtained from patients at different hospitals in Mexico. These drug resistant isolates were selected based on their drug sensitivity pattern to the antimycobacterial drugs isoniazid, rifampin, rifabutin, ethambutol, ofloxacin, clarithromycin, streptomycin and ethionamide, previously determined. 3.2. Inoculum preparation for microplate Alamar Blue Assay All the strains were cultured at 37 ◦ C in Middlebrook 7H9 broth, supplemented with 0.2% glycerol and 10% OADC enrichment (oleic acid, albumin, dextrose, catalase; DIFCO) until log phase growth was achieved. The inoculum for the microcolorimetric assay for screening was prepared by diluting log phase growth cultures with sterile Middlebrook 7H9 to MacFarland No. 1 turbidity standard and then further diluted 1:10. This suspension was prepared just before inoculation of the microplate. For the non tuberculous mycobacteria, a 1:50 dilution was prepared. The working suspension was prepared just prior to inoculation of the microplate. 3.3. Antimycobacterial screening by microplate Alamar Blue Assay The mycobacterial effect was determined by a microcolorimetric assay with Alamar Blue dye, according to Collins and Franzblau (1997) with some modifications (Jimenez-Arellanes et al., 2003). The test was performed in 96-well sterile microplates (Nunc). Six different extract solutions with concentrations in the range 200–6.25 g/mL were tested in each plate in duplicate, reference compounds were tested at concentrations of 50, 25 and 12.5 g/mL. Stock solutions of extracts and reference compounds were prepared in dimethylsulfoxide at a concentration of 20 mg/mL in sterile conditions and stored at −70 ◦ C until use. Working standard solutions were prepared by diluting stock solutions to 800 g/mL in 7H9. Then 200 L of sterile water was added to the outer-perimeter wells. Then 100 L of 7H9 broth, 100 L of each extract or standard solution and 100 L of each solution of tuberculous and non tuberculous mycobacterial suspensions were added to the other wells and to the drug-free control wells. Simultaneously a 1:10 diluted control was prepared from the bacterial suspension representing the growth of 10% of the bacteria population tested. The plates were incubated at 37 ◦ C. After 5 days of incubation for M. tuberculosis
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Table 1 Collection sites of Hypericum species growing on mainland Portugal, Azores and Madeira. Hypericum species
Collection site
Voucher number
H. calycinum L. H. androsaemum L. H. foliosum Aiton H. hircinum subsp. majus (Aiton) N.Robson H. grandifolium Choisy H. perforatum L. H. undulatum Schoub ex Willd. H. perfoliatum L. H. humifusum L. H. linarifolium Vahl. H. canariense L. H. tomentosum L. H. pubescens Boiss. H. glandulosum Aiton H. elodes L.
Serra de Sintra (mainland Portugal) Serra do Gerês (mainland Portugal) Furna de Enxofre, Terceira (Azores) Serra de Sintra (mainland Portugal) Levada Faja Nogueira (Madeira) Serra de Sintra (mainland Portugal) Aljezur (mainland Portugal) Alter do Chão (mainland Portugal) Serra de Sintra (mainland Portugal) Braganc¸a (mainland Portugal) Quinta Grande (Madeira) Serra de Sintra (mainland Portugal) Serpa (mainland Portugal) Curral das Freiras (Madeira) Alcácer do Sal (mainland Portugal)
LISI:193/2003 LISI:186/2003 LISI:371/2009 LISI:131/2003 MADJ: 10193 LISI:174/2003 LISI:168/2003 LISI:109/2003 LISI:158/2003 LISI:149/2003 MADJ: 10188 LISI:120/2003 LISI:118/2003 MADJ: 10187 LISI:127/2003
and after 2 days for the non tuberculous mycobacteria, one control was developed with 20 L of Alamar blue solution (Trek Diagnostics, Westlake, OH) and 12 L of sterile 10% Tween 80. The plates were reincubated at 37 ◦ C for 24 h. After this incubation, if the well turned pink, all the wells received Alamar Blue and Tween solutions in the same way and were incubated for an additional 24 h. Wells with a well-defined pink colour or a shade that was deeper than the 10% growth well were scored positive for growth. The minimal inhibitory concentration (MIC) was defined as the lowest extract concentration that prevented a colour change to pink. Extracts were considered active if they gave a MIC ≤ 200 g/mL, standards were considered active if they presented a MIC ≤ 50 g/mL. 3.4. Bacteria The following strains of bacteria were used: Agrobacterium tumefasciens 2, Escherichia coli CCMI 270, Staphylococcus aureus CCMI 335 and Streptococcus faecium CCMI 338. The bacteria are from the Culture Collection of Industrial Microbiology LMI (INETI, Lisbon). Brain heart infusion (Merck, Darmstadt, Germany) was used to grow bacteria. 