Journul
of Ethnopharmacobgy,
Elsevier Scientific Publishers
111
29 (1990) 111- 116 Ireland Ltd.
Short Communication
ANTIMICROBIAL
R.M. PEREZ
ACTIVITY
G., J.G. AVILA
OF SOME AMERICAN
A., S. PEREZ
G., A. MARTINEZ
ALGAE
C. and G. MARTINEZ
C.
Escuela Superior de Ingenieria Quimica e Indust& Extractivas, Instituto Polite’cnico National and Escuela Nacionul de Estudios Profesionales Ixtacalo, Universidad Autbnoma MetropolitanaXochimilco, Punto Fijo 16, C.P. 07050, Me’xico D.F. lMe’xicol (Accepted
November
9, 19891
Introduction
In the decade of the forties, interest was developed in marine flora and fauna as potential sources for the production of sustances with antimicrobial, antiviral and antifungicidal properties (Baslow, 19691. A number of algae of the Chlorophyceae, Rhodophyceae and Phaeophyceae are capable of producing antimicrobial sustances such as hydroquinones, sesquiterpenic phenols, brominated phenols, polyphenols, etc. (Faulkner, 19781. This study was designed to screen several marine and continental algae with the purpose of finding those which have the capacity to inhibit microbial growth in vitro. Materials and methods
Plant materials Plant material was collected in the Gulf of Mexico (Veracruzl, Pacific Ocean (Zihuatanejo, Ixtapa, Michoadn and Peru), Gulf of Cortes (La Paz) and Caribbean Sea (Venezuela). The continental algae were collected in Lake Cuitzeo (Guanajuatol, Lake Media Luna (San Luis Potosil, River Las Estacas (Morelosl and Lake Titicaca (Peru). The plants were identified by Dr Maria de Jesus Flores Departimento de Algas, Escuela National de Ciencias Biologicas, IPN (Mexico). Herbarium sheets were deposited in the same intitution. Extract preparation One hundred grams of dried plants material were ground and extracted in a Soxhlet for 6 h with 500 ml of 95% ethanol. The ethanol extract was taken to dryness under reduced pressure at 40°C. Extract yields are noted in 0 1990 Elsevier 037%87411502.45 Published and Printed in Ireland
Scientific Publishers
Ireland Ltd.
1
SEA ALGAE
australe
Spyridia filamentosa Vidalia obtusifolia
Polysiphonia subtilissima Prionitis decipiens Scinata jo fins toniue
Lithophyllum
Gracikwia cerwicornis Gracilaria cervicornis Gracilati sjoestedtii Gymnogon grus marhnensis Hypnea cervicornis Hypnea musciformis Hypnea spinella Lauren& obtusa
Centroceras clavalutum Geranium fastigiatum Corallina subuluta Graciluria cervicornis
Amphiroa hancoki Bostrychia montagne Bryochdia cuspidata Bryocludia thysigera
Rhodophyceae Amansia multifida
Species
AMERICAN
TABLE
Venezuela Peru Peru La Paz Veracruz
La Paz Veracruz Venezuela Veracruz Zihuatanejo
Veracruz La Paz La Paz Michoacan
Michoacan Veracruz Veracruz Venezuela Veracruz Venezuela Veracruz Verezuela
6.9 6.4 9.4 9.7 10.7
10.9 11.1 10.4 12.3 8.5 9.7 11.8 7.8 11.7
9.6 11.5 8.3 12.4 8.9 10.8 9.6 7.4 10.6
Extract yield (%I
16.3 (25) 22.1 (20)
17.8 (25) 26.5 (5)
27.1 (20)
24.3 (10) 21.6 (15)
I
Antimicrobial
EFFECTS
22.8 (10)
FOR ANTIMICROBIAL
Zihuatanejo
Sample origin
TESTED
24.2 (10)
17.3 (15) 17.8 (20)
14.5 (15)
18.2 (20)
II
activity III
zone inhibition
19.4 (20)
IV
V
mm (cone. mg/mlP VI
VII
La Paz Michoacan Venezuela Veracruz Peru
Ulva Ulva Ulva Ulva Ulva L&a
*I: S. aureus, II: S. pyogenes,
26.2
(5)
4.5 (25)
26.5 (501
16.3 (50) 17.8 (10)
16.8 (50)
22.1 (25)
27.8 (10)
15.5 (20)
IV: Pr. vulgaris, V: E. colt VI: A. fumigatus,
10.7 6.3 8.1 9.4 8.7 10.2
8.8 12.9 11.8 7.4 12.8 10.3 9.7 9.4 10.3 7.6 11.7 12.3 11.9 12.8 11.6
III: Ps. aeruginosa,
Zihuatanejo Zihuatanejo Peru La Paz
Enteromorpha flelucia Enteromorpha lengulata Halimeda opuntia Rhizoclonium kerneru
