Preliminary pharmacological screening of Bixa orellana L. leaves

Journal of Ethnopharmacology 108 (2006) 264–271

Preliminary pharmacological screening of Bixa orellana L. leaves Jamil Ahmad Shilpi a , Md. Taufiq-Ur-Rahman b , Shaikh Jamal Uddin c , Md. Shahanur Alam c , Samir Kumar Sadhu d , V´eronique Seidel a,∗ a

Phytochemistry Research Laboratories, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, UK b Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, UK c Pharmacy Discipline, Life Science School, Khulna University, Khulna 9208, Bangladesh d Laboratory of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan Received 29 September 2005; received in revised form 26 April 2006; accepted 13 May 2006 Available online 12 June 2006

Abstract Preliminary pharmacological studies were performed on the methanol extract of Bixa orellana L. (Bixaceae) leaves to investigate neuropharmacological, anticonvulsant, analgesic, antidiarrhoeal activity and effect on gastrointestinal motility. All studies were conducted in mice using doses of 125, 250 and 500 mg/kg of body weight. In the pentobarbitone-induced hypnosis test, the extract statistically reduced the time for the onset of sleep at 500 mg/kg dose and (dose-dependently) increased the total sleeping time at 250 and 500 mg/kg dose. A statistically significant decrease in locomotor activity was observed at all doses in the open-field and hole-cross tests. In the strychnine-induced anticonvulsant test, the extract increased the average survival time of the test animals (statistically significant at 250 and 500 mg/kg). The extract significantly and dosedependently reduced the writhing reflex in the acetic acid-induced writhing test. Antidiarrhoeal activity was supported by a statistically significant decrease in the total number of stools (including wet stools) in castor oil-induced diarrhoea model. A statistically significant delay in the passage of charcoal meal was observed at 500 mg/kg in the gastrointestinal motility test. The extract was further evaluated in vitro for antioxidant and antibacterial activity. It revealed radical scavenging properties in the DPPH assay (IC50 = 22.36 ␮g/ml) and antibacterial activity against selected causative agents of diarrhoea and dysentery, including Shigella dysenteriae. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Bixa orellana; Neuropharmacological activity; Analgesic activity; Antioxidant activity; Antidiarrhoeal activity; Antibacterial activity; Charcoal motility test

1. Introduction Bixa orellana L. (Bixaceae), known as the annatto plant, is a small evergreen tree native of the Central and Southern American rain forests (Perry, 1980). In Bangladesh, it is commonly called ‘Latkan’ and is cultivated to a small extent in the forests of Dhaka and Chittagong (Yusuf et al., 1994). This species enjoys wide folkloric uses in India, China, the Philippines, Brazil and Guiana (Kirtikar and Basu, 1999). The plant is alexipharmic; useful in headaches, blood disorders, as an anti-emetic and to

∗ Correspondence to: Department of Pharmaceutical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, UK. Tel.: +44 141 548 2751; fax: +44 141 552 6443. E-mail address: [email protected] (V. Seidel).

0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.05.008

allay thirst. The seeds are cordial, astringent, febrifuge and a good remedy for gonorrhoea (Yusuf et al., 1994; Kirtikar and Basu, 1999). The root bark is also useful in gonorrhoea (Yusuf et al., 1994; Ghani, 2003) and as an antiperiodic and antipyretic (Kirtikar and Basu, 1999). An infusion of the leaves and roots is useful in epilepsy, dysentery, fever and jaundice (Perry, 1980; Joshi, 2000; Ghani, 2003). Local people in the district of Khulna in Bangladesh use the leaves of the plant for a range of illnesses including diarrhoea, sleeplessness and skin diseases. Previous phytochemical investigations have revealed the presence of several carotenoid derivatives including bixin and norbixin (Satyanarayana et al., 2003), some terpenoids, tocotrienols, arenes and flavonoids (including luteolin and apigenin) in Bixa orellana seeds (Jondiko and Pattenden, 1989; Frega et al., 1998; Pino and Correa, 2003). Investigations on

