- Email: [email protected]
In vitro antibacterial activity of Argentine folk medicinal plants against Salmonella typhi
ELSEVIER
Journal
In vitro antibacterial
activity of Argentine folk medicinal plants against Salmonella typhi
of Ethnopharmacology
44 (1994) 41-46
Cristina Pkrez*, Claudia Anesini Cbredra de Farmacologia.
Far&ad
Received
de Odonrologia.
I8 March
Universidad de Buenos Aires. M. T. de Alvear 2142. ( 1122) Buenos Aires. Argennrina
1994; revision received 17 May
1994; accepted 20 May
1994
Abstract Boiling water extracts of 132 samples from 54 plant families. commonly used in Argentine folk medicine, were screened for antibacterial activity against Salmonella fyphi. The agar-well diffusion method was used. A reference concentration-response curve for ampicillin was used to estimate the apparent activity of the samples. Twenty four species showed antibacterial activity. Cassia occidenralis roots, Heimia salic@lia aerial parts, Punica granatum fruit pericarp and Rosa horhoniana flowers produced some of the more active extracts. Taking into account the multiple resistance of Salmonella t&i. these findings could be useful in the search for new clinically useful antimicrobials.
Keywords:
Antibacterial/antimicrobial
activity; Salmonella f~$~i; Argentine folk medicinal plants
1. Introduction
In a previous paper, this laboratory reported the antimicrobial activity of plants used in Argentine folk medicine against Escherichiu coli, Staphylococcus aureus and Aspergillus niger (Anesini and Perez, 1993). The present study deals with the screening of those plants for antibacterial activity against Salmonella typhi. Bacteria of the Sulmonellae genus are parasites that infect a number of animal species (Furowicz and Terzolo, 1975). Sulmonella typhi is a serotype that infects only humans, producing typhoid fever. In Sulnwnellu * Corresponding
0378-8741/94/.%07.00
author
0
SSDI 037%8741(94)01163-T
1994 Elsevier Science Ireland
gastroenteritis, ampicillin (or chloramfenicol) does not seem of major clinical benefit, either in the treatment of the disease or in the therapy of the asymptomatic Sufmonellu carrier (Winshell et al., 1970). The acetone-treated, heat-killed Sulmonellu lyphi vaccine provides only partial protection, at best. These facts encourage research leading to the discovery of more effective antibiotics. 2. Materials and methods 2. I.
Plunrs
A list of 132 samples that include different plant parts corresponding to 122 species from 54 families was used, as reported previously (Anesini
Ltd. All rights reserved
C.
42
Pkrez,
Table I Antibacterial activity of plant extracts against
C. Anesini / J. Ethnophormocol.
44 (1994)
41-46
Table I (continued) Solmonello ryphi
Part(s) tested
Antimicrobial equivalence (Wnl)”
L. (36)
Fruit pericarp
73.4 f 19.6
Desp.
Flower
37.9 f 2.8
Root
IO.1 l 0.5
Fruit pericarp
10.3 f 0.4
Leaf/branch
10.0 f 0.5
Seed
10.4 f 0.5
Leaf
15.5 * 0.0
Leaf
16.0 f I.6
Family/species Family/species
Part(s) tested
Antimicrobial equivalence (&ml)’
Fruiting body
21.0 f 6.5
Bark
10.3 f 0.7
PUNICACEAE
AGARICACEAE Coprinus
sp. (104)
Punica gronatum
ROSACEAE
BIGNONIACEAE Tobebuio heptophyllo
(Veil.) Tol. (95)
Rosa Borboniona
(64) RUBIACEAE
CALYCERACEAE Leaf
Acicorpho tribuloides
10.4 f 0.5
Borreria eupororioides
Cham. et Schlechtend. (94)
Juss. (IO) COMPOSITAE
sp. (67)
Achyrocline
Aerial parts
12.1 f 1.4
RUTACEAE Citrus sinensis
EUPHORBIACEAE Croron urucurono Baill. (12)
Dsbeck
(44) Bark resin
13.2 zt I.0 SOLANACEAE Solonum sisymbriifolium
Lam. (20)
LABIATAE Origonum X opplii
Leaf
18.0 zt I.0
Leaf
9.8 f 0.4
UMBELLIFERAE
(Domin.) Boros (3) Rosmorinus ofjicinolis L.(52)
Foeniculum vulgare
Mill.
(13)
LEGUMINOSAE Cassio occidenrolis
Root
31.0 f 6.2
Root
16.9 f 2.5
URTICACEAE Vrrico urens L. (49)
L. (Ill) Glycyrrhka
astragalino
Gill. ex Hook et Am (19) Myroxilon
peruiJerum
Bark
21.2 f 3.5
Fruit
10.1
Seed
12.6 zt I.0
Aerial parts
46.3 f 3.7
Leaf
34.9 f 4.1
Leaf
17.0 f 2.5
Fruit
10.3 f 0.5
L. Fil. (88) Prosopis srrombulijero
l
0.9
(Lam.) Benth. (5) LINACEAE Linum usilorissimum
L.
(11) LYTHRACEAE Heimio solicijolia
(HBK.)
