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Antiinflammatory and antipyretic activities of Clausena anisata Jude Efiom Okokon1, Anwanga Effiong Udoh1, Ukeme Essien Andrew1, Louis Uchechukwu Amazu2 1Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria. 2Department of Pharmacology and Therapeutics, College of Medicine, Envans Envwerem University, Orlu Campus, Nigeria. *Corresponding author: judeefiom@yahoo.com; Tel: +234-802-3453678 Received: 5 July 2012, Revised: 16 July 2012, Accepted: 19 July 2012

Abstract Anti-inflammatory and antipyretic activities of leaf extract of Clausena anisata were evaluated to ascertain the folkloric claim of its anti-inflammatory and antipyr-etic effects. The crude leaf extract (39–117mg/kg) of C. anisata was investigated for anti-inflammatory and antipyretic activities using various experimental models. The extract caused a significant (p<0.05–0.001) dose-dependent reduction of infla-mmation and fever induced by different agents used. The anti-inflammatory and antipyretic effects of this plant may in part be mediated through the chemical cons-tituents of the plant Keywords: Clausena anisata, Anti-inflammatory, antipyretic

Introduction Clausena anisata (Wild) Hook .F. ex Benth (Rutaceae)(syn. Clausena abyssinica

(Engl.) Engl., Clausena inequalis (DC.) Benth.) is a tropical shrub or tree up to 10 meters high growing in and on evergreen forests. It is commonly known as ‘mbiet ekpene’ by the Ibibios of Niger Delta region of Nigeria. The plant is traditionally use as effective remedies for worms infections, respiratory ailments, hypertension, malaria, fever, rheumatism, arthritis and other inflammatory conditions, headaches, pains, toothaches, convulsions and others (Hutchings et al.,1996). The Ibibios use the plant to treat measles (Ajibesin et al.,2005), ma-laria, pains and inflammations (Uwaifo, 1984; Philip Ikpe, personnal communication).The plant has been reported to contain coumarins, limonoids, carbazole alkaloids, monoterpeno-ids furanocoumarin lactones (Lakashmi et al.,1984) and essential oils (Cakrabirty and Chow-dhury, 1995; Ngadjui et al.,1989; Ito et al., 2000; Usman et al., 2010). Reports of antimicrob-ial (Gundidza et al.,1994), antibacterial (Senthikumar and Venkatesalu,2009), antidiabetic (Ojewole, 2002), anticonvulsant (Makanju, 1983), antitumor promoting (Ito et al., 2000) and in vivo antiplasmodial and analgesic (Okokon et al., 2012) activities have been published. This study was carried out to evaluate the anti-inflammatory and antipyretic activities of this

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plant to confirm its use traditionally to treat inflammatory diseases and fever. Materials and methods Plant Materials

The fresh leaves of Clausena anisata were collected from the Ukap in Ikono area of

Akwa Ibom State and were identified and authenticated as Clausena anisata (Wild) Hook .F. ex Benth (Rutaceae) by Dr. (Mrs.) Margaret Bassey of the Department of Botany and Ecolo-gical studies, University of Uyo and deposited at University of Uyo herbarium (UUH 653). Extraction

The leaves of the plant were air-dried, pulverized using pestle and mortar and cold-

macerated for 72 hours using ethanol. The liquid ethanolic extract that was obtained by filtra-tion was concentrated and evaporated to dryness in vacuo at 40C using rotary evaporator.. The ethanolic extract was stored at -4OC until used. Phytochemical Screening

Phytochemical screening of the crude leaf extract was carried out employing standard

procedures and tests (Trease and Evans, 1989, Sofowora, 1993), to reveal the presence of ch-emical constituents such as alkaloids, flavonoids, tannins, terpenes, saponins, anthraquinon-es, reducing sugars, cardiac glycosides among others. Animals

