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Evaluation of the anti-stress and anticonvulsant activities of leaf extract of Alchornea cordifolia in mice
Journal of Ethnopharmacology 127 (2010) 768–770
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Evaluation of the anti-stress and anticonvulsant activities of leaf extract of Alchornea cordifolia in mice Solomon Umukoro ∗ , Aderemi Caleb Aladeokin Department of Pharmacology and Therapeutics, University of Ibadan, Ibadan, Nigeria
a r t i c l e
i n f o
Article history: Received 20 August 2009 Received in revised form 19 November 2009 Accepted 24 November 2009 Available online 29 November 2009 Keywords: Alchornea cordifolia Forced swimming test Anti-stress Convulsions
a b s t r a c t Aim of the study: The extract of the leaves of Alchornea cordifolia (AC) is extensively used in ethnomedicine for ulcers, rheumatic pains, febrile convulsions and for enhancing physical performance. In this study, the anti-stress and anticonvulsant activities of the aqueous leaf extract of Alchornea cordifolia were investigated in mice. Materials and methods: The anti-stress activity was assessed based on the ability of the extract to alter the duration of immobility, in the forced swim endurance test, whilst a picrotoxin-treated animal, was employed as the model for convulsive seizures. Results: The extract (100–400 mg/kg) given orally was found to significantly (p < 0.05) reduce the duration of immobility, which suggest an anti-stress/anti-fatigue property. However, AC when tested at doses between 100 and 400 mg/kg did not prevent convulsions induced by picrotoxin in mice. The acute toxicity study carried out in mice revealed that the extract was well tolerated by the animals, as no death was observed at oral doses of 500–4000 mg/kg. Conclusions: The results of this preliminary study provide evidence, which may support the use of Alchornea cordifolia against stress or fatigue in ethnomedicine. © 2009 Published by Elsevier Ireland Ltd.
1. Introduction Alchornea cordifolia Schum (Euphorbiaceae) commonly known as Christmas bush, is a multi-stemmed shrub of about 5 m in height, found in the tropical regions of Africa. The plant has gained wide popularity, as a remedy for cough, gonorrhoea, ulcers, rheumatic pains, fever, wounds and diarrhoea (Ogungbamila and Samuelsson, 1999; Tona et al., 2000). The decoction of the leaves is also taken 2–3 times daily as central nervous system stimulant and in enhancing sexual performance in males that are relatively impotent (Defeo, 1992). The rural populace of Southern Nigeria also employs the decoction of the leaves in the management of febrile convulsions in children. The leaf extract has been shown to contain alchornine, alchorneinone, gentisnic acid and yohimbine (Ogungbamila and Samuelsson, 1999). Phytochemical studies of the leaf extract also revealed the presence of flavonoid, alkaloids, saponins, phenols and tannins (Ogungbamila and Samuelsson, 1999; Tona et al., 2000). Previous studies have established the spasmolytic, antiinflammatory, anti-microbial, anti-diarrhoeal and analgesic properties of the extract of Alchornea cordifolia (Defeo, 1992; Ogungbamila and Samuelsson, 1999; Osadede and Okeye, 2003).
∗ Corresponding author. E-mail address: [email protected] (S. Umukoro). 0378-8741/$ – see front matter © 2009 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2009.11.023
However, there is no existing pharmacological data that indicate the usefulness of the leaves extract of AC against stress or fatigue and convulsions. Therefore, this preliminary study was carried out to evaluate the anti-stress and anticonvulsant activities of an aqueous leaf extract of Alchornea cordifolia in mice. 2. Materials and methods 2.1. Plant material Fresh leaves of Alchornea cordifolia were purchased from Mushin market, Lagos and authenticated by Prof. J.D. Olowokudejo of the Department of Botany and Microbiology, University of Lagos, Nigeria. A voucher specimen with number UIH22266 has been deposited in the herbarium in the Department of Botany and Microbiology, University of Ibadan, Nigeria. 2.2. Preparation of plant extract The leaves were dried in an oven at 40 ◦ C and the dried leaves were ground into fine powder. One hundred grams of the powered leaves was soaked in 500 ml of distilled water for 24 h at room temperature with occasional stirring. The solution was thereafter filtered and the filtrate was evaporated to a brownish sticky residue in an oven at 40 ◦ C. The yield of the extract was 10.6% with respect to the powdered leaves and 400 mg of the residue was dissolved in
S. Umukoro, A.C. Aladeokin / Journal of Ethnopharmacology 127 (2010) 768–770
20 ml of distilled water to obtain the concentrations used for the study. 2.3. Laboratory animals Swiss albino mice (18–25 g) of either sex obtained from the Animal Unit, University of Ibadan, Ibadan, Nigeria were used for the study. The animals were kept and maintained under standard laboratory conditions and were allowed free access to food and water. The ethical guidelines for the handling of laboratory animals were followed in the study.