Link (29) MYRTACEAE Eucalyptus comandulensis
Denth. (31) Psidium luridum
(Spreng.) Burret (18) OLEACEAE Olea europaea
L. ( 132)
ZYGDPHY LLACEAE Lorrea divoricala Cav. (38)
“Data represent mean equivalence to ampicillin f S.E.M. calculated from 3 experiments by interpolation to the reference concentration-response curve of ampicillin.
and Perez, 1993). It had been prepared on the basis of plant use in Argentine folk medicine (Ratera and Ratera, 1980; Toursarkissian, 1980; MartinezCrovetto, 1981). Commercially procured materials were identified by employing morphological, anatomical and histochemical techniques, in comparison with voucher-specimens of herbarium reference standards. One voucher set was deposited at our own Institution, the other set at the Pharmacobotanic Museum (Faculty of Pharmacy and Biochemistry, University of Buenos Aires). These voucher samples are cited in Tables 1 and 2 as 1 through 132 into brackets.
43
C. Perez. C. Anesini/ J. Eihnopharmacol. 44 (1994) 41-46
Table 2 Plants showing no antibacterial activity against typhi (experimental conditions as in Table I) Family/species ADIANTACEAE Pellaea nivea (Pair.)
Table 2 (continued) Sa/monel/a
Part(s)
Prantl
ANACARDIACEAE Lirhrea rernifolia (Gillies) Schinus molle L. ( I 19)
Leaf
(71)
AMARANTHACEAE Alrernanrhera poronychioides Ahernanrhera pungens H.B.K.
St. Hill (69) (70)
Barkley
tested
(80)
Whole plant Whole plant
Leaf Leaf
Whole plant Leaf/branch Root
BERBERIDACEAE Berheris buxifolia Lam. (34) Berberis bu.w$oiia Lam. (26)
Bark Leaf
ex DC.)
BORAGINACEAE Bvrrago officinalis L. (53)
CELASTRACEAE Ma_rrenus ilicifolia Mart.
Mikanja mirracanrha Kunth (61) Parrhenium hysreriophorus L. (98)
Leaf Aerial
parts
Plerocaulon sp. ( I 16) Senecio graveolens Wedd. (40) Schkuhria pinnata (Lam.) D.Kuntze Tageres minura L. (I 07) Xanrhium cavanillesii Schouw (93) Xanthium spinosum L. ( 17)
Leaf/branch Aerial parts Leaf/branch Leaf/branch Seed Leaf/branch
(59)
var. sericea (16)
Seed Seed
CHENOPODIACEAE Chenopodium graveolens Willd. var. bangii
Aerial
Aellen (I
Aerial
(76) (27)
COMPOSITAE Ambrosia tenurfolia Sprengel (33) Artemisia absinrhium L. (65) Arfemisia copa Phil. (92) Baccharis ar/ic.u/ota (Lam.) Pers. (83) Baccharis gaudiehaudiana DC. (8 I ) Bacchoris gaudichaudiana DC. (2I ) Boccharis incarum Wedd. ( 106) Baccharis microdonra DC. ( I 18) Carihamus linctorius L. ( 125) Cenlaurea melifensis L. (60) Cnicus benedicms L. (46) Cichorium imybus L. (47) Cichorium inrybus L. (48) Euparorium inulaefolium Kunth ( 120) Gamochoera americana (Mill.) Wedd. (101) Haplopappus rigidus Phil. (6) Manicaria recutita L. (66)
parts
parts
)
EQUISETACEAE Anemia romemosa (Sav.) Swartz
Leaf/branch Leaf/branch
Leaf/branch Whole plant
(97)
Equisetum giganteum L. (43)
(58 )
Aerial
CUCURBITACEAE Cucurbita maxima Duschesne ( 126) Cirrullus vulgaris Schrad. (85)
EPHEDRACEAE Ephedra ochreafa Miers ( 100) Ephedra trianda Tul. Emond J.H. Hunziker (8)
Bark
Flower
Mayytenus vitis-idaea Griseb.
tested
(Murr.)
BUDDLEJACEAE Buddleja globosa Hope
Part(s)
CONVOLVULACEAE Dichondra sericea Swartz
ARISTOLOCHIACEAE Arisrolochia angusfijolia Cham. (91) Arisrolochia macroura Gomez (35) Aristolochia macroura Gomez ( I 13)
BIGNONIACEAE Tabebuia imperiginosa (Mart. Standl. (68)
Family/species
parts
Leaf Leaf
Aerial parts Leaf/branch Whole plant Leaf Leaf Branch Whole plant Leaf Aerial parts Leaf/flower Root Leaf Leaf Whole plant Branch Flower
GENTIANACEAE Gentiana achalensis Hieron. Genriana parvi’ora (Griseb.)
ex Gilg (I 17) Gilg (108)
GRAMINEAE Cynodon dactylon (L.) Pers. (54)
Root Fruit
Hordeum vu/gore L. (5 I )
LABIATAE Hedeoma mulrijlorum Benth. (89) Lavandula sp. (41) Marrubium v&gore L. (24) Menrho x piperita L. (78) Menrha x rotundijolia L. (77) Minrhosrachys mollis (Kunth) Ocimum basilicum L. (129) Salvia gilliesii Benth (109) So/via sp. (28)
LAURACEAE Laurus nobihs L. (I 28)
Griseb.
