Traditional uses, phytochemistry, and pharmacological activities of Cochlospermum tinctorium A. Rich (Cochlospermaceae): a review

The plant Cochlospermum tinctorium A. Rich is a sub-shrub that belongs to the family Cochlospermaceae. The plant has been used in traditional medicine for the treatment of malaria, rickets, stomachache, diarrhea, gastric ulcer, parasitic infestations, liver diseases, fever, pain, inflammation, infectious diseases, epilepsy, snake bite, burns, orchitis, labour, menstrual problems, and many other diseases. This review summarizes the traditional uses, phytochemistry, and pharmacological activities of Cochlospermum tinctorium. To date, few bioactive molecules have been identified and isolated from the plant such as 7,3-dimethyldihydroquercelin, 5,4-dimethylquercelin, cochloxanthine, dihydrocochloxanthine, arjunolic acid, 3-O-E-p-coumaroylalphitolic acid, alphitolic acid, 1-hydroxytetradecan-3-one, 3-bisabolen, 2-tridecanone, 3-hexadecanone, 1-dodecanol, l-tetradecanol, 2-pentdecanone, 3-octadecanone, 1-hydroxy-3-hexadecanone, 1-nonadecanol, l-O-acetyl-3-hexadecanone, and l-hydroxy-3-oetadecanone. The literature related some of the reported ethnomedicinal uses of the plant to these compounds found in the different parts of the plant. The comprehensive information documented in this review about the importance of the C. tinctorium may provide an opportunity for research advancement in drug discovery and a better understanding of the medicinal benefits of the plant.


Background
For decades, humans majorly rely on plants for food and management of diseases [1]. It is estimated that approximately 75% of the global population relies on herbal medicines for their basic health care needs. Indeed, many drugs that are currently in use in modern medicine are obtained from plants [2]. Given the predominant uses of medicinal plants in traditional medicine, there is an upsurge in research to investigate the active medicinal compounds, efficacy, and safety of such plants [3]. The literature suggests that the search for novel therapeutic compounds based on traditional uses and folkloric information about medicinal plants obtained from the community could guide and serve as a potential strategy for the development of new therapeutic compounds. Therefore, there is need for data and high quality-research on medicinal plants to provide stronger scientific evidence and confirm their medicinal uses and safety in traditional medicines.
The plant Cochlospermum tinctorium A. Rich (Cochlospermaceae) has been used for years in traditional medicine for the treatment of various ailments in many African countries such as Ivory Coast, Ghana, Cameroon, Nigeria, Gambia, Guinea, Senegal, Burkina Faso, and many others [4]. The plant is predominantly available in the savannah and throughout the dried parts of West Africa. It is locally called Oja Ikoko or Sewutu (Yoruba), Obazi or Abanzi (Igbo), and Rawaya or Kyamba (Hausa) languages of Nigeria [5]. Recently, C. tinctorium has gained attention from scientific community as a result of its traditional and wide range of medicinal uses. A review by Haidara et al. focused on the antimalarial and hepatoprotective activities of C. tinctorium and other medicinal plants used in Malian traditional medicine for the management of malaria and liver diseases [6]. However, studies reporting the traditional uses, phytochemical profile, and pharmacological activities of the plant are limited. Therefore, we conducted a review of the literature to provide comprehensive information related to the traditional uses, phytochemical contents, and pharmacological activities of the plant with a mission to stimulate further research on its potentials for the development of novel therapeutic compounds.

Botanical description of Coclospermum tinctorium
The plant C. tinctorium belongs to the family Cochlospermaceae. The family consists of seven genera which include Amoreuxia, Azeredia, Cochlospermum, Euryanathe, Lachnocistus, Maximilianea, and Wittelsbachia [7]. The genus Cochlospermum Kunth (syn. Maximilianea Marth., Bixaceae) consists of some tropical species such as C. tinctorium, C. planchonii, C. angolense, C. vitifolium, C. regium, C. orinocense, C. religiosum, C. gillivraei, C. wittei, C. tetraporum, C. intermedium, and C. fraseri [8]. The two species C. planchonii and C. tinctorium are widely available in West Africa [9]. The plant C. tinctorium is a sub-shrub which is about 80 cm in length with a woody subterranean root stock that produces its shoots annually [10]. The leaves of the plant are alternate with lobes lanceolate to oblong, basally connate for up to 25% of their length [10]. The plant possesses few inflorescences and flowered panicle or raceme, usually produced at ground level from the rootstock, sometimes appearing on top of leafy shoots [10] (Fig. 1). The fruits of C. tinctorium are elongated about 3-5 valve and its capsules containing seeds are embedded in the cotton foam. The seeds are colored ranging from brown to black with a bean-shape. The endosperm of the plant is oily with broad cotyledon [12].