3.5. Antibacterial assays The MICs of the active extracts and compounds were determined by the broth dilution method (Muroi and Kubo, 1996). A 105 mL−1 cfu suspension was obtained by measurement of the cell concentration in a spectrophotometer (UNICAM 8700), at 620 nm wavelength and successive dilutions in saline solution. The following concentrations (g/mL) were tested: 200, 100, 50, 25 and 12.5. After incubation for 24 h at 30–37 ◦ C the microbial growth was examined and the MIC was defined as lowest concentration of the test compound yielding no visible growth. The MIC of each extract and compound was determined in duplicate. Samples with no visible growth were inoculated into the agar culture medium in order to detect bactericidal/bacteriostatic activity. Rifampicin (Sigma) was used as a positive control. 4. Results and discussion The ethanol extracts from 15 Hypericum species and three reference compounds were evaluated for their antimycobacterial and other antibacterial activities (Table 2). The most active extracts against M. tuberculosis H37 Rv were those obtained from H. elodes and H. hircinum subsp. majus, with MICs ranging from 25 to 50 g/mL. The screening of those active extracts against drug-resistant variants of the H37 Rv was also performed and a potent activity
was observed from H. foliosum, H. hircinum subsp. majus, H. grandifolium, H. humifusum and H. elodes with MICs ranging from 25 to 50 g/mL. H. elodes and H. hircinum subsp. majus were also active against drug resistant clinical isolates with MICs ranging from 12.5 to 50 g/mL. In spite of the weak activity of H. humifusum, H. foliosum and H. grandifolium against the reference strain H37 Rv, with MICs of 200 g/mL, they presented higher activities against the drug-resistant reference strains and clinical isolates. H. hircinum subsp. majus showed the higher activity only against M. fortuitum (MICs 50 g/mL), a non tuberculous Mycobacterium species. The lack of activity of H. perforatum observed in this work is contrary to that described by McCutcheon et al. (1997) using the disc diffusion method. This could be attributed to the differences in the screening methods and to the extremely high concentration used in the McCutcheon study. The activity observed for the H. calycinum extract is in accordance with the qualitative preliminary results of Gottshall et al. (1949). The highest level of Gram-negative antibacterial activity (MICs 2.5–200 g/mL) was found towards Agrobacterium tumefaciens [Rhizobium radiobacter] (UniProt; Young et al., 2001), the causal agent of crown gall disease (the formation of tumours) in over 140 species of dicotyledons, A. tumefaciens is still a serious pathogen of walnuts, grape vines, stone fruits, nut trees, sugar beets, horse radish and rhubarb. The chemical isolation to establish the phytochemical profiling and to identify the active constituents may open new perspectives for lead compounds. Staphylococcus aureus was the more susceptible Gram-positive bacteria (MICs 6–50 g/mL). H. perfoliatum, H. elodes, H. glandulosum, H. canariense, H. grandifolium, H. foliosum, H. hircinum subsp. majus, H. humifusum and H. calycinum strongly inhibited S. aureus (MICs 6–25 g/mL). Gibbons et al. (2002) and Gottshall et al. (1949) have reported no activity for the extract of H. calycinum. The positive results obtained for H. foliosum and H. hircinum subsp. majus against this microorganism are in accordance with those found by Gibbons et al. (2002) and Pistelli et al. (2000). The remarkable activity of H. glandulosum, H. grandifolium and H. canariense is in accordance with results formerly reported by Rabanal et al. (2002). Worthy of note is the potential for these active Hypericum spp. to be investigated for synergistic effects in treating staphylococcal infections, since there are currently being performed studies for combination therapies (Smirnova et al., 2011). Five Hypericum extracts, namely H. androsaemum, H. foliosum, H. grandifolium, H. canariense and H. glandulosum, also showed significant activity against Streptococcus faecium (MICs 12.5–50 g/mL). In Table 3 the composition data is gathered on the quantification of biologically active phenolic compounds, such as anthraquinones, flavonoids, flavonoid glycosides and phenolic
Table 2 Antimycobacterial and antibacterial activity of ethanol crude extracts of Hypericum spp. and pure compounds. Hypericum spp.