Bark Root
(45)
Leaf/ flower Flower Leaf/branch Leaf/flower Leaf/flower Leaf/branch Leaf Leaf/branch Leaf
Leaf
C. PPrez. C. Anesini/ J. Ethnophormocol. 44 (1994) 41-46
44
Table 2 (continued)
Table 2 (continued) Family/species
Part(s)
LEGUMINOSAE Acacia oromo Gillies ex Hook. Acacia oromo Gillies ex Hook. Acocio aroma Gillies ex Hook.
et Am. (23) et Am. (86) et Am. (87)
Eryrhrino crisro-golli L. (30) Geoflroeo decorticons (Gill. ex. H. et Am.) Burkart (32) Gledifsio rrioconros L. ( I IO) Kromerio friondro Ruiz et Pavon ( I21 ) Prosopis o/finis Spreng. (90)
tested
Leaf Branch Leaf/branch/ fruit Bark Bark Fruit Root Leaf
LlLlACEAE Allium sorivum L. (I 34)
Bulb
MALVACEAE Molvo sylvestris L. (55) MENISPERMACEAE Cissompelos poreiro L.
Leaf/flower
( I 12)
Root/branch
Part(s)
PIPERACEAE Piper nigrum L. (I 22)
Fruit
RUTACEAE Citrus sp (39) Pilocorpus pennotijolius Lem. (42) Ruto cholepensis L. ( I3 I ) Ruro cholepensis L. (73)
Leaf Leaf Leaf Banch
SALICACEAE Salix humboldriono Willd. (7)
Bark
SANTALACEAE Jodino rhombifolio Hook.
Leaf
(Looser)
MORACEAE Cecropio odenopus Mart.
Leaf
Fritsch
grisebochii
Hook.
leprosw
Tilia europoea
Flower
L. (57)
Berg (63) Eugenio uniJoro L. (I 14) Psidium guineense SW. ( 102)
ULMACEAE Celis rule Gill. ex Planch.
Leaf Fruit pericarp
UMBELLIFERAE
Leaf
(56)
Anelhum gruveolens L. (4) sellowiunum Wolff. (22) Azorella compacfa Phil. ( I 15) Cuminum cyminum L. ( 123)
Seed Leaf Whole plant Fruit Fruit
Apiutn
Leaf
Pimpinella
fil. et
Bulb
Porieruria Urricu
Aerial
PHYTOLACCACEAE Periveriu olliocea L. (96)
Leaf
PLANTAGINACEAE Plunrago Iomenloso Lam. (75)
Leaf
onisutn
L.
( 14)
URTICACEAE
PASSIFLORACEAE
POLYGONACEAE Rumes crispus L. (25)
Resin
TILIACEAE
Leaf
Possijloro coeruleu L. (37)
Whole plant
(9)
et Am. (103)
MYRTACEAE Blephorocolyx rweedeei (H. et Am.)
ORCHIDACEAE Cyrtopodium pulmifrons Rchb. Warm (105)
Fruit Seed Leaf
STY RACACEAE
Leaf
OLEACEAE Oleo europoeo L. ( 130)
tested
SCROPHULARIACEAE
Sryrox
(SO)
et Am. (72)
SOLANACEAE Copsicum annum L. ( 124) Copsicum onnum L. (I 27) Fobiono imbricato Ruiz et Pav. (79)
Scoporio
MONIMIACEAE Lclureliopsis philippiono Schodde (62)
Family/species
parts
debilis
G. Forsk.
Whole plant
(99)
Branch
urens L. (84)
USNEACEAE (Jsnea hurhuru (L.) Vigg. var. hieronymii (Krempelh)
Whole plant
Mull. Arg. (74)
VERBENACEAE
Leaf/root
Verbenu lifrorulis
Leaf/branch
H. B.K. (2)
ZYGOPHYLLACEAE Bulnesiu .wrmien/oi Lorentz
ex Griseb.
(I 5)
Bark
C. Pkez,
C. Anesini / J. Ethnophannacol.
2.2. Plant extracts Extracts were made from air-dried samples of the whole plant or from the portion specified as being used medicinally. Each 500 mg of dry material was steeped for 30 min with 8 ml boiling water according to the method of Anesini and Perez (1993). Each extract was filtered and the volume adjusted so that each ml of extract was equivalent to 62.5 mg of dry starting material. 2.3. Determination of the antibacterial activity The agar-well diffusion method (Perez et al., 1990) was modified. Nutrient agar was used as a culture medium. It was inoculated with a clinical strain of Salmonella typhi, previously suspended in tryptose broth. Wells 6 mm in diameter were punched into the agar and filled with plant extracts and solvent blanks or standard solutions of ampicillin (1 l- 1000 &ml). The antibiotic standards were obtained from Laboratorios Beta Sociedad Anonima Industrial y Comercial, Argentina. The antimicrobial activity was evaluated by measuring the inhibition-zone diameter observed at 48 h. The mean values were interpolated by comparison to a reference concentration-response curve of ampicillin.
