Research in Microbiology 153 (2002) 647–652 www.elsevier.com/locate/resmic
Antimicrobial and antiviral activities of polyphenolics from Cocos nucifera Linn. (Palmae) husk fiber extract Daniele Esquenazi a,∗ , Marcia D. Wigg b , Mônica M.F.S. Miranda b , Hugo M. Rodrigues c , João B.F. Tostes c , Sonia Rozental a , Antonio J.R. da Silva c , Celuta S. Alviano d a Laboratório de Biologia Celular de Fungos, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (UFRJ),
21941-590, Ilha do Fundão, Rio de Janeiro, RJ, Brazil b Laboratório Experimental de Drogas Antivirais e Citotóxicas, Instituto de Microbiologia Professor Paulo de Góes, UFRJ,
21941-590, Ilha do Fundão, Rio de Janeiro, RJ, Brazil c Núcleo de Pesquisas de Produtos Naturais, UFRJ, 21941-590, Ilha do Fundão, Rio de Janeiro, RJ, Brazil d Laboratório de Estruturas de Superfície de Microrganismos, Instituto de Microbiologia Professor Paulo de Góes, UFRJ,
21941-590, Ilha do Fundão, Rio de Janeiro, RJ, Brazil Received 17 July 2002; accepted 2 October 2002 First published online 2 October 2002
Abstract The decoction of Cocos nucifera L. husk fiber has been used in northeastern Brazil traditional medicine for treatment of diarrhea and arthritis. Water extract obtained from coconut husk fiber and fractions from adsorption chromatography revealed antimicrobial activity against Staphylococcus aureus. The crude extract and one of the fractions rich in catechin also showed inhibitory activity against acyclovirresistant herpes simplex virus type 1 (HSV-1-ACVr). All fractions were inactive against the fungi Candida albicans, Fonsecaea pedrosoi and Cryptococcus neoformans. Catechin and epicatechin together with condensed tannins (B-type procyanidins) were demonstrated to be the components of the water extract. 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Cocos nucifera; Antimicrobial activity; Antiviral activity; Catechins; Condensed tannins
1. Introduction The use of natural products with therapeutic properties is as ancient as human civilization and, for a long time, mineral, plant and animal products were the main source of drugs [7]. Plants can produce antimicrobial compounds to protect themselves from biotic attack that could be essential for microbial infection resistance [32]. Alternative mechanisms of infection prevention and treatment should be included in the initial activity screenings [5]. One approach that has been used for the discovery of antimicrobial agents from higher plants is based on the evaluation of traditional medicinal plant extracts. Cocos nucifera L. (Palmae) species is widely distributed on the Brazilian northeastern coast. When the fruit is fully * Correspondence and reprints.
E-mail address:
[email protected] (D. Esquenazi).