The animals (Swiss albino mice) both male and female that were used for these expe-

riments were obtained from University of Uyo animal house. The animals were housed in standard cages and were maintained on a standard pelleted Feed (Guinea Feed) and water ad libitum. Permission and approval for animal studies were obtained from College of Health Sciences Animal Ethics committee, University of Uyo. Determination of Median Lethal dose (LD50) The median lethal dose (LD50) of the extract was estimated using albino mice by intr-aperitoneal (i.p) route using the method of Miller and Tainter (1944). This involved intraperi-toneal administration of different doses of the extract (100 – 1000 mg/kg) to groups of three mice each. The animals were observed for manifestation of physical signs of toxicity such as writhing, decreased motor activity, decreased body/limb tone, decreased respiration and dea-th. Evaluation of anti-inflammatory activity of the extract Carrageenin-induced mice hind paw oedema

Increase in the mice hind paw linear circumference induced by planar injection of the

phlogistic agent was used as the measure of acute inflammation (Winter et al., 1962). Adult albino mice of either sex were used after 24 hours fast and deprived of water only during

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experiment. Inflammation of the hind paw was induced by injection of 0.1ml of freshly prep-ared carrageenin suspension in normal saline into the sub planar surface of the hind paw. The linear circumference of the injected paw was measured before and 0.5, 1, 2, 3, 4 and 5 hrs after administration of phlogistic agent. For routine drug testing, the increase in paw circumf-erence 0.5, 1, 2, 3, 4 and 5 hrs after administration of phlogistic agent was adopted as the par-ameter for measuring inflammation (Winter, et al., 1962; Akah and Nwambie, 1994; Ekpen-du et al., 1994, Besra et al., 1996). Edema (inflammation) was assessed as difference in paw circumference between the control and 0.5, 1, 2, 3, 4 and 5 hrs after administration of phlogi-stic agent [Hess and Milonig, 1992]. The extract (39, 78 and 117 mg/kg i.p) was administer-ed to various groups of mice, 1 hr before inducing inflammation. Control mice received carr-ageenin while reference group received ASA (100 mg/kg). The average (mean) oedema was assessed by measuring with vernier calipers. Egg-albumin induced inflammation

Inflammation was induced in mice by the injection of egg albumin (0.1ml, 1% in nor-

mal saline) into the sub planar tissue of the right hind paw (Akah and Nwambie, 1994). The linear circumference of the injected paw was measured before and 0.5, 1, 2, 3, 4 and 5hrs after the administration of the phlogistic agent. The leaf extract (39, 78 and 117 mg/kg i.p) and ASA (100 mg/kg orally) were administered to 24 hrs fasted mice 1 hr before the inducti-on of inflammation. Control group received 10 ml/kg of distilled water orally. Edema (infla-mmation) was assessed as the difference in paw circumference between the control and 0.5, 1, 2, 3, 4 and 5 hrs after the administration of the phlogistic agent (Hess and Milonig, 1972). The average (mean) edema was assessed by measuring with vernier calipers. Xylene–induced ear oedema

Inflammation was induced in mice by tropical administration of 2 drops of xylene at

the inner surface of the right ear. The xylene was left to act for 15 mins. C. anisata leaf extra-ct (39, 78, and 117 mg/kg i.p), dexamethasone (4 mg/kg) and distilled water (0.2 ml/kg) were orally administered to various groups of mice 30 minutes before the induction of inflammati-on. The animals were sacrificed under light anaesthesia and the left ears cut off. The differen-ce between the ear weights was taken as the oedema induced by the xylene (Tjolsen et al., 1992). Evaluation of antipyretic activity of the extract 2,4–Dinitrophenol (DNP) induced pyrexia

Adult albino rats (120–165 g) of both sexes fasted for 24 hours but allowed water ad

libitum were used for the experiment. They were randomized into groups of 6 rats each. DNP (10 mg/kg, i.p) was administered to the rats after obtaining the basal rectal temperatures. Hy-perthermia developed within 30 min of DNP administration. Different doses of extract (39, 78, and 117 mg/kg i.p), aspirin (100 mg/kg) and distilled water (10 ml/kg, orally) were admi-nistered respectively to the treatment and control groups of animals. Rectal temperatures of the animals were obtained at an hour interval for 5 hrs (Backhouse et al., 1994; Winter et al., 1962; Mbagwu et al., 2007).