44 (1994)
DIAMETER
45
41-46
OF lNHlBlflON
(mm)
60-
40 -
30 -
01 -2
-1.5
-1
-0.6
0
log concentration (ampicillin, mg/ml) Fig. I. Reference curve of Salmonella
fyphi sensitivity to am-
picillin. Shown are means f
S.E.M.
from 4 experiments.
of them made in duplicate.
Slope (b) = 17.21 mm/log
tercept (i) = 43.47 mm and correlation
coefficient
each C; in-
(r) = 0.99.
3. Results 3.1. Optimization of the experimental conditions After preliminary studies, the following conditions appeared to be optimal for antibacterial screening against Salmonella typhi: (i) inoculum concentration: suspensions in tryptose phosphate broth exhibiting 0.52-0.57 units of absorbance at 580 nm; (ii) suspension: dilution of the inoculum; l/20 v/v in nutrient agar medium; (iii) inoculum amount: 15 ml of nutrient agar medium inoculated as described above was poured into a single Petri plate; (iv) test sample size: 50 ~1 samples were placed in each well; (v) incubation temperature: 25°C and (vi) incubation time: 48 h. 3.2. Reference curve Linear regression analysis was applied to data. As it can be observed, a good fitting of data to a linear function (antibacterial activity versus logarithm of the concentration of ampicillin) can be
achieved. The calculated parameters and experimental data are reported in Fig. 1, which was used as a reference to evaluate the activity of the test samples by interpolation. 3.3. Antibacterial activity Twenty four extracts were found to exhibit antibacterial activity on Salmonella typhi (Table l), whereas 108 extracts were inactive (Table 2). 4. Discussion and conclusions Research on natural resources has been encouraged by the World Health Organization from 1978 to date. The finding of 24 active plant extracts may be interesting in the search for new efficacious and safe antimicrobials against Salmonella typhi. Considering the multiple resistance of Salmonella typhi, clinically important
46
C. P&es,
C. Anesini / J. Ethnopharmacol.
both in the treatment of diseases and in the case of asymptomatic carriers (Winshell et al., 1970), our results could stimulate further pharmacological studies seeking new antibiotics from the higher plants. Since Salmonella typhi is also involved in contamination of food and drugs (Thatcher and Clark, 1973; Christopulos et al., 1980), our extracts may also be useful as conservation agents. Further investigation will be necessary to elucidate the pharmacological mechanisms and the clinical usefulness of the compounds contained in the extracts herein studied. Acknowledgements
This work was supported in part by the Third World Academy of Sciences (award TWAS RGBC: 91-027). The authors are grateful to Ing. Ricardo Rossow of the Faculty of Pharmacy and Biochemistry, University of Buenos Aires, for the botanical identification of the plant species used in this study. References Anesini. C. and Perez. C. (1993) Screening of plants used in
44 (1994)
41-46
Argentine folk medicine for antimicrobial activity. Ethnopharmacology
39,
I 19-i
Journal of
28.
Christopulos. M.C., Cinto. 0.. D&ma de Wollf, C., Frade, A.. Maffe, Z.I., Mercado, E.C., Moro. A.A., Raffo Palma. MS.. Rivas, M., Rosetti, F., Torrado, S.S., Varsavsky. E. (1980) Bromatologia
analitica
para
alimentos
y
medicamentos.
Subsecretaria de Medicina Social y Fiscalizacion Sanitaria, lnstituto National de Farmacologia y Bromatologia. Buenos Aires, pp. 25-41. Furowicz. A. and Terzolo. H. (1975) Aislamiento de serotipos de SalmoneNa sp en animales de la region de Balcarce. Boletin Veterinario. Dpto. de Production Animal. Unidad de Patologia Animal, n-17, pp. l-5. Martinez Crovetto, R. (198 I) Plantas utikadas en Medicina en el noroeste de Corrientes. Ministerio de Cultura de la Nation. San Miguel de Tucuman, pp. 13-125. Perez, C., Pauli. M. and Bazerque, P. (1990) An antibiotic assay by the agar-well diffusion method. Acra Biologiae et Medicine kperimentalis 15, I l3- I 15. Ratera, E.L. and Ratera. M.O. (1980) Phntas de luj7ora argentina empleadas en medicina popular. Hemisferio Sur, Buenos Aires, pp. 17-105. Thatcher, F.S. and Clark, D.S. (1973) Anrjlisis microbioldgico de /OS alimentos. Acribia. Zaragoza, pp. 6-8. Toursarkissian, M. (1980) Plantas medicinales de la Argentina: sus nombres
Botcinicos.
Vulgares.
Uses
y
Distribucidn
Hemisferio Sur. Buenos Aires. pp. I-139. Winshell, E.B., Cherubin. C.. Winter, J. and Neu. H.C. (1970) Antibiotic resistance of Sulmonellu in the eastern United States. In: G.L. Hobby (Ed.). Antimicrobial Agents and Chemotherapy-1969. American Society for Microbiology. Bethesda MD, pp. 86-89. geogrrjfica.
