Journal of Ethnopharmacology,

81

36 ( 1992) 8 I 85

Elsevier Scientific Publishers Ireland Ltd.

Parasitologica .1and microbiological evaluation of Mixe Indian medicinal plants (Mexico) Michael Heinrich”, Michaela Kuhnta, Colin W. Wrightb, Horst Rimpler”, J, David Phillipsonb, Alfred Schandelmaier” and David C. Warhurst’ UInstitut fir Pharmazeutische School of Pharmacy, 29-39

Biologic, Universitiit, Schiinzlestr. I, 0-7&W Freiburg (Germutzy), hDepartment of Phurmacognosy, The Bru~wiek Square, London WCIM IAX and ~~epartment of Medical Para.~~tolvgy, ~t~ndon School of Hygiene and Tropical Medicine. Kepper Street, London WCIE 7HT (U, K.)

(Received September 9, 1991; accepted November 21. 1991)

Medicinal plants are an important health resource in many regions of the Americas and are of particular importance to many Indian communities. Based on a recent ethnobotanical study in Mexico, we investigated the activity of 29 plant extracts against Entumoeba histolytira. three bacteria f3aciks sabtilis, Escherichia co& and Micrococcus luteas) and two fungi (Cladosporium cucumerimun and Penjcii~j~ o~u~i~rn). After separation of these extracts between CH,Cl, and HP0 the resulting phases were also evaluated. Key worak antiprotozoal;

Entamoeba; antibacterial; antifungal; gastrointestinal

medicines; dermatological medicines; Mixe Indians

(Mexico)

Introduction The treatment of gastrointestinal disorders is of increasing importance for developing countries such as Mexico in which diarrhoea and dysentery are major health problems. Frequently these illnesses are associated with infections with E&amoeba histolytica or with various bacteria. New lead compounds to treat amoebiasis in particular, are needed (Wright and Phillipson, 1990). We are currently investigating plants with potential biomedical uses in the treatment of gastrointestinal and dermatological infections based on an ethnobotanical collection from a Mixe Indian community in Oaxaca, Mexico and of popular medicinal plants available in many Mexican markets (Heinrich, 1989; Heinrich et al., 1992a,b) To guide our research we use a five-stage process of documentation and evaluation: (1) initial ethnobotanical study to document the medicinal flora; (2) preliminary ethnopha~acological evaluation using published phytochemical and pharmacological information on the plants to select those plants to be passed on to level 3; (3) Correspondence to: Michael Heinrich, Institut mazeutische Biologie, Universitlt, Schlnzlestr. Freiburg, Germany

fur phar1, D-7800

parasitological and microbiological screening of these selected plants; (4) phytochemical investigation of the most active plants; (5) further pharmacological and toxicological evaluation of the latter plants. In this paper we present the results of the parasitological and microbiological investigation of 29 plants (stage 3) which may be of particular interest due to the results of the preliminary ethnopharmacolo~cal evaluation (stage 2). Background and Methods Ethnobotanicai

background

The Mixe live mostly in the cool and humid mountains of the Sierra de Juarez in the Mexican state of Oaxaca. The ethnobotanical study was conducted in the only Mixe speaking community belonging to the subtropical Zstmo de Tehuantepet. The Mixe of this area are mostly peasants and their economy is based on subsistence agriculture (maize) and the production of coffee and citrus fruits for sale. The ethnobotanical study was conducted from November 1985 to December 1986 with two additional stays in February/March f 988 and December 1990IJanuary 199 1 (Heinrich, 1989; Heinrich et al., 1992a,b). The plant material was

Malvaceae Mimosaceae Annonaceae Compositae Asclepiadaceae Loganiaceae Simaroubaceae Scrophulariaceae Compositae Chenopodiaceae Menispermataceae Myrtaceae Rhamnaceae Sterculiaceae Hippocrateaceae

Abelmoschus moschatus Medikus Acucia corn&era L.

Acanthaceae

Labiatae Polemoniaceae Onagraceae Malvaceae Mimosaceae Myrtaceae Fagaceae Labiatae Gesneriaceae Smilacaceae Ancardiaceae Sterculiaceae

Justicia spicigera Schldl.

Leonurus sibiricus L.

