PHYTOTHERAPY RESEARCH, VOL. 10,364-366 (1996)

SHORT CO MM UNlCATlON Antimicrobial Activity of Extracts from Endemic to the Canary Islands

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R. M. Herrera, M. Nrez, D. A. Marfin-Herrera*, R. Lbpez-Garcia and R. M. Rabanal Departamento de Farmacologia, Facultad de Farmacia, Universidad de La Laguna, Tenerife, Spain A. Arias Departamento de Higiene y Medicina Preventiva, Facultad de Medicina. Universidad de La Laguna, Tenerife, Spain

The antibacterial and antifungal activities of several extracts and fractions from four endemic Canary plants, Ceropegia fusca Bolle (Asclepiadaceae), Hypericum reflexum L. f. (Hypericaceae or Guttiferae), Maytenus canariensis (loes)Kunkel8t Sunding (Celastraceae) and pendulo Ait. (), were determined. The antimicrobial study was carried out by the dilution and diffusion methods and the results showed good antibacterial activity but no antifungal activity. The MeOH extract from Hypericum reflexurn was active against gram-positive bacteria Bacillus cereus, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Staphylococ- cus epidennidis and against the gram-negative bacterium Klebsiella pneumoniae.

Keywords: Ceropegia fusca; Hypericum refexum; Maytenus canariensis; Plocama pendula; endemic Canary plants; antimicrobial activities.

INTRODUCTION MATERIALS AND METHODS

Ceropegiu fusca Bolle (Asclepiadaceae), Hypericum material. The plants chosen were collected in April reflexurn L. f. (Hypericaceae or Guttiferae), Muyfenus 1991 in Tenerife (Canary Islands, Spain); Ceropegiufuscu canuriensis (loes) Kunkel & Sunding (Celastraceae) and Bole in ‘Montaiia de 10s Riscos’ (San Isidro, 100-300 m), Plocuma pendulu Ait. (Rubiaceae) are four endemic Hypericum reflexurn in ‘Bmanco de Badajoz’ (Guimar, of the Canary Islands (Bramwell and Bramwell, 1990; Wm), Maytenus cunuriensis in ‘Barranco de Badajoz’ Kunkel. 1992). The pharmacological activities of extracts (Guimar, 500-600 m) and Plocumu pendulu in ‘Barranco from these plants have not previously been investigated. de San Andris’ (San Andris, 20 m). The first three of these species have been used in the The plants’ identity was confirmed by Dr E. Barquin islands’ popular medicines as healing balms (Darias et ul., (Dpto. Biologia Vegetal, EUITA, Universidad de La 1986; Darias et al., 1989; Perez de Paz and Medina Medina, Laguna). A voucher specimen of each plant was deposited 1988). The aim of this study was to determine their possible in the Herbarium of the Department of Pharmacology, antimicrobial activities, and to correlate these activities with University of La Laguna. the traditional use. The last species, Plocumu pendulu, was chosen on the basis of its chemical composition, since the Preparation of extracts. The plants, previously dried and presence of anthraquinones (Gonzllez et al., 1977a) sug- powdered, were macerated sequentially in n-hexane and gested the presence of an antimicrobial type of activity methanol, for 3 days, three times each, at room temperature. (Fairbain, 1980). The resulting extracts being filtered, bound together and Ceropegiu fuscu is known by the popular name of dried under pressure at a temperature below 45 “C. ‘cardoncillo’ or ‘mataperros’ (Bramwell and Bramwell, Afterwards with the methanol extract a distribution 1990), cicatrice and wound healing properties have been between CHCl,/H,O was made and the chloroform and attributed to its popularity (Darias et al., 1986). Hypericum aqueous fractions obtained. Finally the aqueous fraction reflexurn is known popularly as ‘cruzadilla’ or ‘hierba cmz was again subjected to a separation between n-ButOHl de 10s isleiios, (Bramwell and Bramwell, 1990) and its aerial H,O. part is employed as a healing agent (Darias et al., 1986). The yield for each extract and fraction with respect to the Maytenus canuriensis is the only representative of its family initial dry material, was: Ceropegiu fusca: 5.1% for hexane in the Archipelago. This plant is known by the popular name and 12.0% for MeOH (CHCI, 37.5%, ButOH 29.0% and of ‘peralillo’ (Bramwell and Bramwell, 1990) and some aqueous 35.0%). Hypericum reflexum: 2.7% for hexane medicinal properties have been attributed to it (anti- and 14.7% for MeOH (CHCl, 26.6% ButOH 37.9% and rheumatic, emollient, treatment of cutaneous abscess and aqueous 35.8%). Maytenus cunuriensis: 2.1% for hexane papillomas) (Darias er ul., 1989). Plocama pendulu is the and 9.8% for MeOH (CHCl, 17.7%, ButOH 38.9% and only species of the genus and an endemic plant of Canary aqueous 43.6%). Plocumu pendulu: 1.2% for hexane Archipelago known popularly as ‘balo’ (Bramwell and and 28.3% for MeOH (CHCl, 10.3%, ButOH 42.8% and Bramwell, 1990). aqueous 47.3% Author to whom correspondence should be addressed. Infusions were prepared with 1OOg of leaves and

