Studies on the anti-inflammatory and analgesic properties of Jatropha curcas leaf extract

The anti-inflammatory and analgesic properties of the aqueous extract of Jatropha curcas leaves were investigated. Anti-inflammatory effect was studied using acute rat model (carrageenan -induced rat paw oedema) in which aspirin at 150 mg·kg-1, indomethacin at 10 mg·kg-1 (both are standard anti-inflammatory drugs) and aqueous extract of Jatropha curcas leaves at 150 mg·kg-1 were administered orally to 24 Wistar rats 1 h before induction of oedema and compared with a negative control given 10 ml·kg-1 saline. Using the same dose of the plant extract, the analgesic effect of the aqueous extract of Jatropha curcas leaves was also investigated by measuring the number of acetic acid-induced writhing in 18 mice. The mean percentage inhibition of paw volume of rats treated with indomethacin was 83.9% followed by aspirin treated group with 64.3%, and the Jatropha curcas treated group with 60.7%. The mean number of writhing was significantly lower (P < 0.05) in mice given aqueous extract of Jatropha curcas leaves (34.0) compared to the control group (55.8) but higher than that of the standard analgesic, paracetamol (23.5). This result suggests that Jatropha curcas has anti-inflammatory and analgesic properties comparable with those of standard drugs and may be useful for the treatment of painful inflammatory conditions. Plant extract, anti-nociceptive, carrageenan, oedema, writhing Inflammation is a complex biological response of vascular tissue to harmful stimuli caused by injury, infection, environmental agents, malignancy and cellular changes. It is a protective attempt by the body to remove the injurious stimuli as well as initiate the healing process for the tissue (Denko 1992). The inflammatory response is a complex process that includes activation of white blood cells, the release of immune system chemicals such as complements and cytokines, and the production and release of inflammatory mediators and prostaglandins (Cotran et al. 2001). Inflammation may be acute or chronic depending on the disease course. Acute inflammation is characterized by heat, erythema, pain, swelling and loss of function. Pain is a common and distressing feature of many diseases and analgesics relieve pain by acting in the central nervous system or on peripheral pain mechanisms, without significantly altering consciousness. Chronic inflammation on the other hand results in a progressive shift in inflammatory cells characterized by simultaneous destruction and healing of the injured tissue. Many plants have long been recognized as important sources of therapeutically effective medicines (Newman et al. 2003). Herbs such as Tithonia diversifolia and Carum copticum (Owoyele et al. 2003; Thangam and Dhanajayan 2003) have been shown to possess anti-inflammatory and analgesic effects and such plants can be used as sole therapy in managing inflammatory conditions or as complementary therapy allowing patients to take smaller doses of conventional anti-inflammatory drugs, thereby minimizing the side effects of these drugs. ACTA VET. BRNO 2011, 80: 259–262; doi:10.2754/avb201180030259 Address for correspondence: Johnny Olufemi Olukunle Department of Veterinary Physiology and Pharmacology College of Veterinary Medicine University of Agriculture, Abeokuta, Ogun State, Nigeria Phone: 234-080-33807215 E-mail: drfaks@yahoo.com http://www.vfu.cz/acta-vet/actavet.htm Jatropha curcas, belonging to the family Euphorbiaceae is an herbal plant known as “lapalapa” in the South Western part of Nigeria and widely distributed in Central and South America, Africa, India and South East Asia (Cano-Asseleih 1986; Cano-Asseleih et al. 1989). It has been used traditionally in the treatment of wounds, burns, and cuts for its antimicrobial and antifungal properties. These claims have not been scientifically analyzed. Furthermore, most conventional anti-inflammatory drugs act by suppressing rather than abolishing inflammation and are well known for their long-term side effects. This study is therefore aimed at investigating the anti-inflammatory and analgesic properties of the aqueous extract of Jatropha curcas leaves. Materials and Methods Plant material and preparation of extract The leaves of J. curcas were obtained from Apakila area in Abeokuta, Ogun State and verified by botanists at the Forestry Research Institute of Nigeria, (FRIN) Ibadan, Oyo State, Nigeria; a specimen of this plant was deposited at this institute. The leaves were air-dried, pulverized, finely sieved and soaked in 2 l of distilled water for 24 h, after which it was filtered. Thereafter, the filtrate obtained was freeze-dried and the percent yield was 2.4%. This sample was stored in the refrigerator until used in the experiment. Drugs Aspirin, indomethacin, and paracetamol were purchased from a registered chemist in Abeokuta, Ogun State. Animals Eighteen specified pathogen free Swiss mice of either sex weighing 25 g and 24 specified pathogen free Wistar rats of either sex weighing 150-200 g were used for this study. The animals were kept at the Animal House, College of Veterinary Medicine, University of Agriculture, Abeokuta. They were acclimatized to laboratory conditions for 2 weeks prior to the experiment during which they were introduced to growers mash (Vital feeds Limited, Ibadan, Nigeria). The animals were fed this mash and provided water ad libitum throughout the study. The housing provided had the following conditions: controlled lighting of 12:12 h of light: dark, temperature of 25 °C and relative humidity of approximately 50%. Animal experimentation protocols conformed to the Institutional Animal Ethics Committee’s guidelines. Carrageenan-induced rat paw oedema Acute inflammation was induced using the carrageenan induced oedema model (Winter et al. 1962). The rats were starved for 24 h after which they were divided into 4 groups (Groups A-D) of 6 animals each. Rats in group A (negative control, untreated) were given saline at 10 ml·kg-1 1 h before 0.1 ml of 1% freshly prepared suspension of carrageenan (Sigma Chemical Co.) was injected into the plantar surface of the right hind paw of each rat. Group B rats were given aqueous extract of J. curcas leaves orally at the dose of 150 mg·kg-1 1 h before carrageenan injection. Rats in group C were given aspirin, a standard anti-inflammatory drug at the dose of 150 mg·kg-1 orally 1 h before carrageenan injection while group D rats were administered indomethacin, another standard anti-inflammatory drug at a dose of 10 mg·kg-1 orally 1 h before carrageenan injection. The linear circumference of the paw was measured after carrageenan injection (0 h) and 3 h after carrageenan injection using a loop of thread tied round the paw such that it was neither too loose nor too tight. The length of the thread around the paw was then measured on a ruler and rounded off to the nearest centimetre. The percentage inhibition was calculated according to the formula: Percentage inhibition = [C1-C0]control– [C1-C0]test × 100 [C1-C0]control Where: Co = Mean paw size at 0 h after carrageenan injection, C1 = Mean paw size at 3 h after carrageenan injection Acetic acid-induced abdominal writhing in mice This was done as described by Koster et al. (1959). The mice were divided into 3 groups (groups 1–3) of 6 mice each. Distilled water at 5 ml·kg-1 was administered orally to the mice of the control group (group 1), 50 mg·kg-1 paracetamol orally to the mice of group 2 and 150 mg·kg-1 of aqueous extract of J. curcas leaves orally to mice of group 3, 1 h before intraperitoneal injection of acetic acid (0.6%, volume/volume in distilled water, 10 ml·kg-1). The number of writhing exhibited by each animal was counted for 10 min, starting 10 min after acetic acid injection and computed according to the following formula: Percentage inhibition = Mean of writhing (control)– Mean of writhing (test) × 100 Mean number of writhing (control) 260