ripe, the husk turns brown, dry and very fibrous with a high content of pentosans, cellulose and lignin [9]. An extensive range of popular medicinal uses of this plant has been reported [9]. The husk fiber decoction has been used in northeastern Brazil traditional medicine for treatment of diarrhea and arthritis. Heating the coconut shells gives an oil that is used against ringworm infections in the popular medicine of India. The alcoholic extract of ripe dried coconut shell has antifungal activity against Microsporum canis, M. gypseum, M. audouinii, Trichophyton mentagrophytes, T. rubrum, T. tonsurans and T. violaceum. The activity was mainly attributed to the high content of phenolic compounds [30]. These facts and the emergence of drug-resistant strains of many infectious microorganisms prompted us to start investigations on the composition and activities of the aqueous extract obtained from the husk fiber of C. nucifera. In order to study the therapeutic properties of C. nucifera extract, we used antibacterial, antifungal and antiviral assays to determine active compounds. As part of our contin-
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ued program on antimicrobial activity of Brazilian plants, we started a biomonitored assay of C. nucifera extracts. The microorganisms were chosen for the following reasons: The bacterium Staphylococcus aureus was used because of its importance as a human pathogen since S. aureus rapidly develops resistance to many antimicrobial agents that cause therapeutic problems [14]. S. aureus can cause superficial skin lesions (boils and furuncles) and more serious infections such as pneumonia, meningitis and urinary tract infections. It is a major cause of hospital-acquired (nosocomial) infection of surgical wounds and infections associated with indwelling medical devices. S. aureus may also cause food poisoning and toxic shock syndrome. Candida albicans is responsible for mucosal and systemic mycoses and it is one of the most pervasive pathogenic fungi, especially infecting immunocompromised hosts, in which it can invade various tissues [1]. Cryptococcus neoformans, the agent of cryptococcosis, is an increasingly important disease that has a worldwide distribution and is a frequent opportunistic pathogen in AIDS patients [8]. Fonsecaea pedrosoi is the major etiologic agent of chromoblastomycosis, a chronic disease usually limited to the infected subcutaneous tissue of humans and animals even in immunocompetent hosts [29]. The herpes simplex virus (HSV) is an important pathogen that causes morbid diseases, such as mucocutaneous lesions, ophthalmic and neonatal infections. None of the current drugs used in the treatment of herpetic infections eliminate the latent virus in the ganglion, nor prevent its recurrence [24]. The emergence of resistant mutants is easily identified after prolonged therapy with the available synthesized antiviral drugs. AIDS patients are more susceptible to severe infections caused by these mutants, although resistant mutants have already been reported in lesions of an immunocompetent individual [16]. In the present work, we used an acyclovir-resistant herpes simplex virus type 1 (ACVr-HSV-1) to study the antiviral activity of C. nucifera.
2. Materials and methods
University of São Paulo, Brazil. The strain was isolated from human with meningoencephalitis and AIDS. Cells of C. neoformans were grown in a BHI (brain heart infusion) medium, cultivated at room temperature for 5 days. C. albicans 7173 serotype B was obtained from a human case of mucocandidiasis at the Laboratory of Medical Mycology, Hospital Evandro Chagas, FIOCRUZ, Rio de Janeiro. It was cultivated in a BHI medium at room temperature for 5 days. Stock cultures of all microorganisms were maintained on Sabouraud dextrose agar under mineral oil and kept at 4 ◦ C. Acyclovir-resistant herpes simplex virus type 1 (ACVrHSV-1) was isolated from a typical labial lesion from the Virology Department of Federal University of Rio de Janeiro [17]. The virus was acyclovir-resistant up to the concentration of 25 µg/ml [18]. 2.2. Plant material C. nucifera L. (Palmae) var. typica A, commonly known as ‘Olho-de-Cravo’, was collected in Aracaju, Brazil and authenticated by Dr. Benedito Calheiros Dias, Centro de Pesquisas do Cacau, Bahia, Brazil. 2.3. Extract and fraction preparation The husk fiber of coconut (414 g) was dried in the sun, finely ground and the powder soaked for 3 h in 6 l of boiling distilled water. The extract was filtered and lyophilized, yielding 21 g of crude water extract. 500 mg of the lyophilized crude water extract was dissolved in water (1 l) and chromatographed on Amberlite XAD-2. The separation was developed with 500 ml methanol/water (1:1) and 500 ml pure methanol giving fractions I to V. TLC chromatography (silica; solvent system: AcOEt/AcOH/HCO2 H/H2 O, 100:11:11:27) was used to evaluate the separation. Spots were visualized by spraying vanillin-sulfuric acid reagent. Alternatively, the aqueous extract was partitioned with ethyl acetate.