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D-amphetamine induced pyrexia Adult albino rats (120–175 g) of both sexes fasted for 24 hours but allowed water ad

libitum were used for the experiment. They were randomized into groups of 6 rats each. Am-phetamine (5 mg/kg, i.p) was administered to the animals after obtaining basal temperatu-res. Hyperthermia developed 0.5hrs following amphetamine administration. The extract (39, 78 and 117 mg/kg, i.p) aspirin (100 mg/kg orally) and distilled water (10 ml/kg orally) were administered to the animals at peak hyperthermia. Rectal temperatures were obtained at 1hr interval for 5hrs (Blackhouse et al., 1994; Bamgbose and Noamesi, 1981; Mbagwu et al., 2007). Yeast-induced pyrexia

Adult albino rats (130–170 g) of both sexes fasted for 24 hours but allowed water ad

libitum were used for the experiment. They were randomized into groups of 6 rats each. At zero hour, the basal temperature of the rats was taken using digital clinical thermometer. Thereafter, each animal was administered subcutaneously with 20% W/V aqueous suspensi-on of yeast at a volume of 10 ml/kg (Gural et al., 1955, Okokon and Nwafor, 2010). At suita-ble intervals beginning one hour after yeast injection, rectal temperature of animals were taken, animals with increase of 1C were selected and grouped for the study. The extract un-derstudy was administered i.p. after the pyrogen at doses of 39, 78 and 117 mg/kg to respec-tive groups of rats. The control group received distilled water (10 ml/kg) and the reference group administered with ASA (100 mg/kg) both intraperitoneally. The rectal temperature of the groups was taken at 1hr interval for 5hrs. Statistical analysis and data evaluation

Data obtained from this work were analyzed statistically using Students’ t-test and

ANOVA (One- or Two-way) followed by a post test (Tukey-Kramer multiple comparison test). Differences between means will be considered significant at 1 % and 5 % level of sign-ificance i.e P ≤ 0.01and 0.05. Results Phytochemical screening

The phytochemical screening of the ethanolic extract of the leaves of Clausena anis-

ata revealed the presence of cardiac glycosides, tannins, saponins, terpenes and flavonoids. Acute toxicty

The median lethal dose (LD50) was calculated to be 393.7± 25.64 mg/kg. The physic-

al signs of toxicity included excitation, paw licking, increased respiratory rate, decreased mo-tor activity, gasping and coma which was followed by death.

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Anti-inflammatory activity Carragenin-induced oedema in mice

The effect of ethanolic leaf extract of C. anisata on carragenin-induced oedema is sh-

own in figure 1. The extract exerted a significant (P<0.05– 0.001) antiinflammatory effect in a dose–dependent manner which was comparable to the standard drug, ASA,100mg/kg. Egg albumin- induced oedema

Administration of leaf extract of C. anisata on egg albumin - induced oedema in mice

caused a significant (p<0.05–0.001) dose-dependent anti-inflammatory effect against oede-ma caused by egg albumin .The effect was comparable to that of standard drug, ASA (100 mg/kg) (Figure 2).

Figure 1: Effect of Clausena anisata leaf extract on carrageenin- induced oedema in mice.

Figure 2. Effect of Clausena anisata leaf extract on egg- albumin induced oedema in mice.

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Xylene-induced ear edema

Anti-inflammatory effect of leaf extract of C. anisata against xylene-induced ear oed-ema in mice is shown in Table 1. The extract exerted a dose-dependent anti-inflammatory effect which was significant (P<0.0 01) when compared to control. The effect was incompar-able to that of the standard drug, dexamethasone (4.0 mg/kg).