Main text
Traditional uses of Cochlospermum tinctorium The plant C. tinctorium is the most commonly used among all the species of the family Cochlospermaceae [8]. The roots or rhizome are mixed with shea butter oil or other oils and applied for the management of burns [13].
The rhizomes of C. tinctorium are used as a decoction for the treatment of rickets, stomach pain, helminthiasis, beriberi [21], fever, hepatitis, abdominal pain, and bilharzia [18]. The leaves of C. tinctorium are used for the management of diarrhea [22], abscess, and boils [8], while the flowers are used against constipation [8]. The summary of the documented traditional uses of different parts of C. tinctorium and scientific confirmation of their pharmacological activity is presented in Table 1.

Phytochemistry of Cochlospermum tinctorium
The biological activities of medicinal plants mainly depend on the presence of phytochemical contents and other bioactive compounds that contribute to the discovery and development of novel therapeutic agents. These secondary metabolites include alkaloids, glycosides, flavonoids, proanthocyanidins, tannins, terpenoids, phenylpropanoids, resins, lignans, furocoumarins, naphthodianthrones, proteins, and peptides [35]. Essential oils sourced from medicinal and other aromatic Fig. 1 The plant Cochlospermum tinctorium in its natural habitat adopted from [11] Ahmad et al. Future Journal of Pharmaceutical Sciences (2021) 7:20 Page 2 of 13 plants have also been described as a potential source of compounds for pharmaceutical and industrial use because of their efficacy and safety [35].

Preliminary phytochemical constituents presence in Cochlospermum tinctorium
Nergard et al. reported the presence of polyphenols, polysaccharides, gallotannins, and ferullic acid in the aqueous root extract of C. tinctorium [15]. Indeed, Tijjani et al. reported the presence of tannins, cardiac glycosides, and flavonoids in the methanol rhizome extract of C. tinctorium [24]. In 2012, Musa reported the presence of anthraquinones, cardiac glycosides, flavonoids, saponins, and carbohydrates in the 80% acetone fraction of C. tinctorium, while its n-butanol fraction showed the presence of cardiac glycosides, saponins, and carbohydrates [13]. The aqueous methanol (leaf, root, and root bark) extracts of the plant revealed the presence of saponins, flavonoids, tannins, steroids, cardiac glycosides, and alkaloids [20]. Etuk et al. also reported the presence of alkaloids, tannins, cardiac glycosides, saponins, flavonoids, triterpenes, cyanogenic glycosides, and volatile oils in the aqueous root extract of C. tinctorium [28]. Ndouyang and co-workers reported the presence of secondary metabolites such as tannins (total, hydrolysable, and condensed), phytates, oxalates, carotenoids, cyanides, alkaloids, flavonoids, and phenols in the root of C. tinctorium [18].

Carbohydrate composition of Cochlospermum tinctorium
The crude water extract of C. tinctorium revealed the presence of starch, glucose, arabinose, rhamnose, galactose, xylose, glucoronic acid, galacturonic acid, mannose, and fructose [14]. The isolated pectin polysaccharides from the aqueous extract were reported to be majorly rich in galactose A, arabinogalactan, homogalacturonan, rhamnogalacturonan I, and substituted galacturonan such as rhamnogalacturonan II [14].

Pharmacological Activities of Cochlospermum tinctorium
Different extracts obtained from various parts of C. tinctorium have been screened for biological activities including antiplasmodial, anti-ulcer, antioxidant, cytotoxic, antimicrobial, antitumor, antihelmintic, hepatoprotective and hepatocurative, antinociceptive, antidiarrheal, and antiepileptic potentials. The summary of the documented pharmacological activities of different extracts and fractions obtained from different parts of the plant is presented in Table 3.
