78 – 82 Received: June 2014 Accepted: September 2014 ISSN 2006 – 6996

Bajopas Volume 7 Number 2 December, 2014 http://dx.doi.org/10.4314/bajopas.v7i2.15

Bayero Journal of Pure and Applied Sciences, 7(2): 78 – 82 Received: June 2014 Accepted: September 2014 ISSN 2006 – 6996

ANALGESIC AND ANTI-INFLAMMATORY EFFECTS OF AQUEOUS LEAFEXTRACT OF COMBRETUM MICRANTHUM G. DON ( COMBRETACEAE )

1Abdullahi, M.H., 2Anuka, J.A., 1Yaro, A.H., and 1Musa, A. 1Department of Pharmacology, Bayero University, Kano 2Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria Correspondence author: [email protected]

ABSTRACT The analgesic and anti-inflammatory effects of the aqueous leaf extract of Combretum micranthum were studied in mice and rats. The extract was screened for analgesic activity; using acetic acid induced writhing in mice and formalin induced paw licking test in rats. Anti-inflammatory effect was evaluated using formalin induced hind paw oedema in rats. Results showed that, at a dose of 200 mg/kg the extract significantly (p < 0.05) reduced the number of abdominal constrictions in mice and at doses of 100 and 200 mg/kg, the extract significantly (p < 0.05) reduced the licking time in rats in the formalin induced paw licking test. The extract at doses of 50,100 and 200 mg/kg significantly (p < 0.05) reduced hind paw oedema in rats from the first hour of formalin administration. The intraperitoneal LD 50 value of the extract was found to be 2,154.1mg/kgin mice and 2,852.l mg/kg in rats. The analgesic and anti-inflammatory activities of the plant extract may probably be due to the presence of phytochemical contents. Keywords: Combrentum micranthum, Analgesic, Anti-inflammatory, Mice, Rats.

INTRODUCTION shown Combretum micranthum to possess strong The practices of traditional medicine are based on antimalarial activity against both chloroquine sensitive beliefs that have been in existence for hundreds of and resistant strain of Plasmodium falciparium (Kola years, but still prevalent today and people of all and Benjamin, 2002).The methanol leaves extract of continents have this old tradition(Selby, 1998).The C. glutinosum was studied for anti-inflammatory earliest use of plants for medicinal uses probably activity in rats (Abdul-Fattah et al., 2000). Tannins, began as far back as 300BC, during this periodthe flavones and amines were also reported (Burkill, early Greek naturalist described plant and classified 1985). Combretummicranthum is shown to contain them by use as herbs, shrubs and trees (Klein, tannins, carbohydrates, saponins, alkaloids, and 1979).However, since ancient time, man was able to flavonoids but other Combretum species are reported know that, some fruits, stem, and leaves of many to contain anthraquinones, terpenoids and sterols plants can be used to cure some diseases including (Trease and Evans, 1997).Kola and Benjamin (2002) treatment of wounds as done by our rural folks reported that, the methanol leaves extract of (Sofowora, 1982). The knowledge that plants can cure C.micranthum contain alkaloids. Generally diseases is probably instinctive because, even animals Combretaceae family is rich in tannins, saponins, seek out appropriate herbs when they are ill. sterols, carbohydrates, glycoside and trace of alkaloid Herbalists used leaves, flowers, stems and roots of (Trease and Evans, 1996). The objective of this study plant to prevent, relieve and treat illnesses. is to establish analgesic and anti-inflammatory activity Combretum micranthum of the Family of the aqueous leaf extract of Combretum micranthum Combretaceae is widely distributed in savannah in mice and rats. regions and in some places near the coast as a shrub or a tree and its may grow up to 10 m in length, with MATERIALS AND METHODS opposite, ovate and acuminate leaves, the flowers are Plant collection and extraction borne as auxiliary cluster on scaly stalks and the fruits Fresh leaves of Combretummicranthum was collected are small with scaly and four winged (Burkill, from Malumfashi L.G.A, Katsina State. Plant was 1985).Combretum micranthum is locally known as identified and authenticated in the herbarium of the farargeza (Hausa), Okan (Yoruba) and Nzaotego Department of Biological Sciences, Ahmadu Bello (Igbo) (Burkill, 1985).Verbal discussion with herbalist University, Zaria, Nigeria, by comparing with voucher revealed that, leaf of C. micranthum is used in the specimen number 900257. Leaves were air dried management of bleeding, fever, pain and tumor. The under the shade at room temperature (30 oC) for 28 ashes of burnt wood of C. micranthum are used in days and then grounded into a fine powder using Northern Nigeria, as a dehairing agent in preparation pestle and mortar. About 700 g of powdered material of skin for tanning (Burkill, 1985). It was also found was soaked in water for two weeks and maceration that, C. micranthum plant contains potent anti- method was used in the extraction. The extract was microbial constituents (Sofowora, 1993). Studies have concentrated on water bath at temperature of 60 oC.

