- Email: [email protected]
Contribution to the ethnobotanical, phytochemical and pharmacological studies of traditionally used medicinal plants in the treatment of dysentery and diarrhoea in Lomela area, Democratic Republic of Congo (DRC)
Journal of Ethnopharmacology 71 (2000) 411 – 423 www.elsevier.com/locate/jethpharm
Contribution to the ethnobotanical, phytochemical and pharmacological studies of traditionally used medicinal plants in the treatment of dysentery and diarrhoea in Lomela area, Democratic Republic of Congo (DRC) A. Longanga Otshudi *, A. Vercruysse, A. Foriers Laboratory for Pharmacognosy, Phytochemistry and Toxicology, Department of Pharmaceutical Sciences, Vrije Uni6ersiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium Received 5 January 1998; received in revised form 20 December 1999; accepted 10 January 2000
Abstract In order to collect ethnobotanical information about antidiarrhoeal plants, we performed inquiries among traditional healers, community leaders, and native people of Lomela villages in Congo. Six medicinal plants widely used in this region were designated as having antidysenteric and antidiarrhoeal properties. These six medicinal plants were screened for groups of phytochemical compounds with antibacterial and antiamoebic activities. They were found to contain tannins, alkaloids, saponins, flavonoids, sterols and/or triterpenes and reducing sugars. Of the six tested plants, three showed prominent antibacterial activity whereas two acted against Entamoeba histolytica. The usefulness of the phytochemical bases and biological activities of these plants as potential source of antidiarrhoeal remedies is discussed. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Folk medicine; Ethnopharmacology; Antidiarrhoeal plants; Antidysenteric plants; Democratic Republic of Congo; Lomela area
1. Introduction Diarrhoea and dysentery epidemics are mostly common where living conditions are crowded and hygiene is poor. Dysentery is a microbial infection of gastrointestinal tract. Symptoms include fever, vomiting, abdominal pain and diarrhoea which often contains blood and pus. The onset of the * Corresponding author.
disease usually occurs within 2–3 days after infection and lasts for up to several weeks. Dehydration occurs rapidly, especially in children and can cause death if treatment is not given. Parasitic diseases such as dysentery and diarrhoea are the main causes of high mortality rate in developing countries. In these countries, over five million children under the age of five die annually from severe diarrhoeal diseases (WHO, 1996). The majority of rural people has limited access to formal and adequate health services,
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 1 6 7 - 7
412
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
thus people seek help from traditional healers who provide alternative health care services. Indigenous plant remedies are widely used in the treatment of a variety of disorders. However, only few of them have been controlled clinically, or studied chemically and biologically to identify their active constituents. In DRC, numerous plants are traditionally used as medicinal tools with antibacterial and antiparasitic properties in the treatment of diarrhoea and dysentery (Penge et al., 1988, 1989, 1990; Kambu et al., 1990). Since ethnobotanical field research is always the first step in any phytomedicinal research and development work (Mitscher et al., 1987; Rojas et al., 1992), inquiries were studied out in Lomela villages. This study is the first one performed in this region and to the best of our knowledge, most of the plants hereby investigated have not been previously reported to possess antidiarrhoeal activity. Therefore, the present study gives specific and useful information about; (i) which plants and/or plant parts are used in the treatment of diarrhoea and dysentery, (ii) how the plant material is provided and processed for remedies, and (iii) how these remedies are sued for treatments by traditional healers and traditional people of the Lomela region. In addition, a preliminary phytochemical screening has been carried out in order to have an idea of the different classes of organic compounds present in the different extracts of each plant. Finally, we have screened crude extracts derived form these plants for their antibacterial and antiamoebic activities, so as to generate some preliminary data on the biological activity of the plant extracts.
2. Materials and methods
2.1. Study area Inquiries have taken place in the area of Lomela, sub-district of Sankuru, district of Oriental Kasaı¨ (Congo). This region was chosen due to severe epidemics of dysentery that occurred in 1986 and 1994 which were treated essentially with
endemic medicinal plants in complete absence of modern medicine.
2.2. Methods used in the inter6iews with informants Inquiries were executed from 2 March 1996 to 16 April 1996 among the traditional healers and people of Lomela area, mainly in the villages where epidemics of diarrhoea and dysentery occurred. These places are: Lomela centre, in Batetela community; Mukumari, in Batetela community, approximately 70 km from Lomela centre, in the South of Lomela area; Kutusongo, in Bahamba I community, 50 km from Lomela centre, in the South of Lomela area; Wony’Angondo, in Bahamba I community, 40 km from Lomela centre, in the South Lomela area; Betembo, in Bakela community, 25 km from Lomela centre, in the West of Lomela area; Osangamanga, in Bahamba II community, 30 km from Lomela centre, in the North of Lomela area. Interviews based on pre-prepared series of questions were administered to the informants. Such questions included among others: Researcher’s name, Informant: name, tribe, village, community, zone, sub-region, age, sex and religion, Plant: vernacular name, uses in popular medicine, plant part, plant combinations, additional ingredients, preparation, application and dosage, rites, side effects, interdicts, no medicinal uses. These interviews were administered by one of the authors (A. Longanga Otshudi) in local languages, with the help of the political authority of each village. In total, 104 interviews were conducted among healers and native people of Lomela villages, of which 33 interviews were carried out in Lomela centre, 30 in Mukumari, ten in Kutusongo, 21 in Wony’Angondo, six in Osangamanga and four in Betembo. The plants were collected from all the different parts of the investigated Lomela villages, and their botanical identification was carried out with
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
the help of the INERA (Institut National d’Etudes et de Recherches Agronomiques) herbarium at Kinshasa University and the specialists of the ‘Herbarium of National Botanical Garden of Meise, Belgium’. Confirmation was obtained by comparing the collected species with the already identified herbarium specimens. A voucher specimen of each species was deposited in the Department of Pharmaceutical Sciences, Laboratory for Pharmacognosy and Phytochemistry, Vrije Universiteit Brussel.
2.3. Preparation of extracts Each air-dried plant material (10 g) was ground into a fine powder and exhaustively percolated after maceration for 24 h, with solvents having an increasing polarity (diethylether, 70% methanol and H2O). The resulting extracts were evaporated to dryness in vacuum at 40°C, yielding three crude extracts: F1, F2 and F3, respectively. These extracts were subjected to phytochemical analysis and biological activities.
2.4. Antibacterial and antiamoebic testings The antimicrobial testing was performed by the two-fold serial broth microdilution method (Mitscher et al., 1972; Vanden Berghe and Vlietinck, 1982) whereas the antiamoebic assay was carried out using the microdilution technique in Diamond TPS-1-monophasic medium (Wright Colin et al., 1988).
2.5. Test organisms Test microorganisms used in the preliminary antibacterial and antiamoebic testings included both enteropathogens and protozoan. As enteropathogens, we have used: Shigella dysenteriae B-1995/2-3 Shigella sonnei ATTC 25931 Shigella boydii USA 91/D1
:external Belgian quality assessment strain :reference strain :external USA quality assessment strain (C.A.P)
Shigella flexneri AZ 749 Salmonella typhimurium ATTC 14028 Escherichia coli ATTC 25922 Vibrio parahaemolyticus USA 94/DO7 Vibrio cholerae B1994/3-3 Campylobacter jejuni ATCC 24428 Campylobacter coli ATCC 33559
413
:clinical isolate :reference strain
:reference strain :external USA quality assessment strain (C.A.P) :external Belgian quality assessment strain :reference strain :reference strain
As protozoan, we have used Entamoeba histolytica ATCC 30459 (reference strain). All the enteropathogens were provided by the department of microbiology of the AZ-VUB (Academisch Ziekenhuis — Vrije Universiteit Brussel) while the reference strain of Entamoeba histolytica was provided by the Institute for Tropical Medicine ‘Prins Leopold’, Antwerpen (Belgium).
2.6. Preliminary phytochemical analysis Preliminary phytochemical screening of the plants was undertaken using described methods (Sofowora, 1982; Harborne, 1988; Wagner and Bladt, 1996). The screening covered mainly alkaloids, sterols and/or triterpenes, flavonoids, saponins, tannins, anthraquinones and reducing sugars.
2.6.1. Test for alkaloids Alkaloids were tested in all three extracts, ether, methanol, and water. In ether extracts 10 mg of ether residue was macerated with HCl (2% 1.5 ml). The resulting acid solution was divided into three parts, to a portion of which was added Mayer’s reagent, to another Dragendorff’s reagent while the third acted as blank. In alcoholic extracts, 10 mg of methanol residue was macerated with HCl (2%, 1.5 ml), filtered, basified with NH4OH (25%) and extracted with
414
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
CHCl3. The chloroform soluble portion was evaporated, dissolved in HCl (2%, 1.5 ml), divided into three parts and tested as in the ether extracts. In water extracts, 10 mg of aqueous residue was dissolved in distilled water (1.5 ml), basified with NH4OH (25%) and extracted with CHCl3. The chloroform solution was extracted with HCl (10%) and the acid aqueous solution was divided into three parts and tested as in the ether extract. A positive ( +) reaction was recorded if the addition of both reagents produced a faint turbidity; + + reaction was recorded if a light opalescent precipitate was observed; and + + + reaction was recorded if a heavy yellowish-white precipitate was observed with Mayer’s reagent and a heavy yellowish-orange precipitate with Dragendorff’s reagent. Alkaloids detection in plants showing preliminary positive test by the above screening was further confirmed using TLC (Wagner and Bladt, 1996). Powdered drug (2 g) was mixed thoroughly with 1 ml 10% ammonia solution and then extracted twice with 5 ml CHCl3. The chloroformic solution was evaporated to 1 ml and 10 ml of this solution was chromatographed on silica gel G Plates in toluene-ethyl acetate-diethylamine (70:20:10). The presence of alkaloids was detected on the plate, first by UV fluorescence (l=254 nm) and then by the application of Dragendorff and iodoplatinate reagents. The only plant (E. 6illosum) which gave preliminary positive tests showed at least seven alkaloid spots.