fasciata lactuca lactuca lactuca lactuca rigida
Veracruz Verezuela
Zihuatanejo Peru Michoacan Venezuela La Paz
Venezuela La Paz Lxtapa
Codium caneatum Codium taylorii Cymopolia barbata Enteromorpha flexuosa
Caulerpa sertubwioides Chaetomorpha antennina Chaetomorpha media Chaetomorpha media
Chlorophyceae Caulerpa racemosa Caulerpa sertularioides Caulerpa sertularioides
Sargassum macdogalu Sargassum semicola Sargassum vulgare
10.6
Ixtapa
Zihuatanejo La Paz La Paz Veracruz
Padina viekersiae
Sargassum lie bmannii 12.5 9.8 10.9 11.6
6.9 10.3 11.7
Zihuatanejo La Paz Michoacan
Ectocarpus breviarticulatus Padina durvillaei Padina vickersiae
11.7 7.8 9.2
Zihuatanejo Venezuela Veracruz
Phaeophyceae Chnoospora minima Dictyopteris delecatula Dictyopteris delacatula
VII: C. albicans.
24.2 (5) 23.4 (15)
18.2 (15)
20.1 (20)
19.3 (10)
19.3 (25)
18.6 (10)
12.1 (20)
114
Table 1. Thick tarry residues were obtained, parts of which were dissolved in distilled water at six concentrations (5, 10, 15, 20, 25 and 50 mglmll. Antimicrobial screening Nutrient media was prepared according to manufactures instructions (trypticase soy broth, TSB, Squibb and Sabouraud-dextrose, Merck) and sterilized for 20 min at 12OOC. The following microorganisms were used for this test: (11 Staphylococcus aureus, (111Staphyloccus pyrogenes, (III) Pseudomonas aeruginosa, (IV) Proteus vulgaris, (Vl Escherichia ~0.5, (VI) Aspergillus fumigatus, (VII) Candida albicans. S. aureus, S. pyrogenes, Pr. vulgaris, E. coli and Ps. aeruginosa were cultured overnight at 37OC in TSB. C. albicans was cultured for 48 h at 27OC in Sabouraud-dextrose. Each culture was diluted with sterile 0.9% saline solution (1 : 10001 to give a suspension of about lo8 microorganisms per ml. An amount of 0.1 ml of this suspension was spread on the test plate. Four wells, 1 cm in diameter, were made in each plate, into which 0.1 ml of test solution was pipetted. All determinations were made at least in duplicate. For the bacteria, a positive control with streptomycin (Sigma) was used (0.1 ml of a 10 mglml solution1 and for the mold and yeast, nystatin (Squibb) was used (0.1 ml of a 50 mglml solution). For the negative control, 0.1 ml of distilled water was used. Inhibition zones were read 1 day after incubation at 37OC for the bacteria and after 3-4 days at 27OC for the mold and yeast. Results and discussion The antimicrobial effects of sea water algae are summarized in Table 1. Tested fresh water algae are listed in Table 2. Of the 65 plant samples tested, 9 showed activity against S. aureus, 9 against S. pyogenes, 4 against Ps. aeruginosa, 1 against Pr. vulgaris, 3 against E. coli, 3 against A. fumigatus and 2 against C. albicans. Activity was found against both Gram-positive and Gram-negative bacteria. Three algae showed activity against the mold A. fumigatus and 2 algae on the yeast C. albicans. It is interesting to note that most active extracts were those of the Rhodophytae. None of the extracts from the fresh water algae showed activity against microorganisms at concentrations up to 50 mglml. Samples of Chaetomorpha media from Venezuela showed inhibition of Ps. aeruginosa, but the same algae from Michoacan did not have antimicrobial activity. No activity was observed for Gracilaria cerviconis from Veracruz and La Paz while the collection obtained from Venezuela had activity against S. aureus. Dictyopteris delacatula had considerable variation in activity depending on the place it was obtained. The sample from Veracruz inhibited the growth of S. aureus and P. pyogenes, while the sample obtained from
2
TESTED
“Key to organisms
1.
