Toxicity studies of medicinal plants used in sub-Saharan Africa
Graphical abstract
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 non-infectious 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 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.
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, 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 is an indicator of general cytotoxicity towards normal, healthy cells (Da Silva et al., 2004).
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 anti-parasitical, 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), (Fig. 3, Fig. 4).
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 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 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 well-formed 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).
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<0.05) 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).
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 concentrations. The test tubes were kept in an incubator at 22 ± 1 °C. 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 °C. 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 < 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 extract-treated groups. In addition, ghost cells were detected in 10 mg/ml (Tulay and Ozlem, 2010).
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).
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 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 µg/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<0.05) 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<0.05). Less than 50% of caudal epididymal sperm were motile. Also, the semen volume was significantly reduced (p<0.05) and percentage of sperm cells with abnormal morphology was higher (p<0.05). In-vitro studies to verify these results showed that increasing doses (50–500 µg/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%±3.97% of sperm cells obtained from rats treated with Q. amara failed to be capacitated and their acrosome remained intact. About 33%±3.12 failed to undergo the acrosome reaction, (although they were capacitated) while only 27%±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)
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), 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).
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).
Section snippets
General conclusion
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 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
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2022, Journal of King Saud University - ScienceCitation Excerpt :However, there is a need to investigate the safety of using a high dose of these spices over a long time to avoid creating problems while trying to solve another. The need to confirm the safety of this plant is because a handful of plants are toxic, majorly when consumed at higher doses (Mounanga et al., 2015), including the common spice, Myristica fragrans Houtt. ( Myristicaceae) (Benzeid et al., 2018).