2.6.2. Test for fla6onoids Flavonoids were tested in all the three extracts. Ten milligrams of each plant residue was dissolved in 1.5 ml appropriate solvent and tested for flavonoids using Shinoda’s reaction. A positive ( +) reaction was recorded when a slight red coloration was observed; + + reaction was recorded when a medium red coloration was observed; and + + + reaction was recorded when a strong red coloration was observed.
2.6.3. Test for tannins Tannins were tested in the methanol and water extracts by gelatin-salt block test. After dissolving 10 mg of water and methanol residues in distilled water and methanol, respectively, water and methanol extracts were separately divided into three parts. A sodium chloride solution was added to one portion of each test extract, 1% gelatin solution to each second portion, and the gelatin– salt reagent to each third portion. Precipitation with the latter reagent, or with both the gelatinsalt reagents was indicative of the presence of tannins. Precipitation with salt solution (control) indicated a false-positive test. Positive tests were further confirmed by the addition of a few drops of dilute ferric chloride (1% FeCl3) to the test extracts which gave blue black or green black coloration. A positive (+) reaction was recorded when a slight precipitate was recorded; + + reaction was recorded when a medium precipitate was observed; and + + + reaction was recorded when a heavy precipitate was observed. 2.6.4. Test for saponins The presence of saponins was tested in the water extract. The honey comb froth which persisted for at least 30 min after vigorous shaking of the water plant extract and a positive Liebermann-Buchard’s reaction in the hydrolysed water extract indicated the presence of saponins. A + sign was recorded when froth reached a height of 0.5 cm; + + sign with a height up to 1 cm; + + + sign with a height more than 1 cm. 2.6.5. Test for anthraquinones Borntrager’s test was used for the detection of anthraquinones in all the three extracts. Free anthraquinones were tested in the ether extracts whereas combined anthraquinones were tested in the hydrolysed methanol and water extracts. A positive (+ ) reaction was recorded when a light pink, red or violet colour was observed; + + sign was indicated when a medium pink, red or violet colour was observed; + + + sign was indicated when a strong pink, red or violet colour was observed.
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
2.6.6. Test for sterols and/or triterpenes Sterols and/or triterpenes were tested in the ether extracts by Salkowski test and/or Libermann’s test. For Salkowski test, the presence of a reddish-brown colour indicated a positive reaction while a colour change from violet to blue to green was considered as positive reaction for Liebermann’s test. A + sign was recorded when a light colour was observed; + + sign for a medium colour and + + + for a strong colour. 2.6.7. Test for reducing sugars Reducing sugars were tested in the methanolic extract. To a heated methanolic plant extract was added drop by drop a mixture of equal parts of Fehling’s solution No. 1 and No. 2. In most cases reduction took place near the boiling point and was shown by a brick-red precipitate of cuprous oxide. Reducing sugars include all monosaccharides and many dissacharides (e.g. lactose, maltose, cellobiose and gentiobiose). A positive (+ ) reaction was recorded when a slight precipitate was recorded; + + reaction was recorded when a medium precipitate was observed; and + + + reaction was recorded when a heavy precipitate was observed.
3. Results and discussion The rural area of Lomela is located in the district of Oriental Kasai, Sub-district of Sankuru in the DRC. It is the widest area of Sankuru with a surface area of 26 346 km2 and population estimated to be ca. 45.874 in 1958. This area is located approximately at 22 – 25° longitude East, 1°30% –3° longitude South at an altitude of 500 m. The area lies entirely in the region of shady equatorial forests, where it rains all year round with less rainfall observed in March, June and August. There is around 1729 mm annual rainfall. The temperatures are tropical, with 30.9°C at the maximum and 19.5°C as a minimum. The area has a variety of different ethnic groups. It is inhabited by the Batetela (in majority), Bakela, Bahamba and Djonga. The main vernacular language spoken by the majority of the population is Otetela
415
followed by Okela and Djonga. The majority of the population is nominally christian, protestant or kimbanguist. The population lives mainly from the income of agriculture (rice, maize, coffee…), hunting and fishing. An ethnobotanical study of antidiarrhoeal plants used by traditional healers in six communities of Lomela area (DRC) has been carried out. During the course of the study, a total of 104 informants were interviewed for their knowledge on the uses of plants for the treatment of diarrhoea. The average age of the interviewed informants showed that they were predominantly older members of Lomela communities. The group of interviewed persons had a larger number of males, 83, as compared to 21 females. Educationally, more than half of the informants, 59, had no formal education. The rest have had either primary or secondary education. Six medicinal plants widely used in this area for the treatment of diarrhoea and dysentery were collected by one of the authors (A. Longanga Otshudi). These plants are listed in Table 1. The result of the preliminary phytochemical screening of these plants is reported in Table 2. The frequency of usage of collected plants and the proportion of detected organic compounds are summarized in Table 3. In Tables 4 and 5, we report the results of biological testings. The ethnobotanical, phytochemical, antibacterial and antiparasitological information of the collected plants in relation to gastrointestinal disorders is as follows.
3.1. Roureopsis obliquifoliolata. (Gilg) Schellenb (Connaracea) A climbing shrub with brown-pubescent branchlets present in rain forest. Native to South of Nigeria, also in French Cameroon, Spanish Guinea, Congo (DRC) and Angola (Troupin, 1952). In Lomela area (DRC), roots are grounded into fine bits and added to cold water. The mixture is filtered by using a fine clean cloth, then, a small quantity (half a cup) is taken orally twice a day for ten days for the treatment of diarrhoea and dysentery. This plant is also used for the treatment of toothache and elephantiasis (Troupin, 1952).
416
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
The phytochemical screening showed flavonoids, sterols and/or triterpenes, saponins, reducing sugars and tannins. The antibacterial screening showed activity against all the tested enteropathogens. The methanolic and aqueous extracts showed the highter potency, with a MIC value of 31.25 mg/ml for Vibrio cholerae. These extracts also acted weakly against E. histolytica with a MIC value of 500 mg/ml after 72 and 144 h incubation. Biological activity of this plant could be attributed to tannins. Most frequently tannin containing plants are used to treat diarrhoea and dysentery (Heinrich et al., 1992).
Preliminary phytochemical analysis showed alkaloids, sterols and/or triterpenes, saponins and reducing sugars. The isolation of alkaloids in E. 6illosum has been previously described (Parvez, 1994). In vitro antibacterial screening demonstrated activity in methanolic and water extracts against Campylobacter coli and Campylobacter jejuni with a MIC value of 62.5 mg/ml. They also acted against E. histolytica at 125 mg/ml. The antibiotic and antiparasitic activities of this plant are thought to be due mainly to alkaloids.
3.2. Epinetrum 6illosum. (Excell) Troupin (Menispermaceae)
An elegant tree, 12–20 feet. Native to Congo (DRC), Angola and Guinea (Leondard, 1962). In DRC, the maceration of root is drunk (1 cup) three times per day for 7–10 days for the treatment of diarrhoea and dysentery. The stem bark decoction is taken orally for abdominal pains and fever. In contrast, young fruits added to palm wine are taken orally as a laxative. Preliminary phytochemical screening showed the presence of flavonoids, sterols and/or triterpenes, saponins and reducing sugars.
A twining lianca, ca. 2 m high, found in secondary forests in the costal area of Congo (DRC) and Angola (Parvez, 1994). In Lomela area, root decoction of the plant is taken orally for the treatment of diarrhoea and dysentery. The crushed leaves are used as a wound dressing for fast healing. The plant is known to have abortive activity (Troupin, 1951).
3.3. Croton mubango. Mu¨ll (Eurphobiaceae)
Table 1 Antidiarrhoeal investigated plants, their preparation and traditional uses Family/species Menispermaceae Epinetrum 6illosum. (Excell) Troupin Connaraceae Roureopsis obliquifoliolata. (Gild) Schellenb Euphorbiaceae Croton mubango. Mu¨ll Vitaceae Cissus rubiginosa (Welw. Ex. Bak) Planch Asteraceae Vernonia amygdalina. Del
Simaroubaceae Quassia africana. Baill
Vernacular name Plant part
Preparation
Application
Uses
Dipapoma
Root bark Leaves
Decoction
Oral
Diarrhoea, dysentery, burns
Lungangula
Root bark
Decoction
Oral
Diarrhoea, dysentery, elephantiasis
Unganga
Stem bark Root bark
Maceration
Oral
Diarrhoea, dysentery, abdominal pains, fever
Sume
Stem bark Root bark
Decoction
Oral
Diarrhoea, dysentery
Mpasi nyioso
Leaves Root bark
Maceration
Oral
Diarrhoea, dysentery, gastroenteritis, malaria, hepatitis, worms
Dokotele
Root bark
Decoction
Oral
Diarrhoea, dysentery, fever, malaria
a
+/+ +/+ +/+ −
−/− +/+ −/− −
−/− −/− +/+ −
++/++ ++/++ ++/+++ ++
E, diethylether extract; M, methanol extract; W, water extract; RB, rootbark; L, leaves; SB: stembark.