769
Table 1 Effect of aqueous leaf extract of Alchornea cordifolia on the duration of immobility in mice. Treatment
Dose (mg/kg)
Duration of immobility (s)
Saline Alchornea cordifolia Alchornea cordifolia Alchornea cordifolia ␣-Methyldopa Amphetamine
– 50 100 200 10 5
370 336 174.5 100.0 467.40 33.20
± ± ± ± ± ±
18.50 20.1 10.50* 5.10* 21.60* 5.20*
2.4. Drugs
The animals were forced to swim in water at room temperature after post-drug treatment and the duration of immobility(s) was measured at interval of 30 min. Each value represents the mean ± S.E.M for 6 animals per group. * p < 0.05 compared with saline-treated group (ANOVA).
Amphetamine sulphate (Sigma, USA), ␣-methyldopa hydrochloride (Sigma, USA) and picrotoxin (Sigma, USA) were used as reference drugs in the study.
Table 2 Effect of aqueous leaf extract of Alchornea cordifolia on picrotoxin-induced convulsion in mice.
2.5. Acute toxicity study Mice (6/group) were treated with the extract (0.5–4.0 g/kg, p.o) or saline (10 ml/kg, p.o) after 6 h fasting period. The animals were observed for toxic symptoms and mortality was assessed after 24 h post-treatment (Miller and Tainter, 1944). 2.6. Anti-stress test The anti-stress property of AC was assessed as previously described (Bhattacharya and Ghosal, 2000). The animals (6 mice/group) were forced to swim individually in a transparent tank (30 cm × 45 cm × 40 cm) containing water at room temperature 30 min after treatment with the extract (100–400 mg/kg, orally), ␣-methyldopa (10 mg/kg, i.p), amphetamine (5 mg/kg, i.p) or saline (10 ml/kg, orally). The duration of immobility was measured over 30 min and the animals were judged immobile whenever they remained floating passively in the water (Subarnas et al., 1993). 2.7. Anticonvulsant test Mice (6/group) were pretreated with oral doses of the extract (100–400 mg/kg), or saline (10 ml/kg) 30 min before induction of convulsions with i.p injection of picrotoxin (10 mg/kg). The animals were observed for the onset of convulsions or death for a period of 30 min post-injection of picrotoxin. 2.8. Data analysis Data were expressed as mean ± SEM and one-way ANOVA was used to analyze the significance of the data. Further post hoc analyses were performed by the Fisher’s LSD t-test. Differences with p < 0.05 were considered significant. 3. Results and discussion The extract (500–4000 mg/kg, orally) had a high safety profile, as no death was observed at the tested dose levels. The behavioral changes observed at high doses of AC (2000–4000 mg/kg) were sedation, hyperventilation and motor in co-ordination. The extract (100–400 mg/kg, orally), in a similar manner to amphetamine (5 mg/kg, i.p), was found to shorten the duration of immobility in mice. In contrast, as shown in Table 1, ␣-methyldopa (10 mg/kg, i.p) prolonged the duration of immobility in the animals. Whilst, as shown in Table 2, AC (200–400 mg/kg, orally) failed to prevent convulsions in the picrotoxin-treated animals. The forced swimming test is one of the most suitable paradigms, widely used for the routine evaluation of anti-stress and antidepressant property of novel compounds (Bhattacharya and Ghosal,
Drug
Dose (mg/kg)
Convulsions (%)
Mortality (%)
Saline Alchornea cordifolia Alchornea cordifolia Alchornea cordifolia
– 100 200 400
100 100 100 100
100 100 100 100
The animals (6 per group) were treated with the extract or saline 30 min before induction of convulsions with intraperitoneal injection of 10 mg/kg dose of picrotoxin.