MIC (g/mL) Antimycobacterial activity
Antibacterial activity
Drug-resistant strains
Mycobacterium spp.
H37Rv
H37Rv INH-R
H37Rv ETAM-R
H37Rv SM-R
H37Rv RIF-R
Is.cl. SIN-3
Is.cl. SIN-4
Is.cl. HG8
Is.cl. MMDO
M. fortuitum
M. smeg.
M. avium
M. chelonae
E. coli
S. aureus
S. faecium
100 >200 200 25 200 >200 >200 >200 200 >200 >200 >200 >200 100 50
100 >100 50 25 50 >100 >100 >100 100 >100 100 >100 >100 100 50
>100 >100 50 100 50 >100 >100 >100 50 >100 100 >100 >100 100 50
100 >100 100 50 100 >100 >100 >100 50 >100 >100 >100 >100 100 25
100 >100 100 25 100 >100 >100 >100 50 >100 >100 >100 >100 100 50
100 n.t. 100 12.5 100 >100 >100 >100 >100 >100 >100 >100 >100 >100 50
>100 >100 100 100 100 >100 >100 >100 >100 >100 >100 >100 >100 >100 50
100 n.t. 100 50 100 n.t. n.t. n.t. 100 n.t. n.t. n.t. n.t. >100 50
100 >100 100 50 100 >100 >100 >100 >100 >100 >100 >100 >100 >100 50
>100 >100 >100 50 >100 >100 >100 >100 100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 100
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
>200 >200 n.t. >200 n.t. >200 >200 >200 >200 >200 n.t. >200 >200 n.t. >200
20 >200 12.5a 25 12.5 50 50 6 25 >200 12.5 >200 50 12.5 12
>200 50a 12.5a >200 50a >200 200 >200 200a >200 25a >200 >200 12.5a >200
>200 >200 25 ≤0.06
>50 >50 25 ≤0.06
>50 >50 25 ≤0.06
>50 >50 25 ≤0.06
>50 >50 25 >2
n.t. n.t. n.t. >2
>50 >50 25 >2
n.t. n.t. n.t. n.t.
>50 >50 25 0.125
>50 >50 >50 n.t.
50 >50 >50 n.t.
>50 >50 >50 n.t.
>50 >50 50 n.t.
n.t. n.t. n.t. 1
100a >100 50a 0.001
25a 100a 50a 0.5a
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H. calycinum H. androsaemum H. foliosum H. hircinum ssp. majus H. grandifolium H. perforatum H. undulatum H. perfoliatum H. humifusum H. linarifolium H. canariense H. tomentosum H. pubescens H. glandulosum H. elodes Compounds Hypericin Pseudohypericin Hyperforin Rifampicin
Drug-resistant clinical isolates
n.t.: not tested. Antimycobacterial activity: H37Rv – reference strain; drug resistant strains: H37Rv INH-R, H37Rv ETAM-R, H37Rv SM-R, H37Rv RIF-R – strains resistant to isoniazid, ethambutol, streptomycin and rifampin, respectively. Drug resistant clinical isolates: Is.cl.SIN-3 – resistant to streptomycin, isoniazid, rifampin, rifabutin, ethambutol, clarithromycin and ofloxacin, Is.cl.SIN-4 – resistant to streptomycin, isoniazid, rifampin, rifabutin, ethambutol, ethionamide and ofloxacin, Is.cl.HG8 – resistant to ethambutol, clarithromycin and ethionamide, Is.cl.MMDO – resistant to isoniazid and ethambutol. Mycobacteria species: M. fortuitum, M. smegmatis, M. avium, M. chelonae. Antibacterial activity: Gram− (Escherichia coli) and Gram+ (Staphylococcus aureus, Streptococcus faecium); rifampicin – positive control. a Bacteriostatic.
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−b −b n.c. 0.3g 0.012h 0.02g n.c. n.c. +e n.c. +c − − −; 0.12f 0.07 0.04 0.04 0.24 0.07 0.04 0.02 −; +c 0.002 n.c. 0.004 0.004 0.01 0.001 0.002 0.005 0.004 0.004 0.002 0.07 0.002 − 0.13 0.11 0.03 0.06 0.19 0.0009 0.0007 0.007
5. Conclusion Industrial development of medicinal plants play an important role in the discovery of new drugs. Natural products are directly or indirectly responsible for almost 40% of the drugs used in modern therapeutics. Considering that the development of new synthetic drugs demand for high costs the ethnopharmacology may constitute a precious guide for the selection of plant extracts and compounds (Grando et al., 2008). On the other hand the increasing incidence of multi-drug resistant strains worldwide determines the urgent need for new anti-multidrug resistant bacterial drugs. The assays performed in the present study describe the antimicrobial activity of fifteen Hypericum extracts, some of them herein investigated for the first time, with MICs ranging from 2.5 to 50 g/mL. In the assays with reference compounds the results obtained suggest that they might contribute to the antimicrobial activities observed in the extracts. From this study, new insights and evidence are open on the potential of Hypericum species as a source of new leads towards the discovery of either new candidates for the treatment of multidrug resistant tuberculosis and as potent antimicrobials for drug design.