2.1. Microorganisms 2.4. Compound identification Strains of S. aureus isolated from human infections were obtained from University Hospital of the Federal University of Rio de Janeiro (UFRJ) and one strain used was obtained from American Type Culture Collection (ATCC 2592). They were grown on Mueller–Hinton agar plates. A pathogenic strain of F. pedrosoi was isolated from a human case of chromoblastomycosis [20]. Mycelium was inoculated in Czapeck–Dox chemically defined medium [2] and for conidium formation, cultures were inoculated for 5 days under shaking conditions at room temperature. Conidia were isolated by filtration, centrifugation and resuspended in the medium. C. neoformans var. neoformans T1 -444, serotype A was kindly provided by Olga Fisherman from the Federal
Ethyl acetate extract was used for HPLC/DAD analyses, made according to a protocol devised by Peng and coworkers to analyze procyanidins in grape seeds [23]. Electrospray LC/MS analysis was made with a reverse phase RP-18 column (125 × 2 mm, 5 µm particles). Elution was achieved with solvents A (0.05% TFA in water) and B (acetonitrile), linear gradient from 0 to 50% B in 20 min, held isocratic at 50% B up to 45 min and then reduced to 0% B from 45 to 55 min. Flow rate was 1 ml/min and 5 µl of sample were injected. The apparatus was operated in positive mode, scanning from 300 to 2000 amu. Mass chromatograms were also obtained by monitoring peaks between masses 550–600, 850–900 and 900–2000 amu.
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A crude tannin sample was submitted to chromatography (Toyopearl HW-40F 26 × 2.5 cm, eluent: EtOH/H2 O from 30:70 to 60:40 v/v and Sephadex LH-20, 100 cm × 1 cm, eluent: ethanol) to separate the catechins from oligomeric/ polymeric fraction. Catechin and epicatechin were also isolated and their structures confirmed by comparison with standards. Catechin and epicatechin were also identified by co-injection of the sample plus their standards. The oligomeric/polymeric fraction was then submitted to degradation with phloroglucinol in 1% HCl for 30 min, under N2 [10]. The resulting products were analyzed by HPLC on a RP-18 column (250 × 4 mm, 5 µm particles). Elution was made with solvents A (1% acetic acid in water) and B (methanol/1% acetic acid in water, 6:4), linear gradient from 0 to 60% B in 60 min and then to 100% B from 60 to 65 min. Flow rate was 1 ml/min. 2.5. Antibacterial and antifungal activity The agar diffusion method was carried out to evaluate antibacterial and antifungal activity of the crude extract and fractions. Each extract was evaluated for its ability to inhibit the growth of three species of fungi and one species of bacteria. The extract was diluted in water (10 mg/ml) and sterilized by filtration using a 0.22-µm membrane. 10-µl aliquots of crude extract and fractions I–V were applied to newly inoculated Mueller–Hinton agar plates for S. aureus and to separate BHI agar plates containing either the yeast C. albicans, or C. neoformans or conidia of F. pedrosoi. Zones of inhibition were measured after incubation at room temperature for 24 h (bacteria) and for 48 h (fungi). Chloramphenicol and ketoconazol were tested concomitantly on each occasion as references for evaluation of antibacterial and antifungal activities respectively, in identical conditions. The results are reported as the average of three experiments. 2.6. Cytotoxicity and antiviral tests The lyophilized plant extract and fraction II were prepared at a final concentration of 200 µg/ml, immediately diluted with Eagle’s minimum essential medium (MEMEagle) without serum and sterilized by filtration using a 0.22-µm membrane. HEp-2 cells (human larynx carcinoma cell line) and Vero cells (Cercopitheccus aethiops kidney epithelial cells) were cultivated in MEM-Eagle supplemented with 5% fetal bovine serum, garamicin and fungizon. The maximum non-toxic concentrations (MNTC) of each sample were determined by the method described by Walker et al. [31], with small modifications [18], based on cellular morphologic alterations. Several concentrations of each sample were placed in contact with confluent cell monolayers and incubated in 5% CO2 atmosphere at 37 ◦ C for five days. At the end of this time the cells were examined using an inverted optical microscope (Leitz), comparing treated and control untreated cultures, and determining the MNTC.