Inflammation is a complex biological response of vascular tissue to harmful stimuli caused by injury, infection, environmental agents, malignancy and cellular changes. It is a protective attempt by the body to remove the injurious stimuli as well as initiate the healing process for the tissue (Denko 1992). The inflammatory response is a complex process that includes activation of white blood cells, the release of immune system chemicals such as complements and cytokines, and the production and release of inflammatory mediators and prostaglandins (Cotran et al. 2001).
Inflammation may be acute or chronic depending on the disease course. Acute inflammation is characterized by heat, erythema, pain, swelling and loss of function. Pain is a common and distressing feature of many diseases and analgesics relieve pain by acting in the central nervous system or on peripheral pain mechanisms, without significantly altering consciousness. Chronic inflammation on the other hand results in a progressive shift in inflammatory cells characterized by simultaneous destruction and healing of the injured tissue.
Many plants have long been recognized as important sources of therapeutically effective medicines (Newman et al. 2003). Herbs such as Tithonia diversifolia and Carum copticum (Owoyele et al. 2003;Thangam and Dhanajayan 2003) have been shown to possess anti-inflammatory and analgesic effects and such plants can be used as sole therapy in managing inflammatory conditions or as complementary therapy allowing patients to take smaller doses of conventional anti-inflammatory drugs, thereby minimizing the side effects of these drugs.
Jatropha curcas, belonging to the family Euphorbiaceae is an herbal plant known as "lapalapa" in the South Western part of Nigeria and widely distributed in Central and South America, Africa, India and South East Asia (Cano-Asseleih 1986;Cano-Asseleih et al. 1989). It has been used traditionally in the treatment of wounds, burns, and cuts for its antimicrobial and antifungal properties. These claims have not been scientifically analyzed. Furthermore, most conventional anti-inflammatory drugs act by suppressing rather than abolishing inflammation and are well known for their long-term side effects. This study is therefore aimed at investigating the anti-inflammatory and analgesic properties of the aqueous extract of Jatropha curcas leaves.