0.08 0.03 0.02 0.41 0.17 0.03 0.31 0.21 0.12 0.27 0.003 0.05 0.13 0.65 0.97 0.70 0.19 0.88 1.02 0.003 − − 0.04 n.c. 0.005 0.001 0.001 − − 0.004 − 0.08 n.c. − 0.0006 − − 0.0001 − − − 0.0007 0.07 −
Isoquercitrina Rutina Caffeic acida
Hyperosida
0.12 0.0004 0.36 0.14 0.07 0.13 0.07 0.21 1.01 0.70 0.003
0.06 0.03 0.05 0.03 0.14 0.04 0.04 0.08 0.02 0.003 0.03
Amentoflavonea
Pseudohypericin
+d −e 0.14f 0.55g −h 0.03g n.c. n.c. n.c. n.c. +c
acids, including previous studies (Farinha et al., 2002). Significant differences of composition among Hypericum species were observed which might be related with the differences of biological activities verified in the present study. A patent on extracts from some Hypericum species useful for the treatment of persistent tuberculosis was previously registed (Nogueira et al., 2009). Three reference compounds isolated from H. perforatum, hypericin, pseudo-hypericin and hyperforin, were active against all the drug-resistant strains and some of the clinical isolates tested, with MICs from 25 to up to 50 g/mL. However only hyperforin showed activity against the strain H37 Rv at a MIC of 25 g/mL. All of them showed also some activity against non tuberculous Mycobacterium species (MICs ≥ 50 g/mL). The antibacterial activity was also assayed against Gram+ bacteria, Staphylococcus aureus and Streptococcus faecium, with MICs of hypericin and hyperforin ranging from 25 to 50 g/mL. Prior QSAR studies described quinones (Dimov et al., 2001) and biflavonoids (Dias et al., 2004) as active compounds against tuberculosis. Hypericin and pseudohypericin, two anthraquinones, are present in some Hypericum species (Farinha et al., 2002; Mathis and Ourisson, 1964; Kartnig et al., 1996; Umek et al., 1999). However, none of these compounds presented a huge activity against the whole group of Mycobacteria tested.
Acknowledgement Collaborative work was performed under the auspices of the Iberoamerican Program for Science and Technology (CYTED), Project X.11: PIBATUB.
n.c.: not confirmed; −: not present. a Farinha et al. (2002). b Kitanov (2001). c Piovan et al. (2004). d Klingauf et al. (2005). e Rainha et al. (2011). f Sagratini et al. (2008). g Hosni et al. (2010). h Rainha et al. (2012).
H. calycinum H. androsaemum H. hircinum ssp. majus H. perforatum H. undulatum H. perfoliatum H. humifusum H. linarifolium H. tomentosum H. pubescens H. elodes
0.9 1.46 − 0.06 0.19 0.91 4.18 4.81 0.97 0.84 0.006
References Chlorogenic acida
Compounds (mg/100 mg of plant) Species
Table 3 Cumulative data on quantitative HPLC analysis of extracts of 11 portuguese Hypericum species.
Quercitrina
Quercetina
Kaempferola
Hypericina
Hyperforin
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Barnes, J., Andersen, L.A., Phillipson, J.D., 2001. Hypericum perforatum L. A review of its chemistry, pharmacology and clinical properties. J. Pharm. Pharmacol. 53, 583–600. Collins, L.A., Franzblau, S.G., 1997. Microplate Alamar Blue Assay versus BACTEC 460 system for high-throughput screening of compound against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob. Agents Chemother. 41, 1004–1009. Costa, A.F., 1994. Farmacognosia, vol II. Fundac¸ão Calouste Gulbenkian, Lisboa1021–1023. Dias, J.C., Rebelo, M.M., Alves, C.N., 2004. A semi-empirical study of biflavonoid compounds with biological activity against tuberculosis. J. Mol. Struct. (Theochem) 676, 83–87. Dimov, D., Nedyalkova, Z., Haladjova, S., Schüürmann, G., Mekenyan, O., 2001. QSAR Modeling of antimycobacterial activity and activity against other bacteria of 3-formyl rifamycin SV derivatives. Quant. Struct. Act. Relat. 20, 298–318.
