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In this study, the effect of the methanol extract of Indigofera pulchra Willd. (Papillionaceae) was investigated against castor oil induced diarrheoa. Its effects on perfused isolated rabbit jejunum and guinea pig ileum were also evaluated. The extract produced a dose-dependent protection against the castor oil-induced diarrheoa in mice with the highest protection (100%), obtained at 200 mgkg−1 comparable to that of loperamide (5 mgkg−1), a standard antidiarrhoeal drug. The extract (0.4 – 6.4 mgml−1) produced a concentration relaxation of the rabbit jejunum. However, no observable effect was noticed when the guinea pig ileum was treated. The extract blocked the contractile effect of acetylcholine (2 × 10−8 gml−1) and histamine (4 × 10−7 gml−1) on both rabbit jejunum and guinea pig ileum. Phytochemical screening revealed the presence of flavonoids, tannins, saponins and steroids. The intraperitoneal median lethal dose (LD50) value for the extract was found to be 2154.0 mgkg−1. The results obtained revealed that the extract possesses pharmacologically active compounds with gastrointestinal relaxant and antidiarrhoeal activities and may possibly explain the use of the plant in traditional medicine for the treatment of gastrointestinal disorder.
Indigofera pulchra (Willd) family: Papilionaceae is an erect stiff, grey-pubescent softly wooded under-shrub that grows up to 1–1.5m high. It is widely distributed from Senegal to Southern Nigeria and over Eastern and central Africa from Ethiopia to Angola (Burkhill, 1995). In ethnomedicine, the leaves are used to treat infected wound (Herper, 1976; Burkill, 1995), while the decoction of the aerial part is used as prophylactic against snake-bite (Sule et al., 2003), anti-inflammatory (Abubakar et al., 2007) and to treat gastrointestinal disturbances (Personal communication). Previous pharmacological studies on the methanol extract of this plant revealed that it exhibited venom detoxifying (Abubakar et al., 2006) and antimicrobial (Musa et al., 2007) activities. To our knowledge, there is no report in the literature on antispasmodic and antidiarrhoeal activities of the methanol extract of Indigofera pulchra aerial parts. The present study was therefore carried out to investigate the effects of the extract on gastrointestinal smooth muscles, so as to validate the use of the plant in traditional medicine for the treatment of diarrhoea and other gastrointestinal disturbances.
Indigofera pulchra sample was collected from Samaru-Zaria, Kaduna state, Nigeria in the month of September, 2005 and was authenticated by Mallam Musa of the Biological Sciences Department, Ahmadu Bello University Zaria-Nigeria where a voucher specimen (No.410) was deposited. The aerial part was cut, dried under shade and made into powder using pestle and mortar and subsequently referred to as powdered plant material.
The powdered plant material (500 g) was extracted with 80% methanol using soxhlets apparatus, the solvent was removed in-vacuo to yield a residue (56 g) referred to as Indigofera pulchra extract (IPE).
New Zealand rabbit (2.25 kg), Guinea-pig (0.75 Kg) and Swiss albino mice (18–25 g) maintained in the animal house facility of the Department of Pharmacology and Clinical Pharmacy, Ahmadu Bello University, Zaria, Nigeria, were used in these experiments. The animals were maintained on standard animal feeds and water ad libitum. This research was carried out in Ahmadu Bello University, Zaria-Nigeria in accordance with the rules governing the use of laboratory animals as accepted internationally.
Acetylcholine (Sigma, USA), Histamine (Sigma, USA), castor oil (Bell Sons and Co., England) and loperamide (Janssen, Germany).
The methanol extract of Indigofera pulchra was subjected to phytochemical screening using standard procedures (Silva et al., 1998).
The method previously described by Lorke (1983) was adopted. Briefly, 12 mice were used. In the first phase, three doses of the Indigofera pulchra extract (10, 100 and 1000 mgkg−1) were administered to three groups each containing three mice. In the second phase, three doses of the extract 1600, 2900 and 5000 mgKg−1 were administered to three groups each containing one mouse. All treatment was by intraperitoneal route .The median Lethal dose (LD50) was determined as the geometric mean of the highest non lethal dose and the lowest lethal dose of which there is 1/1 and 0/1 survival.
The rabbit was sacrificed by a blow on the head. Segment of the jejunum, about 3.0 cm long was removed and dissected free of adhering mesentery. The intestinal contents were removed by flushing with Tyrode solution of the following compositions in millimoles (mM): NaCl, 136.8; KCl, 2.7; CaCl2, 1.3; NaHCO3, 12.0; MgCl2, 0.5; NaPO4, 0.14; glucose, 5.5. The tissue was mounted in a 25ml organ bath containing Tyrode solution maintained at 37°C and aerated with air. An initial tension of 0.5 g was applied to the segments and 60 minutes equilibration period was allowed with the physiological solution changed every 15 minutes. At the end of equilibration period, the effect of acetylcholine (2.0 × 10−8 – 1.6 × 10−7 gml−1), histamine (4.0 × 10−4 – 1.6 × 10−3 mgml−1) and the methanol extract of Indigofera pulchra (0.04 – 0.64 mgml−1) were investigated non-cumulatively. The contact time for each concentration was one minute which was followed by washing three times. The effect of the extract on the contractile response induced by acetylcholine (2 × 10−5 gml−1) and histamine (4 × 10−4 gml−1) were also determined. The tissue was allowed a resting period of 15 minute before the next addition (Amos et al., 1998).