J.A. Shilpi et al. / Journal of Ethnopharmacology 108 (2006) 264–271

Bixa orellana leaves have revealed the presence of flavonoid bisulfates (Harborne, 1975) and of an essential oil comprising mainly sesquiterpenes with ishwarane as the major compound (Lawrence and Hogg, 1973). The roots have been found to contain the triterpene tomentosic acid (Schneider et al., 1965). Previous pharmacological studies have revealed that Bixa orellana extracts possess antiprotozoal, anthelmintic and platelet antiaggregant activity (Villar et al., 1997; Barrio et al., 2004). Root extracts have been reported to have spasmolytic activity (Mans et al., 2004). Extracts of leaves and branches have shown to be effective at neutralising the effects of snake venoms (Nunez et al., 2004). Extracts from different parts (leaves and seeds in particular) have displayed in vitro antimicrobial activity (Irobi et al., 1996; Castello et al., 2002; Fleischer et al., 2003). The seed extracts have been reported to exhibit chemopreventive (Agner et al., 2005) and antioxidant activity (Martinez-Tome et al., 2001). Bixin has also been found to have anticlastogenic activity (Antunes et al., 2005). With a view to find the pharmacological rationale for some of the reported and traditional uses of the plant, the methanol extract of Bixa orellana leaves was evaluated for neuropharmacological, anticonvulsant, analgesic, antidiarrhoeal activity and for its effect on gastrointestinal transit in mice. It was also studied for its antibacterial activity, against selected bacterial species known to be associated with diarrhoea and dysentery, and for its ability to neutralise free radicals in vitro. 2. Materials and methods 2.1. Plant material and extraction The leaves of Bixa orellana L. (Bixaceae) were collected from plants growing in the district of Khulna, Bangladesh, in October 2003 and identified by the experts in the Bangladesh National Herbarium where a voucher specimen has been deposited (DACB 30554). The dried and coarsely powdered leaves (400 g) were extracted with methanol at room temperature for 72 h. The filtrate was evaporated to dryness under reduced pressure (45 ◦ C) to afford the crude extract (yield ca. 6%) used in pharmacological screening. 2.2. Animals Swiss Albino mice (weighing 20–25 g) of either sex, obtained from the Animal Resources Branch of the International Center for Diarrhoeal Disease and Research, Dhaka, Bangladesh (ICDDR, B), were used for the evaluation of neuropharmacological, anticonvulsant, analgesic and antidiarrhoeal activity and for the evaluation of the effect on gastrointestinal motility. The animals were housed under standard laboratory conditions (relative humidity 55–65%, room temperature 23.0 ± 2.0 ◦ C and 12-h light:12-h dark cycle). The animals were fed with a standard diet and water ad libitum. In all animal experiments, the guidelines of the Animal Experimentation Ethics Committee, ICDDR, B were followed.

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2.3. Microorganisms The test organisms, Escherichia coli, Staphylococcus aureus, Shigella dysenteriae, Shigella boydii, Shigella flexneri and Shigella sonnei, obtained from laboratory stocks of the Microbiology Section of the Pharmacy Discipline, Life Science School, Khulna University, were used to evaluate antibacterial activity. 2.4. Preliminary phytochemical analysis The crude extract of Bixa orellana leaves was subjected to a preliminary phytochemical screening for the presence of steroids, flavonoids, tannins and saponins (Harborne, 1984). 2.5. Acute toxicity test Test animals were divided into groups (n = 6 per group) which were administered different doses of the crude extract (62.5, 125, 250, 500, 1000, 2000 and 4000 mg/kg p.o.), while the control group received only the vehicle (1% Tween 80 in water, p.o.). The general signs and symptoms of toxicity were observed for 24 h and mortality was recorded for each group at the end of this period (Lorke, 1983). 2.6. Evaluation of neuropharmacological activity 2.6.1. Evaluation of effect on pentobarbitone-induced sleeping time The pentobarbitone-induced hypnosis test was used following the method described by Ramirez et al. (1998). The animals were randomly divided into control, positive control and test groups (n = 6 per group). The test group received the crude extract (at the doses of 125, 250 and 500 mg/kg p.o.). The positive control group was treated with diazepam (1 mg/kg i.p.) and the control group received vehicle (1% Tween 80 in water at the dose of 10 ml/kg p.o.). Thirty minutes later, pentobarbitone (50 mg/kg i.p.) was administered to each mouse to induce sleep. The animals were observed and the latent period (time between the pentobarbitone administration and the onset of sleep) as well as the duration of sleep (time between the loss and recovery of the righting reflex) were recorded. 2.6.2. Open- field test This experiment was carried out as described by Gupta et al. (1971). The animals were divided into control and test groups (n = 6 per group). The control group received vehicle (1% Tween 80 in water at the dose of 10 ml/kg p.o.). The test group received the crude extract (at the doses of 125, 250 and 500 mg/kg p.o.). The animals were placed on the floor of an open field (100 cm × 100 cm × 40 cm h) divided into a series of squares. The number of squares visited by each animal was counted for 3 min on 0, 30, 60, 90, 120, 180 and 240 min during the study period.

