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Toxicity studies of medicinal plants used in sub-Saharan Africa
Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Toxicity studies of medicinal plants used in sub-Saharan Africa Marlaine Boukandou Mounanga a, Ludovic Mewono b, Sophie Aboughe Angone a,n a
Pharmacopeia and Traditional Medicine Institute: National Center of Technological and Scientific Research, Libreville, Gabon Groupe de Recherche en Immunologie, Microbiologie appliquée, Hygiène et Physiologie (GRIMAH.P), Département des Sciences de la Vie et de la Terre- Ecole Normale Supérieure, BP 17009 Libreville, Gabon
b
ar t ic l e i nf o
a b s t r a c t
Article history: Received 15 March 2015 Received in revised form 13 May 2015 Accepted 7 June 2015
In sub-Saharan Africa, traditional medicine is widely used in rural and urban areas also. This is essentially due to the prohibitive cost of pharmaceutical-based medicine and the low incomes of a major part of the population. In addition, the efficacies of many of these traditional and plant-based medicines are proven, but the fact remains that certain plants used in traditional medicine have toxic effects. It is in this perspective that we investigated by bibliographic literature on the toxicity of plants used in traditional medicine. It is crucial to gain knowledge on these plant-based medicines prepared and prescribed by practitioners, particularly in terms of toxicity, composition, specific efficacy of disease and to advise practitioners of this alternative medicine on the protection and security of patients. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Plants Traditional medicine Toxicity Sub-Saharan Africa
1. Introduction The use of plants is as old as human kind itself. It has always been part of human culture. In Africa, for example, plants are used in traditional medicine to treat different infectious and noninfectious diseases (Diallo et al., 2003; Gurib-Fakim, 2006). These treatments include body-washes, massages, ingestions, etc. (Walker and Sillans, 1961). Plants provide a wide variety of biochemicals useful to mankind. Their uses include food, colors, fragrances and agricultural chemicals and pharmaceuticals. According to the World Health Organization (WHO), about 40–90% of people living in developing countries frequently use traditional medicine (van Andel and Carvalheiro, 2013). This situation is the consequence of a limited access to modern health services and also researchers explained the high prevalence of traditional medicine use by massive rural-to-urban migration, the influence of cultural and social surroundings, and the belief that natural products pose no risk (van Andel and Carvalheiro, 2013; Lins Brandão et al., 2006; Bussmann et al., 2007; Oreagba et al., 2011). Though the use of medicinal plants can have deleterious effects on health as reported elsewhere (Fall et al., 2011). Indeed, of about 1,500,000 plants investigated, most of them contain toxic substances (Ishii et al., 1984) like some secondary metabolites. Plants produce a variety of secondary metabolites that are subdivided by chemists into several classes based on their molecular
n Correspondence to: Pharmacopeia and Traditional Medicine Institute, National Center of Technological and Scientific Research, Po. Box: 5011, Libreville, Gabon. E-mail addresses: [email protected] (M. Boukandou Mounanga), [email protected] (L. Mewono), [email protected] (S. Aboughe Angone).
structure. Among secondary metabolites are saponins, terpenoids, cyanogenic, tannins, toxic amino acids, glycosides, alkaloids (Dai and Mumper, 2010). It has been shown that the toxicity of a given plant depends on various factors, including the strength of secondary metabolites, the quantity consumed, the time of exposure, different parts of the plant (root, oil, leaves, stem bark and seeds), individual body chemistry, climate and soil, and genetic differences within the species (Tülay, 2012). This said it clearly appears that medicinal plants should be used with precautions and toxicology studies conducted to increase the knowledge on the plant or plants preparation given to populations. For this purpose, in-vitro and in-vivo models are available to study the toxicity of medicinal plants. Regardless of the type of extract, the parts of plant used, the concentration of the extract, the mode of administration, and the organism under consideration the lethal dose 50 (LD50) who represent the dose who kill 50% of a tested population is used to appreciate the toxicity of the plant (Amy et al., 2002). In addition, for chronic or sub-chronic toxicity histological or genetic modifications are the most relevant indicators. In this paper, we aimed to review, on the basis of 9 studies, medicinal plant extract induced cellular or organ dysfunction (Table 1) and to show the importance of toxicity studies. 1.1. Hemolytic effect Chrysophyllum pruniforme (synonym: Donella pruniformis) (Pierre ex Engl.) is a tall tree who belongs to the family of Sapotaceae. This plant is widely spread out from West to East Africa and Central Africa. In Gabon and Congo, C. pruniforme's bark infusion is used to treat coughs. Phytochemical studies showed that it contains reducing sugars, phenolic compounds, flavonoids,
http://dx.doi.org/10.1016/j.jep.2015.06.005 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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Table 1 Recapitulative table of plants and their toxicity. Plant scientific name
Family name
Botanical description
Chrysophyllum pruniforme (Pierre ex Engl.) Semecarpus anacardium Linn.
Sapotaceae
Anacardiceae
Quassia amara L. Radlk.
Simaroubaceae
Artemisia Asteraceae maciverae Linn. Nerium oleander Apocynaceae L.
Parts used
Adverse effect
Model of experimentation
References
Tall tropical tree growing to 10–20 m or more in height. The leaves are Barks oval, 3–15 cm long, green above, densely golden pubescent below.
Hemolytic
Human blood samples
Aboughe et al., 2013
Medium-to-large size tree, 15–25 m in height with gray bark exuding Leaves an irritant secretion on incising and exfoliating in small irregular flakes. Leaves are simple alternate, obviate – oblong, 30–60 cm long and 12–30 cm broad, rounded at the apex coriaceous glabrous above and more or less pubescent, beneath. Small tropical tree, growing only 2–6 m in height. It has beautiful red Stem flowers and fruits that turn red as they mature. bark
Neurotoxic
Intraperitoneal administration on mice
Chaudhary et al., 2010
Spermatotoxic
Oral administration of rats
Small herbaceous.
Whole plant
Nephrotoxic
Leaves
Toxic for lung and heart
Intraperitoneal administration on Swiss albinos rats Oral administration on mice
Obembe and Raji, 2012 Atawodi et al., 2013
Leaves
Hepatotoxic
Leaves
Toxic for spleen Intraperitoneal administration on mice
Nusaibah, 2012.
Whole plant
Toxic for gastrointestinal tract
Oral administration on Wistar rats
Sokar et al., 2003
Leaves
Genotoxic
On Alium cepa
Tulay and Ozlem, 2010
An evergreen shrub reaching 4 m in height. Leaves are 10–22 cm long, narrow, untoothed and short-stalked, dark or gray-green, white or yellow. All leaves have a prominent mid rib, are leathery in texture and usually arise in groups of three from the stem. Aphania Sapindaceae A forest tree to 55 ft high, with short bole and dense drooping foliage. senegalensis The flowers are greenish-white and fragrant. The ripe fruits are red (Juss. Ex Poir.) and edible. Cupressus Cupressaceae A medium-sized coniferous evergreen tree to 35 m tall, with a conic sempervirens crown. The foliage grows in dense sprays, dark green in color. The L. leaves are scale-like, 2–5 mm long, and produced on rounded (not flattened) shoots. Herniaria Caryophyllaceae A yellowish green, prostrate, hairy, spreading herb. Stem and branches cinerea DC. with 3–9 mm long internodes, with short spreading hairs. Leaves are opposite, sessile, hirsute, blade narrow elliptic to lanceolate or oblanceolate, usually 4–7 mm long and 1.5–2.3 mm wide, acute, margins entire with straight spreading hairs. Inula viscosa L. Asteraceae Small shrub. The stem is dark brown. The leaves have an elongated Aiton. lanceolate shape with a pointed tip. The larger leaves measure about 70 mm 18 mm.