−/− −/− −/− −
−/− −/− −/− −
RB/SB RB/SB RB/L RB −/− −/− −/− −
++/+ +++/+ +++/+ −/− −/− −/− +/+ − − − + + − +
E
RB/L RB
W
Sterols and/or triterpenes
Epinetrum 6illosum Roureopsis obliquifoliolata Croton mubango Cissus rubiginosa Vernonia amygdalina Quassia africana
M
E
W
E
M
Flavonoids
Alkaloids
Plant part
Name of plant
Preliminary phytochemical screening of some plants used in Congolese phytogherapya
Table 2
+/+ −/− −/− +
++/+ +
W
Saponins
M
−/− −/− −/− −
−/− −/− −/− −
−/− −/− − −
E
Anthraquinones
−/− −/− ++/++ −
−/− −
W
++/++ ++/+ ++/++ +
++/+ +++
M
Reducing sugars
−/− ++/++ ++/++ −
−/− +++
M
Tannins
−/− +/+ ++/++ −
−/− ++
W
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423 417
418
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
Table 3 Frequency of used plants in popular medicine and the proportion of detected organic compounds Family/species
Frequency of usage
Menispermaceae Epinetrum 6illosum. + (Excell) Connaraceae Roureopsis + obliquifoliolata (Gild) Schellenb Euphorbiaceae ++ Croton mubango. Mu¨ll Vitaceae Cissus rubiginosa. + (Welw. Ex. Bak) Planch Asteraceae +++ Vernonia amygdalina. Del Simaroubaceae Quassia africana. +++ Baill
Proportion of detected organic compounds
*4/7
5/7
Antibacterial screening showed activity in methanolic and aqueous extracts against several tested enteropathogens. The highest activity was observed towards Shigella dysenteriae and Shigella boydii with MIC values of 31.25 mg/m. In contrast the plant extracts showed weak activity against E. histolytica (MIC values of 500 mg/ml after 144 h incubation). Activity against tested enteropathogens could be due to the presence of tannin and flavonoids in the plant.
3.5. Vernonia amygdalina. Del (Asteracea) 4/7
4/7
5/7
3/7
* Of the seven screened organic compounds, four were detected in Epinetrum 6illosum. + Plant cited in one or two investigated villages. ++ Plant cited in three to five investigated villages. +++ Plant cited in all the six investigated villages.
In vitro antibacterial screening showed no activity against tested enteropathogens. Furthermore, the three tested extracts showed no activity against E. histolytica.
3.4. Cissus rubiginosa. (Welw.ex Bak) Planch (Vitaceae) A climbing or trailing herb, present in savannah and secondary forests. The plant is widespread in tropical Africa (Dewit, 1960). In Lomela area, root or stem decoction is drunk for the treatment of diarrhoea and dysentery (1 cup, 2 ×1 day for 7 days). Leaf infusion is used for the same purpose. Preliminary phytochemical analysis showed the presence of flavonoids, sterols and/or triterpenes, reducing sugars and tannins.
A shrub or small tree 6–10 feet. high. The plant is common and often planted, from Guinea to Cameroon. Extending to Congo (DRC), Uganda and Tanganyika (Oliver, 1877). In Lomela area, the leaf maceration is orally used for the treatment of diarrhoea and dysentery whereas the root decoction is drunk for gastroenteritis, malaria, hepatitis and worms. The use of the root decoction for the treatment of gastrointestinal disorders also has been reported from Rwanda (Vlietinck et al., 1995). Preliminary phytochemical screening showed the presence of flavonoids, sterols and/or triterpenes, saponins, reducing sugars and tannins. The isolation of saponins in V. amygdalina has been previously described (Jisaka et al., 1992). Antibacterial testing of the plant extracts showed no activity against tested enteropathogens. Furthermore, a weak activity was observed against E. histolytica. This result validates previous reports on the antibacterial activity of extracts derived from V. amygdalina towards Escherichia coli (Vlietinck et al., 1995).
3.6. Quassia africana. Baill (Simaroubaceae) A glabrous shrub, 8–12 feet high; present in rain forest. The plant is native to Cameroon, Congo (DRC) and Angola (Gilbert, 1958). In Lomela area, the root bark decoction is drunk for the treatment of diarrhoea and dysentery. Some traditional healers use Quassi africana in combination with Vernonia amygdalina for the
R R R R R R S S S S S S R R R L L L
Epinetrum 6illosum F1 F2 F3 F1 F2 F3 F1 F2 F3 F1 F2 F3 F1 F2 F3 F1 F2 F3
Extracts
\1000 \1000 1000 \1000 250 250 \1000 \1000 \1000 \1000 1000 250 \1000 \1000 \1000 \1000 \1000 \1000
S.s.
Bacteria
\1000 \1000 1000 \1000 31.25 62.5 \1000 \1000 \1000 \1000 31.25 31.25 \1000 \1000 \1000 \1000 \1000 \1000
S.d. \1000 \1000 \1000 \1000 62.5 62.5 \1000 \1000 \1000 \1000 \1000 31.25 \1000 \1000 \1000 \1000 \1000 \1000
S.f. \1000 \1000 \1000 \1000 125 62.5 \1000 \1000 \1000 \1000 \1000 31.25 \1000 \1000 \1000 \1000 \1000 \1000
S.b. \1000 62.5 62.5 \1000 500 1258 \1000 1000 \1000 \1000 500 250 \1000 1000 \1000 \1000 1000 \1000
C.c. \1000 62.5 62.5 500 31.25 500 \1000 1000 1000 \1000 1000 250 \1000 1000 1000 \1000 1000 \1000
C.j.
\1000 \1000 \1000 \1000 500 31.25 \1000 \1000 \1000 \1000 \1000 125 \1000 \1000 \1000 \1000 \1000 \1000
V.c.
\1000 \1000 \1000 \1000 \1000 500 \1000 1000 \1000 \1000 \1000 250 \1000 \1000 \1000 \1000 \1000 \1000
V.p.
\1000 \1000 \1000 \1000 \1000 250 \1000 \1000 \1000 \1000 500 125 \1000 \1000 \1000 \1000 \1000 \1000
S.t.
500 \1000 \1000 \1000 \1000 \1000 \1000 \1000 \1000 \1000 \1000 \1000 \1000
\1000 \1000 \1000 \1000
E.c.
a R, Root bark; S, stem bark; L, leaves; F1, deithyl ether extract; F2, mehanol extract; F3, aqueous extract; S.s, Shigella sonnei; S.d, S. dysenteriae; S.f, S. flexneri; S.b, S. boydii; C.c, C. coli; C.j, C. jejuni; V.c, Vibrio cholerae; V.p, V. parahaemolyticus; S.t, S. typhimurium; E.c, E. coli; MIC, minium inhibitory concentration (mg/ml).
Vernonia amygdalina
Quassia africana
Cissus rubiginosa
Croton mubango
Roureopsis obliquifoliolata
Parts
Plants
Table 4 MIC values (mg/ml) of tested plant extractsa
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423 419
420
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
All the plant materials were collected during the rainy season and early in the morning, since most of interviewed traditional people indicated that some plants have their full therapeutic effect only if collected at certain time or during a certain season. Most of the investigated plants were designated several times by different informants and multiple citations were particularly found within the same study area, exception made for Vernonia amygdalina and Quassia africana which were cited in all the investigated villages. Most of the collected plants were used separately except Quassia africana which is used in combination with Vernonia amygdalina for treating malaria and fever. Root is the plant part most frequently used by traditional people of Lomela for preparing traditional medicine as observed in this study. This agrees with a study carried out in Tanzania, where Chhabra et al. (1993) also found that roots were the most prominent plant part used to prepare remedies. The concentration and the nature of the active compounds can vary significantly
treatment of fever and malaria. The use of this plant for the treatment of diarrhoea and dysentery in Kinshasa (DRC) has been previously reported (Kambu et al., 1990). Preliminary phytochemical screening showed the presence of sterols and/or triterpenes, saponins and reducing sugars. The isolation of quassinoids in this plant has been previously described (Cabral et al., 1993). Antibacterial screening showed no activity against tested enteropathogens whereas the plant extracts demonstrated good activity against E. histolytica with MIC value of 125 mg/ml after 72 h incubation. Certainly the quassinoids in the extracts are effective against E. histolytica, since these organic compounds are known to have antiamoebic properties (Wright Colin et al., 1988). In contrast, we did not find out in this study the previous antibacterial activity reported on extracts derived from Quassia africana against Escherichia coli and Shigella flexneri (Kambu et al., 1990). Table 5 Antiamoebic activities (mg/ml) of tested plant extractsa Plants
Epinetrum 6illosum
Roureopsis obliquifoliolata
Cissus rubiginosa
Croton mubango
Quassia africana
Vernonia amygdalina
Parts
R R R R R R S S S S S S R R R L L L
Extracts
F1 F2 F3 F1 F2 F3 F1 F2 F3 F1 F2 F3 F1 F2 F3 F1 F2 F3
Concentrations 500 mg/ml
250 mg/ml
125 mg/ml
Control of growth
72 h
144 h
72 h
144 h
72 h
144 h
72 h
144 h
+++ − − ++ − − + + + ++ ++ +++ + − − ++ − −
++ − − ++ − − − − − ++ ++ ++ + − − + − +
+++ − − ++ + + + ++ ++ +++ +++ +++ + − − ++ + +
++ − − ++ + + + ++ ++ ++ ++ ++ + − − + + +
+++ − − ++ ++ ++ + ++ ++ +++ +++ +++ ++ − − ++ ++ ++
++ − − ++ ++ ++
+++ +++ +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
++ +++ +++ +++ +++ +++ ++ +++ +++ +++ ++ ++ ++ ++ ++ +++ ++ ++
++ ++ +++ ++ ++ + + + ++ ++ ++
a R, Root bark; S, stem bark; L, leaves; F1, diethylether extract; F2, methanol extract; F3, aqueous extract. −, All amoebae killed; +, 100–80% amoebae killed; ++, 80–20% amoebae killed; +++, 20–0% amoebae killed.