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2.6.3. Hole-cross test The method used was done as described by Takagi et al. (1971). A steel partition was fixed in the middle of a cage (30 cm × 20 cm × 14 cm h). A hole (diameter 3 cm) was made in the steel partition at a height of 7.5 cm above the floor of the cage. The animals were divided into control and test groups (n = 6 per group). The control group received vehicle (1% Tween 80 in water at the dose of 10 ml/kg p.o.) whereas the test group received the crude extract (at the doses of 125, 250 and 500 mg/kg p.o.). Each animal was then placed on one side of the chamber and the number of passages of each animal through the hole from one chamber to the other was recorded for 3 min on 0, 30, 60, 90, 120, 180 and 240 min during the study period. 2.7. Evaluation of anticonvulsant activity The anticonvulsant activity test was followed according to the method described by Gupta et al. (1999). The animals were divided into control and test groups (n = 6 per group). The control group received vehicle (1% Tween 80 in water at a dose of 10 ml/kg p.o.) while the test group received the crude extract (at the doses of 125, 250 and 500 mg/kg p.o.) 45 min before i.p. injection of strychnine (2 mg/kg). The average survival time (min) and the percentage of mortality after 24 h were recorded. 2.8. Evaluation of analgesic activity The acetic acid-induced writhing test according to the method of Koster et al. (1959) was used. The crude extract (at the doses of 125, 250 and 500 mg/kg) was orally administered to mice (n = 6) 45 min before i.p. injection of 0.6% (v/v) acetic acid at a dose of 10 ml/kg, while vehicle (1% Tween 80 in water) was used as control treatment. The positive control group received acetylsalicylic acid at the dose of 100 mg/kg p.o. After a 5 min interval for proper absorption of acetic acid, animals were observed for specific body contractions (writhings) which were recorded for 15 min.

each) were mixed with an ethanolic solution of DPPH (0.004%, 1 ml) and allowed to stand at room temperature for 30 min. The UV absorbance of the resulting solutions was recorded at λ = 517 nm (Yan et al., 1998). The experiment was performed in triplicate and the average absorption was noted for each concentration. Ascorbic acid was used as the positive control. The free radical-scavenging activity was calculated as a percentage inhibition of the DPPH radical by a sample or ascorbic acid according to the formula:   AC(o) − AA(t) × 100 % Inhibition = AC(o) where AC(o) is the absorbance of the background control at t = 0 min and AA(t) is the absorbance of the solution containing the antioxidant at a particular concentration at t = 30 min. The IC50 value is the concentration of the sample required to scavenge 50% DPPH. 2.10. Evaluation of antidiarrhoeal activity The castor oil-induced diarrhoea test was used for this study according to the method described by Abdullahi et al. (2001). Animals were divided into control, positive control and test groups (n = 6 per group). The control group received vehicle (1% Tween 80 in water at a dose of 10 ml/kg p.o.). The positive control group received loperamide (at a dose of 3 mg/kg p.o.). The test group received the crude extract (at the doses of 125, 250 and 500 mg/kg p.o.). This was carried out 30 min prior to oral administration of the diarrhoea-inducing castor oil (0.5 ml to each mouse). Each animal was placed in an individual cage, the floor of which was lined with blotting paper. The floor lining was changed every hour. During an observation period of 4 h, the total number of faecal output and the number of diarrhoeic faeces excreted by the animals were recorded. A numerical score based on stool consistency was assigned as follows: normal stool = 1, semi solid stool = 2 and watery stool = 3. 2.11. Evaluation of effect on gastrointestinal motility