Journal
In vitro antibacterial
activity of Argentine folk medicinal plants against Salmonella typhi
of Ethnopharmacology
44 (1994) 41-46
Cristina Pkrez*, Claudia Anesini Cbredra de Farmacologia.
Far&ad
Received
de Odonrologia.
I8 March
Universidad de Buenos Aires. M. T. de Alvear 2142. ( 1122) Buenos Aires. Argennrina
1994; revision received 17 May
1994; accepted 20 May
1994
Abstract Boiling water extracts of 132 samples from 54 plant families. commonly used in Argentine folk medicine, were screened for antibacterial activity against Salmonella fyphi. The agar-well diffusion method was used. A reference concentration-response curve for ampicillin was used to estimate the apparent activity of the samples. Twenty four species showed antibacterial activity. Cassia occidenralis roots, Heimia salic@lia aerial parts, Punica granatum fruit pericarp and Rosa horhoniana flowers produced some of the more active extracts. Taking into account the multiple resistance of Salmonella t&i. these findings could be useful in the search for new clinically useful antimicrobials.
Keywords:
Antibacterial/antimicrobial
activity; Salmonella f~$~i; Argentine folk medicinal plants
1. Introduction
In a previous paper, this laboratory reported the antimicrobial activity of plants used in Argentine folk medicine against Escherichiu coli, Staphylococcus aureus and Aspergillus niger (Anesini and Perez, 1993). The present study deals with the screening of those plants for antibacterial activity against Salmonella typhi. Bacteria of the Sulmonellae genus are parasites that infect a number of animal species (Furowicz and Terzolo, 1975). Sulmonella typhi is a serotype that infects only humans, producing typhoid fever. In Sulnwnellu * Corresponding
0378-8741/94/.%07.00
author
0
SSDI 037%8741(94)01163-T
1994 Elsevier Science Ireland
gastroenteritis, ampicillin (or chloramfenicol) does not seem of major clinical benefit, either in the treatment of the disease or in the therapy of the asymptomatic Sufmonellu carrier (Winshell et al., 1970). The acetone-treated, heat-killed Sulmonellu lyphi vaccine provides only partial protection, at best. These facts encourage research leading to the discovery of more effective antibiotics. 2. Materials and methods 2. I.
Plunrs
A list of 132 samples that include different plant parts corresponding to 122 species from 54 families was used, as reported previously (Anesini
Ltd. All rights reserved
C.
42
Pkrez,
Table I Antibacterial activity of plant extracts against
C. Anesini / J. Ethnophormocol.
44 (1994)
41-46
Table I (continued) Solmonello ryphi
Part(s) tested
Antimicrobial equivalence (Wnl)”
L. (36)
Fruit pericarp
73.4 f 19.6
Desp.
Flower
37.9 f 2.8
Root
IO.1 l 0.5
Fruit pericarp
10.3 f 0.4
Leaf/branch
10.0 f 0.5
Seed
10.4 f 0.5
Leaf
15.5 * 0.0
Leaf
16.0 f I.6
Family/species Family/species
Part(s) tested
Antimicrobial equivalence (&ml)’
Fruiting body
21.0 f 6.5
Bark
10.3 f 0.7
PUNICACEAE
AGARICACEAE Coprinus
sp. (104)
Punica gronatum
ROSACEAE
BIGNONIACEAE Tobebuio heptophyllo
(Veil.) Tol. (95)
Rosa Borboniona
(64) RUBIACEAE
CALYCERACEAE Leaf
Acicorpho tribuloides
10.4 f 0.5
Borreria eupororioides
Cham. et Schlechtend. (94)
Juss. (IO) COMPOSITAE
sp. (67)
Achyrocline
Aerial parts
12.1 f 1.4
RUTACEAE Citrus sinensis
EUPHORBIACEAE Croron urucurono Baill. (12)
Dsbeck
(44) Bark resin
13.2 zt I.0 SOLANACEAE Solonum sisymbriifolium
Lam. (20)
LABIATAE Origonum X opplii
Leaf
18.0 zt I.0
Leaf
9.8 f 0.4
UMBELLIFERAE
(Domin.) Boros (3) Rosmorinus ofjicinolis L.(52)
Foeniculum vulgare
Mill.
(13)
LEGUMINOSAE Cassio occidenrolis
Root
31.0 f 6.2
Root
16.9 f 2.5
URTICACEAE Vrrico urens L. (49)
L. (Ill) Glycyrrhka
astragalino
Gill. ex Hook et Am (19) Myroxilon
peruiJerum
Bark
21.2 f 3.5
Fruit
10.1
Seed
12.6 zt I.0
Aerial parts
46.3 f 3.7
Leaf
34.9 f 4.1
Leaf
17.0 f 2.5
Fruit
10.3 f 0.5
L. Fil. (88) Prosopis srrombulijero
l
0.9
(Lam.) Benth. (5) LINACEAE Linum usilorissimum
L.
(11) LYTHRACEAE Heimio solicijolia
(HBK.)