The in vivo antimalarial activity of dichloromethane extract of C. tinctorium investigated against plasmodium berghei has shown a significant antiplasmodial activity (IC 50 = 17.59 mg/kg) [33]. The aqueous extract of C. tinctorium also produced effective antiplasmodial activity possibly due to the presence of triterpenes, carotenoids, and flavonoids [33].
The petroleum ether fraction of C. tinctorium (50, 100, and 200 mg/kg) produced non-dose-dependent antiplasmodial activity (80.67%, 64.14%, and 69.71%) respectively against Plasmodium berghei [34]. Indeed, the lowest dose of the fraction (50 mg/kg) demonstrated the highest antiplasmodial activity [34]. In the same study, the ethanolic fraction of C. tinctorium (25, 50, and 100 mg/kg) produced a significant and dose-dependent antiplasmodial activity (62.42%, 65.70%, and 98.17%) respectively [34]. The highest dose of the ethanol fraction (100 mg/kg) produced antiplasmodial activity in a similar manner to the standard drug, chloroquine (5 mg/kg) [34]. The mean percentage antiplasmodial activity of the ethanol fraction (62.90%) was slightly higher than the activity of the petroleum ether fraction (62.26%) [34]. Therefore, the ethanol fraction was reported to be more effective and potent than the petroleum ether fraction [34]. Additionally, the petroleum ether fraction (50, 100, and 200 mg/kg) produced non-dose-dependent mean packed cell volume (PCV) of 36.83%, 32.42%, and 37.96% respectively [34]. Similarly, the ethanolic fraction (25,50, and 100 mg/kg) revealed a mean PCV of 40.22%, 27.50,% and 43.04% respectively [34]. Therefore, it was suggested that the two fractions possess anemia ameliorating properties that may be associated with complication of malaria [34]. Benoit-Vical et al. reported the antiplasmodial activity of the identified compounds (3-tetradecanone and 3hexadecanone) in the essential oils obtained from whole tubercles, tubercle epiderma, and the central cylinder of C. tinctorium against two strains of Plasmodium falciparum (chloroquine resistant and chloroquine sensitive) [39]. The oil obtained from the tubercle epiderma produced the highest antiplasmodial activity (IC 50 = 5-53 μg/ml) [36]. In addition, the essential oil obtained from the C. tinctorium showed insecticidal activity against mosquitoes in West Africa [36]. These findings validate the traditional use of C. tinctorium as herbal medicine against malaria [5].
Anti-ulcer activity The antiulcer activity of C. tinctorium was investigated using hydrochloric acid (HCl)/ ethanol-induced gastric ulceration model in mice [15]. The mice were administered the crude aqueous extract of C. tinctorium 1 h prior to the administration of 0.24 ml of 0.3 M HCl-60% ethanol orally. The polysaccharide enriched crude root extract of C. tinctorium (25, 50, and 250 mg/kg) significantly inhibited the HCl/ethanol-induced gastric ulcer (11%, 20%, and 55%) respectively [15]. Additionally, the aqueous root extract of C. tinctorium and its most acidic fraction further demonstrated radical scavenging activities against 1,1-diphenyl-2picryl-hydrazyl (DPPH), which is associated with the anti-ulcer activity of the extract [15]. It was suggested that the ferulic acid and polysaccharide content of C. tinctorium may be responsible for the antiulcer and the radical scavenging activities of the extract [15]. The purified pectins of the extract also demonstrated a moderate induction of B cell proliferation, which demonstrated a possible immune-modulating function of the extract in its ulcer healing effect [15].
Inngjerdingen et al. reported the anti-adhesive activities of the aqueous root extract and isolated pectic polysaccharide fractions of C. tinctorium against Helicobacter pylori by in vitro flow cytometric assay using human gastric adenocarcinoma epithelial cells [14]. The extract and isolated fractions demonstrated significant anti-adhesive potential against H. pylori which is associated with anti-ulcer activity of C. tinctorium [14]. The ulcer-healing potential exhibited by the C. tinctorium confirmed its ability and usefulness as antiulcer agent in traditional medicine [14].