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The antiviral activity was achieved by the reduction of the virus titers using TCID50 determinations. HEp-2 and Vero cell monolayers cultivated in 96 well microplates were treated with two-fold dilutions of the extracts and fractions beginning from the maximum non-toxic concentration. Immediately after, logarithmic dilutions of ACVr-HSV-1 were added to the treated and untreated cell cultures and incubated in 5% CO2 atmosphere at 37 ◦ C. After five days of incubation the virus titer was calculated by the Reed and Muench statistical method and expressed in TCID50 [27]. The results were expressed in viral inhibition index (VII) calculated by the formula VII = B − A, where B is the virus titer in virusinfected control (no extract), and A is the virus titer in the test samples. The percentage of inhibition (PI) [19] using antilogarithmic values of TCID50 was calculated as follows: PI = (1 − T antilogarithm/C antilogarithm) × 100. In another experiment, the extract and fraction II, at the MNTC, were either added to ACVr-HSV-1 (105,3 TCID50/ ml) or MEM-Eagle [3]. The mixtures were incubated at 37 ◦ C for 2 h, with shaking every 20 min. Next, the mixtures were diluted and inoculated in HEp-2 and Vero cells cultivated in microplates. The microplates were incubated for five days at 37 ◦ C in an atmosphere of 5% CO2 . At the end of this time the residual virus titer was determined by the Reed and Muench [27] statistical method and expressed in TCID50 . The virucidal index (VI) and percentage of inhibition were calculated as mentioned above.
3. Results Fractions II–V produced in vitro antimicrobial activity in assays against all strains of S. aureus tested. The zones of inhibition produced in agar diffusion assays ranged from 0 to 13 mm, as shown in Table 1. No growth inhibition was observed when the extracts were tested with all three species of fungi. Table 2 shows the MNTC and inhibitory effect of the crude extract and fraction II. As can be observed, the crude extract had a protective effect displaying a viral inhibition index (VII) > 3.0 (PI > 99.9%) in HEp-2 and Vero cells, at Table 1 Antibacterial activity of the C. nucifera husk fiber crude extract and fractions against S. aureus Materiala Crude extract Fraction I Fraction II Fraction III Fraction IV Fraction V
Zones of inhibitionb ATCC2592c
D063d
D065d
D075d
8e
8e
7e
0 12 11 9 8
0 13 12 10 9
8e 0 12 11 10 9
0 13 12 11 10
a 10 µl of a 10 mg/ml solution made with each extract or fraction. b Diameter, in mm, of the zone of inhibition. c S. aureus standard. d Strains of S. aureus isolated from patients at University Hospital (UFRJ). e Partial
inhibition.
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Table 2 Cytotoxicity and antiviral activity of C. nucifera husk fiber crude extract and fraction II Cell
Material
MNTC (µg/ml)
Inhibitory effect VI
PI
VII
PI
3.2 5.0
> 99.9 > 99.9
3.13 4.59
> 99.9 > 99.9
HEp-2
Crude extract Fraction II
100 25
1.81 1.0
98.4 90.0
VERO
Crude extract Fraction II
100 100
3.0 3.25
> 99.9 > 99.9
MNTC: maximum non-toxic concentration; VII: viral inhibition index; PI: percentage of inhibition; VI: virucidal index.
Fig. 1. Trace of reverse phase HPLC analysis of ethyl acetate partition extract. Captions in figure indicate catechin (1), epicatechin (2) and polymeric procyanidins (3). The remaining peaks belong to oligomeric (trimers and dimers) procyanidins.