Peru Peru Guanajuato Guanajuato
Cyanophyceae Lyngb ya sp. Oscellatoria sp. Phormidium sp. Spirulina sp,
Sample origin (Ok11
ALGAE
San Luis Potosi San Luis Potosi Morelos Morelos Morelos Peru
as in Table
FRESH WATER
Chlorophyceae Fissidens sp. Hydrocotyle sp. Najas maritimas Najas sp. Odegonium sp. Spyrogyru sp.
Species
AMERICAN
TABLE
9.3 10.7 12.6 13.1
7.9 8.4 9.1 9.8 10.7 11.6
Extract yield (o/o)
FOR ANTIMICROBIAL
-
_ -
I
Antimicrobial
-
-
-
-
II
EFFECTS
activity
-
-
-
-
-
-
-
IV
-
III
zone inhibition
-
-
-
-
V
mm (cone. mg/ml)8
-
-
-
VI
-
-
-
VII
116
Venezuela was active only on S. pyogenes. Difference in activity was notable for Cauterpa sertuhoides, as material obtained in La Paz was active on A. fumigatus while the sample obtained from Zihuatanejo was active on C. albicans, and the one obtained from Ixtapa had no detectable antimicrobial activity. In the case of Ulva lactuca, samples from Michoacan, Venezuela and Veracruz had no antimicrobial activity, but the one obtained from La Paz had strong activity on Ps. aeruginosa. These results suggest, that the production of antimicrobial substances in algae are influenced by the degree of sea water contamination and certain environmental parameters, as the algae produce antibiotics as a means of defense. Plants showing antimicrobial activity in this screening will further be examined for their active ingredients. References Baslow, M.H. (1969) Marine Phamacology. A Study of Toxins and Other Biologically Active Substances of Marine Origin. Williams & Willkins Co., Baltimore. Faulkner, D.J. (1978) Topics in Antibiotic Chemistry. Ellis Horwood, Cilichester, England, pp. 299.
of Ethnopharmacobgy,
Elsevier Scientific Publishers
111
29 (1990) 111- 116 Ireland Ltd.
Short Communication
ANTIMICROBIAL
R.M. PEREZ
ACTIVITY
G., J.G. AVILA
OF SOME AMERICAN
A., S. PEREZ
G., A. MARTINEZ
ALGAE
C. and G. MARTINEZ
C.
Escuela Superior de Ingenieria Quimica e Indust& Extractivas, Instituto Polite’cnico National and Escuela Nacionul de Estudios Profesionales Ixtacalo, Universidad Autbnoma MetropolitanaXochimilco, Punto Fijo 16, C.P. 07050, Me’xico D.F. lMe’xicol (Accepted
November
9, 19891
Introduction
In the decade of the forties, interest was developed in marine flora and fauna as potential sources for the production of sustances with antimicrobial, antiviral and antifungicidal properties (Baslow, 19691. A number of algae of the Chlorophyceae, Rhodophyceae and Phaeophyceae are capable of producing antimicrobial sustances such as hydroquinones, sesquiterpenic phenols, brominated phenols, polyphenols, etc. (Faulkner, 19781. This study was designed to screen several marine and continental algae with the purpose of finding those which have the capacity to inhibit microbial growth in vitro. Materials and methods
Plant materials Plant material was collected in the Gulf of Mexico (Veracruzl, Pacific Ocean (Zihuatanejo, Ixtapa, Michoadn and Peru), Gulf of Cortes (La Paz) and Caribbean Sea (Venezuela). The continental algae were collected in Lake Cuitzeo (Guanajuatol, Lake Media Luna (San Luis Potosil, River Las Estacas (Morelosl and Lake Titicaca (Peru). The plants were identified by Dr Maria de Jesus Flores Departimento de Algas, Escuela National de Ciencias Biologicas, IPN (Mexico). Herbarium sheets were deposited in the same intitution. Extract preparation One hundred grams of dried plants material were ground and extracted in a Soxhlet for 6 h with 500 ml of 95% ethanol. The ethanol extract was taken to dryness under reduced pressure at 40°C. Extract yields are noted in 0 1990 Elsevier 037%87411502.45 Published and Printed in Ireland
Scientific Publishers
Ireland Ltd.