Antioxidant activity The antioxidant activity of the aqueous root extract of C. tinctorium was evaluated using DPPH radical scavenging activity, hydroxyl radical scavenging activity, and ferric iron-reducing activity [18]. The extract produced significant antioxidant activity against DPPH radical which could be related to the presence of soluble oxalates and phytates in the plant [18]. The extract also produced remarkable hydroxyl radical scavenging activity possibly due to the presence of phenolic compounds such as hydrolysable tannins, flavonoids, alkaloids, and carotenoids [18]. Also, the ferric ironreducing activity elicited by the extract was suggested to be due to the presence of oxalates, phytates, tannins, flavonoids, alkaloids, and carotenoids [18].
The methanol root extract of C. tinctorium produced minimum inhibitory concentration (MIC) against S. dysentriae (100 μg/ml), S. aureus and C. ulcerans (500 μg/ml), E. coli and P. mirabilis (1000 μg/ml), and K. pneumoniae (1500 μg/ml) [24]. Also, the extract revealed minimum bactericidal concentration (MBC) against S. dysentriae (500 μg/ml), S. aureus and C. ulcerans (1000 μg/ml), E. coli and P. mirabilis (1500 μg/ml), and K. pneumoniae (2000 μg/ml) [24]. The rate of bactericidal activity of the extract was further determined on the two most susceptible bacterial isolates (S. aureus and S. dysentriae) [24]. There was no viable S. aureus and S. dysentriae after 10 and 6 h of exposure to the extract respectively. Therefore, the extract showed higher rate of bactericidal activity against S. dysentriae than S. aureus [24]. The antibacterial activity of the methanol root extract of the plant could be due to the presence of alkaloids, flavonoids, tannins, and cardiac glycosides [24]. Flavonoids are known to possess effective antimicrobial activity against a wide range of microorganisms due to their ability to form complex with cellular proteins and bacterial cell walls [45]. Diallo et al. further reported the antimicrobial activity of carotenoids obtained from C. tinctorium against C. albicans, A. fumigatus, and E. coli [42]. The antibacterial activity of the methanol root extract of C. tinctorium was also evaluated against resistant food-borne strains of S. aureus and Listeria monocytogene isolated from onion, cabbage, lettuce, and tomato using agar diffusion method [25]. The extract produced the highest activity against antibiotic-resistant S. aureus isolates (22.00 mm) from tomato and L. monocytogene (21.00 mm) at a concentration of 10 mg/ml, whereas the lowest zone of inhibition was observed against S. aureus (12.00 mm) isolated from spring onion and L. monocytogene (13.00 mm) at a concentration of 2.5 mg/ml. The MIC and MBC of the extract against S. aureus and L. monocytogene isolates were found to be within the range of 0.625-2.5 mg/ml and 2.5-5 mg/ml respectively [25].
The above investigations suggested that the extracts of C. tinctorium and its fractions possess potential antimicrobial activities. However, the studies carried out concentrated on in vitro evaluations which cannot give assurance that the extracts and fractions of the plant could show same in vivo antimicrobial activities in an intact animal. Therefore, antimicrobial evaluations using in vivo animal models infected with specific microbial agents and other clinically isolated microbial strains are required to further validate these claims. Additionally, further investigations are necessary to investigate the effects of the plant against multi-drug resistant microbial strains and elucidate the specific cellular and molecular mechanisms of antimicrobial activities of the extracts and fractions to develop novel antimicrobial agents.
Furthermore, the antitumor activity of the arjunolic acid triacetate and arjunolic acid triacetate methylester were evaluated on two-stage carcinogenesis test using dimethylbenz[a]anthracen and 12-0-tetradecanoyl-phorbol-13-acetate as tumor-initiating and tumor-promoting compounds respectively [41]. The arjunolic acid compounds caused a substantial delay in the occurrence of papillomas, and reduced the number and the rate of the papillomas appearance compared to control group which demonstrated their tumor inhibitory effects [41].