CCC 0951418X/96/040364-03 0 1996 by John Wiley & Sons, Ltd. Accepted (revised) 9 May I995 ANTIMICROBIAL ACTIVITY OF PLANTS FROM CANARY ISLANDS 365 lo00 mL of water and the volume reduced until finally Table 1. Antimicrobial activity by disc-diffusion method adjusted to a concentration of 1 g/mL dry material. Dose (3.75 mg/disc) Bacteria 123456C Preparation of samples. In the study of antimicrobial activity, Bacillus cereus 15 14 15 15 15 10 24 the different extracts and fractions were diluted in di- Bacillus subtilus 24 10 12 12 13 14 29 methylsulphoxide (DMSO). The corresponding concentra- Micrococcus luteus 22 9 10 13 17 14 29 tions are expressed in terms of mg of extract or fraction per Staphylococcus aureus 25 9 11 13 13 13 26 mL of solvent, with the exception of the infusion whose Staphylococcusepidermidis 21 9 10 14 15 14 24 Bordetella bronchiseptica 22 10 10 14 15 14 30 concentration is expressed in terms of mg/mL of initial dry -- material. Escherichia coli 9 7 10 7 31 Klesbsiella pneumoniae 23 10 11 13 16 14 28 Microorganisms. The following strains were used as test Pseudomonas aeruginosa - - 7- 8-11 organisms: Values represent inhibition diameter in mrn; (' Peptide nucleic Bacteria: Klebsiella pneumoniae (CECT 440), Pseudomo- acid with C-terminal amide. Net charge is + in solution. 1, MeOH extract of Hypericum reflexum; 2, CHCI, fraction of nus aeruginosa (CECT 1lo), Staphylococcus aureus (CECT Hypericum refleum;3, ButOH fraction of Hypericum reflexurn; 4, 240), Staphylococcus epidennidis (CECT 23 l), Bordetella MeOH extract of Maytenus canariensis; 5, ButOH fraction of bronchiseptica (CECT 142), Micrococcus luteus (CECT Maytenus canariensis; 6, Hexane extract of Ceropegia fusca; C, 247), Bacillus cereus var. mycoides (CECT 193), Bacillus chlorarnphenicol (30 pg/disk). subtilis (CECT 356), Escherichia coli (CECT 405). Fungi: Candida albicans (CECT Candida tropicalis 1001), Table 1. No antifungal activity was detected for any plant. (CECT Candida guilliermondii (CECT 1400), 1019), Plocamu pendula was the only species with wholly inactive Saccharomyces cerevisiae (CECT 1 Cryprococcus 193), extracts. The results express the diameter of inhibition albidus (CECT 1081). zones in mm of these extracts against different bacteria at Antimicrobial methods. The antibacterial and antifungal doses 3.75 mg. The results indicated that the MeOH, CHCl, effects were tested by disc-diffusion method (Bauer et al., and ButOH extracts from Hypericum refrexum gave a clear 1969) and the minimum inhibitory concentration (MIC) antibacterial activity against the majority of organisms determined by microdilution in microplates method (Jones tested. (except gram-negatives: Escherichia coli and Pseu- et al., 1987). domonas aeruginosa). The inhibition zones obtained with In the first method, three concentrations were prepared the MeOH extract are similar to those shown by chlor- for each extract: 125,250 and 375 mg/mL, and blank discs amphenicol. The inhibition zones of MeOH and ButOH were impregnated with 10 JLL of sample (final doses extracts from Maytenus canariensis and of the Ceropegia obtained: 1.25,2.50and 3.75 mg). Blank discs impregnated fusca hexane extract were modest. Likewise, in Table 2 the with DMSO were used as negative controls and chlor- MIC values (dilution method) of those active extracts, amphenicol (30 p,g-Difco) and amphotericin B expressed in mg/mL, are presented. The MIC values (100 Fg-Pasteur) disks were used as appropriate positive obtained confirm the existence of activity in the Hypericum controls. The inhibition zones were measured at 24 h. The refrexum MeOH extract against Bacillus cereus, Bacillus culture media employed were Muller-Hinton agar (Difco) subrilis, Micrococcus luteus, Staphylococcus aureus, Staph- for bacteria and Bacto Yeast Morphology agar (Difco) for ylococcus epidennidis and Klebsiella pneumoniae. fungi. In the second method, from an initial solution extract of 50mg/mL, doubling dilutions in the culture medium, DISCUSSION AND CONCLUSIONS Miiller-Hinton broth (Difco) for bacteria and Yeast Nitrogen Base (Difco) for fungi, were prepared, buffered at pH 7, and tested at a concentration range from 5 to 0.05 mg/mL. Hypericum refrexum showed antibacterial activity and the results obtained confirm the type bioactivity presented by most of the Hypericum species studied (Barbagallo and RESULTS Chisari, 1987; Sakar et al., 1988; Decosterd et al., 1990; Jayasurikab et al., 1991). The values reached are important Inhibition results of the diffusion-disc method only from with both methods, above all for the MeOH extract, against plant extracts that had antibacterial activity are shbwn in all gram-positive bacteria, and also against gram-negative