2000). This test is based on the observation that animals forced to swim in water, eventually assumed a characteristic immobile posture, devoid of any activity (Subarnas et al., 1993). The appearance of immobility therefore, reflects a state of tiredness, fatigue, reduced stamina or a lowered mood (hopelessness or despair) (Bhattacharya and Ghosal, 2000; Subarnas et al., 1993). These signs represent some of the core symptoms observed in depressed patients or in individuals under intense stress (Subarnas et al., 1993). The ability of AC to reduce the duration of immobility in this study suggests that it possesses phytochemically active compound(s) with anti-stress property. The presence of yohimbine in the leaves of Alchornea cordifolia (Ogungbamila and Samuelsson, 1999) might be playing a significant role in its anti-stress activity, as this alkaloid has been found to enhance physical performance, through the blockade of ␣2 -adrenergic receptors (Goldberg et al., 1983). Picrotoxin-induced convulsions in rodents still enjoy wide popularity as a suitable laboratory model, for screening anticonvulsant property of drugs (Zetler, 1981). In this study, the extract did not exhibit anticonvulsant activity, as it could not prevent seizures or death induced by picrotoxin, a known antagonist of gamma aminobutyric acid chloride ionophore complex (Akaike et al., 1985). In conclusion, the results of the study suggest that Alchornea cordifolia possesses phytoactive compound(s) that might be useful against stress or fatigue. Acknowledgement We thanked Mrs. B. Arinola of the Department of Pharmacology and Therapeutics, University of Ibadan, Nigeria for her technical assistance. References Akaike, W., Hzattori, K., Ocmura, Y., Czarpenter, D.O., 1985. Bicculline and picrotoxin block gamma-aminobutyric acid-gated chloride ion conductance by different mechanisms. Experiential 41, 70–71. Bhattacharya, K., Ghosal, S., 2000. Experimental evaluation of the anti-stress activity of a herbal formulation, zetress. Journal of Natural Remedies 1, 1–7.
770
S. Umukoro, A.C. Aladeokin / Journal of Ethnopharmacology 127 (2010) 768–770
Defeo, V., 1992. Medicinal and magical plants in the northern Peruvian Andes. Fitoterapia 63, 417–440. Goldberg, M.R., Robertson, D., Yohimbine, 1983. A pharmacological probe for study of the ␣2 -adrenoceptor. Pharmacological Review, 143–180. Miller, L.C., Tainter, M.C., 1944. Estimation of the LD50 and its errors by means of logarithmic-probit graph paper. Proceedings of the Society for Experimental Biology and Medicine 57, 261–264. Ogungbamila, F.O., Samuelsson, G., 1999. Smooth muscle relaxing flavonoids from Alchornea cordifolia. Acta Pharmaceutica Nordica 2, 421–422. Osadede, P.O., Okeye, F.B., 2003. Anti-inflammatory effects of crude methanolic extract and fractions of Alchornea cordifolia leaves. Journal of Ethnopharmacology 89, 19–24.