A research conducted by Benoit et al. demonstrated effective cytotoxic activity (IC 50 = 360-500 μg/ml) of the aqueous leaf extract of C. tinctorium [32]. In the same study, the essential oils obtained from the leaves of C. tinctorium also produced cytotoxic activity (IC 50 = 1600-2000 μg/ml) [32].
The cytotoxic effects of 80% acetone and n-butanol extracts of C. tinctorium rhizomes (62.5, 125, 250, 500, and 1000 μg/ml) were reported using brine shrine lethality assay [13]. The n-butanol extract (LC 50 = 437 ± 8 μg/ml) was found to be twice less potent than the 80% acetone extract (LC 50 = 240 ± 3 μg/ml) due to the presence of more secondary metabolites responsible for the cytotoxic activities in the acetone extract compared to the nbutanol fraction [13]. The presence of flavonoids and tannins in the acetone fraction was suggested to be responsible for its higher potency and cytotoxic activity [13].
Muhammad et al. also reported the cytotoxic activities of the methanol root extract of C. tinctorium and its factions (n-hexane, ethyl acetate, and aqueous) using brine shrimp lethality test [26]. The n-hexane and ethyl acetate fractions demonstrated the highest potency (LC 50 = 1.175 μg/ml) followed by the methanol extract (LC 50 = 3.165 μg/ml). However, the aqueous fraction was the least potent (LC 50 = 15.019 μg/ml) [26].
Antihelminthic activity The antihelmintic activity of the ethanol shoot extract of C. tinctorium was evaluated against Terrestris lumbricoides, Taenia saginata, and Taenia solium using adult worm motility test with albendazole as a standard drug [23]. The extract (25, 50, and 100 mg/cm 3 ) produced an average paralysis time of 35.3, 57.0, and 72.0 min and death time of 47.2, 74.5, and 91.9 min respectively in a concentration-dependent manner against these parasites [23]. The extract demonstrated lower antihelmintic activity compared to the standard drug albendazole used at the same concentrations [23]. This pharmacological investigation is of importance in the traditional use of C. tinctorium as herbal drug against parasitic diseases such as schistosomiasis [21].
Hepatoprotective activity The aqueous roots extract of C. tinctorium (36 mg/kg, p.o) produced a promising hepatoprotective activity against aflatoxin B 1 (AFB 1 )-induced hepatotoxicity in albino rats [29]. The liver marker enzymes sorbitol dehydrogenase (SDH), serum glutamate pyruvic transaminase (SGFT), and serum glutamate oxalacetate transaminase (SGOT) significantly increased in the AFB 1 -treated rats when compared with the control group. The pretreatment of the rats with the C. tinctorium (36 mg/kg, p.o) for 72 h produced a significant reduction in the level of AFB 1 -induced elevation of the activities of the serum enzymes (SDH and SGFT) [29]. However, the extract produced non-significant decrease in the level of SGOT which is widely available in other tissues [29]. Also, the pretreatment of the rats with the extract produced significant protection against the AFB 1 -induced inhibition of microsomal benzphetamine N-demethylase and aniline hydroxylase [29]. However, the extract only produced considerable but nonsignificant protection against the loss of microsomal cytochrome P-450 induced by AFB 1 [29].
The methanol, ethanol, and aqueous extracts of C. tinctorium rhizomes were reported to have significant hepatoprotective activity against carbon tetrachloride (CCl 4 ) and galactosamine-induced cytotoxicity in cultured rat hepatocytes [21]. The methanol and ethanol extracts demonstrated higher antihepatotoxic effect than the aqueous extract as a result of the presence of tannins and polyphenol compounds (gallic and ellagic acid) [21]. Additionally, carotenoids were also present in the methanol fraction which also significantly prevented CCl 4 -induced hepatotoxicity [21]. It was suggested that the polyphenol compounds and carotenoids may account for the antihepatotoxic activity of the plant [21].