Plant material and preparation of extract
The leaves of J. curcas were obtained from Apakila area in Abeokuta, Ogun State and verified by botanists at the Forestry Research Institute of Nigeria, (FRIN) Ibadan, Oyo State, Nigeria; a specimen of this plant was deposited at this institute.
The leaves were air-dried, pulverized, finely sieved and soaked in 2 l of distilled water for 24 h, after which it was filtered. Thereafter, the filtrate obtained was freeze-dried and the percent yield was 2.4%. This sample was stored in the refrigerator until used in the experiment.

Drugs
Aspirin, indomethacin, and paracetamol were purchased from a registered chemist in Abeokuta, Ogun State.

Animals
Eighteen specified pathogen free Swiss mice of either sex weighing 25 g and 24 specified pathogen free Wistar rats of either sex weighing 150-200 g were used for this study. The animals were kept at the Animal House, College of Veterinary Medicine, University of Agriculture, Abeokuta. They were acclimatized to laboratory conditions for 2 weeks prior to the experiment during which they were introduced to growers mash (Vital feeds Limited, Ibadan, Nigeria). The animals were fed this mash and provided water ad libitum throughout the study. The housing provided had the following conditions: controlled lighting of 12:12 h of light: dark, temperature of 25 °C and relative humidity of approximately 50%. Animal experimentation protocols conformed to the Institutional Animal Ethics Committee's guidelines.

Carrageenan-induced rat paw oedema
Acute inflammation was induced using the carrageenan induced oedema model (Winter et al. 1962). The rats were starved for 24 h after which they were divided into 4 groups (Groups A-D) of 6 animals each. Rats in group A (negative control, untreated) were given saline at 10 ml·kg -1 1 h before 0.1 ml of 1% freshly prepared suspension of carrageenan (Sigma Chemical Co.) was injected into the plantar surface of the right hind paw of each rat. Group B rats were given aqueous extract of J. curcas leaves orally at the dose of 150 mg·kg -1 1 h before carrageenan injection. Rats in group C were given aspirin, a standard anti-inflammatory drug at the dose of 150 mg·kg -1 orally 1 h before carrageenan injection while group D rats were administered indomethacin, another standard anti-inflammatory drug at a dose of 10 mg·kg -1 orally 1 h before carrageenan injection.
The linear circumference of the paw was measured after carrageenan injection (0 h) and 3 h after carrageenan injection using a loop of thread tied round the paw such that it was neither too loose nor too tight. The length of the thread around the paw was then measured on a ruler and rounded off to the nearest centimetre. The percentage inhibition was calculated according to the formula: Where: Co = Mean paw size at 0 h after carrageenan injection, C 1 = Mean paw size at 3 h after carrageenan injection Acetic acid-induced abdominal writhing in mice This was done as described by Koster et al. (1959). The mice were divided into 3 groups (groups 1-3) of 6 mice each. Distilled water at 5 ml·kg -1 was administered orally to the mice of the control group (group 1), 50 mg·kg -1 paracetamol orally to the mice of group 2 and 150 mg·kg -1 of aqueous extract of J. curcas leaves orally to mice of group 3, 1 h before intraperitoneal injection of acetic acid (0.6%, volume/volume in distilled water, 10 ml·kg -1 ). The number of writhing exhibited by each animal was counted for 10 min, starting 10 min after acetic acid injection and computed according to the following formula: Percentage inhibition = Mean of writhing (control) -Mean of writhing (test) × 100 Mean number of writhing (control)