AP AP LE LEIFLISE BA RO BA AP TU RO RO AP

LE

LE

BA

SE LE BA LE SA LE AP LE LE AP RO LEIBA LE BA/FR

Plant parts”

Gastrointestinal pain Purgative, diuretic, emetic, sudorific Skin infections, insect-bites Diarrhoea, labour problems Skin infections Dysentery, diarrhoea Diarrhoea, gastric pain Gastrointestinal problems Diarrhoea, dysentery, contraception Dysentery, haemorrhage Diarrhoea Purgative, scabies, cough, fever

Dysentery, diarrhoea, stomachache, scabies

Stomachache, skin infections, headache

Stomachache, snakebites, induce labour Strong skin infections Diarrhoea Stomachache, vomiting Purgative, antiparasitic Stomachache Dysentery, stomachic, fever Stomachic Skin infections, toothache Stomachache, antiparasitic Diarrhoea, dysentery Dysentery Skin infections Diarrhoea, haemorrhage, pain in the uterus Skin infections, gastic ulcers

USeS

TE TE TO TE TO TE TE TE TE CI TE TE

TE

TO TE TE DR EX TE TE TO TE TE TE TO TE TO TE TE TO

Cl

MI MI MA MI MI MA MI MI MI MA

MA

EXrrE MA TO MI

MI

MI MA MI MI MI MI MA

MA

MI MI MI MI MI MI MA

Origin a

plants and on Bye

Application”

aAbbreviations for plant parts used: AP, aerial parts; BA, bark; FL, flower; FR, fruit; LE, leaves; RO, root; SA, Xylem-Phloem sap; SE, seed; TU, tuber. bAbbreviations for applications used: CL cold infusion; DR, drops, oral; EX, external massage; TE, tea; TO, topical. CAbbreviations for origin: MA, plants available on regional markets and Ml, Mixe medicinal plants. Data based on Heinrich (1989) in the case of the Mixe medicinal et al. (1988), Heinrich et al. (1992b) and Martinez (1969) for the commercially available plants.

Smilax hmceolata L. Spondias purpurea L. Waltheria americana L.

Psidium guineense SW. Quercus oleoides Schldl. et Cham. Satureja macrosterna Bricq. Sinningia incarnata (Aublet) D. Dehnh.

Loeselia mexicana Brand Ludwigia octavalis (Jacq.) Ravan Malvaviscus arboreus Cav. Mimosa tenuijlora Willd.

Labiatae

Hyptis verticillara Jacq.

Hippocrafea excelsa H.B.K.

Cissampelos pareira L. Eugenia acapulcensis Steud. Gouania polygama (Jacq.) Urban Guazuma ulmifolia Lam.

Castela texana (Tom et Gray} Rose Castilleja tenuifolia Benth. Chapralia nutants (L.) Pollack Chenopodium graveolens Wild.

Annona muricata L. Artemisia i~oviciana ssp. mexicana Nutt. Asclepias curassiviaca L. Buddleja americana L.

Family

Species

ETHNOBOTANICAL DATA OF THE PLANTS TESTED AGAINST ENTAMOEBA HISTOL YTICA. BACTERIA AND FUNGI

TABLE 1

83

collected during these periods and air dried in a field dryer. Additionally several of the plants are being grown at the botanical garden in Freiburg. Voucher specimens (collection numbers Heinrich and Antonio GUI I-265 and MAR Z-60) are deposited at the herbarium of the Institut fiir Pharmazeutische Biologie at Freiburg (FB) and also at the Mexican National Herbarium of the National Autonomous University (MEXU, Mexico DF). Extraction of the plant material