Oral administration on Wistar rats
Ali Hussien, 2010 Fall et al., 2011
is an indicator of general cytotoxicity towards normal, healthy cells (Da Silva et al., 2004).
Fig. 1. Hemolytic activity of aqueous and phenolic extracts on human erythrocytes (Aboughe et al., 2013).
saponins, and catechic tannins (Aboughe et al., 2013). In this study the author shows that in vivo studies, the LD50 was 90 mg/kg in mice by oral administration of the aqueous extract, what according to the scale of toxicity of Hodge ant Sterner classifies it among the moderately toxic ones. In addition, aqueous and total phenolic extract of C. pruniforme show a hemolytic effect on human erythrocytes obtained from healthy donors. At the end of the experimentation, the aqueous extract showed hemolytic activities about 0%, 3%, 7%, 20%, 33%, and 47% in 1.25; 2.5; 3.75; 5; 7.5; and 10 mg/mL, respectively. At the same time total phenolic extract showed hemolytic activities about 7%, 79%, 99.6%, 99.9%, and 100% for the same plant concentrations. The hemolytic activity of total phenolic extracts was significantly higher compared to the one of aqueous extracts (Fig. 1) (Aboughe et al., 2013). Hemolytic activity
1.1.1. Hepatotoxic effect Aphania senegalensis (Juss. Ex Poir.) (Sapindaceae) is a medicinal plant widely used in Senegal in folk medicine. But, this tree can be found in several other African countries and in Asia. A phytochemical screening of leaves revealed that, they contain flavonoids, tannins and saponins (Faye, 2008). Its pharmacological activity was described mainly for the leaves and relied on antiparasitical, analgesic and anti-inflammatory activities (Fall et al., 2009). Tests of acute and sub-acute toxicity were carried out in male and female Wistar rats. The LD50 could not be determinate because of no deaths or observable clinical signs occurred after acute oral administration of a dose of 5.000 mg/kg. For sub-acute toxicity was carried out for a 28-day period with the administration of increasing doses of 500, 1000 and 2000 mg/kg of aqueous extract of plants. Biochemical analyses revealed a significant increase in aminotransferase activity. Levels of aminotransferase in treated animals were found to be twice higher than normal values. Further histological examination of liver of treated animals confirmed the existence of various liver lesions like degenerated hepatocytes with pyknotic nuclei, congestion, the central veins and the surrounding sinusoids in some lobules were filled with blood and fatty body (Fig. 2) (Fall et al., 2011), (Figs. 3 and 4). 1.1.2. Neurotoxic effect Semecarpus anacardium Linn. Belonging to the family of Anacardiaceae is a moderate-sized deciduous tree found in found in abundance in Assam, Bihar, Bengal and Orissa, Chittagong, central
Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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Fig. 2. Deleterious effects of Aphania senegalensis on liver cells. (a) Degenerated hepatocyte with pycnotic nuclei. (b) Lipidic droplet. (c) Inflammation (Fall et al., 2011).
Fig. 3. Transmission electron micrograph of brain cell (a) control; (b) low dose; (c) high dose.
Fig. 4. Histopathological appearances of the kidney of rats showing tubular epithelial necrosis N (b) compared to normal kidney (a).
India and Western peninsula of Eastern Archipelago and Northern Australia (Kirtikar and Basu, 1975). Different parts of this plant are used to treat many diseases like arthritis, tumors, and infections (Semalty et al., 2010). The plant is useful in skin diseases, piles, dysentery, fever, loss of appetite, urinary discharges, heals ulcers, and asthma (Basavaraj et al., 2011). The oil is a tonic, good for leucoderma, coryza, lessens, inflammation, and it is useful in superficial pain (Kirtikar and Basu, 1998). Raman and Pednekar (2012) found that the plant possesses biomolecules like alkaloids, saponins, tannins, flavonoids, steroids, glycosides, hexose sugars,
diterpenes, mucilages and gum extracts whereas, carbohydrates, phenols, proteins, monosaccharides, starch were found in negligible amount. Because of it belonging to Anacardiaceae family, it has potential to drive allergic manifestations through contact dermatitis. Choudhari and Deshmukh (2007) reported that the S. anacardium extract induced severe anemia in rat. The crude chloroform extract was found to be very toxic according to Patwardhan et al. (1988). In addition, traditional use of latex of leaf and stem has been found to be toxic to the skin (Choudhury et al., 2011b). The aqueous extract of plant was administered
Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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through ip. injection in two different doses to mice during 5 days. Several harmful effects were then observed following injections of two doses: mainly akinesia, catalepsy, decreasing of body weight and hyperpnoea for the high doses were observed. Biochemical parameters like serum glutamate oxaloacetate transminase (GOT) and serum (glutamate pyruate transminase), cerebral cortex lipoperoxidation (LPO) and midbrain LPO showed an elevation for both the treatment. The authors also found that the cell have wellformed nuclei, whereas in low dose there are shrunken nuclei and fragmented chromatin and high dose showing more ruptured and increased fragmented chromatin (Choudhury et al., 2011a).
1.1.3. Nephrotoxic effect Artemisia maciverae Linn. is a small herbaceous plant belonging to the Asteraceae family. The phytochemicals present in the chloroform extract of A. maciverae are flavonoids, triterpenes, terpenoids, glycosides, phlobatannins and tannins. Various pharmacological studies have been carried out on this plant product which showed that, the chloroform extract possesses anti-malarial activity (Ene et al., 2008). The chloroform extract of A. maciverae at 0; 50; 100 and 200 mg/kg were given to adult male Swiss albino rats through the ip route for 90 days. Administration of chloroform extract of A. maciverae produced signs of toxicity like loss of appetite, loss of agility, dizziness and convulsion. These signs of toxicity were found to increase in severity with increasing dose of extract. Mortality was recorded in 50 and 100 mg/kg treatment group in week one and four of treatment. All the animals in 200 mg/kg treatment group died within the first week of treatment with convulsions as a major observable sign of toxicity. There were statistically significant (p o0.05) elevation in the levels of creatinine and urea in the treated groups. Gross and histopathological observations indicated congestion, tubular epithelial necrosis in the kidneys of animals treated. The toxic effect of this plant extract was reversed within 4 weeks after treatment withdrawal, suggesting that the nephrotoxic effect of chloroform extract of A. maciverae may not be permanent. A hepatotoxicity study carried out with the chloroform extract of this plant, in Swiss albino rats indicate that long term exposure to therapeutic doses of chloroform extract of this plant is relatively safe, but high dose
exposure may result in hepatocellular injury (Ene et al., 2011). In another study by Ene and al. (2012), this chloroform extract caused elevation in the hemoglobin and lymphocyte counts of the rats treated with 200 mg/kg of extract at the onset of treatment. This suggests that there was any possible breach of the integrity of blood synthesis and regulatory systems with high doses of extract compared with the lower doses (Atawodi et al., 2013).