2.9. Evaluation of radical scavenging activity 2.9.1. Qualitative determination by thin-layer chromatography (TLC) The antioxidant activity was evaluated by measuring the reduction of the free radical 2,2-diphenyl-1-picryl-hydrazyl (DPPH). Samples (0.2 mg in 10 ␮l) were applied on a TLC plate (Si gel 60 F254 , 0.2 mm thickness, Merck) developed in n-hexane–ethyl acetate (2:1) and sprayed with an ethanolic solution of DPPH (Aldrich Chemicals, USA) of a concentration sufficient to give a violet colour to the plate (approximately 0.4 mg/ml). Samples containing radical scavengers were identified as yellow spots on the plate (Xiong et al., 1996). 2.9.2. Quantitative determination A stock solution (5 mg/ml) of the crude extract was prepared in ethanol. Serial dilutions were carried out to obtain concentrations of 1, 5, 10, 50, 100 and 500 ␮g/ml. Diluted solutions (1 ml

The method described by Abdullahi et al. (2001) was adopted to evaluate the effect of the crude methanol extract on the gastrointestinal transit in mice. The test animals were starved for 24 h prior to the experiment but were allowed free access to water. The animals were divided into control, positive control and test groups (n = 6 per group). Control group received vehicle (1% Tween 80 in water at a dose of 10 ml/kg p.o.). Positive control group received atropine sulphate (at the dose of 0.1 mg/kg i.p.), test groups received the crude extract (at the doses of 125, 250 and 500 mg/kg p.o.). After 30 min, mice of each group were fed with 1 ml of charcoal meal (3% suspension of deactivated charcoal in 0.5% aqueous methyl cellulose). After 30 min of the administration of charcoal meal, the animals of each group were sacrificed. The length of the intestine (pyloric sphincter to caecum) and the distance traversed by charcoal as a fraction of that length was measured. The charcoal movement in the intestine was expressed as a percentage.

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2.12. Evaluation of antibacterial activity Sterile 6.0 mm diameter blank discs (BBL, Cocksville, USA) were impregnated with test substances at the dose of 500 ␮g/disc. These discs, along with standard discs (Kanamycin, Oxoid Ltd., UK) and control discs, were placed in Petri dishes containing a suitable agar medium seeded with the test organisms and kept at 4 ◦ C to facilitate maximum diffusion. Following incubation at 37 ◦ C, antibacterial activity of the test agents was determined by measuring the diameter of the zone of inhibition around the discs (recorded in millimeters) (Bauer et al., 1966).

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Table 1 Effect of Bixa orellana leaves extract on pentobarbitone-induced sleeping time in micea Treatment

Dose (mg/kg, route of administration)

Latent period (min)

Duration of sleep (min)

Control Diazepam

10b , p.o. 1, i.p.

6.38 ± 0.28 4.07 ± 0.36**

58.45 ± 1.70 87.25 ± 2.70**

Bixa orellana extract

125, p.o.

5.57 ± 0.31

63.98 ± 1.49

250, p.o. 500, p.o.

5.07 ± 0.28 3.73 ± 0.22**

76.70 ± 2.47** 90.82 ± 3.18**

** P < 0.01

2.13. Statistical analysis

a b

vs. control; one-way ANOVA followed by Dunnett’s test. Values are mean ± S.E.M. (n = 6). In ml/kg.