For all assays, 15 g of the dried plant material were extracted once with EtOH (96%) and twice with EtOH (70%). The resulting combined extract was concentrated and freeze dried. Two-thirds of the total extract was dissolved in approximately 100 ml of HzO. In most cases CH& was used to separate the lipophilic and hydrophilic fractions. Tannin containing drugs (Eugenia acapuicensis, Guazuma ulmtfolia, Quercus oleoides, Psidium guineense, Spondias purpurea) were extracted by per-

colation with acetone (70%). Biological assays

The antiamoebic assay was carried out as described previously using a microdilution assay in Diamond TPS-1 medium (Wright et al., 1988). To evaluate the antibacterial and antifungal activity we used a method utilising silica gel plates (Merck 5549). The test organisms were Bac~Z~ussubtilis DSM 347, Escherichia coii DSM 1077, Micrococcus luteus DSM 348 (bacteria), Cladosporium cucumerinum CBS 108.23 and Penicillium oxalicum CBS 219.30 (fungi). Different amounts of a solution of the extract were applied as spots on TLC plates. This method for the antibacterial assay is similar to the one described by Hamburger and Cordell (1987) and Baumgartner et al. (1990) with the following modifications. An overnight culture (10 ml) of bacteria was mixed with an equal amount of medium (Difco LB) and incubated for another 3-4 h. The final optical density of the bacteria in solution was 0.25 or higher (600 nm, Zeiss PM2A). To start incubation on the TLC plates the bacterial suspension was mixed with 60 ml of medium to yield a total of 80 ml. This was filled into a staining chamber and the TLC plates were dipped twice into the solution for 5 s, to assure complete soaking of the plates. The plates were incubated for 6 h (Bacillus subtilis) or 8 h (Escherichia coli and Micrococcus luteus) at 37°C in a water satured atmosphere. To detect bacteria, tetrazoliumchloride ( I%, Aldrich I- 1,040-6) was sprayed onto the plates and the resulting white

inhibition zones were detected after 1 h ~Baci~~us s&i/is} or 3-4 h (Escherichia co/i and Micrococcus luteus) of incubation. The TLC plates may also be developed using standard solvent systems to separate the compounds. Fungal cultures were incubated for 2 days in Czapek Dox medium and applied to the TLC plates by dipping the plates into 80 ml of inoculated medium. Detection was direct by observing white inhibition zones against a grey fungal background (for Cladosporium cucumerinum; Homans and Fuchs, 1970) or against the fungal mycelium (Penicil~ium oxalicurn; Baumgartner et al., 1990) after 3 days. Analysis of the results

All experiments were run in duplicate and the following levels of inhibition documented: (-) no inhibition, good growth of the organisms in the zone of application; (f ) weak inhibition, zone of inhibition up to 1 mm in diameter; (+) significant inhibition, diameter of inhibition zone between l-2 mm; (++) inhibition zone of 2-5 mm; and (+++) inhibition zone larger than 5 mm. In Table 3 we list the minimal amount applied to the TLC plates that produce an inhibition zone of 1 mm or larger. Results and Discussion In a previous ethnobotanical study in the community of San Juan Guichicovi we documented a total of 213 plants. Sixty-six are used as teas or occasionally cold decoctions or drops to treat gastrointestinal illnesses and 72 are applied topically against dermatological problems (Heinrich, 1989; Heinrich et al., 1992a,b). For parasitological and microbiological screening we selected those 29 plants with the greatest ethnopha~acolo~~l relevance by complementing the ethnobotani~l data with published phytochemical and pharmacological information. Most of the selected plants are also among those widely used in the area (Heinrich, 1989). Of the 29 plants, 20 are used for gastrointestinal problems including dysentery, stomachache, diarrhoea, vomiting, gastric ulcers, parasitic infections etc.; rive plants are employed against skin infections and four plants are used for both groups of indications. Nine plants are available on regional markets and the remaining 20 plants originate from the Mixe medical system (Table 1). In the anti-amoebic assay the ethanolic extract of five plants showed an IC,, value of 125-250

84 TABLE

of Castela texana the activity is thought to be due to quassinoids. A number of quassinoids have previously been isolated from related species (Mitchell et al., 1971) and some of these compounds have been shown to have potent antiamoebic properties (Wright et al., 1988). With A. muricata the activity is presumably due to isoquinolinealkaloids (Leboeuf et al., 1982). Also the CH2C1, phase of Chenopodium graveolens has a rather low ICsO value of 31-62 &ml. In this case and in all the others the chemical basis of the activity is still uncertain. Extracts from many of the plants show activity against the three bacteria (Bacillus subtilis, Escherichia coli and Micrococcus luteus) and the two fungi (Cladosporium cucumerinum and Penicillium oxalicum) (Table 3). We disregarded activities of amounts higher than 20 &spot in the case of the bacteria (10 pg/spot in the case of B. subtilis) and 30 pg/spot for the two fungi, since we consider it unlikely that activities above these