The hepatoprotective activity of the rhizome of C. tinctorium was further investigated using CCl 4 -induced toxicity and tert-butyl hydroperoxide in vitro induction of lipid peroxidation and hepatocyte lysis [27]. The aqueous, hydro-ethanolic, and ethanolic extracts of the plant showed significant hepatoprotective activities in a dose-dependent manner [27]. The ethanolic extract was found to be more potent than the standard drug, silymarin [27]. The ethanolic and hydro-ethanolic extracts also showed remarkable activities against the induction of lipid peroxidation and hepatocyte lysis, while the aqueous extract demonstrated a weaker activity [27].
The aqueous root extract of C. tinctorium (100, 200, and 300 mg/kg) significantly and dose-dependently reduced the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and blood clotting time, which indicated its ability to preserve hepatic function [30]. However, the levels of serum albumin, total protein, and reduced glutathione in the groups treated with the extract significantly elevated compare to the CCl 4 -treated group [30]. The highest hepatoprotective activity was observed in the group treated with the highest dose of the extract (300 mg/kg) which was better than the standard drug, silymarin [30]. In addition, the pretreatment of the animals with the aqueous extract of C. tinctorium progressively reduced the hepatic lesion and cellular damage [30]. The observed hepatoprotective action could possibly be related to the antioxidant activity of the phytochemical compounds such as flavonoids and tannins present in the extract [46].
Etuk et al. further reported the hepatocurative action of the aqueous root extract of C. tinctorium on experimentally induced liver damage following intraperitoneal administration of CCl 4 for 5 days in Wistar rats [28]. The oral administration of the aqueous root extract of C. tinctorium (200 mg/kg) for 7 days after induction of hepatotoxicity with CCl 4 caused a significant reduction in the blood clotting time, ALT, AST, ALP, and bilirubin [28]. The extract also increased the level of serum total protein, albumin, and reduced glutathione compared to the CCl 4 -treated group [28]. Also, the extract reversed the CCl 4 -induced hepatic damage [28].
The hepatocurative activity of the aqueous leaves extract of C. tinctorium (50-150 mg/kg) against acute liver damage following single intraperitoneal injection of CCl 4 was reported by Adam et al. [31]. The CCl 4 produced elevated level of ALT, AST, and cholesterol as a result of impairment of the structural integrity of the liver [31]. The treatment of the animals with the extract remarkably decreased the levels of the serum enzymes (ALT, AST, and ALP) and bilirubin and significantly increased the level of albumin [31]. The histopathological examination of the liver also revealed a dose-dependent regeneration and improvement on the structural integrity of the liver cells [31]. It was suggested that the hepatocurative potential of the extract may be attributed to the presence of tannins, carotenoids, gallic, and ellergic acids [31].
In 2012, Akinloye and Ayankojo reported a significant reduction in the levels of SGFT, SGOT, cholesterol, urea, and bilirubin after oral administration of methanol leaf extract of C. tinctorium (200 mg/kg) in CCl 4 -induced hepatotoxicity [6]. The extract also remarkably prevented lipid peroxidation of hepatocytes demonstrated by a significant reduction in the level of malondialdehyde (MDA) compared to the CCl 4 -treated [6]. Additionally, the extract demonstrated evidence of hepatocytes regeneration which gives an evidence of its moderate hepatoprotective activity against CCl 4 -induced liver damage [6].
Analgesic activity The analgesic activity of the aqueous methanol leaf extract (AMLE), root bark extract (AMRBE), and root extract (AMRE) of C. tinctorium were reported by Ahmed and colleagues using acetic acid-induced writhing and hot plate tests in mice [20]. The extracts produced significant and dose-dependent protection against the acetic acid-induced abdominal writhing [20]. The AMLE showed the highest protection against the acetic acid-induced abdominal constrictions at the dose of 80 mg/kg which was greater than of the standard drug, ketoprofen (82.30%) [20]. The AMRBE produced a non-dose-dependent increase in the mean latency of pain response in hot plate test which was only significant at the dose of 20 mg/kg [20].
Anti-inflammatory activity Ahmed et al. reported the anti-inflammatory activity of C. tinctorium using carrageenan-induced paw edema in rats. The AMRE and AMRBE of C. tinctorium produced a non-dose-dependent protection against the carrageenan induced-paw edema [20]. Also, the AMLE significantly and dose-dependently inhibited the carrageenan induced-hind paw edema. The highest inhibitory effect of the extract was observed in the group treated with the highest dose of the extract (80 mg/kg) [20].