All data were expressed as mean ± S.E.M. One-way ANOVA followed by Dunnett’s multiple comparison tests was used to analyse the data obtained from in vivo experiments, except for the open-field and hole-cross experiments. For the latter two tests, two-way ANOVA followed by Bonferroni post tests was adopted. All statistical analyses were performed with Prism 4.0 (GraphPad software Inc., San Diego, CA). P < 0.05 was considered to be significant. 3. Results 3.1. Preliminary phytochemical analysis and acute toxicity Results of the preliminary phytochemical analysis carried out on the crude methanol extract indicated the presence of sterols, flavonoids, tannins and saponins. No lethal effects were observed within 24 h after the administration of the extract at any of the doses used, even at the highest dose tested (4000 mg/kg). Therefore, the lethal dose (LD50 ) of the extract in mice could not be determined. Dose levels of 125, 250 and 500 mg/kg body weight were chosen for the pharmacological screening.

Fig. 1. Effect of Bixa orellana leaves extract on locomotor activity in the openfield test. Each bar represents values as mean ± S.E.M. (n = 6). *** P < 0.001 vs. control; two-way ANOVA followed by Bonferroni post tests.

3.2. Neuropharmacological activity 3.2.1. Pentobarbitone-induced hypnosis test In the pentobarbitone-induced hypnosis test, Bixa orellana leaves extract was found to reduce the time for onset of sleep compared to the control with results statistically significant only at 500 mg/kg dose levels. The extract was also found to increase the duration of sleep in the test animals compared to the control with statistical significance at 250 and 500 mg dose groups (Table 1). 3.2.2. Open-field test In the open-field test, Bixa orellana leaves extract exhibited a decrease in the movements of the test animals at all dose levels tested. The results were statistically significant for all doses and followed a dose-dependent response (Fig. 1). 3.2.3. Hole-cross test Results of the hole-cross test followed a similar trend to the ones observed in the open-field test. They were statistically significant for all dose levels and followed a dose-dependent

Fig. 2. Effect of Bixa orellana leaves extract on locomotor activity in the hole-cross test. Each bar represents values as mean ± S.E.M. (n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control; two-way ANOVA followed by Bonferroni post tests.

response. The depressing effect was most intense during the second (60 min) and third (90 min) observation periods (Fig. 2). 3.3. Anticonvulsant activity In the anticonvulsant activity study, Bixa orellana leaves extract significantly increased the average survival time of the

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Table 2 Effect of Bixa orellana leaves extract on average survival time in strychnineinduced convulsions in micea Treatment

Dose (mg/kg, route of administration)

Survival time (min) after strychnine administration

Control

10b , p.o.

5.38 ± 0.49

Bixa orellana extract

125, p.o. 250, p.o. 500, p.o.

5.60 ± 0.32 7.33 ± 0.21* 10.68 ± 0.73**

Table 4 In vitro radical scavenger activity of Bixa orellana leaves extract and ascorbic acid at different concentrations (1, 5, 10, 50, 100 and 500 ␮g/ml) on 2,2-diphenyl1-picrylhydrazyl (DPPH) free radical Sample

Concentration (␮g/ml)

% Inhibitiona

Bixa orellana extract

01 05 10 50 100 500

17.59 26.67 39.13 60.07 82.29 90.58

± ± ± ± ± ±

0.0031 0.0037 0.0017 0.0018 0.0026 0.0035

22.36

01 05 10 50 100 500

31.99 51.67 68.50 94.71 96.67 98.53

± ± ± ± ± ±

0.0015 .0023 0.0064 0.0026 0.0029 0.0026

4.33

* P < 0.05, ** P < 0.01, a b

vs. control; one-way ANOVA followed by Dunnett’s test. Values are mean ± S.E.M. (n = 6). In ml/kg.