Link (29) MYRTACEAE Eucalyptus comandulensis
Denth. (31) Psidium luridum
(Spreng.) Burret (18) OLEACEAE Olea europaea
L. ( 132)
ZYGDPHY LLACEAE Lorrea divoricala Cav. (38)
“Data represent mean equivalence to ampicillin f S.E.M. calculated from 3 experiments by interpolation to the reference concentration-response curve of ampicillin.
and Perez, 1993). It had been prepared on the basis of plant use in Argentine folk medicine (Ratera and Ratera, 1980; Toursarkissian, 1980; MartinezCrovetto, 1981). Commercially procured materials were identified by employing morphological, anatomical and histochemical techniques, in comparison with voucher-specimens of herbarium reference standards. One voucher set was deposited at our own Institution, the other set at the Pharmacobotanic Museum (Faculty of Pharmacy and Biochemistry, University of Buenos Aires). These voucher samples are cited in Tables 1 and 2 as 1 through 132 into brackets.
43
C. Perez. C. Anesini/ J. Eihnopharmacol. 44 (1994) 41-46
Table 2 Plants showing no antibacterial activity against typhi (experimental conditions as in Table I) Family/species ADIANTACEAE Pellaea nivea (Pair.)
Table 2 (continued) Sa/monel/a
Part(s)
Prantl
ANACARDIACEAE Lirhrea rernifolia (Gillies) Schinus molle L. ( I 19)
Leaf
(71)
AMARANTHACEAE Alrernanrhera poronychioides Ahernanrhera pungens H.B.K.
St. Hill (69) (70)
Barkley
tested
(80)
Whole plant Whole plant
Leaf Leaf
Whole plant Leaf/branch Root
BERBERIDACEAE Berheris buxifolia Lam. (34) Berberis bu.w$oiia Lam. (26)
Bark Leaf
ex DC.)
BORAGINACEAE Bvrrago officinalis L. (53)
CELASTRACEAE Ma_rrenus ilicifolia Mart.
Mikanja mirracanrha Kunth (61) Parrhenium hysreriophorus L. (98)
Leaf Aerial
parts
Plerocaulon sp. ( I 16) Senecio graveolens Wedd. (40) Schkuhria pinnata (Lam.) D.Kuntze Tageres minura L. (I 07) Xanrhium cavanillesii Schouw (93) Xanthium spinosum L. ( 17)
Leaf/branch Aerial parts Leaf/branch Leaf/branch Seed Leaf/branch
(59)
var. sericea (16)
Seed Seed
CHENOPODIACEAE Chenopodium graveolens Willd. var. bangii
Aerial
Aellen (I
Aerial
(76) (27)
COMPOSITAE Ambrosia tenurfolia Sprengel (33) Artemisia absinrhium L. (65) Arfemisia copa Phil. (92) Baccharis ar/ic.u/ota (Lam.) Pers. (83) Baccharis gaudiehaudiana DC. (8 I ) Bacchoris gaudichaudiana DC. (2I ) Boccharis incarum Wedd. ( 106) Baccharis microdonra DC. ( I 18) Carihamus linctorius L. ( 125) Cenlaurea melifensis L. (60) Cnicus benedicms L. (46) Cichorium imybus L. (47) Cichorium inrybus L. (48) Euparorium inulaefolium Kunth ( 120) Gamochoera americana (Mill.) Wedd. (101) Haplopappus rigidus Phil. (6) Manicaria recutita L. (66)
parts
parts
)
EQUISETACEAE Anemia romemosa (Sav.) Swartz
Leaf/branch Leaf/branch
Leaf/branch Whole plant
(97)
Equisetum giganteum L. (43)
(58 )
Aerial
CUCURBITACEAE Cucurbita maxima Duschesne ( 126) Cirrullus vulgaris Schrad. (85)
EPHEDRACEAE Ephedra ochreafa Miers ( 100) Ephedra trianda Tul. Emond J.H. Hunziker (8)
Bark
Flower
Mayytenus vitis-idaea Griseb.
tested
(Murr.)
BUDDLEJACEAE Buddleja globosa Hope
Part(s)
CONVOLVULACEAE Dichondra sericea Swartz
ARISTOLOCHIACEAE Arisrolochia angusfijolia Cham. (91) Arisrolochia macroura Gomez (35) Aristolochia macroura Gomez ( I 13)
BIGNONIACEAE Tabebuia imperiginosa (Mart. Standl. (68)
Family/species
parts
Leaf Leaf
Aerial parts Leaf/branch Whole plant Leaf Leaf Branch Whole plant Leaf Aerial parts Leaf/flower Root Leaf Leaf Whole plant Branch Flower
GENTIANACEAE Gentiana achalensis Hieron. Genriana parvi’ora (Griseb.)
ex Gilg (I 17) Gilg (108)
GRAMINEAE Cynodon dactylon (L.) Pers. (54)
Root Fruit
Hordeum vu/gore L. (5 I )
LABIATAE Hedeoma mulrijlorum Benth. (89) Lavandula sp. (41) Marrubium v&gore L. (24) Menrho x piperita L. (78) Menrha x rotundijolia L. (77) Minrhosrachys mollis (Kunth) Ocimum basilicum L. (129) Salvia gilliesii Benth (109) So/via sp. (28)
LAURACEAE Laurus nobihs L. (I 28)
Griseb.