1
SEA ALGAE
australe
Spyridia filamentosa Vidalia obtusifolia
Polysiphonia subtilissima Prionitis decipiens Scinata jo fins toniue
Lithophyllum
Gracikwia cerwicornis Gracilaria cervicornis Gracilati sjoestedtii Gymnogon grus marhnensis Hypnea cervicornis Hypnea musciformis Hypnea spinella Lauren& obtusa
Centroceras clavalutum Geranium fastigiatum Corallina subuluta Graciluria cervicornis
Amphiroa hancoki Bostrychia montagne Bryochdia cuspidata Bryocludia thysigera
Rhodophyceae Amansia multifida
Species
AMERICAN
TABLE
Venezuela Peru Peru La Paz Veracruz
La Paz Veracruz Venezuela Veracruz Zihuatanejo
Veracruz La Paz La Paz Michoacan
Michoacan Veracruz Veracruz Venezuela Veracruz Venezuela Veracruz Verezuela
6.9 6.4 9.4 9.7 10.7
10.9 11.1 10.4 12.3 8.5 9.7 11.8 7.8 11.7
9.6 11.5 8.3 12.4 8.9 10.8 9.6 7.4 10.6
Extract yield (%I
16.3 (25) 22.1 (20)
17.8 (25) 26.5 (5)
27.1 (20)
24.3 (10) 21.6 (15)
I
Antimicrobial
EFFECTS
22.8 (10)
FOR ANTIMICROBIAL
Zihuatanejo
Sample origin
TESTED
24.2 (10)
17.3 (15) 17.8 (20)
14.5 (15)
18.2 (20)
II
activity III
zone inhibition
19.4 (20)
IV
V
mm (cone. mg/mlP VI
VII
La Paz Michoacan Venezuela Veracruz Peru
Ulva Ulva Ulva Ulva Ulva L&a
*I: S. aureus, II: S. pyogenes,
26.2
(5)
4.5 (25)
26.5 (501
16.3 (50) 17.8 (10)
16.8 (50)
22.1 (25)
27.8 (10)
15.5 (20)
IV: Pr. vulgaris, V: E. colt VI: A. fumigatus,
10.7 6.3 8.1 9.4 8.7 10.2
8.8 12.9 11.8 7.4 12.8 10.3 9.7 9.4 10.3 7.6 11.7 12.3 11.9 12.8 11.6
III: Ps. aeruginosa,
Zihuatanejo Zihuatanejo Peru La Paz
Enteromorpha flelucia Enteromorpha lengulata Halimeda opuntia Rhizoclonium kerneru
fasciata lactuca lactuca lactuca lactuca rigida
Veracruz Verezuela
Zihuatanejo Peru Michoacan Venezuela La Paz
Venezuela La Paz Lxtapa
Codium caneatum Codium taylorii Cymopolia barbata Enteromorpha flexuosa
Caulerpa sertubwioides Chaetomorpha antennina Chaetomorpha media Chaetomorpha media
Chlorophyceae Caulerpa racemosa Caulerpa sertularioides Caulerpa sertularioides
Sargassum macdogalu Sargassum semicola Sargassum vulgare
10.6
Ixtapa
Zihuatanejo La Paz La Paz Veracruz
Padina viekersiae
Sargassum lie bmannii 12.5 9.8 10.9 11.6
6.9 10.3 11.7
Zihuatanejo La Paz Michoacan
Ectocarpus breviarticulatus Padina durvillaei Padina vickersiae
11.7 7.8 9.2
Zihuatanejo Venezuela Veracruz
Phaeophyceae Chnoospora minima Dictyopteris delecatula Dictyopteris delacatula
VII: C. albicans.