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
between different parts of the same plant. We observed that the stem bark of Croton mubango is used against diarrhoea while the seeds (fruits) of the same plant cause diarrhoea and are sometimes used for treating constipation. Another observation made is the way by which remedies are administered for treating diarrhoea. In most cases, the oral route is preferred. The usual modes of preparing remedies are decoction, infusion and maceration, with water and/or palm wine as solvents. The bacteriological quality of these remedies is solely dependent on their mode of preparation (decoction, infusion) or the antimicrobial properties of palm wine, sometimes used as solvent. No rites or incantations are required to give the desired effect from a remedy. The dosage of a remedy depends mainly upon the nature of the drug (plant part) to be administered, the sex of the patient and the age. In general, pregnant women and children get small doses. The duration of the treatment varies, depending on the type of remedy and the severity of diarrhoea. In general, at least 10 – 14 days are assumed to be necessary for treatment. Normally, if after 2 weeks of treatment dysentery persists, patients die of anemia and dehydration. No interdicts are required during the treatment, but since one knows that dysentery is a very contagious disease, traditional healers recommended no contact with the patient. That is why, sometimes, even food was given to the patients by means of sticks. When asked about secondary effects, only a few of the respondents reported vomiting, abdominal pains, loss of appetite and abortion for Epinetrum 6illosum, but we can not overlook the fact that some of these effects (vomiting and abdominal pains) also occur during dysentery episode. The preliminary phytochemical screening carried out on the six collected plant materials covered mainly alkaloids, saponins, sterols and/or triterpenes, tannins, flavonoids, anthraquinones and reducing sugars. Out of the six screened plants, only one (1/6), Epinetrum 6illosum, gave a positive test for alkaloids which are reputed to have important physiological activities. Three plant materials (3/6) gave a positive test for tan-
421
gent action; this provokes their usefulness in preventing diarrhoea and controlling haemorrhage. In addition, tannins have been reported to possess cytotoxic and antineoplastic activities. Four plant materials (4/6) gave a positive test for flavonoids which are known to possess antiviral, antiinflammatory and cytotoxic activities. Five plant materials (5/6) gave a positive test for saponins which are well known to have expectorant and antitussive activities. The economical importance of saponins lies in their easy conversion to medicinally used steroidal hormones. All the six plant materials (6/6) gave a positive reaction for reducing sugars which are known to be useful in providing a means of storing energy and transport of energy. In addition, many classes of plant constituent (e.g. the nucleic acids and the plant glycosides) contain sugars as essential features of their structures. Sugars also play a number of ecological roles in protection form wounding and infection and in the detoxification of foreign substances. All the six plant materials (6/6) also gave positive reaction for sterols and/or triterpenes. The therapeutic and industrial interest of triterpenes and steroids make them a group of secondary metabolites of outstanding importance. No anthraquinones were detected in all the investigated plants. We were also interested in the frequency of usage of a given plant. Since the observations on the frequency could not be quantified, popularity of the plant was used as an indicator. Thus, considering the frequency of usage of a given plant and the proportion of the organic compounds it contains, we observed that the most frequently used medicinal plant does not, in most instances, contain the highest proportion of organic compounds (Table 3). The results of the antibacterial screening indicated that methanol and aqueous extracts derived from Roureopsis obliquifoliolata, Cissus rubiginosa and Epinetrum 6illosum produced in vitro antibacterial activity against one or more tested enteropathogens (Table 4). This could partly be explained by the widespread occurrence of polyphenolic compounds, which are soluble in water and methanol. Of great importance is Epinetrum 6illosum which also showed high activ-
422
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
ity against Entamoeba histolytica (Table 5). Although Quassia africana showed no activity against tested enteropathogens, the antiamoebic activity of its extracts was comparable to those derived from Epinetrum 6illosum (Table 5). Croton mubango and Vernonia amygdalina did not show any prominent antibacterial and antiamoebic activities in this study. We must point out that sometimes, traditional healers prefer a combination of several plants to treat disease. This combination could have a pharmacological activity whereas separate constituents might not. Evidence is hereby presented that some of the plants used for the treatment of diarrhoea and dysentery by the population of Lomela area (DRC) really have some biological activities (antibiotic and antiparasitic activities) which might explain partly the properties claimed in the treatment of gastrointestinal diseases. Some of these interesting plants are now being subjected to indepth study for chemical constituents. Such substances in the purified state may be useful for the treatment of diarrhoea and dysentery.
Acknowledgements The authors are grateful to Professor Penge On’Okoko (Kinshasa University) for his collaboration. Our sincere thanks go to all the technicians of INERA herbarium (Kinshasa University) and the ‘Herbarium of National Botanical Garden of Meise, Belgium’ for the botanical identification of the plant species. We also thank the informants who provided information about antidiarrhoeal plants used in Lomela area for the treatment of diarrhoea and dysentery.
References Chhabra, S.C., Mahunnah, R.L.A., Mshiu, E.N., 1993. Plants used in traditional medicine in Eastern Tanzania. VI. Angiosperms (Sapotacea-Zingiberaceae). Journal of Ethnopharmacology 39, 83–103. Cabral, J.A., McChesney, J.D., Milhous, W.K., 1993. A new antimalarial quassinoid from Simaba guianesis. Journal of Natural Products 56 (11), 1954–1961.
Dewit, 1960. Spermatophytes. Flore du Congo Belge et du Ruanda-Urundi, 9, 538. Gilbert, G., 1958. Spermatophytes. Flore du Congo Belge et du Ruanda-Urundi 7, 125. Harborne, J.B., 1988. Phytochemical methods. A Guide to Modern Techniques of Plants Analysis, first ed. Chapman and Hall, London, pp. 1 – 226. Heinrich, H., Rimpler, H., Barrera, N., Antonio, 1992. Indigenous phytotherapy of gastrointestinal disorders in a low land mixed community (Oaxaca, Mexico): ethnopharmacologic evaluation. Journal of Ethnopharmacology 36, 63 – 80. Jisaka, M., Kawanaka, M., Sugiyama, H., Takegawa, K., Huffman, M.A., Ohigashi, H., et al., 1992. Antischistosomal activities of sesquiterpene lactones and steroid glucosides from Vernonia amygdalina, possibly used by wild chimpanzees against parasite-related diseases. Biosciences – Biotechnology– Biochemistry 56 (5), 845 – 846. Kambu, K., Tona, L., Luki, N., Cimanga, K., et Uvoya, A., 1990. Activite´ antibacte´rienne d’extraits a` partir de plantes utilise´es en pre´paration comme antidiarrhe´iques a` Kinshasa, Zaı¨re. Annales Pharmaceutiques Franc¸aise 48 (5), 255 – 263. Leondard, J., 1962. Spermatophytes. Flore du Congo Belge et du Ruanda-Urundi. 8 (1), 57. Mitscher, L.A., Drake, S., Gollapudi, S.R., Okwute, S.K., 1987. A modern look at folkloric use of anti-infective agents. Journal of Natural Products 50, 1025 – 1040. Mitscher, L.A., Leu, R.P., Bathela, M.S., Wu, W.-K., 1972. Antimicrobial agents from higher plants. I. Introduction, rationale and methodology. Lloydia 35, 157 – 176. Oliver, D., 1877. Compositae. Flora of Tropical Africa 3, 284. Parvez, M., 1994. Bis benzylisoquinoline alkaloids from Epinetrum 6illosum. Journal of Chemical Society of Pakistan 16 (4), 257 – 260. Penge, O., Bashengezi, M., Longanga, O., Foemi, K., 1988. Evaluation des activite´s antimicrobienne et antiambienne de Syzygium guineense DeWild. (Myrtaceae). Revue Zaı¨roise des Sciences Nucle´aires 3 (1 – 2), 211 – 222. Penge, O., Bashengezi, M., Longanga, O., Foemi, K., 1989. Evaluation des effets antibacterien et antiamibien de Triclisia giletti DeWild. (Menispermaceae). Bulletin de Me´decine Traditionnelle et Pharmacope´e 3 (1), 31 – 39. Penge, O., Bashengezi, M., Foemi, K., Longanga, O., 1990. Etude chimique et Bacteriologique de Tetracera poggei Gilg. (Dilleniaceae). Bulletin de Me´decine Traditionnelle et Pharmacope´e 4 (2), 147 – 153. Rojas, A., Hermandez, L., Pereda-Miranda, R., Mata, R., 1992. Screening for antimicrobial activity of crude drug extracts and pure natural products from Mexican medicinal plants. Journal of Ethnopharmcology 35, 275 – 283. Sofowora, A., 1982. Medicinal plants and traditional medicine in Africa, first edition. John Wiley, Chichester, pp. 142 – 146.
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423 Troupin, G., 1951. Spermatophytes. Flore du Congo Belge et du Ruanda-Urundi 2, 220. Troupin, G., 1952. Spermatophytes. Flore du Congo Belge et du Ruanda-Urundi 3, 77. Vanden Berghe, A.D., Vlietinck, J.A., 1982. Screening methods for antibacterial and anitviral agents from higher plants. Methods in Plant Biochemistry 6, 47–67. Vlietinck, A.J., Van Hoof, L., Totte´, J., Lasure, A., Vanden Berghe, D., Rwangabo, P.C., et al., 1995. Screening of hundred Rwandese plants for antimicrobial and antiviral properties. Journal of Ethnopharmcology 46, 31– 47.
.
423
Wagner, H., Bladt, S., 1996. Plant drug analysis. In: A Thin Layer Chromatography Atlas, 2nd edn. Springer-Verlag, Berlin, pp. 1 – 327. WHO, Scientific working Group, 1996. Resistance to antimicrobial agents. Bulletin of the World Health Organization 71, 335 – 336. Wright Colin, W., O’Neill Melanie, J., Phillipson David, J., Warhurst David, C., 1988. Use of microdilution to assess in vitro antiamoebic activities of Brucea ja6anica fruits, Simarouba amara stem, and a number of quassinoids. Antimicrobial Agents and Chemotherapy 32 (11), 1725 – 1729.