2

PLANTS WITH SIGNIFICANT ANTIAMOEBIC ACTIVITY (LESS THAN 250 &ml) AND THEIR CORRESPONDING ICseVALUES (&ml) Plant

Inhibitory

Annona muricata Caslela texana Chenopodium graveolens Gouania polygama Quercus oleoides

concentration

Crude extract

CH,Cl, fraction

H,O fraction

63 3l- 63 125-250 125-250 125-250

31-63 4-8 31-63 125-250 125-250

250 63 > 250 > 250 >250

pg/ml or less (Table 2). Of particular interest are Castela texana and Annona muricata, which showed activity at 63 pg/ml and 31-63 pg/ml, respectively. In both cases the activity is mainly concentrated in the more lipophilic fraction upon separation between CHzClz and H20. In the case

TABLE

3

ANTIBACTERIAL SPOT IN j&

AND ANTIFUNGAL

Species

ACTIVITY

OF THE PLANT

EXTRACTS

TESTED

Antibacterial activity (&spot)

(AMOUNT

E. coli

M. luteus

Acacia cornigera Annona muricata Artemisia huioviciana Castela texana Chaptalia nutans

5.0 5.0 1.0 10.0 5.0

20.0 10.0 30.0

Chenopodium graveolens Cissampelos pareira

10.0 -

Eugenia acapulcensis Gouania polygama Guatuma ulmifolia Hyptis verticillata Justicia spicigera Leonurus sibiricus Loeselia mexicana

5.0 10.0 10.0 7.0 10.0 9.0 -

20.0 20.0

Ludwigia octavalis Malvaviscus arboreus Mimosa tenuiflora Psidium guineense Quercus oleoides

5.0 5.0 5.0 1.0 1.0

20.0 20.0 10.0 1.0

15.0 22.0 15.0 15.0 4.0

-

Satureja macrostema Smilax lanceolata Spondias purpurea Waltheria americana

10.0 1.0 5.0 5.0

20.0 20.0 20.0

15.0 10.0 10.0 10.0

25.0 10.0

0.01

PER

Antifungal activity (@pot)

B. subtilis

Ampicillin Nystatin

OF EXTRACT

20.0

c. cucum.

P. oxalicum

15.0 10.0 5.0

1.0 20.0

5.0 10.0

15.0

-

20.0

10.0 25.0

25.0 -

15.0 20.0 20.0 10.0

25.0 10.0

10.0 25.0 25.0

30.0 15.0 30.0

10.0 10.0 25.0

25.0

25.0 20.0 10.0 25.0

-

10.0 -

0.001

20.0

5.0 10.0

0.001 0. I

0.1

Organisms used: B. subtilis. Bacillus subtilis DSM 347; E. coli, Escherichia coli DSM 1077; M. luteus, Micrococcus luteus DSM 348; C. cucum.. Cladosporium cucumerinum CBS 108.23; P. oxalicum, Penicillium oxalicum CBS 219.30.

85

levels are of any practical therapeutical interest. Of all 29 extracts 22 inhibited at least one of the three bacteria at levels below these amounts. Eighteen inhibit at least one of the two fungi. Particularly strong antibacterial activity was found with extracts of Gouania polygama, Hyptis verticillata, Quercus oleoides, Psidium guineense, Smilax lanceolata and Waltheria americana. Upon separa-

tion between CH2C12 and HI0 we did not observe any significant concentration of the activity in one of the two phases with the exception of Psidium guineense. In this case the activity was clearly concentrated in the CH$lz phase. Leonurus sibiricus and Annona muricata, Waltheria americana show strong antifungal activity. The activity tended to be concentrated in the CHzClz phase. It is also noteworthy that Castela texana, which is the most active plant in our antiamoebic assays, has low activity against bacteria and fungi. This evaluation is a further step towards understanding the pharmacopoeia of the Mixe (for a similar example from a completely different region see Cox et al., 1989). Plants with the indication listed in Table 1 may produce various physiological effects, which could contribute to the healing process. Other plants may not produce any significant physiological effect at all. Accordingly a rather broad spectrum of biological assays ought to be used to evaluate these plants. In concentrating on potential effects of infectious processes in the gastrointestinal tract we were able to select the most promising candidates for phytochemical studies on plants which may yield lead compounds against important gastrointestinal infections (Caceres et al., 1990). The number of plants that show interesting activity is much higher than one would expect if randomly selected plants were tested. These biological assays are also necessary for understanding the rationale of the Mixe plant use. The results presented here will be pursued further and the relevant compounds of the most interesting plants will be isolated and their structures elucidated. Acknowledgements