1.1.4. Genotoxic effect Inula viscosa (L.) Aiton (Compositae) is a perennial weed that is found mostly in the mediterranean basin (Al-Dissi et al., 2001). I. viscosa contains some pharmacologically active compounds including sesquiterpenes, sesquiterpenes acids (Ulubelen et al., 1987, Marongiu et al., 2003), lactones, flavonoids, and essential oils (Lauro and Rolih, 1990, Wollenweber et al., 1991). It has been used for its antiinflammatory (Barbetti et al., 1985), antipyretic, antiseptic, antiphlogistic activities (Lauro and Rolih, 1990, Lev and Amar, 2000) and diabetes (Yaniv et al., 1987). It has been demonstrated that aqueous extracts of this plant exhibit antifungal activity in vitro (Cohen et al., 2002, Maoz et al., 1999, Qasem et al., 1995) and some of its organic solvent extracts are antibacterial (Debat, 1991). In Spain, this herb has been used for treating gastroduodenal disorders (Lastra et al., 1993). I. viscosa has antiulcerogenic effects (Alkofah and Atta, 1999) causes abortion (Farnsworth et al., 1975; Karim et al., 1990), prevents zygote implantation in mammals (Al-Dissi et al., 2001), prevents growth of pathogenic fungi (Maoz and Neeman, 2000), has a strong antioxidant activity (Schinella et al., 2002), and has also nematicidal and antihelminth properties (Oka et al., 2001). Several researchers validated the tests which use plant test systems in vivo, tests on the animals jointly performed in vitro often confirm the results obtained (Vicentini, et al. 2001; Teixeira et al. 2003), and can be extrapolated to other organisms such as humans. For this study small bulbs (1.5–2.0 cm in diameter) of Alium cepa was used to initiate the test, the outer scales of the bulbs and the dry bottom plate were removed without destroying the root. For each extract sample, six bulbs were placed in tap water (pH 7.3) for 48 h and then onion roots were treated with the leaves aqueous extracts of I. viscosa at 2.5; 5; and 10 mg/ml
Fig. 5. Mitotic and chromosomal aberrations after the Inula viscose leaf extract treatments in Alium cepa root tip meristem cells visualized with microscopy. (a) Fragments; (b) stickiness; (c) polar deviation and chromatid bridge; (d) membrane damage; (e) binucleated cell; (f) apoptotic bodies; (g and h) cells with damaged nucleus (Tulay and Ozlem, 2010).
Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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Fig. 6. (a) There is odema ( ), congestion (-) in the lung (H&E 10). (b) The lung was suffering from congestion, hyperplasia of epithelial lining of bronchiol ( ), ) and thickening of alveolar wall due to lymphatic infiltration (-) also around bronchiole (H&E 10). (c) There is only thrombosis (-) in the odema, thrombosis ( heart (H&E 10) (Hussien, 2011).
concentrations. The test tubes were kept in an incubator at 22 7 1 1C. Several of the newly formed root tips were then cut from each bulb and examined for any visible morphological abnormalities. The bulbs with satisfactory root lengths (2–2.5 cm) were used in the study. Therefore, individual sets of five bulbs were used for each extract sample. Tap water (pH 7.3) was used as a negative control and ethyl methane sulfonate (EMS, M) used as a positive control mutagen. After 24 h of exposure, several root tips were removed from the bulbs, fixed in 3: 1 (v/v) ethanol: glacial acetic acid and stored overnight at 4 1C. The next day they were placed in 70% (v/v) aqueous alcohol and refrigerated until used. An average of five slides was made for each bulb using five root tips hydrolyzed in 1 N hydrochloric acid (HCl) for 3 min and microscope slides were prepared by squashing the stained root tips in 2% (w/v) acetic orcein. Five slides were prepared per bulb, and each slide was examined at a total magnification of 40 10. The authors show that: I. viscosa leaf extracts caused strong inhibition of mitotic index positively correlated with increasing concentration of I. viscosa leaf extracts. The extracts induced chromosome and cytological alterations both in a clastogenic effect. The occurrence of chromosome fragments allows observation of statistically significant differences on I. viscosa leaf extracts. In addition to the chromosome fragments, sticky metaphase and polar deviations (wrong directions of chromosome movement) were also observed (Fig. 5b and c). Statistical analysis showed that the genotoxic activities of extract induced micronuclei in the root tip meristem cells of A. cepa. Micronucleus formation in 1000 cells per slide (% MNC value) was also increased in extract concentrations compared with negative and positive control, which is statistically significant (p o 0.05). In addition, cells with membrane damage (Fig. 5d), binucleate cells (Fig. 5e), and nucleus damage (Fig. 5g and h) were found at various frequencies.
Moreover, apoptotic cells (Fig. 5f) were detected in the extracttreated groups. In addition, ghost cells were detected in 10 mg/ml (Tulay and Ozlem, 2010).
1.1.5. Lung and heart injury Nerium oleander L. belongs to the Apocynaceae family. It is an evergreen shrub or small tree distributed in the Mediterranean, tropical region and subtropical Asia. The phytochemical screening of plant revealed the presence of alkaloids, tannins, terpenoid, saponins, cardiac glycosidase and carbohydrates (Suganya et al., 2012). This plant has antioxidant, hepatoprotective, antimicrobial and cytotoxicity activity (Singhal and Gupta, 2012; Mohadjerani, 2012; El Sawi et al., 2010). This plant has been reported to be extremely toxic. Hensley (1997) revealed that animals can suffer a reaction or death from this plant. N. oleander sap can cause skin irritation, sever eye inflammation and irritation, allergic reactions characterized by dermatitis. Twelve male mice (albino, Sprigue Dawlly) were used for the study. The animals were treated orally with LD50 dose (520 mg/kg b.w.) of alcoholic extract of N. oleander leaves and normal saline for the control. Necropsy of animals was performed immediately after death and gross pathology of organs were recorded. The observation of animals showed abdominal pain, manifested by restlessness, pawing the ground, looking at the flank, frequent lying down and getting up, and humped posture, diarrhea, salivation with foam in the mouth, depression, weakness, in coordination of movements, tachypnea, convulsion and death. Histopathological examination showed edema, congestion and endothelial proliferation in the lung (Fig. 6a), hyperplasia of epithelial lining of bronchiol, thrombosis and thickening of alveolar wall due to lymphatic infiltration also around bronchiol (Fig. 6b), whilst Fig. 6c shows thrombosis in the heart (Ali Hussien, 2010).
Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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1.1.6. Testicular injury Quassia amara L. is a neotropical forest shrub or small tree belonging to the Simaroubaceae family. It is a source of several compounds alkaloids like and terpens. Antimalarial, antifeedant and antiulcerogenic activities are attributed to quassinoids, a group of substances belonging to the terpens class (Ajaiyeoba et al., 1999; Toma et al., 2002). The hydromethanolic extract (100 mg/kg) of stem bark was administered daily (per os) during 6 weeks to an experimental group composed by 5 male rats while the control group received distilled water for same duration. At the end of treatment, all male rats were sacrificed by decapitation.
Reproductive and visceral organs were placed on a pre-warmed slide and two drops of warm 2.9% sodium citrate was added. Progressive forward motility was examined and scored to the nearest. Viability study (percentage of live spermatozoa) was performed using eosin/nigrosin stain. Sperm motility and movement characteristics were assessed. Sperm acrosomal status was then evaluated using coomassie brilliant blue staining technique. Pre-extension motility was determined by mixing one drop of sperm cells with one drop of 2.9% sodium citrate and observed under the light microscope. The freshly collected sperm cells were carefully aspirated and then dispersed into a 50 ml conical flask
Fig. 7. Transverse sections through the Epididymis of normal control rats and rats treated with 100 mg/kg Quassia amara stem bark extract. Arrow indicates loosely packed epididymal ducts in the treated group when compared with control.
Fig. 8. Transverse sections through the seminal vesicle of normal control rats and rats treated with 100 mg/kg Quassia amara stem bark extract. Arrow indicates epithelial erosion of the treated group when compared with control.
Fig. 9. Coomassie brilliant blue (CBB) staining patterns of rat spermatozoa during capacitation. (A) Uncapacitated cell with uniform head staining. (B) Acrosome intact capacitated sperm with staining in the acrosomal region and (C) acrosome-reacted capacitated sperm cells with no head staining (400 magnification).
Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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Fig. 10. (a) A sectional view of control spleen showed normal white pulp and normal red pulp (100 ). (b) A sectional view of 150 mg/ml alcoholic extract of Cupressus sempervirens treated mice spleen showed moderate periarterial hyper plasma (2) (400 ). (c) A sectional view of 150 mg/ml aquatic extract of C. sempervirens treated mice spleen showed depletion of white pulp mainly neutrophil and macrophage (2) (400 ). (d) A sectional view of 350 mg/ml alcoholic extract of C. sempervirens treated mice spleen showed sever amyloid deposition in red pulp (Δ)and around white pulp, (400 ). (e) A sectional view of 350 mg/ml aquatic extract of C. sempervirens treated mice spleen showed amyloid like substance deposition in red pulp and around white pulp (Δ), (400 ) (Nusaibah, 2012). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 11. Gastric lesions observed; (a) stomach of control rats; (b) stomach of treated rats: ulceration of gastric mucous (arrows). Note the extensive area of gastric glandular mucosal necrosis. Bar 80 mm.
Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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Fig. 12. Intestine lesions observed on rats which received orally the butanolic extract of Herniaria cinerea at 2 g/kg. (a) Intestine of control rats; (b) intestine of treated rats: necrosis of intestinal mucous (arrows). Note the severe necrosis of intestinal villosity on small intestine. Bar: 50 m.
containing the extender solution (80% buffer (Trisodium citrate) and 20% egg yolk). The mixture was then gently shaken and 5 ml was dispersed into series of test tubes each. Graded doses (50– 500 mg/ml) of crude methanol extract of Q. amara were then added to the series of extender solutions. Sperm motility, viability and morphology were observed and recorded immediately and thereafter hourly over a period of four hours. Results showed that there were no changes in animal behavior or body and also treatment had non-significant effect on the visceral organs. The weight of testes, caudal epididymis and seminal vesicle was significantly lower (p o0.05) in the treated group when compared with the control. Caudal epididymal sperm parameters showed evidence of toxicity. The sperm motility, viability and count were significantly reduced (p o0.05). Less than 50% of caudal epididymal sperm were motile. Also, the semen volume was significantly reduced (p o0.05) and percentage of sperm cells with abnormal morphology was higher (p o0.05). Invitro studies to verify these results showed that increasing doses (50–500 mg/ml) of methanol extract of Q. amara stem bark decreased sperm motility and viability in a dose dependent manner. The percentage of sperm cells with abnormal morphology increased with treatment dose. Histopathological examination of treated rats revealed that the epididymis was made up of fewer ducts, which were loosely packed together when compared with the control and the ducts contained less amounts of ductular eosinophilic material (Fig. 7). Transverse section through the seminal vesicles of treated rats shows erosion of epithelial membrane (Fig. 8). Over 97% of caudal epididymal sperm of control rats were capacitated and their acrosome reacted after in vitro induction. However, 39%7 3.97% of sperm cells obtained from rats treated with Q. amara failed to be capacitated and their acrosome remained intact. About 33%73.12 failed to undergo the acrosome reaction, (although they were capacitated) while only 27%7 1.98 of sperm collected from the caudal epididymis were capacitated and their acrosome reacted. All the incapacitated sperm had their acrosome intact. Fig. 9 shows the different staining patterns of rat spermatozoa during capacitation (Obembe and Raji, 2012)
and also contains some of phenolic compounds (anthocyanidin, catechines flavones, flavonols and isoflavones), tannins (gallic acid, caffeic acid, coumaric acid), lignans, catchol, essential oil (Kassem et al., 1991), piperitone, and camphore (Hussein, 1985). The cones and young branches are anthelmintic, antipyretic, anti-rheumatic, astringent, balsamic, vasoconstrictive, anti-inflammatory, and hair tonic. The fruits of plant were used traditionally to treat diabetes and as antiseptic (Said et al., 2002) such as antioxidant (Emami et al., 2007), and it is effective in the treatment of hyperlipidemia (Karkabounas et al., 2003). For the experiment 30 males Swiss albino mice were divided into five groups, and received through i. p. different doses (150 and 350 mg/ml) of ethanol and aqueous extract of seed of C. sempervirens. The results revealed that, at 150 mg/ml the alcoholic extract caused moderate periarterial hyper plasma (Fig. 10d) on spleen, while the same dose of aqueous extract showed depletion of white pulp cells, mainly neutrophils and macrophages (Fig. 10e). At the dose of 350 mg/ml the alcoholic extract of C. sempervirens caused severe amyloid deposition in red pulp and around white pulp (Fig. 10b) on spleen, while the 350 mg/ml aqueous extract showed amyloid like substance deposition in red pulp and around white pulp (Fig. 10c) (Nusaibah, 2012).
1.1.7. Spleen injury Cupressus sempervirens L. is a tree which belongs to the family Cupressaceae. The plant is distributed in Turkey and throughout the mediterranean region. The plant is rich in flavonoids constituents such as quercitrin, quercetin, myricitrin (Harborne, 1993),
2. General conclusion
1.1.8. Gastrointestinal tract injury Herniaria cinerea DC. belongs to the family of the Caryophyllaceae. It is used as a traditional remedy for kidney stone diseases (Bellakhdar, 1997) and it has an antilithiatic activity (Grases et al., 1995). The butanolic extract of the whole plant was administered orally at 0.1; 0.5; 1; 2; 2.5; and 4 g/kg to Wistar rats. Examination of results revealed that abnormal signs start at 1 g/kg and increased with doses. The recorded symptoms are abnormal digestive signs like anorexia and bloody diarrhea and respiratory dysfunction appeared in the form of nasal hemorrhage with polypnea. Comparison of stomach structure in the control group and treated rats (Fig. 11) showed total necrosis of gastric crypts. In the small intestine, necrosis of intestinal villosity has been observed (Fig. 12) (Sokar et al., 2003).
Are medicinal plants are always safe? This question is the key point of this review. The use of medicinal plants is consubstantial to human history. For example, the Bible advises the use of hyssop
Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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for purification and the treatment of various affections. In addition, the herbals have been usually considered to be safe and nontoxic compared to synthetic compounds (Tülay, 2012; Bussmann et al., 2007; Oreagba et al., 2011). If true in most cases, plant extracts can also induce deleterious effects to the users. This is supported by numerous studies on medicinal plant pharmacological properties which show that, the aqueous extracts seem overall to be less toxic than organic extracts. The phytochemical composition of both type of extract (aqueous and alcoholic) is responsible of this observation. The relative low toxicity the aqueous extract relies on the fact they contain the total family of medicinal compounds (known and unknown) just as they are found in their natural source and hence offer less risk of side effects, and also to the weakness of water to extract strong links to the example of covalent bonding (Tülay, 2012). In general, from one plant to another, a dose that can cause damage varies according to both the chemical compounds present in the plant and their quantity. As a matter of fact, the degree of toxicity is actually proportional to the dose administered; thereby the more the dose increases the more the lesions caused are important especially if the consumption is regular. But, the question remains on the damage caused by the chronic exposition to an even low toxic extract as folk medicine are potentially toxic, mutagenic, and carcinogenic (Mengs, 1988; Ferreira-Machado et al., 2004). Overall, medicinal plants are double edge sword, and as such one much be cautious when using them. Finally, effort must be done to gather traditional healers, scientist and health authorities to establish safe and affordable procedures to make safer the use of medicinal plants. And, clinical trials can be conducted to evaluate the safety of medicinal plants with an emphases on the type of extract (aqueous and alcoholic).