Contribution to the ethnobotanical, phytochemical and pharmacological studies of traditionally used medicinal plants in the treatment of dysentery and diarrhoea in Lomela area, Democratic Republic of Congo (DRC) A. Longanga Otshudi *, A. Vercruysse, A. Foriers Laboratory for Pharmacognosy, Phytochemistry and Toxicology, Department of Pharmaceutical Sciences, Vrije Uni6ersiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium Received 5 January 1998; received in revised form 20 December 1999; accepted 10 January 2000
Abstract In order to collect ethnobotanical information about antidiarrhoeal plants, we performed inquiries among traditional healers, community leaders, and native people of Lomela villages in Congo. Six medicinal plants widely used in this region were designated as having antidysenteric and antidiarrhoeal properties. These six medicinal plants were screened for groups of phytochemical compounds with antibacterial and antiamoebic activities. They were found to contain tannins, alkaloids, saponins, flavonoids, sterols and/or triterpenes and reducing sugars. Of the six tested plants, three showed prominent antibacterial activity whereas two acted against Entamoeba histolytica. The usefulness of the phytochemical bases and biological activities of these plants as potential source of antidiarrhoeal remedies is discussed. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Folk medicine; Ethnopharmacology; Antidiarrhoeal plants; Antidysenteric plants; Democratic Republic of Congo; Lomela area
1. Introduction Diarrhoea and dysentery epidemics are mostly common where living conditions are crowded and hygiene is poor. Dysentery is a microbial infection of gastrointestinal tract. Symptoms include fever, vomiting, abdominal pain and diarrhoea which often contains blood and pus. The onset of the * Corresponding author.
disease usually occurs within 2–3 days after infection and lasts for up to several weeks. Dehydration occurs rapidly, especially in children and can cause death if treatment is not given. Parasitic diseases such as dysentery and diarrhoea are the main causes of high mortality rate in developing countries. In these countries, over five million children under the age of five die annually from severe diarrhoeal diseases (WHO, 1996). The majority of rural people has limited access to formal and adequate health services,
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 1 6 7 - 7
412
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
thus people seek help from traditional healers who provide alternative health care services. Indigenous plant remedies are widely used in the treatment of a variety of disorders. However, only few of them have been controlled clinically, or studied chemically and biologically to identify their active constituents. In DRC, numerous plants are traditionally used as medicinal tools with antibacterial and antiparasitic properties in the treatment of diarrhoea and dysentery (Penge et al., 1988, 1989, 1990; Kambu et al., 1990). Since ethnobotanical field research is always the first step in any phytomedicinal research and development work (Mitscher et al., 1987; Rojas et al., 1992), inquiries were studied out in Lomela villages. This study is the first one performed in this region and to the best of our knowledge, most of the plants hereby investigated have not been previously reported to possess antidiarrhoeal activity. Therefore, the present study gives specific and useful information about; (i) which plants and/or plant parts are used in the treatment of diarrhoea and dysentery, (ii) how the plant material is provided and processed for remedies, and (iii) how these remedies are sued for treatments by traditional healers and traditional people of the Lomela region. In addition, a preliminary phytochemical screening has been carried out in order to have an idea of the different classes of organic compounds present in the different extracts of each plant. Finally, we have screened crude extracts derived form these plants for their antibacterial and antiamoebic activities, so as to generate some preliminary data on the biological activity of the plant extracts.
2. Materials and methods
2.1. Study area Inquiries have taken place in the area of Lomela, sub-district of Sankuru, district of Oriental Kasaı¨ (Congo). This region was chosen due to severe epidemics of dysentery that occurred in 1986 and 1994 which were treated essentially with
endemic medicinal plants in complete absence of modern medicine.
2.2. Methods used in the inter6iews with informants Inquiries were executed from 2 March 1996 to 16 April 1996 among the traditional healers and people of Lomela area, mainly in the villages where epidemics of diarrhoea and dysentery occurred. These places are: Lomela centre, in Batetela community; Mukumari, in Batetela community, approximately 70 km from Lomela centre, in the South of Lomela area; Kutusongo, in Bahamba I community, 50 km from Lomela centre, in the South of Lomela area; Wony’Angondo, in Bahamba I community, 40 km from Lomela centre, in the South Lomela area; Betembo, in Bakela community, 25 km from Lomela centre, in the West of Lomela area; Osangamanga, in Bahamba II community, 30 km from Lomela centre, in the North of Lomela area. Interviews based on pre-prepared series of questions were administered to the informants. Such questions included among others: Researcher’s name, Informant: name, tribe, village, community, zone, sub-region, age, sex and religion, Plant: vernacular name, uses in popular medicine, plant part, plant combinations, additional ingredients, preparation, application and dosage, rites, side effects, interdicts, no medicinal uses. These interviews were administered by one of the authors (A. Longanga Otshudi) in local languages, with the help of the political authority of each village. In total, 104 interviews were conducted among healers and native people of Lomela villages, of which 33 interviews were carried out in Lomela centre, 30 in Mukumari, ten in Kutusongo, 21 in Wony’Angondo, six in Osangamanga and four in Betembo. The plants were collected from all the different parts of the investigated Lomela villages, and their botanical identification was carried out with
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
the help of the INERA (Institut National d’Etudes et de Recherches Agronomiques) herbarium at Kinshasa University and the specialists of the ‘Herbarium of National Botanical Garden of Meise, Belgium’. Confirmation was obtained by comparing the collected species with the already identified herbarium specimens. A voucher specimen of each species was deposited in the Department of Pharmaceutical Sciences, Laboratory for Pharmacognosy and Phytochemistry, Vrije Universiteit Brussel.
2.3. Preparation of extracts Each air-dried plant material (10 g) was ground into a fine powder and exhaustively percolated after maceration for 24 h, with solvents having an increasing polarity (diethylether, 70% methanol and H2O). The resulting extracts were evaporated to dryness in vacuum at 40°C, yielding three crude extracts: F1, F2 and F3, respectively. These extracts were subjected to phytochemical analysis and biological activities.
2.4. Antibacterial and antiamoebic testings The antimicrobial testing was performed by the two-fold serial broth microdilution method (Mitscher et al., 1972; Vanden Berghe and Vlietinck, 1982) whereas the antiamoebic assay was carried out using the microdilution technique in Diamond TPS-1-monophasic medium (Wright Colin et al., 1988).
2.5. Test organisms Test microorganisms used in the preliminary antibacterial and antiamoebic testings included both enteropathogens and protozoan. As enteropathogens, we have used: Shigella dysenteriae B-1995/2-3 Shigella sonnei ATTC 25931 Shigella boydii USA 91/D1
:external Belgian quality assessment strain :reference strain :external USA quality assessment strain (C.A.P)
Shigella flexneri AZ 749 Salmonella typhimurium ATTC 14028 Escherichia coli ATTC 25922 Vibrio parahaemolyticus USA 94/DO7 Vibrio cholerae B1994/3-3 Campylobacter jejuni ATCC 24428 Campylobacter coli ATCC 33559
413
:clinical isolate :reference strain
:reference strain :external USA quality assessment strain (C.A.P) :external Belgian quality assessment strain :reference strain :reference strain
As protozoan, we have used Entamoeba histolytica ATCC 30459 (reference strain). All the enteropathogens were provided by the department of microbiology of the AZ-VUB (Academisch Ziekenhuis — Vrije Universiteit Brussel) while the reference strain of Entamoeba histolytica was provided by the Institute for Tropical Medicine ‘Prins Leopold’, Antwerpen (Belgium).
2.6. Preliminary phytochemical analysis Preliminary phytochemical screening of the plants was undertaken using described methods (Sofowora, 1982; Harborne, 1988; Wagner and Bladt, 1996). The screening covered mainly alkaloids, sterols and/or triterpenes, flavonoids, saponins, tannins, anthraquinones and reducing sugars.
2.6.1. Test for alkaloids Alkaloids were tested in all three extracts, ether, methanol, and water. In ether extracts 10 mg of ether residue was macerated with HCl (2% 1.5 ml). The resulting acid solution was divided into three parts, to a portion of which was added Mayer’s reagent, to another Dragendorff’s reagent while the third acted as blank. In alcoholic extracts, 10 mg of methanol residue was macerated with HCl (2%, 1.5 ml), filtered, basified with NH4OH (25%) and extracted with
414
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
CHCl3. The chloroform soluble portion was evaporated, dissolved in HCl (2%, 1.5 ml), divided into three parts and tested as in the ether extracts. In water extracts, 10 mg of aqueous residue was dissolved in distilled water (1.5 ml), basified with NH4OH (25%) and extracted with CHCl3. The chloroform solution was extracted with HCl (10%) and the acid aqueous solution was divided into three parts and tested as in the ether extract. A positive ( +) reaction was recorded if the addition of both reagents produced a faint turbidity; + + reaction was recorded if a light opalescent precipitate was observed; and + + + reaction was recorded if a heavy yellowish-white precipitate was observed with Mayer’s reagent and a heavy yellowish-orange precipitate with Dragendorff’s reagent. Alkaloids detection in plants showing preliminary positive test by the above screening was further confirmed using TLC (Wagner and Bladt, 1996). Powdered drug (2 g) was mixed thoroughly with 1 ml 10% ammonia solution and then extracted twice with 5 ml CHCl3. The chloroformic solution was evaporated to 1 ml and 10 ml of this solution was chromatographed on silica gel G Plates in toluene-ethyl acetate-diethylamine (70:20:10). The presence of alkaloids was detected on the plate, first by UV fluorescence (l=254 nm) and then by the application of Dragendorff and iodoplatinate reagents. The only plant (E. 6illosum) which gave preliminary positive tests showed at least seven alkaloid spots.