This research would not have been possible without the help of the healers and other informants from San Juan Guichicovi, who are the

original keepers of this botanical knowledge. Help by H. Bunz in growing of the plants is also gratefully acknowledged. We thank the Deutsche Forschungsgemeinschaft (Bonn, Germany) for short term tinancial support to Prof. Dr. H. Rimpler. C.W.W. is supported by a C.W. Maplethorpe fellowship of the University of London. D.C.W. is funded by the Public Health Laboratory service, U.K. References Baumgartner, B., Erdelmaier, C.J., Wright, A.D., Rali, T. and Sticher, 0. (1990) An antimicrobial alkaloid from Ficus sepfica. Phyrochemistry 29, 3327-3330. Caceres, A., Cano, O., Samayoa, B. and Aguilar, L., (1990) Plants used in Guatemala for the treatment of gastrointestinal disorders. Journal of Ethnopharmacology 30, 55-73. Cox, P.A., Sperry, R., Tuominen, M. and Bohlin, L. (1989) Pharmacological activity of the Samoan ethnopharmacopoeia. Economic Botany 43, 487-497. Hamburger, M.O. and Cordell, G.A. (1987) A direct bioautographic TLC assay for compounds possessing antibacterial activity. Journal of Natural Products 30, 19-22. Heinrich, M. (1989) Ethnobotanik der Tieflandmixe (Oaxaca, Mexico) und phytochemische Untersuchung von Capraria biflora L. (Scrophulariaceae). Dissertationes Boranicae 144, Berlin. Cramer. Heinrich, M., Antonio B., N. and Kuhnt, M. (1992a) Arzneipflanzen in Mexiko: Der Markt von Matias Romero (Oaxaca). Deutsche Apotherzeitung 132, in press. Heinrich, M., Rimpler, H. and Antonio B., N. (1992b) Indigenous phytotherapy of gastrointestinal disorders in a Mixe lowland community. Journal of Ethnopharmacology, 36, 63-80. Homans, A.L. and Fuchs, A. (1970) Direct bioautography on thin-layer chromatograms as a method for detecting fungitoxic substances. Journal of Chromatography 51, 327-329. Leboeuf, M., Cave, A., Bhaumik, P.K., Mukherjee, B. and Kukherjee, R. (1982) The chemistry of the Annonaceae. Phyiochemistry 21, 2783-2813. Linares, E., Flares, P., B.y. and Bye, R. (1988) Selecion de Plantas Medicinales de Mexico. Ed. Limusa, Mexico D.F. Martinez, M. (1969) Las Planras Medicinales de Mexico. Ed. Botas, Mexico D.F. Mitchell, R.E., Stocklin, W., Stefanovic, M. and Geissman, T.A. (1971) Chaparrolide and Castelanolide, new bitter principles from Castela nicholsoni. Phytochemisrry 10, 4 I l-41 7. Wright, C.W., O’Neill, M.I., Phillipson, J.D. and Warhurst, D.C. (1988) Use of microdilution to assess in vitro antiamoebic activities of Brucea javanica fruit, Simarouha amara stem and a number of quassinoids. Antimicrobial Agems and Chemotherapy 32, 1725-1729. Wright, C.W. and Phillipson, J.D. (1990) Natural products and the development of selective antiprotozoal drugs. Phyrotherapy Research 4, 12?- 139.

Parasitological and microbiological evaluation of Mixe Indian medicinal plants (Mexico).

Medicinal plants are an important health resource in many regions of the Americas and are of particular importance to many Indian communities. Based o...
550KB Sizes 0 Downloads 0 Views