Antipyretic test

Dinitrophenol induced pyrexia

The antipyretic effect of the extract on DNP induced pyrexia is shown in Table 2. Ad-ministration of the leaf extract of C. anisata (39, 78 and 117 mg/kg) in the presence of the pyrogen caused a significant (P<0.05–0.001) reduction in the temperatures of the extract tre-ated rats when compared with the control. The antipyretic effect was dose-dependent and c-omparable to that of the standard drug, ASA (100 mg/kg).

Amphetamine–induced pyrexia

The effect of the extract on amphetamine induced pyrexia is shown in Table 3. The extract exerted a significant (P<0.05– 0.001) dose-dependent antipyretic effect when compa-red to control. The antipyretic effect of the extract was comparable to that of the standard, ASA (100 mg/kg). Yeast-induced pyrexia

Table 4 shows the effect of the extract against yeast-induced pyrexia.There was a do-se-dependent reduction in the temperature of rats treated with the leaf extract. The reductions

Table 1: Effect of Clausena anisata leaf extract on xylene-induced ear oedema in mice.

Dose (mg/kg) Weight of right ear (g) Weight of right ear (g) Increase in ear weight (g) % Inhibition

Control 0.075 0.01 0.043 0.00 0.032 0.01 Extract 450 0.050 0.01 0.041 0.01 0.009 0.01a 71.87 Extract 450 0.048 0.01 0.042 0.01 0.006 0.01a 81.25 Extract 450 0.043 0.01 0.038 0.01 0.005 0.00a 84.37 Asa 100 0.040 0.01 0.036 0.01 0.004 0.00a 87.50

Data are expressed as mean SEM. Significant at aP < 0.05, b P < 0.001 when compared to control. n = 6.

Table 2. Effect of Heinsia crinata leaf extract on DNP induced pyrexia in mice.

Time interval (hr) Dose (mg/kg)

0 0.5 1 2 3 4 5

Control 35.01 0.16

36.65 0.13

37.36 0.09

37.23 0.08

36.91 0.21

36.86 0.18

36.50 0.20

Extract 450 34.48 0.10

36.48 0.10

35.84 0.08b

35.75 0.07c

35.28 0.05c

34.66 0.04c

34.46 0.06c

Extract 450 34.31 0.13

36.35 0.19

35.76 0.43b

35.45 0.17c

35.03 0.16c

34.56 0.16c

34.40 0.17c

Extract 450 34.75 0.19

36.75 0.09

36.08 0.12b

35.61 0.10c

35.26 0.10c

34.40 0.13c

34.26 0.14c

Asa 100 35.08 0.10

38.86 0.23

36.40 0.20

36.28 0.19b

35.67 0.20b

34.53 0.20c

34.25 0.17c

Data are expressed as mean SEM. Significant at aP < 0.05, b P < 0.001 when compared to control. n = 6.

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Table 3. Effect of Heinsia crinata leaf extract on amphetamine induced pyrexia in mice.

Time interval (hr) Dose (mg/kg)

0 0.5 1 2 3 4 5

Control 34.50 0.04

36.30 0.12

36.82 0.13

36.80 0.11

36.76 0.10

36.71 0.13

36.30 0.11

Extract 450 34.45 0.16

36.45 0.10

36.30 0.15

35.86 0.15a

35.45 0.14a

35.28 0.17a

34.63 0.17b

Extract 450 34.68 0.23

36.58 0.18

35.98 0.22a

35.75 0.32a

35.25 0.30c

34.55 0.31c

34.46 0.29b

Extract 450 34.38 0.22

36.68 0.29

35.87 0.16

35.53 0.21a

35.18 0.20b

34.46 0.17c

34.40 0.13c

Asa 100 34.55 0.25

36.76 0.17

36.50 0.12a

36.26 0.19a

35.76 0.20a

35.23 0.21s

35.06 0.22a

Data are expressed as mean SEM. Significant at aP < 0.05, b P < 0.001 when compared to control. n = 6.