Statistical analysis
Results were expressed as mean ± SEM. Analysis of the data was done using the one-way Analysis of Variance (ANOVA) followed by the Duncan multiple range test. The P value < 0.05 was considered significant in all cases.

Anti-inflammatory test
Aqueous extract of J. curcas leaves at a dose of 150 mg·kg -1 exhibited significant antiinflammatory activity in carrageenan induced rat paw oedema model (Table 1). The percentage inhibition in an ascending order is J. curcas (60.7%) performed comparably to aspirin (64.3%) but indomethacin had the highest percentage inhibition value (83.9%).
Indomethacin had the highest percentage inhibition of the paw volume (83.9%), aspirin caused (64.3%) inhibition while the extract of J. carcus caused (60.7%) inhibition of increase in paw volume a performance well comparable with the standard drug aspirin.

Acetic acid-induced abdominal writhing in mice
Aqueous extract of J. curcas leaves at a dose of 150 mg·kg -1 also exhibited significant analgesic activity in mice ( Table 2). The mean number of writhing was significantly lower (P < 0.05) in mice given aqueous extract of Jatropha curcas leaves compared to the control but higher than that of the standard analgesic, paracetamol.

Discussion
The result of this study suggests that aqueous extract of J. curcas leaves has antiinflammatory effect comparable to those of the standard drugs such as aspirin and Table 1. Effect of aqueous extract of Jatropha curcas leaves on carrageenan induced paw oedema in Wistar rats.
Values are Mean ± SEM CBF -Circumference of the rat paw immediately after carrageenan injection (cm), CAF -Circumference of the rat paw 3 h after carrageenan injection (cm).

Groups (n = 6)
Dose ( indomethacin. This observation is in line with a similar study carried out by Singh et al. 2008 in which the methanolic root extract of J. curcas exhibited systemic and significant anti-inflammatory activity in carrageenan-induced rat paw oedema. Carrageenan-induced inflammatory process is believed to be biphasic (Vinegar et al. 1969). The initial phase seen at the 1 st h is attributed to the release of histamine and serotonin (Cruckhon and Meacock 1971). The second accelerating phase of swelling is due to the release of prostaglandin, bradykinin and lysozyme. It has been reported that the second phase of oedema is sensitive to both clinically useful steroidal and non-steroidal anti-inflammatory agents (Katzung 1998). The anti-inflammatory activity exerted by aqueous extract of J. curcas leaves suggests that it could affect kinnin, prostaglandin, bradykinin and lysozyme synthesis.
The presence of alkaloids and flavonoids in plants has been found to exert active antiinflammatory effects. Studies on the phytochemical analysis of J. curcas by Singh et al. (2008) have revealed the presence of alkaloids and flavonoids in the plant.
The analgesic effect of J. curcas plant was also noticed. Berkenkopf and Weichman (1988) reported that several chemicals such as phenylquinone and acetic acid could induce a writhing response in laboratory animals. Intraperitoneal injection of acetic acid in this experiment produced abdominal writhing response which was inhibited by aqueous extract of J. curcas leaves by 39.0% as against the standard drug paracetamol which inhibited the writhing by 57.9%.
This result provides a scientific basis for the utilization of this herb in traditional medicine for the treatment of wounds and other conditions that can cause inflammation. Further tests are needed to explore the exact mechanism of action at the molecular level and to know the actual constituents responsible for this activity.
In conclusion, aqueous extract of J. curcas leaves could serve as an alternative antiinflammatory therapy in managing inflammatory conditions or as complementary therapy allowing patients to take smaller doses of conventional anti-inflammatory drugs, thereby minimizing the side effects of these standard drugs.