Ascorbic acid

test animals after strychnine-administration at the doses of 250 and 500 mg/kg. It failed to prevent the mortality of the test animals to any degree (Table 2). 3.4. Analgesic activity In the acetic acid-induced writhing test, Bixa orellana leaves extract, given at the doses of 125, 250 and 500 mg/kg, significantly (P < 0.001) and dose-dependently displayed an analgesic effect by inhibiting the frequency of acetic acid-induced abdominal constrictions in mice (Table 3). 3.5. Radical scavenging activity 3.5.1. Qualitative determination by thin-layer chromatography In the TLC-based qualitative antioxidant assay using DPPH spray, Bixa orellana leaves extract showed considerable free radical scavenging properties indicated by the presence of a yellow/white spot on a purple background on the TLC plates. 3.5.2. Quantitative determination The extract showed a concentration-dependent radical scavenging activity by inhibiting DPPH with an IC50 value of 22.36 ␮g/ml. Ascorbic acid, used as the positive control in this test, had an IC50 value of 4.33 ␮g/ml (Table 4). 3.6. Antidiarrhoeal activity In the castor oil-induced diarrhoea model, Bixa orellana leaves extract was found to reduce significantly (P < 0.01) and Table 3 Effect of Bixa orellana leaves extract on acetic acid induced writhing in mice Treatment Control Acetyl salicylic acid Bixa orellana extract

Writhings (n)a

Inhibition (%)

100

36.67 ± 2.93 6.17 ± 0.79**

– 83.18

125 250 500

18.17 ± 1.40** 10.83 ± 0.85** 7.50 ± 0.85**

50.45 70.45 79.55

Dose (mg/kg, p.o.) 10b

a Values are mean ± S.E.M. (n = 3) and are expressed as percentage inhibition of absorbance at 517 nm compared to control. b The IC 50 value (concentration of the sample required to scavenge 50% DPPH) was determined by extrapolation from linear regression analysis.

dose-dependently the total number of faeces and the total number of wet faeces in mice. The results were comparable with those observed for the standard drug loperamide (Table 5). 3.7. Gastrointestinal motility In the charcoal motility test, Bixa orellana leaves extract was found to delay the intestinal transit of charcoal meal in mice to a statistically significant level (P < 0.01) compared to the control at the dose of 500 mg/kg (Table 6). 3.8. Antibacterial activity In the antibacterial screening assay, Bixa orellana leaves extract showed moderate inhibition of the growth of Escherichia coli, Staphylococcus aureus, and Shigella dysenteriae. However, it failed to inhibit the growth of Shigella boydii, Shigella flexneri and Shigella sonnei. Kanamycin, used as the standard antibacterial drug, displayed strong inhibition of the growth of all test organisms (Table 7). Table 5 Effect of Bixa orellana leaves extract on castor oil-induced diarrhoea in micea Treatment Control Loperamide Bixa orellana extract

** P < 0.01

vs. control; one-way ANOVA followed by Dunnett’s test. Counted for 15 min, starting 5 min after acetic acid administration; values are mean ± S.E.M. (n = 6). b In ml/kg. a

IC50 (␮g/ml)b

** P < 0.01 a b

Dose (mg/kg, p.o.) 10b 3 125 250 500

Total number of faeces in 4 h

Total number of wet faeces in 4 h

15.83 ± 0.87 2.33 ± 0.56**

11.33 ± 0.76 0.83 ± 0.31**

11.83 ± 0.95** 7.50 ± 0.62** 5.67 ± 0.71**

4.67 ± 0.49** 2.83 ± 0.40** 1.67 ± 0.42**

vs. control; one-way ANOVA followed by Dunnett’s test. Values are mean ± S.E.M. (n = 6). In ml/kg.

J.A. Shilpi et al. / Journal of Ethnopharmacology 108 (2006) 264–271 Table 6 Effect of Bixa orellana leaves extract on gastrointestinal transit of charcoal meal in micea Treatment

Dose (mg/kg, route of administration)

Distance traversed by charcoal meal (%)

Control Atropine sulphate

10b , p.o. 0.1, i.p.

54.92 ± 3.45 26.89 ± 2.05**

Bixa orellana extract

125, p.o. 250, p.o. 500, p.o.