2.6.2. Test for fla6onoids Flavonoids were tested in all the three extracts. Ten milligrams of each plant residue was dissolved in 1.5 ml appropriate solvent and tested for flavonoids using Shinoda’s reaction. A positive ( +) reaction was recorded when a slight red coloration was observed; + + reaction was recorded when a medium red coloration was observed; and + + + reaction was recorded when a strong red coloration was observed.
2.6.3. Test for tannins Tannins were tested in the methanol and water extracts by gelatin-salt block test. After dissolving 10 mg of water and methanol residues in distilled water and methanol, respectively, water and methanol extracts were separately divided into three parts. A sodium chloride solution was added to one portion of each test extract, 1% gelatin solution to each second portion, and the gelatin– salt reagent to each third portion. Precipitation with the latter reagent, or with both the gelatinsalt reagents was indicative of the presence of tannins. Precipitation with salt solution (control) indicated a false-positive test. Positive tests were further confirmed by the addition of a few drops of dilute ferric chloride (1% FeCl3) to the test extracts which gave blue black or green black coloration. A positive (+) reaction was recorded when a slight precipitate was recorded; + + reaction was recorded when a medium precipitate was observed; and + + + reaction was recorded when a heavy precipitate was observed. 2.6.4. Test for saponins The presence of saponins was tested in the water extract. The honey comb froth which persisted for at least 30 min after vigorous shaking of the water plant extract and a positive Liebermann-Buchard’s reaction in the hydrolysed water extract indicated the presence of saponins. A + sign was recorded when froth reached a height of 0.5 cm; + + sign with a height up to 1 cm; + + + sign with a height more than 1 cm. 2.6.5. Test for anthraquinones Borntrager’s test was used for the detection of anthraquinones in all the three extracts. Free anthraquinones were tested in the ether extracts whereas combined anthraquinones were tested in the hydrolysed methanol and water extracts. A positive (+ ) reaction was recorded when a light pink, red or violet colour was observed; + + sign was indicated when a medium pink, red or violet colour was observed; + + + sign was indicated when a strong pink, red or violet colour was observed.
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
2.6.6. Test for sterols and/or triterpenes Sterols and/or triterpenes were tested in the ether extracts by Salkowski test and/or Libermann’s test. For Salkowski test, the presence of a reddish-brown colour indicated a positive reaction while a colour change from violet to blue to green was considered as positive reaction for Liebermann’s test. A + sign was recorded when a light colour was observed; + + sign for a medium colour and + + + for a strong colour. 2.6.7. Test for reducing sugars Reducing sugars were tested in the methanolic extract. To a heated methanolic plant extract was added drop by drop a mixture of equal parts of Fehling’s solution No. 1 and No. 2. In most cases reduction took place near the boiling point and was shown by a brick-red precipitate of cuprous oxide. Reducing sugars include all monosaccharides and many dissacharides (e.g. lactose, maltose, cellobiose and gentiobiose). A positive (+ ) reaction was recorded when a slight precipitate was recorded; + + reaction was recorded when a medium precipitate was observed; and + + + reaction was recorded when a heavy precipitate was observed.
3. Results and discussion The rural area of Lomela is located in the district of Oriental Kasai, Sub-district of Sankuru in the DRC. It is the widest area of Sankuru with a surface area of 26 346 km2 and population estimated to be ca. 45.874 in 1958. This area is located approximately at 22 – 25° longitude East, 1°30% –3° longitude South at an altitude of 500 m. The area lies entirely in the region of shady equatorial forests, where it rains all year round with less rainfall observed in March, June and August. There is around 1729 mm annual rainfall. The temperatures are tropical, with 30.9°C at the maximum and 19.5°C as a minimum. The area has a variety of different ethnic groups. It is inhabited by the Batetela (in majority), Bakela, Bahamba and Djonga. The main vernacular language spoken by the majority of the population is Otetela
415
followed by Okela and Djonga. The majority of the population is nominally christian, protestant or kimbanguist. The population lives mainly from the income of agriculture (rice, maize, coffee…), hunting and fishing. An ethnobotanical study of antidiarrhoeal plants used by traditional healers in six communities of Lomela area (DRC) has been carried out. During the course of the study, a total of 104 informants were interviewed for their knowledge on the uses of plants for the treatment of diarrhoea. The average age of the interviewed informants showed that they were predominantly older members of Lomela communities. The group of interviewed persons had a larger number of males, 83, as compared to 21 females. Educationally, more than half of the informants, 59, had no formal education. The rest have had either primary or secondary education. Six medicinal plants widely used in this area for the treatment of diarrhoea and dysentery were collected by one of the authors (A. Longanga Otshudi). These plants are listed in Table 1. The result of the preliminary phytochemical screening of these plants is reported in Table 2. The frequency of usage of collected plants and the proportion of detected organic compounds are summarized in Table 3. In Tables 4 and 5, we report the results of biological testings. The ethnobotanical, phytochemical, antibacterial and antiparasitological information of the collected plants in relation to gastrointestinal disorders is as follows.
3.1. Roureopsis obliquifoliolata. (Gilg) Schellenb (Connaracea) A climbing shrub with brown-pubescent branchlets present in rain forest. Native to South of Nigeria, also in French Cameroon, Spanish Guinea, Congo (DRC) and Angola (Troupin, 1952). In Lomela area (DRC), roots are grounded into fine bits and added to cold water. The mixture is filtered by using a fine clean cloth, then, a small quantity (half a cup) is taken orally twice a day for ten days for the treatment of diarrhoea and dysentery. This plant is also used for the treatment of toothache and elephantiasis (Troupin, 1952).
416
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
The phytochemical screening showed flavonoids, sterols and/or triterpenes, saponins, reducing sugars and tannins. The antibacterial screening showed activity against all the tested enteropathogens. The methanolic and aqueous extracts showed the highter potency, with a MIC value of 31.25 mg/ml for Vibrio cholerae. These extracts also acted weakly against E. histolytica with a MIC value of 500 mg/ml after 72 and 144 h incubation. Biological activity of this plant could be attributed to tannins. Most frequently tannin containing plants are used to treat diarrhoea and dysentery (Heinrich et al., 1992).
Preliminary phytochemical analysis showed alkaloids, sterols and/or triterpenes, saponins and reducing sugars. The isolation of alkaloids in E. 6illosum has been previously described (Parvez, 1994). In vitro antibacterial screening demonstrated activity in methanolic and water extracts against Campylobacter coli and Campylobacter jejuni with a MIC value of 62.5 mg/ml. They also acted against E. histolytica at 125 mg/ml. The antibiotic and antiparasitic activities of this plant are thought to be due mainly to alkaloids.
3.2. Epinetrum 6illosum. (Excell) Troupin (Menispermaceae)
An elegant tree, 12–20 feet. Native to Congo (DRC), Angola and Guinea (Leondard, 1962). In DRC, the maceration of root is drunk (1 cup) three times per day for 7–10 days for the treatment of diarrhoea and dysentery. The stem bark decoction is taken orally for abdominal pains and fever. In contrast, young fruits added to palm wine are taken orally as a laxative. Preliminary phytochemical screening showed the presence of flavonoids, sterols and/or triterpenes, saponins and reducing sugars.
A twining lianca, ca. 2 m high, found in secondary forests in the costal area of Congo (DRC) and Angola (Parvez, 1994). In Lomela area, root decoction of the plant is taken orally for the treatment of diarrhoea and dysentery. The crushed leaves are used as a wound dressing for fast healing. The plant is known to have abortive activity (Troupin, 1951).
3.3. Croton mubango. Mu¨ll (Eurphobiaceae)
Table 1 Antidiarrhoeal investigated plants, their preparation and traditional uses Family/species Menispermaceae Epinetrum 6illosum. (Excell) Troupin Connaraceae Roureopsis obliquifoliolata. (Gild) Schellenb Euphorbiaceae Croton mubango. Mu¨ll Vitaceae Cissus rubiginosa (Welw. Ex. Bak) Planch Asteraceae Vernonia amygdalina. Del
Simaroubaceae Quassia africana. Baill
Vernacular name Plant part
Preparation
Application
Uses
Dipapoma
Root bark Leaves
Decoction
Oral
Diarrhoea, dysentery, burns
Lungangula
Root bark
Decoction
Oral
Diarrhoea, dysentery, elephantiasis
Unganga
Stem bark Root bark
Maceration
Oral
Diarrhoea, dysentery, abdominal pains, fever
Sume
Stem bark Root bark
Decoction
Oral
Diarrhoea, dysentery
Mpasi nyioso
Leaves Root bark
Maceration
Oral
Diarrhoea, dysentery, gastroenteritis, malaria, hepatitis, worms
Dokotele
Root bark
Decoction
Oral
Diarrhoea, dysentery, fever, malaria
a
+/+ +/+ +/+ −
−/− +/+ −/− −
−/− −/− +/+ −
++/++ ++/++ ++/+++ ++
E, diethylether extract; M, methanol extract; W, water extract; RB, rootbark; L, leaves; SB: stembark.