Subarnas, A., Tadano, T., Nakahata, N., Arai, Y., Kinemuchi, H., Oshima, Y., Kisara, K., Ohizumi, Y., 1993. Possible mechanism of antidepressant activity of beta-amyrin palmitate isolated from Lobelia inflata leaves in the forced swimming test. Life Sciences 52, 289–296. Tona, L., Kambu, K., Ngimbi, T., Mesiza, K., Penge, O., Lusakibana, M., Cimanga, K., Debruyne, T., Apersw, S., Totte, J., Pieters, L., Vlietinck, A.J., 2000. Antiamoebic and spasmolytic activities extracts from some antidiarrhoeal traditional preparations used in Congo. Phytomedicine 7, 31–38. Zetler, G., 1981. Central depressant effects of caerulen and cholecystokinin ocatapeptide (CCK-8) differ from those of diazepam and haloperidol. Neuropharmacology 20, 277–283.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Evaluation of the anti-stress and anticonvulsant activities of leaf extract of Alchornea cordifolia in mice Solomon Umukoro ∗ , Aderemi Caleb Aladeokin Department of Pharmacology and Therapeutics, University of Ibadan, Ibadan, Nigeria
a r t i c l e
i n f o
Article history: Received 20 August 2009 Received in revised form 19 November 2009 Accepted 24 November 2009 Available online 29 November 2009 Keywords: Alchornea cordifolia Forced swimming test Anti-stress Convulsions
a b s t r a c t Aim of the study: The extract of the leaves of Alchornea cordifolia (AC) is extensively used in ethnomedicine for ulcers, rheumatic pains, febrile convulsions and for enhancing physical performance. In this study, the anti-stress and anticonvulsant activities of the aqueous leaf extract of Alchornea cordifolia were investigated in mice. Materials and methods: The anti-stress activity was assessed based on the ability of the extract to alter the duration of immobility, in the forced swim endurance test, whilst a picrotoxin-treated animal, was employed as the model for convulsive seizures. Results: The extract (100–400 mg/kg) given orally was found to significantly (p < 0.05) reduce the duration of immobility, which suggest an anti-stress/anti-fatigue property. However, AC when tested at doses between 100 and 400 mg/kg did not prevent convulsions induced by picrotoxin in mice. The acute toxicity study carried out in mice revealed that the extract was well tolerated by the animals, as no death was observed at oral doses of 500–4000 mg/kg. Conclusions: The results of this preliminary study provide evidence, which may support the use of Alchornea cordifolia against stress or fatigue in ethnomedicine. © 2009 Published by Elsevier Ireland Ltd.
1. Introduction Alchornea cordifolia Schum (Euphorbiaceae) commonly known as Christmas bush, is a multi-stemmed shrub of about 5 m in height, found in the tropical regions of Africa. The plant has gained wide popularity, as a remedy for cough, gonorrhoea, ulcers, rheumatic pains, fever, wounds and diarrhoea (Ogungbamila and Samuelsson, 1999; Tona et al., 2000). The decoction of the leaves is also taken 2–3 times daily as central nervous system stimulant and in enhancing sexual performance in males that are relatively impotent (Defeo, 1992). The rural populace of Southern Nigeria also employs the decoction of the leaves in the management of febrile convulsions in children. The leaf extract has been shown to contain alchornine, alchorneinone, gentisnic acid and yohimbine (Ogungbamila and Samuelsson, 1999). Phytochemical studies of the leaf extract also revealed the presence of flavonoid, alkaloids, saponins, phenols and tannins (Ogungbamila and Samuelsson, 1999; Tona et al., 2000). Previous studies have established the spasmolytic, antiinflammatory, anti-microbial, anti-diarrhoeal and analgesic properties of the extract of Alchornea cordifolia (Defeo, 1992; Ogungbamila and Samuelsson, 1999; Osadede and Okeye, 2003).
∗ Corresponding author. E-mail address: [email protected] (S. Umukoro). 0378-8741/$ – see front matter © 2009 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2009.11.023
However, there is no existing pharmacological data that indicate the usefulness of the leaves extract of AC against stress or fatigue and convulsions. Therefore, this preliminary study was carried out to evaluate the anti-stress and anticonvulsant activities of an aqueous leaf extract of Alchornea cordifolia in mice. 2. Materials and methods 2.1. Plant material Fresh leaves of Alchornea cordifolia were purchased from Mushin market, Lagos and authenticated by Prof. J.D. Olowokudejo of the Department of Botany and Microbiology, University of Lagos, Nigeria. A voucher specimen with number UIH22266 has been deposited in the herbarium in the Department of Botany and Microbiology, University of Ibadan, Nigeria. 2.2. Preparation of plant extract The leaves were dried in an oven at 40 ◦ C and the dried leaves were ground into fine powder. One hundred grams of the powered leaves was soaked in 500 ml of distilled water for 24 h at room temperature with occasional stirring. The solution was thereafter filtered and the filtrate was evaporated to a brownish sticky residue in an oven at 40 ◦ C. The yield of the extract was 10.6% with respect to the powdered leaves and 400 mg of the residue was dissolved in
S. Umukoro, A.C. Aladeokin / Journal of Ethnopharmacology 127 (2010) 768–770
20 ml of distilled water to obtain the concentrations used for the study. 2.3. Laboratory animals Swiss albino mice (18–25 g) of either sex obtained from the Animal Unit, University of Ibadan, Ibadan, Nigeria were used for the study. The animals were kept and maintained under standard laboratory conditions and were allowed free access to food and water. The ethical guidelines for the handling of laboratory animals were followed in the study.