50.04 ± 3.26 46.74 ± 2.25 39.81 ± 2.32**

** P < 0.01 a b

vs. control; one-way ANOVA followed by Dunnett’s test. Values are mean ± S.E.M. (n = 6). In ml/kg.

Table 7 Antibacterial activity of Bixa orellana extract Microorganisms

Escherichia coli Staphylococcus aureus Shigella dysenteriae Shigella boydii Shigella flexneri Shigella sonnei

Diameter of zone of inhibition (mm) Bixa orellana extract (500 ␮g/disc)

Kanamycin (30 ␮g/disc)

15 10 11 0 0 0

39 30 34 38 37 29

4. Discussion The leaves of Bixa orellana have been reported to be used traditionally in the treatment of epilepsy (Ghani, 2003). In this study, a methanol extract of Bixa orellana leaves was prepared to investigate whether it had any effect on the central nervous system and any role in controlling seizures in mice. A number of tests were employed to evaluate neuropharmacological and anticonvulsant activity. Neuropharmacological activity was monitored using the pentobarbitone-induced hypnosis, open-field and hole-cross tests. When pentobarbitone (a barbiturate type of hypnotic agent) is given at an appropriate dose, it induces sedation or hypnosis in animals by potentiating the gamma amino butyric acid (GABA)-mediated postsynaptic inhibition through an allosteric modification of the GABA receptors (Ffrench-Mullen et al., 1993). A test substance with CNS-depressant activity can reduce time for the onset of sleep and/or prolong the duration of sleep. In this study, the reduction in time for the onset of sleep and increase in the duration of total sleeping time caused by Bixa orellana leaves extract was almost comparable to that of the standard drug diazepam. This result suggests that Bixa orellana leaves extract has a depressing effect on the CNS. Epilepsies are disorders of neuronal excitability characterised by the periodic and unpredictable occurrence of seizures. In most common forms of epileptic seizures, effective drugs appear to work either by promoting the inactivated state of voltage-activated Na+ channels or enhance GABA-mediated synaptic inhibition (McNamara,

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2001). In this study, anticonvulsant activity was further monitored using the strychnine-induced anticonvulsant test. The extract significantly increased the survival time after strychnine administration at the doses of 250 and 500 mg/kg compared to the control but failed to prevent the mortality of the test animals. In both the open-field and hole-cross tests, which evaluate the behavioural effects of a test substance on the CNS, Bixa orellana leaves extract exhibited a decrease in locomotor activity in test animals. As Bixa orellana leaves extracts have been reported to be useful in headaches, the extract was also investigated for analgesic activity using the acetic acid-induced writhing model. When administrated intraperitoneally to mice, acetic acid causes algesia by liberating noxious endogenous substances, including serotonin, histamine, prostaglandin, bradykinin and substance P that sensitise pain nerve endings (Collier et al., 1968). Among the prostanoids, mainly prostacyclin (PGI2 ) has been held responsible for the causation of pain following acetic acid administration (Murata et al., 1997). It has been suggested that acetic acid stimulates the vanilloid (VR1 ) and bradykinin (B2 ) receptors in the pathway comprising sensory afferent C-fibers (Ikeda et al., 2001). Therefore, the observed activity of Bixa orellana leaves extract might stem from its ability to interfere with the synthesis and/or release of those endogenous substances or desensitisation of the nerve fibers involved in the pain transmission pathway. Antioxidant activity was recently studied in relation to CNS disorders (Perry et al., 2001). For example, the metabolism of phospholipids is associated with neuronal death in Alzheimer’s disease (Fr¨ohlich and Riederer, 1995) and antioxidants such as Vitamin E play an important role in ␤-amyloid aggregation (Murray and Lynch, 1998). It has been discovered that in vivo free radical-catalysed peroxidation of arachidonate gives rise to a novel series of agents termed isoprostanes (Morrow et al., 1990). This pathway of eicosanoids formation links free radicalmediated tissue injury with biaoactive lipid-derived autocoid generation. It has been postulated that these isoprostanes might contribute to the pathophysiology of inflammatory responses insensitive to currently available steroidal or nonsteroidal antiinflammatory agents (Morrow et al., 1999). In this study, the antioxidant capacity of the methanol extract from Bixa orellana leaves was tested using the DPPH assay. Alcoholic solutions of the stable 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH) radical strongly absorb at 517 nm, showing a deep purple colour. These solutions in the presence of any test substance with free radical scavenging (antioxidant) activity can be ‘decolourised’, resulting in bleaching on the TLC plate and a reduction in absorbance measured spectrophotometrically. Bixa orellana leaves extract showed DPPH free radical scavenging activity in a concentration-dependent manner (Table 4). This suggests that the extract could be useful in the management of free radicalmediated CNS disorders or inflammatory responses. Although it is well known that carotenoids (mainly found in the seeds) are particularly good radical scavengers (Junior et al., 2005), further chemical investigations are necessary to identify whether or not they would be the active constituents in the leaves. The reported traditional use of Bixa orellana leaves in treating dysentery (Perry, 1980; Joshi, 2000) prompted us to screen the