Anticonvulsant activity
The anticonvulsant activity of the hydro-alcoholic root bark extract of C. tinctorium (5, 10, and 20 mg/kg) was investigated using maximal electroshock (MES) test in chicks, pentylenetetrazole (PTZ), and strychnine-induced seizures in mice [19]. The extract at all the doses did not produce any protection against MES and strychnine-induced seizures [19]. However, the extract showed non-significant protection (20%) against PTZ-induced seizures at the highest dose (20 mg/kg) [19]. Therefore, the extract may not have potential in the management of generalized tonic-clonic and absence or clonic seizures [19]. Therefore, based on the reported traditional use of C. tinctorium in the management of epilepsy, other parts of the plant are recommended to be thoroughly screened in order to validate its claimed antiepileptic activity as reported in traditional medicine using other different solvents in order to obtain extracts or fractions that may contain potential antiepileptic compounds.
Antidiarrhoeal activity The antidiarrhoeal potential of the aqueous methanol leaf extract of C. tinctorium (20,40, and 80 mg/kg) was evaluated using castor oilinduced diarrhea in mice and isolated rabbit jejunum preparation [22]. The extract demonstrated significant and non-dose-dependent inhibition of diarrhea [22]. The highest (80 mg/kg) and lowest dose (20 mg/kg) of the extract produced 100% protection against diarrhea compared to the standard drug, loperamide (5 mg/kg) [22]. The extract also inhibited spontaneous muscle contraction of the rabbit jejunum at all the concentration tested (0.32-3.2 mg/ml). This study confirmed the traditional use of the plant in the management of diarrhea [22].

Toxicology
Herbal medicines have gained patronage in developing and developed countries as a result of their effectiveness and safety [47]. However, the safety of the medicinal plants could not be guaranteed despite their wide use [47]. Many scientific studies have shown that many of the herbal plants used as food or medicines are potentially toxic, mutagenic, and carcinogenic [47]. Therefore, it is essential to evaluate and document their safety to determine the consequences of their long use for drug development.
Acute oral administration of the aqueous root extract of C. tinctorium did not produce any death and symptoms of toxicity at the dose of 5000 mg/kg which suggested that the extract is relatively safe following acute oral administration [48]. The intraperitoneal median lethal doses (LD 50 ) of the AMRE, AMLE, AMRBE, and hydro-alcoholic root extract were reported to be 118.32, 288.53, 288.53, and 118.32 mg/kg respectively [19,20].
It is well known that researches always focus to discover and develop potent, efficacious, and safe bioactive compounds because chemical substances obtained from the plants play a key role in the development of conventional drugs [35]. Based on the reported therapeutic potentials of C. tinctorium in the treatment of various diseases in African countries, to date, substantial toxicity studies on the extracts, fractions, and isolated compounds from the plant are lacking to support its safety. Therefore, it is highly recommended for a throughput long term (sub-acute, sub-chronic, chronic, carcinogenic, mutagenic, and teratogenic) toxicity studies on the extracts, fractions, and isolated compounds from C. tinctorium to be conducted in animal models to further increase confidence in its use and ascertain its safety for drug development. It is also worthy to note that the pharmacokinetics (absorption, distribution, metabolism, and excretion) studies have not been investigated.

Conclusion
The plant C. tinctorium is an important medicinal plant with promising therapeutic potentials. Previous pharmacological investigations conducted on C. tinctorium provided supportive evidence for some of the documented traditional uses of the plant. However, many of the reported traditional uses of C. tinctorium still lack scientific evidence; hence, the need for more scientific investigations to authenticate the folkloric uses of the plant. Moreover, among the isolated bioactive compounds of C. tinctorium, only few were screened for biological activities. Therefore, extensive phytochemical and further pre-clinical studies could be done in the future to fully establish its therapeutic potentials and elucidate its detailed mechanisms of pharmacological actions. Finally, there is lack of information on safety profile of C. tinctorium. Therefore, toxicological studies on the extracts, fractions, and isolated bioactive compounds of C. tinctorium should be performed to further ascertain their safety aspect.