769
Table 1 Effect of aqueous leaf extract of Alchornea cordifolia on the duration of immobility in mice. Treatment
Dose (mg/kg)
Duration of immobility (s)
Saline Alchornea cordifolia Alchornea cordifolia Alchornea cordifolia ␣-Methyldopa Amphetamine
– 50 100 200 10 5
370 336 174.5 100.0 467.40 33.20
± ± ± ± ± ±
18.50 20.1 10.50* 5.10* 21.60* 5.20*
2.4. Drugs
The animals were forced to swim in water at room temperature after post-drug treatment and the duration of immobility(s) was measured at interval of 30 min. Each value represents the mean ± S.E.M for 6 animals per group. * p < 0.05 compared with saline-treated group (ANOVA).
Amphetamine sulphate (Sigma, USA), ␣-methyldopa hydrochloride (Sigma, USA) and picrotoxin (Sigma, USA) were used as reference drugs in the study.
Table 2 Effect of aqueous leaf extract of Alchornea cordifolia on picrotoxin-induced convulsion in mice.
2.5. Acute toxicity study Mice (6/group) were treated with the extract (0.5–4.0 g/kg, p.o) or saline (10 ml/kg, p.o) after 6 h fasting period. The animals were observed for toxic symptoms and mortality was assessed after 24 h post-treatment (Miller and Tainter, 1944). 2.6. Anti-stress test The anti-stress property of AC was assessed as previously described (Bhattacharya and Ghosal, 2000). The animals (6 mice/group) were forced to swim individually in a transparent tank (30 cm × 45 cm × 40 cm) containing water at room temperature 30 min after treatment with the extract (100–400 mg/kg, orally), ␣-methyldopa (10 mg/kg, i.p), amphetamine (5 mg/kg, i.p) or saline (10 ml/kg, orally). The duration of immobility was measured over 30 min and the animals were judged immobile whenever they remained floating passively in the water (Subarnas et al., 1993). 2.7. Anticonvulsant test Mice (6/group) were pretreated with oral doses of the extract (100–400 mg/kg), or saline (10 ml/kg) 30 min before induction of convulsions with i.p injection of picrotoxin (10 mg/kg). The animals were observed for the onset of convulsions or death for a period of 30 min post-injection of picrotoxin. 2.8. Data analysis Data were expressed as mean ± SEM and one-way ANOVA was used to analyze the significance of the data. Further post hoc analyses were performed by the Fisher’s LSD t-test. Differences with p < 0.05 were considered significant. 3. Results and discussion The extract (500–4000 mg/kg, orally) had a high safety profile, as no death was observed at the tested dose levels. The behavioral changes observed at high doses of AC (2000–4000 mg/kg) were sedation, hyperventilation and motor in co-ordination. The extract (100–400 mg/kg, orally), in a similar manner to amphetamine (5 mg/kg, i.p), was found to shorten the duration of immobility in mice. In contrast, as shown in Table 1, ␣-methyldopa (10 mg/kg, i.p) prolonged the duration of immobility in the animals. Whilst, as shown in Table 2, AC (200–400 mg/kg, orally) failed to prevent convulsions in the picrotoxin-treated animals. The forced swimming test is one of the most suitable paradigms, widely used for the routine evaluation of anti-stress and antidepressant property of novel compounds (Bhattacharya and Ghosal,
Drug
Dose (mg/kg)
Convulsions (%)
Mortality (%)
Saline Alchornea cordifolia Alchornea cordifolia Alchornea cordifolia
– 100 200 400
100 100 100 100
100 100 100 100
The animals (6 per group) were treated with the extract or saline 30 min before induction of convulsions with intraperitoneal injection of 10 mg/kg dose of picrotoxin.