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Toxicity studies of medicinal plants used in sub-Saharan Africa Marlaine Boukandou Mounanga a, Ludovic Mewono b, Sophie Aboughe Angone a,n a
Pharmacopeia and Traditional Medicine Institute: National Center of Technological and Scientific Research, Libreville, Gabon Groupe de Recherche en Immunologie, Microbiologie appliquée, Hygiène et Physiologie (GRIMAH.P), Département des Sciences de la Vie et de la Terre- Ecole Normale Supérieure, BP 17009 Libreville, Gabon
b
ar t ic l e i nf o
a b s t r a c t
Article history: Received 15 March 2015 Received in revised form 13 May 2015 Accepted 7 June 2015
In sub-Saharan Africa, traditional medicine is widely used in rural and urban areas also. This is essentially due to the prohibitive cost of pharmaceutical-based medicine and the low incomes of a major part of the population. In addition, the efficacies of many of these traditional and plant-based medicines are proven, but the fact remains that certain plants used in traditional medicine have toxic effects. It is in this perspective that we investigated by bibliographic literature on the toxicity of plants used in traditional medicine. It is crucial to gain knowledge on these plant-based medicines prepared and prescribed by practitioners, particularly in terms of toxicity, composition, specific efficacy of disease and to advise practitioners of this alternative medicine on the protection and security of patients. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Plants Traditional medicine Toxicity Sub-Saharan Africa
1. Introduction The use of plants is as old as human kind itself. It has always been part of human culture. In Africa, for example, plants are used in traditional medicine to treat different infectious and noninfectious diseases (Diallo et al., 2003; Gurib-Fakim, 2006). These treatments include body-washes, massages, ingestions, etc. (Walker and Sillans, 1961). Plants provide a wide variety of biochemicals useful to mankind. Their uses include food, colors, fragrances and agricultural chemicals and pharmaceuticals. According to the World Health Organization (WHO), about 40–90% of people living in developing countries frequently use traditional medicine (van Andel and Carvalheiro, 2013). This situation is the consequence of a limited access to modern health services and also researchers explained the high prevalence of traditional medicine use by massive rural-to-urban migration, the influence of cultural and social surroundings, and the belief that natural products pose no risk (van Andel and Carvalheiro, 2013; Lins Brandão et al., 2006; Bussmann et al., 2007; Oreagba et al., 2011). Though the use of medicinal plants can have deleterious effects on health as reported elsewhere (Fall et al., 2011). Indeed, of about 1,500,000 plants investigated, most of them contain toxic substances (Ishii et al., 1984) like some secondary metabolites. Plants produce a variety of secondary metabolites that are subdivided by chemists into several classes based on their molecular
n Correspondence to: Pharmacopeia and Traditional Medicine Institute, National Center of Technological and Scientific Research, Po. Box: 5011, Libreville, Gabon. E-mail addresses: [email protected] (M. Boukandou Mounanga), [email protected] (L. Mewono), [email protected] (S. Aboughe Angone).
structure. Among secondary metabolites are saponins, terpenoids, cyanogenic, tannins, toxic amino acids, glycosides, alkaloids (Dai and Mumper, 2010). It has been shown that the toxicity of a given plant depends on various factors, including the strength of secondary metabolites, the quantity consumed, the time of exposure, different parts of the plant (root, oil, leaves, stem bark and seeds), individual body chemistry, climate and soil, and genetic differences within the species (Tülay, 2012). This said it clearly appears that medicinal plants should be used with precautions and toxicology studies conducted to increase the knowledge on the plant or plants preparation given to populations. For this purpose, in-vitro and in-vivo models are available to study the toxicity of medicinal plants. Regardless of the type of extract, the parts of plant used, the concentration of the extract, the mode of administration, and the organism under consideration the lethal dose 50 (LD50) who represent the dose who kill 50% of a tested population is used to appreciate the toxicity of the plant (Amy et al., 2002). In addition, for chronic or sub-chronic toxicity histological or genetic modifications are the most relevant indicators. In this paper, we aimed to review, on the basis of 9 studies, medicinal plant extract induced cellular or organ dysfunction (Table 1) and to show the importance of toxicity studies. 1.1. Hemolytic effect Chrysophyllum pruniforme (synonym: Donella pruniformis) (Pierre ex Engl.) is a tall tree who belongs to the family of Sapotaceae. This plant is widely spread out from West to East Africa and Central Africa. In Gabon and Congo, C. pruniforme's bark infusion is used to treat coughs. Phytochemical studies showed that it contains reducing sugars, phenolic compounds, flavonoids,
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Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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Table 1 Recapitulative table of plants and their toxicity. Plant scientific name
Family name
Botanical description
Chrysophyllum pruniforme (Pierre ex Engl.) Semecarpus anacardium Linn.
Sapotaceae
Anacardiceae
Quassia amara L. Radlk.
Simaroubaceae
Artemisia Asteraceae maciverae Linn. Nerium oleander Apocynaceae L.
Parts used
Adverse effect
Model of experimentation
References
Tall tropical tree growing to 10–20 m or more in height. The leaves are Barks oval, 3–15 cm long, green above, densely golden pubescent below.
Hemolytic
Human blood samples
Aboughe et al., 2013
Medium-to-large size tree, 15–25 m in height with gray bark exuding Leaves an irritant secretion on incising and exfoliating in small irregular flakes. Leaves are simple alternate, obviate – oblong, 30–60 cm long and 12–30 cm broad, rounded at the apex coriaceous glabrous above and more or less pubescent, beneath. Small tropical tree, growing only 2–6 m in height. It has beautiful red Stem flowers and fruits that turn red as they mature. bark
Neurotoxic
Intraperitoneal administration on mice
Chaudhary et al., 2010
Spermatotoxic
Oral administration of rats
Small herbaceous.
Whole plant
Nephrotoxic
Leaves
Toxic for lung and heart
Intraperitoneal administration on Swiss albinos rats Oral administration on mice
Obembe and Raji, 2012 Atawodi et al., 2013
Leaves
Hepatotoxic
Leaves
Toxic for spleen Intraperitoneal administration on mice
Nusaibah, 2012.
Whole plant
Toxic for gastrointestinal tract
Oral administration on Wistar rats
Sokar et al., 2003
Leaves
Genotoxic
On Alium cepa
Tulay and Ozlem, 2010
An evergreen shrub reaching 4 m in height. Leaves are 10–22 cm long, narrow, untoothed and short-stalked, dark or gray-green, white or yellow. All leaves have a prominent mid rib, are leathery in texture and usually arise in groups of three from the stem. Aphania Sapindaceae A forest tree to 55 ft high, with short bole and dense drooping foliage. senegalensis The flowers are greenish-white and fragrant. The ripe fruits are red (Juss. Ex Poir.) and edible. Cupressus Cupressaceae A medium-sized coniferous evergreen tree to 35 m tall, with a conic sempervirens crown. The foliage grows in dense sprays, dark green in color. The L. leaves are scale-like, 2–5 mm long, and produced on rounded (not flattened) shoots. Herniaria Caryophyllaceae A yellowish green, prostrate, hairy, spreading herb. Stem and branches cinerea DC. with 3–9 mm long internodes, with short spreading hairs. Leaves are opposite, sessile, hirsute, blade narrow elliptic to lanceolate or oblanceolate, usually 4–7 mm long and 1.5–2.3 mm wide, acute, margins entire with straight spreading hairs. Inula viscosa L. Asteraceae Small shrub. The stem is dark brown. The leaves have an elongated Aiton. lanceolate shape with a pointed tip. The larger leaves measure about 70 mm 18 mm.