−/− −/− −/− −
−/− −/− −/− −
RB/SB RB/SB RB/L RB −/− −/− −/− −
++/+ +++/+ +++/+ −/− −/− −/− +/+ − − − + + − +
E
RB/L RB
W
Sterols and/or triterpenes
Epinetrum 6illosum Roureopsis obliquifoliolata Croton mubango Cissus rubiginosa Vernonia amygdalina Quassia africana
M
E
W
E
M
Flavonoids
Alkaloids
Plant part
Name of plant
Preliminary phytochemical screening of some plants used in Congolese phytogherapya
Table 2
+/+ −/− −/− +
++/+ +
W
Saponins
M
−/− −/− −/− −
−/− −/− −/− −
−/− −/− − −
E
Anthraquinones
−/− −/− ++/++ −
−/− −
W
++/++ ++/+ ++/++ +
++/+ +++
M
Reducing sugars
−/− ++/++ ++/++ −
−/− +++
M
Tannins
−/− +/+ ++/++ −
−/− ++
W
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423 417
418
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
Table 3 Frequency of used plants in popular medicine and the proportion of detected organic compounds Family/species
Frequency of usage
Menispermaceae Epinetrum 6illosum. + (Excell) Connaraceae Roureopsis + obliquifoliolata (Gild) Schellenb Euphorbiaceae ++ Croton mubango. Mu¨ll Vitaceae Cissus rubiginosa. + (Welw. Ex. Bak) Planch Asteraceae +++ Vernonia amygdalina. Del Simaroubaceae Quassia africana. +++ Baill
Proportion of detected organic compounds
*4/7
5/7
Antibacterial screening showed activity in methanolic and aqueous extracts against several tested enteropathogens. The highest activity was observed towards Shigella dysenteriae and Shigella boydii with MIC values of 31.25 mg/m. In contrast the plant extracts showed weak activity against E. histolytica (MIC values of 500 mg/ml after 144 h incubation). Activity against tested enteropathogens could be due to the presence of tannin and flavonoids in the plant.
3.5. Vernonia amygdalina. Del (Asteracea) 4/7
4/7
5/7
3/7
* Of the seven screened organic compounds, four were detected in Epinetrum 6illosum. + Plant cited in one or two investigated villages. ++ Plant cited in three to five investigated villages. +++ Plant cited in all the six investigated villages.
In vitro antibacterial screening showed no activity against tested enteropathogens. Furthermore, the three tested extracts showed no activity against E. histolytica.
3.4. Cissus rubiginosa. (Welw.ex Bak) Planch (Vitaceae) A climbing or trailing herb, present in savannah and secondary forests. The plant is widespread in tropical Africa (Dewit, 1960). In Lomela area, root or stem decoction is drunk for the treatment of diarrhoea and dysentery (1 cup, 2 ×1 day for 7 days). Leaf infusion is used for the same purpose. Preliminary phytochemical analysis showed the presence of flavonoids, sterols and/or triterpenes, reducing sugars and tannins.
A shrub or small tree 6–10 feet. high. The plant is common and often planted, from Guinea to Cameroon. Extending to Congo (DRC), Uganda and Tanganyika (Oliver, 1877). In Lomela area, the leaf maceration is orally used for the treatment of diarrhoea and dysentery whereas the root decoction is drunk for gastroenteritis, malaria, hepatitis and worms. The use of the root decoction for the treatment of gastrointestinal disorders also has been reported from Rwanda (Vlietinck et al., 1995). Preliminary phytochemical screening showed the presence of flavonoids, sterols and/or triterpenes, saponins, reducing sugars and tannins. The isolation of saponins in V. amygdalina has been previously described (Jisaka et al., 1992). Antibacterial testing of the plant extracts showed no activity against tested enteropathogens. Furthermore, a weak activity was observed against E. histolytica. This result validates previous reports on the antibacterial activity of extracts derived from V. amygdalina towards Escherichia coli (Vlietinck et al., 1995).
3.6. Quassia africana. Baill (Simaroubaceae) A glabrous shrub, 8–12 feet high; present in rain forest. The plant is native to Cameroon, Congo (DRC) and Angola (Gilbert, 1958). In Lomela area, the root bark decoction is drunk for the treatment of diarrhoea and dysentery. Some traditional healers use Quassi africana in combination with Vernonia amygdalina for the
R R R R R R S S S S S S R R R L L L
Epinetrum 6illosum F1 F2 F3 F1 F2 F3 F1 F2 F3 F1 F2 F3 F1 F2 F3 F1 F2 F3
Extracts
\1000 \1000 1000 \1000 250 250 \1000 \1000 \1000 \1000 1000 250 \1000 \1000 \1000 \1000 \1000 \1000
S.s.
Bacteria
\1000 \1000 1000 \1000 31.25 62.5 \1000 \1000 \1000 \1000 31.25 31.25 \1000 \1000 \1000 \1000 \1000 \1000
S.d. \1000 \1000 \1000 \1000 62.5 62.5 \1000 \1000 \1000 \1000 \1000 31.25 \1000 \1000 \1000 \1000 \1000 \1000
S.f. \1000 \1000 \1000 \1000 125 62.5 \1000 \1000 \1000 \1000 \1000 31.25 \1000 \1000 \1000 \1000 \1000 \1000
S.b. \1000 62.5 62.5 \1000 500 1258 \1000 1000 \1000 \1000 500 250 \1000 1000 \1000 \1000 1000 \1000
C.c. \1000 62.5 62.5 500 31.25 500 \1000 1000 1000 \1000 1000 250 \1000 1000 1000 \1000 1000 \1000
C.j.
\1000 \1000 \1000 \1000 500 31.25 \1000 \1000 \1000 \1000 \1000 125 \1000 \1000 \1000 \1000 \1000 \1000
V.c.
\1000 \1000 \1000 \1000 \1000 500 \1000 1000 \1000 \1000 \1000 250 \1000 \1000 \1000 \1000 \1000 \1000
V.p.
\1000 \1000 \1000 \1000 \1000 250 \1000 \1000 \1000 \1000 500 125 \1000 \1000 \1000 \1000 \1000 \1000
S.t.
500 \1000 \1000 \1000 \1000 \1000 \1000 \1000 \1000 \1000 \1000 \1000 \1000
\1000 \1000 \1000 \1000
E.c.
a R, Root bark; S, stem bark; L, leaves; F1, deithyl ether extract; F2, mehanol extract; F3, aqueous extract; S.s, Shigella sonnei; S.d, S. dysenteriae; S.f, S. flexneri; S.b, S. boydii; C.c, C. coli; C.j, C. jejuni; V.c, Vibrio cholerae; V.p, V. parahaemolyticus; S.t, S. typhimurium; E.c, E. coli; MIC, minium inhibitory concentration (mg/ml).
Vernonia amygdalina
Quassia africana
Cissus rubiginosa
Croton mubango
Roureopsis obliquifoliolata
Parts
Plants
Table 4 MIC values (mg/ml) of tested plant extractsa
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423 419
420
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
All the plant materials were collected during the rainy season and early in the morning, since most of interviewed traditional people indicated that some plants have their full therapeutic effect only if collected at certain time or during a certain season. Most of the investigated plants were designated several times by different informants and multiple citations were particularly found within the same study area, exception made for Vernonia amygdalina and Quassia africana which were cited in all the investigated villages. Most of the collected plants were used separately except Quassia africana which is used in combination with Vernonia amygdalina for treating malaria and fever. Root is the plant part most frequently used by traditional people of Lomela for preparing traditional medicine as observed in this study. This agrees with a study carried out in Tanzania, where Chhabra et al. (1993) also found that roots were the most prominent plant part used to prepare remedies. The concentration and the nature of the active compounds can vary significantly
treatment of fever and malaria. The use of this plant for the treatment of diarrhoea and dysentery in Kinshasa (DRC) has been previously reported (Kambu et al., 1990). Preliminary phytochemical screening showed the presence of sterols and/or triterpenes, saponins and reducing sugars. The isolation of quassinoids in this plant has been previously described (Cabral et al., 1993). Antibacterial screening showed no activity against tested enteropathogens whereas the plant extracts demonstrated good activity against E. histolytica with MIC value of 125 mg/ml after 72 h incubation. Certainly the quassinoids in the extracts are effective against E. histolytica, since these organic compounds are known to have antiamoebic properties (Wright Colin et al., 1988). In contrast, we did not find out in this study the previous antibacterial activity reported on extracts derived from Quassia africana against Escherichia coli and Shigella flexneri (Kambu et al., 1990). Table 5 Antiamoebic activities (mg/ml) of tested plant extractsa Plants
Epinetrum 6illosum
Roureopsis obliquifoliolata
Cissus rubiginosa
Croton mubango
Quassia africana
Vernonia amygdalina
Parts
R R R R R R S S S S S S R R R L L L
Extracts
F1 F2 F3 F1 F2 F3 F1 F2 F3 F1 F2 F3 F1 F2 F3 F1 F2 F3
Concentrations 500 mg/ml
250 mg/ml
125 mg/ml
Control of growth
72 h
144 h
72 h
144 h
72 h
144 h
72 h
144 h
+++ − − ++ − − + + + ++ ++ +++ + − − ++ − −
++ − − ++ − − − − − ++ ++ ++ + − − + − +
+++ − − ++ + + + ++ ++ +++ +++ +++ + − − ++ + +
++ − − ++ + + + ++ ++ ++ ++ ++ + − − + + +
+++ − − ++ ++ ++ + ++ ++ +++ +++ +++ ++ − − ++ ++ ++
++ − − ++ ++ ++
+++ +++ +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
++ +++ +++ +++ +++ +++ ++ +++ +++ +++ ++ ++ ++ ++ ++ +++ ++ ++
++ ++ +++ ++ ++ + + + ++ ++ ++
a R, Root bark; S, stem bark; L, leaves; F1, diethylether extract; F2, methanol extract; F3, aqueous extract. −, All amoebae killed; +, 100–80% amoebae killed; ++, 80–20% amoebae killed; +++, 20–0% amoebae killed.