non-toxic concentrations. Fraction II had a selective toxicity to HEp-2 cells and it had higher antiviral activity than the crude extract showing VII of 5.0 and 4.59 for HEp-2 and Vero cells, respectively. When the virus was directly treated with either the extract or fraction II, the virucidal index (VI) was equal to 1.81 (PI = 98.4%) and 1.0 (PI = 90%) for HEp-2 cells, respectively, and 3.0 (PI > 99.9%) and 3.25 (PI > 99.9%) for Vero cells, respectively. The contents of fractions II–V were evaluated (HPLC) and the results indicated the same general composition (catechins and tannins) for fractions II–V, with the highest concentration in fraction II. The ethyl acetate extract displayed the same bulk composition of the mix of fraction II–V. In view of these results we decided to simplify the procedure, using partition with ethyl acetate instead of XAD-2 chromatography. Application of the reverse phase C18 HPLC method devised by Peng and co-workers [23] to the crude ethyl acetate extract produced the chromatogram shown in Fig. 1, where peaks eluting between 10 and 40 min belong to catechin, epicatechin and B-type procyanidin dimers and trimers. The large peak at 55–60 min belongs to a mixture of polymers with more than five monomeric units.
Catechin, epicatechin and epicatechin-(4→2)-phloroglucinol were obtained when the oligomeric/polymeric fraction was submitted to degradation with phloroglucinol in acidic medium [5]. The results suggest that, for the procyanidins studied, both epicatechin and catechin are terminating units and that the extender flavan unit is epicatechin. The electrospray mass chromatograms (Fig. 2) at 579 amu (M + 1, procyanidin, dimers) and 867 amu (M + 1, procyanidin, trimers) indicated that compounds eluting between 10 and 40 min were dimers and trimers of B-type procyanidins. Peaks for tetramers (M + 1 = 1155 amu) or pentamers (M + 1 = 1443 amu) were not detected.
4. Discussion The results presented can be considered very promising for the isolation of compounds with antibacterial and antiviral activities and can be attributed to the presence of catechins and epicatechins on the coconut husk fiber extract. Liquid chromatography coupled with electrospray ionization mass spectrometry has been largely used to analyze condensed tannins [11,12,23]. Both positive [23] and negative [11,12] ion detection has been currently used. We used
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Fig. 2. Plot of mass chromatograms from the electrospray ionization HPLC/MS tannin analysis, mass ranges m/z 550–600 and 850–900, positive ions.
positive ion detection in view of the results of a previous work [23] where this mode of detection led to higher ion counts and less multiply charged ions, improving sensitivity and simplifying interpretation of data. Thus, the mass chromatogram peaks detected at m/z 579 and 867 are consistent with pseudomolecular ions for oligomeric B-type procyanidins of two and three flavan units. The several peaks displayed (Fig. 2) may be attributed to the presence of at least as many isomers. S. aureus was selected as a first bacterial target for C. nucifera fractions due to its importance as a possible pathogen bacterium. The effect of catechin concentrations in each fraction and/or the different catechin compositions of the fractions could explain the different antimicrobial activities of C. nucifera fractions against S. aureus. The results displayed in Table 1 suggest that catechins and the Btype procyanidins present in higher concentration in fraction II were responsible for the antimicrobial activity against S. aureus, demonstrating a higher inhibitory effect for S. aureus. This was supported, in part, by the negative results obtained with fraction I (absence of catechin), the partial inhibition shown by the crude extract (low concentration) and positive inhibition when fractions II, III, IV and V, rich in catechin, were tested. As minor concentrations of these compounds were also detected in the remaining fractions (III–V) this could also explain their activity. The investigation of plant extracts effective against S. aureus provides an example of prospecting for new compounds which may be particularly effective against infections that are currently difficult to treat. These results are in agree-