2000). This test is based on the observation that animals forced to swim in water, eventually assumed a characteristic immobile posture, devoid of any activity (Subarnas et al., 1993). The appearance of immobility therefore, reflects a state of tiredness, fatigue, reduced stamina or a lowered mood (hopelessness or despair) (Bhattacharya and Ghosal, 2000; Subarnas et al., 1993). These signs represent some of the core symptoms observed in depressed patients or in individuals under intense stress (Subarnas et al., 1993). The ability of AC to reduce the duration of immobility in this study suggests that it possesses phytochemically active compound(s) with anti-stress property. The presence of yohimbine in the leaves of Alchornea cordifolia (Ogungbamila and Samuelsson, 1999) might be playing a significant role in its anti-stress activity, as this alkaloid has been found to enhance physical performance, through the blockade of ␣2 -adrenergic receptors (Goldberg et al., 1983). Picrotoxin-induced convulsions in rodents still enjoy wide popularity as a suitable laboratory model, for screening anticonvulsant property of drugs (Zetler, 1981). In this study, the extract did not exhibit anticonvulsant activity, as it could not prevent seizures or death induced by picrotoxin, a known antagonist of gamma aminobutyric acid chloride ionophore complex (Akaike et al., 1985). In conclusion, the results of the study suggest that Alchornea cordifolia possesses phytoactive compound(s) that might be useful against stress or fatigue. Acknowledgement We thanked Mrs. B. Arinola of the Department of Pharmacology and Therapeutics, University of Ibadan, Nigeria for her technical assistance. References Akaike, W., Hzattori, K., Ocmura, Y., Czarpenter, D.O., 1985. Bicculline and picrotoxin block gamma-aminobutyric acid-gated chloride ion conductance by different mechanisms. Experiential 41, 70–71. Bhattacharya, K., Ghosal, S., 2000. Experimental evaluation of the anti-stress activity of a herbal formulation, zetress. Journal of Natural Remedies 1, 1–7.
770
S. Umukoro, A.C. Aladeokin / Journal of Ethnopharmacology 127 (2010) 768–770
Defeo, V., 1992. Medicinal and magical plants in the northern Peruvian Andes. Fitoterapia 63, 417–440. Goldberg, M.R., Robertson, D., Yohimbine, 1983. A pharmacological probe for study of the ␣2 -adrenoceptor. Pharmacological Review, 143–180. Miller, L.C., Tainter, M.C., 1944. Estimation of the LD50 and its errors by means of logarithmic-probit graph paper. Proceedings of the Society for Experimental Biology and Medicine 57, 261–264. Ogungbamila, F.O., Samuelsson, G., 1999. Smooth muscle relaxing flavonoids from Alchornea cordifolia. Acta Pharmaceutica Nordica 2, 421–422. Osadede, P.O., Okeye, F.B., 2003. Anti-inflammatory effects of crude methanolic extract and fractions of Alchornea cordifolia leaves. Journal of Ethnopharmacology 89, 19–24.
Subarnas, A., Tadano, T., Nakahata, N., Arai, Y., Kinemuchi, H., Oshima, Y., Kisara, K., Ohizumi, Y., 1993. Possible mechanism of antidepressant activity of beta-amyrin palmitate isolated from Lobelia inflata leaves in the forced swimming test. Life Sciences 52, 289–296. Tona, L., Kambu, K., Ngimbi, T., Mesiza, K., Penge, O., Lusakibana, M., Cimanga, K., Debruyne, T., Apersw, S., Totte, J., Pieters, L., Vlietinck, A.J., 2000. Antiamoebic and spasmolytic activities extracts from some antidiarrhoeal traditional preparations used in Congo. Phytomedicine 7, 31–38. Zetler, G., 1981. Central depressant effects of caerulen and cholecystokinin ocatapeptide (CCK-8) differ from those of diazepam and haloperidol. Neuropharmacology 20, 277–283.