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
between different parts of the same plant. We observed that the stem bark of Croton mubango is used against diarrhoea while the seeds (fruits) of the same plant cause diarrhoea and are sometimes used for treating constipation. Another observation made is the way by which remedies are administered for treating diarrhoea. In most cases, the oral route is preferred. The usual modes of preparing remedies are decoction, infusion and maceration, with water and/or palm wine as solvents. The bacteriological quality of these remedies is solely dependent on their mode of preparation (decoction, infusion) or the antimicrobial properties of palm wine, sometimes used as solvent. No rites or incantations are required to give the desired effect from a remedy. The dosage of a remedy depends mainly upon the nature of the drug (plant part) to be administered, the sex of the patient and the age. In general, pregnant women and children get small doses. The duration of the treatment varies, depending on the type of remedy and the severity of diarrhoea. In general, at least 10 – 14 days are assumed to be necessary for treatment. Normally, if after 2 weeks of treatment dysentery persists, patients die of anemia and dehydration. No interdicts are required during the treatment, but since one knows that dysentery is a very contagious disease, traditional healers recommended no contact with the patient. That is why, sometimes, even food was given to the patients by means of sticks. When asked about secondary effects, only a few of the respondents reported vomiting, abdominal pains, loss of appetite and abortion for Epinetrum 6illosum, but we can not overlook the fact that some of these effects (vomiting and abdominal pains) also occur during dysentery episode. The preliminary phytochemical screening carried out on the six collected plant materials covered mainly alkaloids, saponins, sterols and/or triterpenes, tannins, flavonoids, anthraquinones and reducing sugars. Out of the six screened plants, only one (1/6), Epinetrum 6illosum, gave a positive test for alkaloids which are reputed to have important physiological activities. Three plant materials (3/6) gave a positive test for tan-
421
gent action; this provokes their usefulness in preventing diarrhoea and controlling haemorrhage. In addition, tannins have been reported to possess cytotoxic and antineoplastic activities. Four plant materials (4/6) gave a positive test for flavonoids which are known to possess antiviral, antiinflammatory and cytotoxic activities. Five plant materials (5/6) gave a positive test for saponins which are well known to have expectorant and antitussive activities. The economical importance of saponins lies in their easy conversion to medicinally used steroidal hormones. All the six plant materials (6/6) gave a positive reaction for reducing sugars which are known to be useful in providing a means of storing energy and transport of energy. In addition, many classes of plant constituent (e.g. the nucleic acids and the plant glycosides) contain sugars as essential features of their structures. Sugars also play a number of ecological roles in protection form wounding and infection and in the detoxification of foreign substances. All the six plant materials (6/6) also gave positive reaction for sterols and/or triterpenes. The therapeutic and industrial interest of triterpenes and steroids make them a group of secondary metabolites of outstanding importance. No anthraquinones were detected in all the investigated plants. We were also interested in the frequency of usage of a given plant. Since the observations on the frequency could not be quantified, popularity of the plant was used as an indicator. Thus, considering the frequency of usage of a given plant and the proportion of the organic compounds it contains, we observed that the most frequently used medicinal plant does not, in most instances, contain the highest proportion of organic compounds (Table 3). The results of the antibacterial screening indicated that methanol and aqueous extracts derived from Roureopsis obliquifoliolata, Cissus rubiginosa and Epinetrum 6illosum produced in vitro antibacterial activity against one or more tested enteropathogens (Table 4). This could partly be explained by the widespread occurrence of polyphenolic compounds, which are soluble in water and methanol. Of great importance is Epinetrum 6illosum which also showed high activ-
422
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423
ity against Entamoeba histolytica (Table 5). Although Quassia africana showed no activity against tested enteropathogens, the antiamoebic activity of its extracts was comparable to those derived from Epinetrum 6illosum (Table 5). Croton mubango and Vernonia amygdalina did not show any prominent antibacterial and antiamoebic activities in this study. We must point out that sometimes, traditional healers prefer a combination of several plants to treat disease. This combination could have a pharmacological activity whereas separate constituents might not. Evidence is hereby presented that some of the plants used for the treatment of diarrhoea and dysentery by the population of Lomela area (DRC) really have some biological activities (antibiotic and antiparasitic activities) which might explain partly the properties claimed in the treatment of gastrointestinal diseases. Some of these interesting plants are now being subjected to indepth study for chemical constituents. Such substances in the purified state may be useful for the treatment of diarrhoea and dysentery.
Acknowledgements The authors are grateful to Professor Penge On’Okoko (Kinshasa University) for his collaboration. Our sincere thanks go to all the technicians of INERA herbarium (Kinshasa University) and the ‘Herbarium of National Botanical Garden of Meise, Belgium’ for the botanical identification of the plant species. We also thank the informants who provided information about antidiarrhoeal plants used in Lomela area for the treatment of diarrhoea and dysentery.
References Chhabra, S.C., Mahunnah, R.L.A., Mshiu, E.N., 1993. Plants used in traditional medicine in Eastern Tanzania. VI. Angiosperms (Sapotacea-Zingiberaceae). Journal of Ethnopharmacology 39, 83–103. Cabral, J.A., McChesney, J.D., Milhous, W.K., 1993. A new antimalarial quassinoid from Simaba guianesis. Journal of Natural Products 56 (11), 1954–1961.
Dewit, 1960. Spermatophytes. Flore du Congo Belge et du Ruanda-Urundi, 9, 538. Gilbert, G., 1958. Spermatophytes. Flore du Congo Belge et du Ruanda-Urundi 7, 125. Harborne, J.B., 1988. Phytochemical methods. A Guide to Modern Techniques of Plants Analysis, first ed. Chapman and Hall, London, pp. 1 – 226. Heinrich, H., Rimpler, H., Barrera, N., Antonio, 1992. Indigenous phytotherapy of gastrointestinal disorders in a low land mixed community (Oaxaca, Mexico): ethnopharmacologic evaluation. Journal of Ethnopharmacology 36, 63 – 80. Jisaka, M., Kawanaka, M., Sugiyama, H., Takegawa, K., Huffman, M.A., Ohigashi, H., et al., 1992. Antischistosomal activities of sesquiterpene lactones and steroid glucosides from Vernonia amygdalina, possibly used by wild chimpanzees against parasite-related diseases. Biosciences – Biotechnology– Biochemistry 56 (5), 845 – 846. Kambu, K., Tona, L., Luki, N., Cimanga, K., et Uvoya, A., 1990. Activite´ antibacte´rienne d’extraits a` partir de plantes utilise´es en pre´paration comme antidiarrhe´iques a` Kinshasa, Zaı¨re. Annales Pharmaceutiques Franc¸aise 48 (5), 255 – 263. Leondard, J., 1962. Spermatophytes. Flore du Congo Belge et du Ruanda-Urundi. 8 (1), 57. Mitscher, L.A., Drake, S., Gollapudi, S.R., Okwute, S.K., 1987. A modern look at folkloric use of anti-infective agents. Journal of Natural Products 50, 1025 – 1040. Mitscher, L.A., Leu, R.P., Bathela, M.S., Wu, W.-K., 1972. Antimicrobial agents from higher plants. I. Introduction, rationale and methodology. Lloydia 35, 157 – 176. Oliver, D., 1877. Compositae. Flora of Tropical Africa 3, 284. Parvez, M., 1994. Bis benzylisoquinoline alkaloids from Epinetrum 6illosum. Journal of Chemical Society of Pakistan 16 (4), 257 – 260. Penge, O., Bashengezi, M., Longanga, O., Foemi, K., 1988. Evaluation des activite´s antimicrobienne et antiambienne de Syzygium guineense DeWild. (Myrtaceae). Revue Zaı¨roise des Sciences Nucle´aires 3 (1 – 2), 211 – 222. Penge, O., Bashengezi, M., Longanga, O., Foemi, K., 1989. Evaluation des effets antibacterien et antiamibien de Triclisia giletti DeWild. (Menispermaceae). Bulletin de Me´decine Traditionnelle et Pharmacope´e 3 (1), 31 – 39. Penge, O., Bashengezi, M., Foemi, K., Longanga, O., 1990. Etude chimique et Bacteriologique de Tetracera poggei Gilg. (Dilleniaceae). Bulletin de Me´decine Traditionnelle et Pharmacope´e 4 (2), 147 – 153. Rojas, A., Hermandez, L., Pereda-Miranda, R., Mata, R., 1992. Screening for antimicrobial activity of crude drug extracts and pure natural products from Mexican medicinal plants. Journal of Ethnopharmcology 35, 275 – 283. Sofowora, A., 1982. Medicinal plants and traditional medicine in Africa, first edition. John Wiley, Chichester, pp. 142 – 146.
A. Longanga Otshudi et al. / Journal of Ethnopharmacology 71 (2000) 411–423 Troupin, G., 1951. Spermatophytes. Flore du Congo Belge et du Ruanda-Urundi 2, 220. Troupin, G., 1952. Spermatophytes. Flore du Congo Belge et du Ruanda-Urundi 3, 77. Vanden Berghe, A.D., Vlietinck, J.A., 1982. Screening methods for antibacterial and anitviral agents from higher plants. Methods in Plant Biochemistry 6, 47–67. Vlietinck, A.J., Van Hoof, L., Totte´, J., Lasure, A., Vanden Berghe, D., Rwangabo, P.C., et al., 1995. Screening of hundred Rwandese plants for antimicrobial and antiviral properties. Journal of Ethnopharmcology 46, 31– 47.
.
423
Wagner, H., Bladt, S., 1996. Plant drug analysis. In: A Thin Layer Chromatography Atlas, 2nd edn. Springer-Verlag, Berlin, pp. 1 – 327. WHO, Scientific working Group, 1996. Resistance to antimicrobial agents. Bulletin of the World Health Organization 71, 335 – 336. Wright Colin, W., O’Neill Melanie, J., Phillipson David, J., Warhurst David, C., 1988. Use of microdilution to assess in vitro antiamoebic activities of Brucea ja6anica fruits, Simarouba amara stem, and a number of quassinoids. Antimicrobial Agents and Chemotherapy 32 (11), 1725 – 1729.