Bark Root
(45)
Leaf/ flower Flower Leaf/branch Leaf/flower Leaf/flower Leaf/branch Leaf Leaf/branch Leaf
Leaf
C. PPrez. C. Anesini/ J. Ethnophormocol. 44 (1994) 41-46
44
Table 2 (continued)
Table 2 (continued) Family/species
Part(s)
LEGUMINOSAE Acacia oromo Gillies ex Hook. Acacia oromo Gillies ex Hook. Acocio aroma Gillies ex Hook.
et Am. (23) et Am. (86) et Am. (87)
Eryrhrino crisro-golli L. (30) Geoflroeo decorticons (Gill. ex. H. et Am.) Burkart (32) Gledifsio rrioconros L. ( I IO) Kromerio friondro Ruiz et Pavon ( I21 ) Prosopis o/finis Spreng. (90)
tested
Leaf Branch Leaf/branch/ fruit Bark Bark Fruit Root Leaf
LlLlACEAE Allium sorivum L. (I 34)
Bulb
MALVACEAE Molvo sylvestris L. (55) MENISPERMACEAE Cissompelos poreiro L.
Leaf/flower
( I 12)
Root/branch
Part(s)
PIPERACEAE Piper nigrum L. (I 22)
Fruit
RUTACEAE Citrus sp (39) Pilocorpus pennotijolius Lem. (42) Ruto cholepensis L. ( I3 I ) Ruro cholepensis L. (73)
Leaf Leaf Leaf Banch
SALICACEAE Salix humboldriono Willd. (7)
Bark
SANTALACEAE Jodino rhombifolio Hook.
Leaf
(Looser)
MORACEAE Cecropio odenopus Mart.
Leaf
Fritsch
grisebochii
Hook.
leprosw
Tilia europoea
Flower
L. (57)
Berg (63) Eugenio uniJoro L. (I 14) Psidium guineense SW. ( 102)
ULMACEAE Celis rule Gill. ex Planch.
Leaf Fruit pericarp
UMBELLIFERAE
Leaf
(56)
Anelhum gruveolens L. (4) sellowiunum Wolff. (22) Azorella compacfa Phil. ( I 15) Cuminum cyminum L. ( 123)
Seed Leaf Whole plant Fruit Fruit
Apiutn
Leaf
Pimpinella
fil. et
Bulb
Porieruria Urricu
Aerial
PHYTOLACCACEAE Periveriu olliocea L. (96)
Leaf
PLANTAGINACEAE Plunrago Iomenloso Lam. (75)
Leaf
onisutn
L.
( 14)
URTICACEAE
PASSIFLORACEAE
POLYGONACEAE Rumes crispus L. (25)
Resin
TILIACEAE
Leaf
Possijloro coeruleu L. (37)
Whole plant
(9)
et Am. (103)
MYRTACEAE Blephorocolyx rweedeei (H. et Am.)
ORCHIDACEAE Cyrtopodium pulmifrons Rchb. Warm (105)
Fruit Seed Leaf
STY RACACEAE
Leaf
OLEACEAE Oleo europoeo L. ( 130)
tested
SCROPHULARIACEAE
Sryrox
(SO)
et Am. (72)
SOLANACEAE Copsicum annum L. ( 124) Copsicum onnum L. (I 27) Fobiono imbricato Ruiz et Pav. (79)
Scoporio
MONIMIACEAE Lclureliopsis philippiono Schodde (62)
Family/species
parts
debilis
G. Forsk.
Whole plant
(99)
Branch
urens L. (84)
USNEACEAE (Jsnea hurhuru (L.) Vigg. var. hieronymii (Krempelh)
Whole plant
Mull. Arg. (74)
VERBENACEAE
Leaf/root
Verbenu lifrorulis
Leaf/branch
H. B.K. (2)
ZYGOPHYLLACEAE Bulnesiu .wrmien/oi Lorentz
ex Griseb.
(I 5)
Bark
C. Pkez,
C. Anesini / J. Ethnophannacol.
2.2. Plant extracts Extracts were made from air-dried samples of the whole plant or from the portion specified as being used medicinally. Each 500 mg of dry material was steeped for 30 min with 8 ml boiling water according to the method of Anesini and Perez (1993). Each extract was filtered and the volume adjusted so that each ml of extract was equivalent to 62.5 mg of dry starting material. 2.3. Determination of the antibacterial activity The agar-well diffusion method (Perez et al., 1990) was modified. Nutrient agar was used as a culture medium. It was inoculated with a clinical strain of Salmonella typhi, previously suspended in tryptose broth. Wells 6 mm in diameter were punched into the agar and filled with plant extracts and solvent blanks or standard solutions of ampicillin (1 l- 1000 &ml). The antibiotic standards were obtained from Laboratorios Beta Sociedad Anonima Industrial y Comercial, Argentina. The antimicrobial activity was evaluated by measuring the inhibition-zone diameter observed at 48 h. The mean values were interpolated by comparison to a reference concentration-response curve of ampicillin.