Table 2. MIC values for the different extracts MIC (mg/mL) Bacteria 1 2 3 4 5 6 Bacillus cereus 0.05 0.4 0.4 0.2 0.2 1.56 Bacillus subtilus 0.05 0.78 0.05 0.4 0.1 6.25 Micrococcus luteus 0.05 0.4 0.2 0.2 0.1 25 Staphylococcus aureus 0.05 0.2 0.05 0.05 0.05 0.78 Staphylococcus epidermidis 0.05 0.2 0.4 0.2 0.1 3.125 Bordetella bronchiseptica 1.56 3.125 0.2 0.4 0.05 3.125 Escherichia coli 1.56 6.25 6.25 1.56 1.56 12.5 Klebsiella pneurnoniae 0.05 0.78 0.1 0.05 0.1 3.125 Pseudomonas aeruginosa 12.5 25 6.25 3.125 3.125 3.125 1, MeOH extract of Hypericum reflexum;2, CHCI, fraction of Hypericum refleum; 3, ButOH fraction of Hypericum reflexum; 4, MeOH extract of Maytenus canariensis; 5, ButOH fraction of Maytenus canariensis; 6, Hexane extract of Ceropegia fusca. 366 R. M. HERRERA ETAL. Klebsiella pneumoniae. The inhibition zones obtained with being found in the ButOH fraction, not corresponding to the MeOH extract are similar to those shown by chlor- those isolated and assayed by Jimenez (1992). amphenicol, which is significant as we are dealing with an The Ceropegia fusca hexane extract was also active, extract and not pure product. The CHCl, and ButOH indicating products are of a lipophilic nature. However, extracts derived from the MeOH extract, showed inferior values obtained in both tests were not significant. Plocamu activity to those of the MeOH extract. The antibacterial pendula was the only species which showed no type of activity of the MeOH extract could result from a number of antimicrobial activity in spite of showing principals of compounds, some being present in the CHCl, fraction and anthraquinonic nature in its composition (GonAez et al., others in the ButOH. This is not unexpected since in the 1977a). majority of Hypericum species studied there are a large In short, we can conclude that the germicide activity of variety of active principals: hypericin, pseudohypericin, the first three plants justifies their popular use in wound flavonoids, essential oils, xanthones, ypyrone, hyperenon healing, as cicatrice and antiulcerogenic agents. Fur- B, n-alkanes, n-1-alkanols, tannins, filicinic acid derivatives, thermore, the important levels of inhibition presented by etc. (Kikuchi et al., 1985; Gunatilanka et al., 1983; Hypericum reflexurn make a more complete study to find the Jayasuriya et al., 1991). chemical agents responsible for their activity worthwhile. Antimicrobial activity from Maytenus canuriensis is also The activity shown by the ButOH fraction of Maytenus centred in the MeOH extract and the ButOH fraction, but canariensis should stimulate a study of the more polar not in CHCl,, suggesting that components responsible for extracts of this species, further supported by the chemical- the antibacterial activity are polar. Several products from pharmacological importance of the Celastraceae family. this species have been studied (Gonzdlez et al., 1989; Gonzhlez et al., 1990; Gonziilez et al., 1992), some of them showing important biological activities as cytostatics, Acknowledgements antifeedants and insecticide drugs (Gonziilez et al., 1977b; Gonzdlez et al., 1993a; Gonzalez et al., 1993b). A study of The'authors thank Dr E. Barquin for his help in the localization and the antimicrobial activities of dihydro-pagarofwan sesqui- identification of plant material. This work was supported by a Grant from terpenes isolated from this plant was carried out, but no the Consejen'a de Educaci6n. CUlNra y Deportes del Gobierno Aut6nomo antimicrobial activity was found (JimCnez., 1992). How- de Canarias (no 53/90). Part of this work was presented at the 2nd ever, these results are not in disagreement with our study, European Colloquium on Ethnopharmacology (ESE)- I lth International since the substances responsible for the antimicrobial Conference on Ethnomedicine (AGEM), in March 1993, Heidelberg. activity of Maytenus canariensis must be of a polar nature

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