T. Nogueira et al. / Industrial Crops and Products 47 (2013) 126–131 Farinha, A., Martins, J.M., Nogueira, T., Tavares, R., Duarte, F.A., 2002. HPLC analysis of Hypericum L. species from Portugal. In: Nat. Prod. in the New Millennium. Kluwer Acad. Pub.125–134. Figueiredo, R., Ramos, D.F., Moiteiro, C., Medeiros, M.A., Marcelo Curto, M.J., Menezes, J.C., Pando, R.H., Silva, P.E.A., Costa, M.C., 2012. Pharmacophore insights into rpoB gene mutations in Mycobacterium tuberculosis rifampicin resistant isolates. Eur. J. Med. Chem. 47, 186–193. Gibbons, S., Ohlendorf, B., Johnsen, I., 2002. The Genus Hypericum – a valuable resource of anti-Staphylococcal leads. Fitoterapia 73, 300–304. Gibbons, S., Moser, E., Hausmman, S., Stavri, M., Smith, E., Clennett, C., 2005. An antistaphylococcal acylphloroglucinol from Hypericum foliosum. Phytochemistry 66, 1472–1475. Gibbons, S., 2008. Phytochemicals for bacterial resistance – strengths, weaknesses and opportunities. Planta Med. 74, 594–602. Gottshall, R.Y., Lucas, E.H., Lickfeldt, A., Roberts, J.M., 1949. The occurrence of antibacterial substances active against Mycobacterium tuberculosis in seed plants. J. Clin. Invest. 28, 920–921. Grando, R., António, M.A., Araújo, C.E.P., Soares, C., Medeiros, M.A., Carvalho, J.E., Lourenc¸o, A.M., Lopes, L.C., 2008. Antineoplastic 31-Norcycloartanones from Solanum cernuum Vell. Z. Naturforsch. 63c, 507–514. Hosni, K., Msaâda, K., Taârit, M.B., Hammami, M., Marzouk, B., 2010. Bioactive components of three Hypericum species from Tunisia: a comparative study. Ind. Crops Prod. 31, 158L 163. Jayasuriya, H., Clark, A.M., McChesney, J.D., 1991. New antimicrobial filicinic acid derivatives from Hypericum drummondii. J. Nat. Prod. 54, 1314–1320. Jimenez-Arellanes, A., Meckes, M., Ramirez, R., Torres, J., Luna-Herrera, J., 2003. Activity against multidrug-resistant Mycobacterium tuberculosis in Mexican Plants used to treat respiratory diseases. Phytother. Res. 17, 903–908. Kartnig, T., Göbel, I., Heydel, B., 1996. Production of hypericin, pseudohypericin and flavonoids in cell cultures of various Hypericum species and their chemotypes. Planta Med. 62, 51–53. Kitanov, G.M., 2001. Hypericin and pseudohypericin in some Hypericum species. Biochem. Syst. Ecol. 29, 171–178. Klingauf, P., Beuerle, T., Mellenthin, A., El-Moghazy, S.A.M., Boubakir, Z., Beerhues, L., 2005. Biosynthesis of the hyperforin skeleton in Hypericum calycinum cell cultures. Phytochemistry 66, 139–145. Luna-Herrera, J., Costa, M.C., González, H.G., Rodrigues, A.I., Castilho, P.C., 2007. Synergistic antimycobacterial activities of sesquiterpene lactones from Laurus spp. J. Antimicrob. Chemother. 59 (3), 548–552. McCutcheon, A.R., Stokes, R.W., Thorson, L.M., Ellis, S.M., Hancock, R.E.W., Towers, G.H.N., 1997. Anti-mycobacterial screening of British Columbian medicinal plants. Int. J. Pharm. 35, 77–83. Mathis, C., Ourisson, G., 1964. Étude chimio-taxonomic du genre Hypericum II. Identification de constituent de diverses Huilles Essentielles D’ Hypericum. Phytochemistry 3, 115–131. Muroi, H., Kubo, I., 1996. Antibacterial activity of anacardic acid and totarol alone and in combination with methicillin, against methicillin-resistant Staphylococcus aureus. J. Appl. Bacteriol. 80, 387–394. Nogueira, T., Costa, M.C., Moiteiro, C.M., Medeiros, M.A., Curto, M.J, Luna-Herrera, J., Rodriguez, E., 2009. Extracts from Hypericum L. species useful for the treatment of persistent tuberculosis. European Patent EP 1803463 B1.