ment with the finding that catechins of olive plant tissue showed bactericidal activity towards Pseudomonas aeruginosa and S. aureus [22]. Chosa et al. [4] suggested that the purified catechins from green tea could be used as prophylactic agents against the important bacterium Mycoplasma pneumoniae infection. In addition, these plant extracts were not active against the fungi C. albicans, F. pedrosoi and C. neoformans when tested in a separate assay, indicating that they were selective in their antimicrobial activity. The negative results displayed when crude extract and all fractions were tested against these fungi were unexpected in view of the antifungal activity reported for the coconut shell exudates with other fungi [30]. The inhibitory effect of extracts containing catechins and condensed tannins on herpes simplex virus replication has already been reported [6,28]. The antiviral activity has been attributed to an interference with virus cell adsorption rather than virus penetration. In fact, in our study, an antiviral activity was observed when the cells were treated either with the extract or fraction II before virus infection. In addition, the extract and the fraction were also able to inactivate the extracellular virus (virucidal effect). Some authors also found that catechins were capable of inhibiting tumor cell lines [21,33]. Kirszberg and coworkers [15] reported that the C. nucifera extract was capable of inhibiting the proliferation of an erythroleukemic cell line (K562). In fact, the extract was more cytotoxic to HEp-2 (larynx carcinoma) culture than to Vero cells. In recent years, there has been growing interest in alternative therapies and the therapeutic use of natural products,
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especially those derived from plants [26]. Researches are increasingly turning their attention to folk medicine, looking for new leads to developing better drugs against viral and microbial infections [13,25]. The present study tends to confirm the use of folk medicine as safe and useful in the treatment of several diseases produced by microorganisms. The selective antibacterial activity of C. nucifera against S. aureus and HSV-1 suggests that this plant may be useful for topical application in wound healing. Since this plant has been used in diverse medicinal uses, other activities may be identified using different biological assays. C. nucifera may be important in the identification of some novel treatment modalities. Acknowledgements The authors wish to thank E. Brand, P.P.F. Ribeiro, R.B. Varella, M.A.A.M. Guimarães and A.C.D. Castro for their kind help. This work was supported by CNPq, FAPERJ, FUJB and PRONEX. References [1] G.P. Bodey, Candidiasis: A growing concern, Am. J. Med. 77 (1984) 1–48. [2] C. Booth, Fungal culture media, in: Methods in Microbiology, Vol. 4, Academic Press, New York, 1971, pp. 49–94. [3] M. Chen, P. Griffith, H.L. Lucia, G.D. Hsiung, Efficacy of S26308 against guinea pig cytomegalovirus infection, Antimicrob. Agents Ch. 32 (1988) 678–683. [4] H. Chosa, M. Toda, S. Okubo, Y. Hara, T. Shimamura, Antimicrobial and microbicidal activities of tea and catechins against Mycoplasma, Kansenshogaku Zasshi 66 (1992) 606–611. [5] M.M. Cowan, Plant products as antimicrobial agents, Clin. Microbiol. Rev. 12 (1999) 564–582. [6] T. De Bruyne, L. Pieters, M. Witvrouw, E. De Clercq, D. Vanden Berghe, A.J. Vlietink, Biological evaluation of proanthocyanidin dimers and related polyphenols, J. Nat. Products 62 (1999) 954–958. [7] A. De Pasquale, Pharmacognosy: The oldest modern science, J. Ethnopharmacol. 11 (1984) 1–16. [8] F. Dromer, B. Dupont, The increasing problem on fungal infections in the immunocompromised host, J. Mycol. Med. 6 (1996) 1–6. [9] J.A. Duke, Handbook of Phytochemical Constituents of Gras Herbs and Other Economic Plants, CRC Press, 1992. [10] L.Y. Foo, J.J. Karchesy, Procyanidin polymers of Douglas fir bark: Structure from degradation with phloroglucinol, Phytochemistry 28 (1989) 3185–3190. [11] H. Fulcrand, S. Remy, J.M. Souquet, V. Cheynier, M. Moutonet, Study of tannin oligomers by on-line liquid chromatography electrospray ionization mass spectrometry, J. Agr. Food Chem. 47 (1999) 1023– 1028. [12] S. Guyot, T. Doco, J.M. Souquet, M. Moutonet, J.F. Drilleau, Characterization of highly polymerized procyanidins in cider apple (Malus sylvestris var. kermerrien) skin and pulp, Phytochemistry 44 (1997) 351–357.
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