Oral administration on Wistar rats
Ali Hussien, 2010 Fall et al., 2011
is an indicator of general cytotoxicity towards normal, healthy cells (Da Silva et al., 2004).
Fig. 1. Hemolytic activity of aqueous and phenolic extracts on human erythrocytes (Aboughe et al., 2013).
saponins, and catechic tannins (Aboughe et al., 2013). In this study the author shows that in vivo studies, the LD50 was 90 mg/kg in mice by oral administration of the aqueous extract, what according to the scale of toxicity of Hodge ant Sterner classifies it among the moderately toxic ones. In addition, aqueous and total phenolic extract of C. pruniforme show a hemolytic effect on human erythrocytes obtained from healthy donors. At the end of the experimentation, the aqueous extract showed hemolytic activities about 0%, 3%, 7%, 20%, 33%, and 47% in 1.25; 2.5; 3.75; 5; 7.5; and 10 mg/mL, respectively. At the same time total phenolic extract showed hemolytic activities about 7%, 79%, 99.6%, 99.9%, and 100% for the same plant concentrations. The hemolytic activity of total phenolic extracts was significantly higher compared to the one of aqueous extracts (Fig. 1) (Aboughe et al., 2013). Hemolytic activity
1.1.1. Hepatotoxic effect Aphania senegalensis (Juss. Ex Poir.) (Sapindaceae) is a medicinal plant widely used in Senegal in folk medicine. But, this tree can be found in several other African countries and in Asia. A phytochemical screening of leaves revealed that, they contain flavonoids, tannins and saponins (Faye, 2008). Its pharmacological activity was described mainly for the leaves and relied on antiparasitical, analgesic and anti-inflammatory activities (Fall et al., 2009). Tests of acute and sub-acute toxicity were carried out in male and female Wistar rats. The LD50 could not be determinate because of no deaths or observable clinical signs occurred after acute oral administration of a dose of 5.000 mg/kg. For sub-acute toxicity was carried out for a 28-day period with the administration of increasing doses of 500, 1000 and 2000 mg/kg of aqueous extract of plants. Biochemical analyses revealed a significant increase in aminotransferase activity. Levels of aminotransferase in treated animals were found to be twice higher than normal values. Further histological examination of liver of treated animals confirmed the existence of various liver lesions like degenerated hepatocytes with pyknotic nuclei, congestion, the central veins and the surrounding sinusoids in some lobules were filled with blood and fatty body (Fig. 2) (Fall et al., 2011), (Figs. 3 and 4). 1.1.2. Neurotoxic effect Semecarpus anacardium Linn. Belonging to the family of Anacardiaceae is a moderate-sized deciduous tree found in found in abundance in Assam, Bihar, Bengal and Orissa, Chittagong, central
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Fig. 2. Deleterious effects of Aphania senegalensis on liver cells. (a) Degenerated hepatocyte with pycnotic nuclei. (b) Lipidic droplet. (c) Inflammation (Fall et al., 2011).
Fig. 3. Transmission electron micrograph of brain cell (a) control; (b) low dose; (c) high dose.
Fig. 4. Histopathological appearances of the kidney of rats showing tubular epithelial necrosis N (b) compared to normal kidney (a).
India and Western peninsula of Eastern Archipelago and Northern Australia (Kirtikar and Basu, 1975). Different parts of this plant are used to treat many diseases like arthritis, tumors, and infections (Semalty et al., 2010). The plant is useful in skin diseases, piles, dysentery, fever, loss of appetite, urinary discharges, heals ulcers, and asthma (Basavaraj et al., 2011). The oil is a tonic, good for leucoderma, coryza, lessens, inflammation, and it is useful in superficial pain (Kirtikar and Basu, 1998). Raman and Pednekar (2012) found that the plant possesses biomolecules like alkaloids, saponins, tannins, flavonoids, steroids, glycosides, hexose sugars,
diterpenes, mucilages and gum extracts whereas, carbohydrates, phenols, proteins, monosaccharides, starch were found in negligible amount. Because of it belonging to Anacardiaceae family, it has potential to drive allergic manifestations through contact dermatitis. Choudhari and Deshmukh (2007) reported that the S. anacardium extract induced severe anemia in rat. The crude chloroform extract was found to be very toxic according to Patwardhan et al. (1988). In addition, traditional use of latex of leaf and stem has been found to be toxic to the skin (Choudhury et al., 2011b). The aqueous extract of plant was administered
Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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through ip. injection in two different doses to mice during 5 days. Several harmful effects were then observed following injections of two doses: mainly akinesia, catalepsy, decreasing of body weight and hyperpnoea for the high doses were observed. Biochemical parameters like serum glutamate oxaloacetate transminase (GOT) and serum (glutamate pyruate transminase), cerebral cortex lipoperoxidation (LPO) and midbrain LPO showed an elevation for both the treatment. The authors also found that the cell have wellformed nuclei, whereas in low dose there are shrunken nuclei and fragmented chromatin and high dose showing more ruptured and increased fragmented chromatin (Choudhury et al., 2011a).
1.1.3. Nephrotoxic effect Artemisia maciverae Linn. is a small herbaceous plant belonging to the Asteraceae family. The phytochemicals present in the chloroform extract of A. maciverae are flavonoids, triterpenes, terpenoids, glycosides, phlobatannins and tannins. Various pharmacological studies have been carried out on this plant product which showed that, the chloroform extract possesses anti-malarial activity (Ene et al., 2008). The chloroform extract of A. maciverae at 0; 50; 100 and 200 mg/kg were given to adult male Swiss albino rats through the ip route for 90 days. Administration of chloroform extract of A. maciverae produced signs of toxicity like loss of appetite, loss of agility, dizziness and convulsion. These signs of toxicity were found to increase in severity with increasing dose of extract. Mortality was recorded in 50 and 100 mg/kg treatment group in week one and four of treatment. All the animals in 200 mg/kg treatment group died within the first week of treatment with convulsions as a major observable sign of toxicity. There were statistically significant (p o0.05) elevation in the levels of creatinine and urea in the treated groups. Gross and histopathological observations indicated congestion, tubular epithelial necrosis in the kidneys of animals treated. The toxic effect of this plant extract was reversed within 4 weeks after treatment withdrawal, suggesting that the nephrotoxic effect of chloroform extract of A. maciverae may not be permanent. A hepatotoxicity study carried out with the chloroform extract of this plant, in Swiss albino rats indicate that long term exposure to therapeutic doses of chloroform extract of this plant is relatively safe, but high dose
exposure may result in hepatocellular injury (Ene et al., 2011). In another study by Ene and al. (2012), this chloroform extract caused elevation in the hemoglobin and lymphocyte counts of the rats treated with 200 mg/kg of extract at the onset of treatment. This suggests that there was any possible breach of the integrity of blood synthesis and regulatory systems with high doses of extract compared with the lower doses (Atawodi et al., 2013).