131
Okuma, K., Iwakawa, K., Turnidge, J.D., Grubb, W.B., Bell, J.M., O‘Brien, F.G., Coombs, G.W., Pearman, J.W., Tenover, F.C., Kapi, M., Tiensasitorn, C., Ito, T., Hiramatsu, K., 2002. Dissemination of new methicillin-resistant Staphylococcus aureus clones in the community. J. Clin. Microbiol. 40, 4289–4294. Osman, K., Evangelopoulos, D., Basavannacharya, C., Gupta, A., McHugh, T.D., Bhakta, S., Gibbons, S., 2012. An antibacterial from Hypericum acmosepalum inhibits ATP-dependent MurE ligase from Mycobacterium tuberculosis. Int. J. Antimicrob. Agents 39, 124–129. Piovan, A., Filippini, R., Caniato, R., Borsarini, A., Maleci, L.B., Cappelletti, E.M., 2004. Detection of hypericins in the red glands of Hypericum elodes by ESI-MS/MS. Phytochemistry 65, 411–414. Pistelli, L., Bertoli, A., Zucconelli, S., Morelli, I., Panizzi, L., Menichini, F., 2000. Antimicrobial activity of crude extracts and pure compounds of Hypericum hircinum. Fitoterapia 71, S138–S140. Paulo, A., Martins, S., Branco, P., Dias, T., Borges, C., Rodrigues, A.I., Costa, M.C., Teixeira, A., Mota-Filipe, H., 2008. The opposing effects of the flavonoids isoquercitrin and Sissotrin, isolated from Pterospartum tridentatum, on oral glucose tolerance in rats. Phytother. Res. 22, 539–543. Rabanal, R.M., Arias, A., Prado, B., Hernández-Pérez, M., Sánchez-Mateo, C.C., 2002. Antimycrobial studies on three species of Hypericum from the Canary Island. J. Ethnopharmacol. 81, 287–292. Rainha, N., Lima, E., Baptista, J., 2011. Comparison of the endemic Azorean Hypericum foliosum with other Hypericum species: antioxidant activity and phenolic profile. Nat. Prod. Res. 25 (2), 123–135. Rainha, N., Lima, E., Baptista, J., Fernandes-Ferreira, M., 2012. Content of hypericins from plants and in vitro shoots of Hypericum undulatum Schousb. ex Willd. Nat. Prod. Res., pp. 1–11, iFirst. Sagratini, G., Ricciutelli, M., Vittori, S., Öztürk, N., Öztürk, Y., Maggi, F., 2008. Phytochemical and antioxidant analysis of eight Hypericum taxa from Central Italy. Fitoterapia 79, 210–213. Smirnova, M.V., Strukova, E.N., Portnoy, Y.A., Dovzhenko, S.A., Kobrin, M.B., Zinner, S.H., Firsov, A.A., 2011. The antistaphylococcal pharmacodynamics of linezolid alone and in combination with doxycycline in an in vitro dynamic model. J. Chemother. 23 (3), 140–144. Tosun, F., Kizilay, C¸.A., Sener, B., Vural, M., Palittapongarnpim, P., 2004. Antimicobacterial screening of some Turkish plants. J. Ethnopharmacol. 95, 273–275. Umek, A., Kreft, S., Katnig, T., Heydel, B., 1999. Quantitative phytochemical analyses of six Hypericum species growing in Slovenia. Planta Med. 65, 388–390. UniProt Taxonomy Rhizobium radiobacter (Agrobacterium tumefaciens) (Agrobacterium radiobacter). http://pir.uniprot.org/taxonomy/358 Valentão, P., Carvalho, M., Fernandes, E., Carvalho, F., Andrade, P.B., Seabra, R.M., Bastos, M.L., 2004. Protective activity of Hypericum androsaemum infusion against tert-butyl hydroperoxide-induced oxidative damage in isolated rat hepatocytes. J. Ethnopharmacol. 92, 79–84. World Health Organization Global, 2008. Tuberculosis Control Surveillance, Planning, Financing. WHO/HTM/TB/2008, 393. Young, J.M., Kuykendall, L.D., Martínez-Romero, E., Kerr, A., Sawada, H., 2001. A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al., 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis. Int. J. Syst. Evol. Microbiol. 51, 89–103.