24.2 (5) 23.4 (15)
18.2 (15)
20.1 (20)
19.3 (10)
19.3 (25)
18.6 (10)
12.1 (20)
114
Table 1. Thick tarry residues were obtained, parts of which were dissolved in distilled water at six concentrations (5, 10, 15, 20, 25 and 50 mglmll. Antimicrobial screening Nutrient media was prepared according to manufactures instructions (trypticase soy broth, TSB, Squibb and Sabouraud-dextrose, Merck) and sterilized for 20 min at 12OOC. The following microorganisms were used for this test: (11 Staphylococcus aureus, (111Staphyloccus pyrogenes, (III) Pseudomonas aeruginosa, (IV) Proteus vulgaris, (Vl Escherichia ~0.5, (VI) Aspergillus fumigatus, (VII) Candida albicans. S. aureus, S. pyrogenes, Pr. vulgaris, E. coli and Ps. aeruginosa were cultured overnight at 37OC in TSB. C. albicans was cultured for 48 h at 27OC in Sabouraud-dextrose. Each culture was diluted with sterile 0.9% saline solution (1 : 10001 to give a suspension of about lo8 microorganisms per ml. An amount of 0.1 ml of this suspension was spread on the test plate. Four wells, 1 cm in diameter, were made in each plate, into which 0.1 ml of test solution was pipetted. All determinations were made at least in duplicate. For the bacteria, a positive control with streptomycin (Sigma) was used (0.1 ml of a 10 mglml solution1 and for the mold and yeast, nystatin (Squibb) was used (0.1 ml of a 50 mglml solution). For the negative control, 0.1 ml of distilled water was used. Inhibition zones were read 1 day after incubation at 37OC for the bacteria and after 3-4 days at 27OC for the mold and yeast. Results and discussion The antimicrobial effects of sea water algae are summarized in Table 1. Tested fresh water algae are listed in Table 2. Of the 65 plant samples tested, 9 showed activity against S. aureus, 9 against S. pyogenes, 4 against Ps. aeruginosa, 1 against Pr. vulgaris, 3 against E. coli, 3 against A. fumigatus and 2 against C. albicans. Activity was found against both Gram-positive and Gram-negative bacteria. Three algae showed activity against the mold A. fumigatus and 2 algae on the yeast C. albicans. It is interesting to note that most active extracts were those of the Rhodophytae. None of the extracts from the fresh water algae showed activity against microorganisms at concentrations up to 50 mglml. Samples of Chaetomorpha media from Venezuela showed inhibition of Ps. aeruginosa, but the same algae from Michoacan did not have antimicrobial activity. No activity was observed for Gracilaria cerviconis from Veracruz and La Paz while the collection obtained from Venezuela had activity against S. aureus. Dictyopteris delacatula had considerable variation in activity depending on the place it was obtained. The sample from Veracruz inhibited the growth of S. aureus and P. pyogenes, while the sample obtained from
2
TESTED
“Key to organisms
1.
Peru Peru Guanajuato Guanajuato
Cyanophyceae Lyngb ya sp. Oscellatoria sp. Phormidium sp. Spirulina sp,
Sample origin (Ok11
ALGAE
San Luis Potosi San Luis Potosi Morelos Morelos Morelos Peru
as in Table
FRESH WATER
Chlorophyceae Fissidens sp. Hydrocotyle sp. Najas maritimas Najas sp. Odegonium sp. Spyrogyru sp.
Species
AMERICAN
TABLE
9.3 10.7 12.6 13.1
7.9 8.4 9.1 9.8 10.7 11.6
Extract yield (o/o)
FOR ANTIMICROBIAL
-
_ -
I
Antimicrobial
-
-
-
-
II
EFFECTS
activity
-
-
-
-
-
-
-
IV
-
III
zone inhibition
-
-
-
-
V
mm (cone. mg/ml)8
-
-
-
VI
-
-
-
VII
116
Venezuela was active only on S. pyogenes. Difference in activity was notable for Cauterpa sertuhoides, as material obtained in La Paz was active on A. fumigatus while the sample obtained from Zihuatanejo was active on C. albicans, and the one obtained from Ixtapa had no detectable antimicrobial activity. In the case of Ulva lactuca, samples from Michoacan, Venezuela and Veracruz had no antimicrobial activity, but the one obtained from La Paz had strong activity on Ps. aeruginosa. These results suggest, that the production of antimicrobial substances in algae are influenced by the degree of sea water contamination and certain environmental parameters, as the algae produce antibiotics as a means of defense. Plants showing antimicrobial activity in this screening will further be examined for their active ingredients. References Baslow, M.H. (1969) Marine Phamacology. A Study of Toxins and Other Biologically Active Substances of Marine Origin. Williams & Willkins Co., Baltimore. Faulkner, D.J. (1978) Topics in Antibiotic Chemistry. Ellis Horwood, Cilichester, England, pp. 299.