1.1.4. Genotoxic effect Inula viscosa (L.) Aiton (Compositae) is a perennial weed that is found mostly in the mediterranean basin (Al-Dissi et al., 2001). I. viscosa contains some pharmacologically active compounds including sesquiterpenes, sesquiterpenes acids (Ulubelen et al., 1987, Marongiu et al., 2003), lactones, flavonoids, and essential oils (Lauro and Rolih, 1990, Wollenweber et al., 1991). It has been used for its antiinflammatory (Barbetti et al., 1985), antipyretic, antiseptic, antiphlogistic activities (Lauro and Rolih, 1990, Lev and Amar, 2000) and diabetes (Yaniv et al., 1987). It has been demonstrated that aqueous extracts of this plant exhibit antifungal activity in vitro (Cohen et al., 2002, Maoz et al., 1999, Qasem et al., 1995) and some of its organic solvent extracts are antibacterial (Debat, 1991). In Spain, this herb has been used for treating gastroduodenal disorders (Lastra et al., 1993). I. viscosa has antiulcerogenic effects (Alkofah and Atta, 1999) causes abortion (Farnsworth et al., 1975; Karim et al., 1990), prevents zygote implantation in mammals (Al-Dissi et al., 2001), prevents growth of pathogenic fungi (Maoz and Neeman, 2000), has a strong antioxidant activity (Schinella et al., 2002), and has also nematicidal and antihelminth properties (Oka et al., 2001). Several researchers validated the tests which use plant test systems in vivo, tests on the animals jointly performed in vitro often confirm the results obtained (Vicentini, et al. 2001; Teixeira et al. 2003), and can be extrapolated to other organisms such as humans. For this study small bulbs (1.5–2.0 cm in diameter) of Alium cepa was used to initiate the test, the outer scales of the bulbs and the dry bottom plate were removed without destroying the root. For each extract sample, six bulbs were placed in tap water (pH 7.3) for 48 h and then onion roots were treated with the leaves aqueous extracts of I. viscosa at 2.5; 5; and 10 mg/ml
Fig. 5. Mitotic and chromosomal aberrations after the Inula viscose leaf extract treatments in Alium cepa root tip meristem cells visualized with microscopy. (a) Fragments; (b) stickiness; (c) polar deviation and chromatid bridge; (d) membrane damage; (e) binucleated cell; (f) apoptotic bodies; (g and h) cells with damaged nucleus (Tulay and Ozlem, 2010).
Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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Fig. 6. (a) There is odema ( ), congestion (-) in the lung (H&E 10). (b) The lung was suffering from congestion, hyperplasia of epithelial lining of bronchiol ( ), ) and thickening of alveolar wall due to lymphatic infiltration (-) also around bronchiole (H&E 10). (c) There is only thrombosis (-) in the odema, thrombosis ( heart (H&E 10) (Hussien, 2011).
concentrations. The test tubes were kept in an incubator at 22 7 1 1C. Several of the newly formed root tips were then cut from each bulb and examined for any visible morphological abnormalities. The bulbs with satisfactory root lengths (2–2.5 cm) were used in the study. Therefore, individual sets of five bulbs were used for each extract sample. Tap water (pH 7.3) was used as a negative control and ethyl methane sulfonate (EMS, M) used as a positive control mutagen. After 24 h of exposure, several root tips were removed from the bulbs, fixed in 3: 1 (v/v) ethanol: glacial acetic acid and stored overnight at 4 1C. The next day they were placed in 70% (v/v) aqueous alcohol and refrigerated until used. An average of five slides was made for each bulb using five root tips hydrolyzed in 1 N hydrochloric acid (HCl) for 3 min and microscope slides were prepared by squashing the stained root tips in 2% (w/v) acetic orcein. Five slides were prepared per bulb, and each slide was examined at a total magnification of 40 10. The authors show that: I. viscosa leaf extracts caused strong inhibition of mitotic index positively correlated with increasing concentration of I. viscosa leaf extracts. The extracts induced chromosome and cytological alterations both in a clastogenic effect. The occurrence of chromosome fragments allows observation of statistically significant differences on I. viscosa leaf extracts. In addition to the chromosome fragments, sticky metaphase and polar deviations (wrong directions of chromosome movement) were also observed (Fig. 5b and c). Statistical analysis showed that the genotoxic activities of extract induced micronuclei in the root tip meristem cells of A. cepa. Micronucleus formation in 1000 cells per slide (% MNC value) was also increased in extract concentrations compared with negative and positive control, which is statistically significant (p o 0.05). In addition, cells with membrane damage (Fig. 5d), binucleate cells (Fig. 5e), and nucleus damage (Fig. 5g and h) were found at various frequencies.
Moreover, apoptotic cells (Fig. 5f) were detected in the extracttreated groups. In addition, ghost cells were detected in 10 mg/ml (Tulay and Ozlem, 2010).
1.1.5. Lung and heart injury Nerium oleander L. belongs to the Apocynaceae family. It is an evergreen shrub or small tree distributed in the Mediterranean, tropical region and subtropical Asia. The phytochemical screening of plant revealed the presence of alkaloids, tannins, terpenoid, saponins, cardiac glycosidase and carbohydrates (Suganya et al., 2012). This plant has antioxidant, hepatoprotective, antimicrobial and cytotoxicity activity (Singhal and Gupta, 2012; Mohadjerani, 2012; El Sawi et al., 2010). This plant has been reported to be extremely toxic. Hensley (1997) revealed that animals can suffer a reaction or death from this plant. N. oleander sap can cause skin irritation, sever eye inflammation and irritation, allergic reactions characterized by dermatitis. Twelve male mice (albino, Sprigue Dawlly) were used for the study. The animals were treated orally with LD50 dose (520 mg/kg b.w.) of alcoholic extract of N. oleander leaves and normal saline for the control. Necropsy of animals was performed immediately after death and gross pathology of organs were recorded. The observation of animals showed abdominal pain, manifested by restlessness, pawing the ground, looking at the flank, frequent lying down and getting up, and humped posture, diarrhea, salivation with foam in the mouth, depression, weakness, in coordination of movements, tachypnea, convulsion and death. Histopathological examination showed edema, congestion and endothelial proliferation in the lung (Fig. 6a), hyperplasia of epithelial lining of bronchiol, thrombosis and thickening of alveolar wall due to lymphatic infiltration also around bronchiol (Fig. 6b), whilst Fig. 6c shows thrombosis in the heart (Ali Hussien, 2010).
Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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1.1.6. Testicular injury Quassia amara L. is a neotropical forest shrub or small tree belonging to the Simaroubaceae family. It is a source of several compounds alkaloids like and terpens. Antimalarial, antifeedant and antiulcerogenic activities are attributed to quassinoids, a group of substances belonging to the terpens class (Ajaiyeoba et al., 1999; Toma et al., 2002). The hydromethanolic extract (100 mg/kg) of stem bark was administered daily (per os) during 6 weeks to an experimental group composed by 5 male rats while the control group received distilled water for same duration. At the end of treatment, all male rats were sacrificed by decapitation.
Reproductive and visceral organs were placed on a pre-warmed slide and two drops of warm 2.9% sodium citrate was added. Progressive forward motility was examined and scored to the nearest. Viability study (percentage of live spermatozoa) was performed using eosin/nigrosin stain. Sperm motility and movement characteristics were assessed. Sperm acrosomal status was then evaluated using coomassie brilliant blue staining technique. Pre-extension motility was determined by mixing one drop of sperm cells with one drop of 2.9% sodium citrate and observed under the light microscope. The freshly collected sperm cells were carefully aspirated and then dispersed into a 50 ml conical flask
Fig. 7. Transverse sections through the Epididymis of normal control rats and rats treated with 100 mg/kg Quassia amara stem bark extract. Arrow indicates loosely packed epididymal ducts in the treated group when compared with control.
Fig. 8. Transverse sections through the seminal vesicle of normal control rats and rats treated with 100 mg/kg Quassia amara stem bark extract. Arrow indicates epithelial erosion of the treated group when compared with control.
Fig. 9. Coomassie brilliant blue (CBB) staining patterns of rat spermatozoa during capacitation. (A) Uncapacitated cell with uniform head staining. (B) Acrosome intact capacitated sperm with staining in the acrosomal region and (C) acrosome-reacted capacitated sperm cells with no head staining (400 magnification).
Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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Fig. 10. (a) A sectional view of control spleen showed normal white pulp and normal red pulp (100 ). (b) A sectional view of 150 mg/ml alcoholic extract of Cupressus sempervirens treated mice spleen showed moderate periarterial hyper plasma (2) (400 ). (c) A sectional view of 150 mg/ml aquatic extract of C. sempervirens treated mice spleen showed depletion of white pulp mainly neutrophil and macrophage (2) (400 ). (d) A sectional view of 350 mg/ml alcoholic extract of C. sempervirens treated mice spleen showed sever amyloid deposition in red pulp (Δ)and around white pulp, (400 ). (e) A sectional view of 350 mg/ml aquatic extract of C. sempervirens treated mice spleen showed amyloid like substance deposition in red pulp and around white pulp (Δ), (400 ) (Nusaibah, 2012). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 11. Gastric lesions observed; (a) stomach of control rats; (b) stomach of treated rats: ulceration of gastric mucous (arrows). Note the extensive area of gastric glandular mucosal necrosis. Bar 80 mm.
Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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Fig. 12. Intestine lesions observed on rats which received orally the butanolic extract of Herniaria cinerea at 2 g/kg. (a) Intestine of control rats; (b) intestine of treated rats: necrosis of intestinal mucous (arrows). Note the severe necrosis of intestinal villosity on small intestine. Bar: 50 m.
containing the extender solution (80% buffer (Trisodium citrate) and 20% egg yolk). The mixture was then gently shaken and 5 ml was dispersed into series of test tubes each. Graded doses (50– 500 mg/ml) of crude methanol extract of Q. amara were then added to the series of extender solutions. Sperm motility, viability and morphology were observed and recorded immediately and thereafter hourly over a period of four hours. Results showed that there were no changes in animal behavior or body and also treatment had non-significant effect on the visceral organs. The weight of testes, caudal epididymis and seminal vesicle was significantly lower (p o0.05) in the treated group when compared with the control. Caudal epididymal sperm parameters showed evidence of toxicity. The sperm motility, viability and count were significantly reduced (p o0.05). Less than 50% of caudal epididymal sperm were motile. Also, the semen volume was significantly reduced (p o0.05) and percentage of sperm cells with abnormal morphology was higher (p o0.05). Invitro studies to verify these results showed that increasing doses (50–500 mg/ml) of methanol extract of Q. amara stem bark decreased sperm motility and viability in a dose dependent manner. The percentage of sperm cells with abnormal morphology increased with treatment dose. Histopathological examination of treated rats revealed that the epididymis was made up of fewer ducts, which were loosely packed together when compared with the control and the ducts contained less amounts of ductular eosinophilic material (Fig. 7). Transverse section through the seminal vesicles of treated rats shows erosion of epithelial membrane (Fig. 8). Over 97% of caudal epididymal sperm of control rats were capacitated and their acrosome reacted after in vitro induction. However, 39%7 3.97% of sperm cells obtained from rats treated with Q. amara failed to be capacitated and their acrosome remained intact. About 33%73.12 failed to undergo the acrosome reaction, (although they were capacitated) while only 27%7 1.98 of sperm collected from the caudal epididymis were capacitated and their acrosome reacted. All the incapacitated sperm had their acrosome intact. Fig. 9 shows the different staining patterns of rat spermatozoa during capacitation (Obembe and Raji, 2012)
and also contains some of phenolic compounds (anthocyanidin, catechines flavones, flavonols and isoflavones), tannins (gallic acid, caffeic acid, coumaric acid), lignans, catchol, essential oil (Kassem et al., 1991), piperitone, and camphore (Hussein, 1985). The cones and young branches are anthelmintic, antipyretic, anti-rheumatic, astringent, balsamic, vasoconstrictive, anti-inflammatory, and hair tonic. The fruits of plant were used traditionally to treat diabetes and as antiseptic (Said et al., 2002) such as antioxidant (Emami et al., 2007), and it is effective in the treatment of hyperlipidemia (Karkabounas et al., 2003). For the experiment 30 males Swiss albino mice were divided into five groups, and received through i. p. different doses (150 and 350 mg/ml) of ethanol and aqueous extract of seed of C. sempervirens. The results revealed that, at 150 mg/ml the alcoholic extract caused moderate periarterial hyper plasma (Fig. 10d) on spleen, while the same dose of aqueous extract showed depletion of white pulp cells, mainly neutrophils and macrophages (Fig. 10e). At the dose of 350 mg/ml the alcoholic extract of C. sempervirens caused severe amyloid deposition in red pulp and around white pulp (Fig. 10b) on spleen, while the 350 mg/ml aqueous extract showed amyloid like substance deposition in red pulp and around white pulp (Fig. 10c) (Nusaibah, 2012).
1.1.7. Spleen injury Cupressus sempervirens L. is a tree which belongs to the family Cupressaceae. The plant is distributed in Turkey and throughout the mediterranean region. The plant is rich in flavonoids constituents such as quercitrin, quercetin, myricitrin (Harborne, 1993),
2. General conclusion
1.1.8. Gastrointestinal tract injury Herniaria cinerea DC. belongs to the family of the Caryophyllaceae. It is used as a traditional remedy for kidney stone diseases (Bellakhdar, 1997) and it has an antilithiatic activity (Grases et al., 1995). The butanolic extract of the whole plant was administered orally at 0.1; 0.5; 1; 2; 2.5; and 4 g/kg to Wistar rats. Examination of results revealed that abnormal signs start at 1 g/kg and increased with doses. The recorded symptoms are abnormal digestive signs like anorexia and bloody diarrhea and respiratory dysfunction appeared in the form of nasal hemorrhage with polypnea. Comparison of stomach structure in the control group and treated rats (Fig. 11) showed total necrosis of gastric crypts. In the small intestine, necrosis of intestinal villosity has been observed (Fig. 12) (Sokar et al., 2003).
Are medicinal plants are always safe? This question is the key point of this review. The use of medicinal plants is consubstantial to human history. For example, the Bible advises the use of hyssop
Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i
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for purification and the treatment of various affections. In addition, the herbals have been usually considered to be safe and nontoxic compared to synthetic compounds (Tülay, 2012; Bussmann et al., 2007; Oreagba et al., 2011). If true in most cases, plant extracts can also induce deleterious effects to the users. This is supported by numerous studies on medicinal plant pharmacological properties which show that, the aqueous extracts seem overall to be less toxic than organic extracts. The phytochemical composition of both type of extract (aqueous and alcoholic) is responsible of this observation. The relative low toxicity the aqueous extract relies on the fact they contain the total family of medicinal compounds (known and unknown) just as they are found in their natural source and hence offer less risk of side effects, and also to the weakness of water to extract strong links to the example of covalent bonding (Tülay, 2012). In general, from one plant to another, a dose that can cause damage varies according to both the chemical compounds present in the plant and their quantity. As a matter of fact, the degree of toxicity is actually proportional to the dose administered; thereby the more the dose increases the more the lesions caused are important especially if the consumption is regular. But, the question remains on the damage caused by the chronic exposition to an even low toxic extract as folk medicine are potentially toxic, mutagenic, and carcinogenic (Mengs, 1988; Ferreira-Machado et al., 2004). Overall, medicinal plants are double edge sword, and as such one much be cautious when using them. Finally, effort must be done to gather traditional healers, scientist and health authorities to establish safe and affordable procedures to make safer the use of medicinal plants. And, clinical trials can be conducted to evaluate the safety of medicinal plants with an emphases on the type of extract (aqueous and alcoholic).
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Please cite this article as: Boukandou Mounanga, M., et al., Toxicity studies of medicinal plants used in sub-Saharan Africa. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.06.005i