Adult guinea pig was starved overnight but had free access to water. The animal was killed by a blow on the head and exsanguinated. The abdomen was opened and segment of about 3cm was taken from portion situated 10 to 30 cm proximal to the ileocaecal junction, was dissected free of adhering mesentery and mounted in a 25ml organ bath maintained at 37 ° C. The tissue was equilibrated for 60 min with the bathing solution replaced every 10 min. At the end of the equilibration period, the effect of the extract (0.04 – 6.4 mgml−1) on the ileum was evaluated. The effect of histamine (0.4–3.2 × 10−3 mgml−1) on the ileum was also determined. The effects of the extract on contractile responses induced by acetylcholine (2 × 10−5 mgml−1) and histamine (4 × 10−4 mgml−1) were also determined. Responses were recorded on Ugo Basile Unirecorder 7050. Determinations were done in quadruplicate (Amos et al., 1998)
Phytochemical screening revealed the presence of tannins, flavonoids, saponins and steroids.
The intraperitoneal median lethal dose (LD50) of the extract was found to be 2154.0 mgkg−1.
The mice were fasted for 12 h prior to the commencement of the study and were randomly divided into five groups each containing five mice. The mice were treated with normal saline (10 mlkg−1), the methanol extract (50–200 mgkg−1) or loperamide (5 mgkg−1). All treatment was by intraperitoneal (i.p) route. 30 minutes post-treatment, castor oil (0.2 ml / mouse) was given intragastrically. The animals were then placed in individual cages on a clean filter paper. Three hours after the castor oil treatment, the cages were inspected for the presence of characteristic diarrhoeal droppings; absence of which was regarded as protection (Diurno et al., 1996).
The result of the castor oil-induced diarrhoea was analysed using Chi-square test while those of the in vitro studies were analysed by ANOVA followed by Dunnet t-test for multiple comparisons. Differences were regarded as significant at P< 0.05.
The extract (0.4 – 6.4 mgml−1) produced a significant reduction in the amplitude of spontaneous contraction of the smooth muscles of the rabbit jejunum (Fig. 1). The extract also blocked the contractile effect of acetylcholine (2 × 10−5 mgml−1) and histamine (4 × 10−4 mgml−1) (Figure 2).
The extract did not produce any noticeable effect on spontaneous contraction of the guinea- pig ileum. However, it produced a reduction of spontaneous contraction of the guinea pig ileum induced by acetylcholine and histamine. The contractile effect of both acetylcholine (2 × 10−5 mgml−1) and histamine (4 × 10−4 mgml−1) were antagonized in a concentration dependent manner by the extract (Figure 3).
The extract at doses of 50, 100 and 200 mgKg−1 protected mice against castor oil induced diarrhoea by 60, 80 and 100% respectively. Loperamide (5mgKg−1) produced 100% protection (Table 1). All values were significant at P<0.05.
The results of this study suggest that Indigofera pulchra extract possesses antidiarrhoeal and antispasmodic activities. The extract exhibited significant and dose-dependent anti-diarrhoeal activity against castor oil-induced diarrhoea in mice. The extract showed similar activity to that of loperamide when tested at 200 mgkg−1. Loperamide, a drug widely used in the management of diarrhoeal disorders effectively antagonizes diarrhoea induced by castor oil (Niemegeers et al., 1974). The major constituent of castor oil is ricinoleate (Mekeon et al., 1999) which is metabolized to its active metabolite, ricinoleic acid. Ricinoleic acid is responsible for the diarrhoea inducing property of castor oil (Gaginella and Philips, 1975). It stimulates peristaltic activity in the small intestine, leading to changes in the electrolyte permeability of the intestinal mucosa. Its action also involves stimulation of release of endogenous prostaglandin (Galvez et al., 1993).
The portal venous PGE2 concentration was found to be significantly increased following oral administration of castor oil to experimental rats (Luderer et al., 1980). Ricinoleic acid markedly increased the PGE2 content in the gut lumen and also caused an increase in the net secretion of water and electrolytes into the small intestine (Beubler and Juan, 1979). It is possible to suggest here that the anti-diarrhoeal effect of the extract may be due to the inhibition of prostaglandin biosynthesis. Early studies have reported that anti-diarrhoeal activity of medicinal plants may be due to alkaloids, saponins, tannins, sterols and reducing sugar (Galvez et al., 1991; Galvez et al., 1993; Longanga et al., 2000). The preliminary phytochemical screening of methanol extract of Indigofera pulchra revealed the presence of saponins, tannins, flavonoids and steroids among other phytochemical constituents. The anti-diarrhoeal activity of the extract is most likely due to the presence of flavonoids, tannins, saponins either singly or in combination. The observed relaxant effects of the extract against contractile response due to histamine and acetylcholine suggests that its antispasmodic effect may involve both histaminergic and cholinergic mechanism. This further corroborates the ability to protect the mice against diarrhoea induced by castor oil. This study therefore suggests that Indigofera pulchra extract possesses anti-diarrhoeal and antispasmodic properties. Further work is going on in our laboratory to elucidate the mechanism of gastro-intestinal activities of the extract.