44 (1994)
DIAMETER
45
41-46
OF lNHlBlflON
(mm)
60-
40 -
30 -
01 -2
-1.5
-1
-0.6
0
log concentration (ampicillin, mg/ml) Fig. I. Reference curve of Salmonella
fyphi sensitivity to am-
picillin. Shown are means f
S.E.M.
from 4 experiments.
of them made in duplicate.
Slope (b) = 17.21 mm/log
tercept (i) = 43.47 mm and correlation
coefficient
each C; in-
(r) = 0.99.
3. Results 3.1. Optimization of the experimental conditions After preliminary studies, the following conditions appeared to be optimal for antibacterial screening against Salmonella typhi: (i) inoculum concentration: suspensions in tryptose phosphate broth exhibiting 0.52-0.57 units of absorbance at 580 nm; (ii) suspension: dilution of the inoculum; l/20 v/v in nutrient agar medium; (iii) inoculum amount: 15 ml of nutrient agar medium inoculated as described above was poured into a single Petri plate; (iv) test sample size: 50 ~1 samples were placed in each well; (v) incubation temperature: 25°C and (vi) incubation time: 48 h. 3.2. Reference curve Linear regression analysis was applied to data. As it can be observed, a good fitting of data to a linear function (antibacterial activity versus logarithm of the concentration of ampicillin) can be
achieved. The calculated parameters and experimental data are reported in Fig. 1, which was used as a reference to evaluate the activity of the test samples by interpolation. 3.3. Antibacterial activity Twenty four extracts were found to exhibit antibacterial activity on Salmonella typhi (Table l), whereas 108 extracts were inactive (Table 2). 4. Discussion and conclusions Research on natural resources has been encouraged by the World Health Organization from 1978 to date. The finding of 24 active plant extracts may be interesting in the search for new efficacious and safe antimicrobials against Salmonella typhi. Considering the multiple resistance of Salmonella typhi, clinically important
46
C. P&es,
C. Anesini / J. Ethnopharmacol.
both in the treatment of diseases and in the case of asymptomatic carriers (Winshell et al., 1970), our results could stimulate further pharmacological studies seeking new antibiotics from the higher plants. Since Salmonella typhi is also involved in contamination of food and drugs (Thatcher and Clark, 1973; Christopulos et al., 1980), our extracts may also be useful as conservation agents. Further investigation will be necessary to elucidate the pharmacological mechanisms and the clinical usefulness of the compounds contained in the extracts herein studied. Acknowledgements
This work was supported in part by the Third World Academy of Sciences (award TWAS RGBC: 91-027). The authors are grateful to Ing. Ricardo Rossow of the Faculty of Pharmacy and Biochemistry, University of Buenos Aires, for the botanical identification of the plant species used in this study. References Anesini. C. and Perez. C. (1993) Screening of plants used in
44 (1994)
41-46
Argentine folk medicine for antimicrobial activity. Ethnopharmacology
39,
I 19-i
Journal of
28.
Christopulos. M.C., Cinto. 0.. D&ma de Wollf, C., Frade, A.. Maffe, Z.I., Mercado, E.C., Moro. A.A., Raffo Palma. MS.. Rivas, M., Rosetti, F., Torrado, S.S., Varsavsky. E. (1980) Bromatologia
analitica
para
alimentos
y
medicamentos.
Subsecretaria de Medicina Social y Fiscalizacion Sanitaria, lnstituto National de Farmacologia y Bromatologia. Buenos Aires, pp. 25-41. Furowicz. A. and Terzolo. H. (1975) Aislamiento de serotipos de SalmoneNa sp en animales de la region de Balcarce. Boletin Veterinario. Dpto. de Production Animal. Unidad de Patologia Animal, n-17, pp. l-5. Martinez Crovetto, R. (198 I) Plantas utikadas en Medicina en el noroeste de Corrientes. Ministerio de Cultura de la Nation. San Miguel de Tucuman, pp. 13-125. Perez, C., Pauli. M. and Bazerque, P. (1990) An antibiotic assay by the agar-well diffusion method. Acra Biologiae et Medicine kperimentalis 15, I l3- I 15. Ratera, E.L. and Ratera. M.O. (1980) Phntas de luj7ora argentina empleadas en medicina popular. Hemisferio Sur, Buenos Aires, pp. 17-105. Thatcher, F.S. and Clark, D.S. (1973) Anrjlisis microbioldgico de /OS alimentos. Acribia. Zaragoza, pp. 6-8. Toursarkissian, M. (1980) Plantas medicinales de la Argentina: sus nombres
Botcinicos.
Vulgares.
Uses
y
Distribucidn
Hemisferio Sur. Buenos Aires. pp. I-139. Winshell, E.B., Cherubin. C.. Winter, J. and Neu. H.C. (1970) Antibiotic resistance of Sulmonellu in the eastern United States. In: G.L. Hobby (Ed.). Antimicrobial Agents and Chemotherapy-1969. American Society for Microbiology. Bethesda MD, pp. 86-89. geogrrjfica.