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extract for antidiarrhoeal and antidysenteric activity. Evaluation of antidiarrhoeal activity was performed using castor oil-induced diarrhoea model and gastrointestinal motility test in mice. Castor oil causes diarrhoea through its active metabolite ricinoleic acid (Ammon et al., 1974), which stimulates the peristaltic activity of small intestine leading to changes in electrolyte permeability of intestinal mucosa. Its action is also associated with stimulation of release of endogenous prostaglandins (Galvez et al., 1993). In this study, Bixa orellana leaves extract significantly and dosedependently decreased the severity of castor oil-induced diarrhoeal episodes in the test animals. The extract also reduced the total number of faeces as well as the total number of wet faeces both significantly and dose-dependently. In the gastrointestinal motility test, the extract was found to reduce the movement of charcoal meal in mice to a statistically significant level (P < 0.01) only at the highest dose tested (500 mg/kg). Therefore, it could be interpreted that the observed antidiarrhoeal activity of Bixa orellana leaves extract may be attributed to a possible inhibition of prostaglandin biosynthesis and to a lesser extent to its retardation of gastrointestinal transit. The extract was further evaluated for its activity in vitro against a range of bacterial species which are known to trigger diarrhoea (Staphylococcus aureus, Escherichia coli) and dysentery (Shigella spp.). Results of the antibacterial screening assay indicated that Bixa orellana leaves extract moderately inhibited the growth of Escherichia coli, Staphylococcus aureus and Shigella dysenteriae. Previously published studies on the antimicrobial activity of Bixa orellana leaves extract using the agar diffusion method have reported activity of ethanolic extracts against a range of Gram-positive, Gram-negative bacteria and fungi. Thus, Irobi et al. (1996) reported activity against Bacillus subtilis, Staphylococcus aureus, and Streptococcus faecalis with little activity against Escherichia coli, Serratia marcescens, Candida utilis, and Aspergillus niger. Castello et al. (2002) reported activity against Bacillus pumilus while Fleischer et al. (2003) reported activity against Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes, Salmonella typhi, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans. This study represents the first report of the activity of Bixa orellana leaves extract against Shigella dysenteriae. It could be suggested from our observations that the antimicrobial activity of the methanol extract of Bixa orellana leaves may contribute to some extent to the management of diarrhoeal and dysenteric diseases caused by Escherichia coli, Staphylococcus aureus and Shigella dysenteriae. 5. Conclusions Our preliminary pharmacological studies on the methanol extract of Bixa orellana leaves provide in part scientific support for the use of this species in traditional medicine, particularly in various ailments related to CNS disorders, pain, diarrhoea and dysentery. However, further pharmacological investigations are required to understand its underlying mode of action on the CNS, mechanism of pain inhibition and antidiarrhoeal activity. In addition, future bioactivity-guided phytochemical work should be carried out to identify any active constituent(s).

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