Table 4. Effect of Heinsia crinata leaf extract on yeast induced pyrexia in mice.

Time interval (hr) Dose (mg/kg)

0 0.5 1 2 3 4 5

Control 35.01 0.11

36.93 0.15

36.93 0.11

36.86 0.08

36.75 0.07

36.58 0.06

36.55 0.03

Extract 450 34.73 0.15

36.91 0.14

36.87 0.11

36.51 0.20

36.01 0.21b

35.61 0.17c

35.21 0.17c

Extract 450 34.78 0.12

36.85 0.14

36.76 0.12

36.50 0.08a

36.10 0.04b

35.55 0.06c

35.33 0.05c

Extract 450 34.83 0.09

36.85 0.08

36.65 0.08

36.23 0.05b

35.76 0.03c

35.43 0.08c

35.18 0.06c

Asa 100 35.03 0.08

36.05 0.11

36.56 0.05

36.10 0.12b

35.66 0.05c

35.46 0.06c

35.24 0.06c

Data are expressed as mean SEM. Significant at aP < 0.05, b P < 0.001 when compared to control. n = 6. caused by the extract was significant (P<0.005 – 0.001) when compared to control and comp-arable to that of the standard drug, ASA (100 mg/kg). Discussion

Clausena anisata is use traditionally by the Ibibios of Niger Delta regions of Nigeria

in the treatment of inflammatory conditions like pains, fever, arthritis and heamorrhoids (Hu-tchings et al., 1996). The present study was carried out to evaluate these properties scientific-ally using different experimental models.

In the carragenin induced oedema, the extract (39 – 117 mg/kg) exerted pronounced

effect at the early stage of inflammation (1-2hr) indicating effect probably on histamine, ser-otonin and kinnins that are involved in the early stage of carragenin induced oedema (Vane and Booting,1987). The extract also reduced later stage of the oedema maybe due to its abili-ty to inhibit prostaglandin which is known to mediate the second phase of carragenin induced inflammation (Vane and Booting,1987). However, ASA (100 mg/kg) a prototype NSAID, a cyclooxygenase inhibitor whose mechanism of action involves inhibition of prost-aglandin, inhibited significantly the paw swelling due to carragenin injection.

The extract also inhibited egg albumin-induced oedema demonstrating that it can

inhibit inflammation by blocking the release of histamine and 5-HT, two mediators that are

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Okokon et al.

released by egg albumin (Nwafor et al., 2007). However, ASA, a cyclooxygenase inhibitor reduced significantly oedema produced by egg albumin.

The leaf extract exerted considerable inhibition of ear oedema caused by xylene in a

dose-dependent manner. This suggest the inhibition of phospholipase A2 which is involve in the pathophysiology of inflammation due to xylene (Lin et al.,1992). However, dexamethaso-ne, a steroidal antiinflammatory agent also produced significant reduction in the mean right ear weight of positive control rats indicating an inhibition of PLA2. Flavonoids are reported to be involved in antiinflammatory activity of plants (Parmer and Gosh,1978). These have been found to be present in the extract.

In this study, the extract was observed to inhibit greatly DNP-, amphetamine and yea-st-induced pyrexia. The extract is likely to reduce pyrexia by reducing brain concentration of prostaglandin E2 especially in the hypothallamus through its action on COX-3 (Botting and Ayoub,2005) or by enhancement of the production of the body’s own antipyretic substances like vasopressin and arginine (Chandrasekharan,2002).

In conclusion, the results of this study support the ethnobotanical use of the plant in

the treatment of febrile illnesses and inflammatory conditions. Further investigation is being advocated especially in elucidating cellular mechanisms and establishing structural compone-nts of the active ingredients with a view of standardizing them. Conflict of interest

There is no conflict of interest associated with the authors of this paper.

Acknowledgement

The authors are grateful to Mr Enefiok Ukpong of Pharmacology and Toxicology

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