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Animals formalin, Groups 1, 2 and 3 were treated with extract Mice (weighing 25 – 30 g) and rats (weighing150 – at doses of 50, 100, and 200 mg/kg body weight ( i.p. ) 180g) of either sex were used for the experiments. respectively. Group 4 was treated with Diclofenac 25 Animals were obtained from animal House of Faculty mg/kg ( i.p. ) while Group 5 was administered with of Pharmaceutical sciences, Ahmadu Bello University normal saline 10 ml/kg ( i.p. ). Increases in linear paw Zaria. Animals were kept in a well-ventilated room, circumferences were taken as an index of increase in fed with a pelletized grower mash (vital) and water paw volume which is a measure of oedema. The paw provided ad -libitum . volume (cm) was measured at 1, 2, 3, 4, and 5 hour after formalin injection using vernier caliper. Acute toxicity study The intraperitoneal median lethal dose ( LD 50 ) Statistical Analysis determination was conducted using the method of The results were expressed as Mean ± SEM. The Lorke (1983). significance of difference between the means was determined by the student’s t-test and results were Acetic acid induced writhing test considered significant when p < 0.05. Method of Koster etal (1959) was used. Thirty mice were divided into five groups of six mice each. Groups RESULTS 1, 2 and 3 were treated with extract at doses of 50, Acute Toxicity Study 100, and 200 mg/kg body weight ( i.p. ) respectively. The median lethal dose ( LD 50 ) afterintraperitoneal Group 4 was treated with piroxicam 10 mg/kg body (I.P) administration of the aqueous leaf extract is weight intraperitoneally while Group 5 received normal reported in Table 1. The LD 50 value of the extract was saline 10 ml/kg body weight intraperitoneally. Thirty found to be 2,154.1mg/kgin mice and 2,852.l mg/kg minutes after treatment, mice in all groups were in rats v administered 0.6% ( /v)freshly preparedacetic acid Analgesic Activities solution ( i.p. ) and the number of abdominal Acetic acid induced writhing in mice contractions were counted for each animal five The anti-abdominal constrictions effect of aqueous minutes post acetic acid administration for 10 leaf extract of C. micranthum in mice induced with minutes. acetic acid is presented in figure 1. The extract Formalin induced paw licking test significantly (P < 0.05) reduced the number of Method of Hunskaar and Hole(1987) was adopted. In abdominal constrictions at the dose of 200 mg/kg this test, licking time in seconds was registered from compared to group administered with normal saline 0-5 minutes (first Phase) and 20 - 25 minutes (second while piroxicam (standard drug) significantly reduced phase) after subplantar administration of formalin (20 abdominal constrictions in mice at P < 0.01. v µl of 1% /v) in the right hind paw of the rat. A total of Formalin induced licking in rats 30 rats were divided into five different treatment The inhibitory effect of aqueous leaf extract of C. groups with six rats in each group; Groups 1, 2 and 3 micranthum on formalin induced paw licking response were treated with extract at doses of 50, 100, and 200 in rat is presented in figure 2. At doses of 50, 100 and mg/kg body weight ( i.p. ) respectively. Group 4 was 200 mg/kg, the extract significantly (P < 0.05) treated with pentazocin 10 mg/kg body weight reduced paw licking time in the second phase intraperitoneally while Group 5 was administered with compared to group treated with normal saline. The normal saline 10 ml/kg body weight intraperitoneally. reduction of paw licking time was dose dependent. Thirty minutes post-treatment 20 µl of freshly prepared1 % solution of formalin was administered at Anti -inflammationactivity the subplantar region of right hind paw of each rat. Formalin induced hind paw oedema in rats Rats were placed individually in a transparent glass The inhibitory effect of aqueous leaf extract of C. cylinder 20 cm in diameter and the time spent licking micranthum on formalin induced hind paw oedema in was recorded during 0 – 5 minutes and 20 – 25 rat is presented in Table 2.The extract significantly (P minutes. < 0.05) inhibit the progressive increase hind paw Formalin induced hind paw oedema test oedema in rats at doses of 50, 100 and 200 mg/kg This test was conducted according to Sayyah et from the first hour and significantly (P < 0.01) showed al (2003).Thirty rats were divided into five different inhibitory response similar to the standard drug treatment groups with six rats in each group. Formalin (Diclofenac) in the fifth hour at the highest dose of solution was injected into subplantar of the right hind 200 mg/kg while at lowest dose of 50 mg/kg showed paw of the rat. Thirty minutes before injection of no significant inhibition in the fourth and fifth hour.

Table 1: Acute Toxicity Studies of aqueous leaf extract of C.micranthum ( LD 50 ) in mice and rats Extracts Route of Animal species LD 50 values administration (mg/kg) ALE I.P Mice 2154.1 ALE I.P Rat 2852.1

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Table 2: Effect of aqueous leaf extract (ALE) of C.micranthum on formalin induced hind paw oedema in rats Dose Paw oedema (cm) at the Treatment (mg/kg) 1hr 2hr 3hr 4hr 5hr ALE 50 0.60+0.01 a 0.61+0.01 b 0.61+0.01 a 0.64+0.01 0.67+0.02 ALE 100 0.59+0.02 a 0.60+0.01 a 0.61+0.01 a 0.63+0.02 a 0.65+0.01 ALE 200 0.57+0.02 a 0.60+0.01 a 0.60+0.01 a 0.62+0.02 b 0.64+0.02 b Diclofenac 25 0.59+0.02 a 0.61+0.02 b 0.61+0.01 a 0.63+0.02 a 0.64+0.02 b N/saline 0.2 0.67+0.01 0.76+0.02 0.74+0.05 0.79+0.03 0.84+0.02 aP< 0.05 and bp< 0.01, Students t-test, Mean + SEM, n =6

a 10

Number of writhes b

0 N/Saline ALE 50 ALE 100 ALE 200 Piroxicam 10 Dose (mg/kg)

a Fig. 1: The effect of ALE of C. micranthum on acetic acid induced writhing in mice. p< 0.05; bp< 0.01 Student’s t-test , Mean ± SEM for n = 6, ALE= Aqueous Leaf Extract

50 First phase Second phase 40

Licking time (sec) a

20 a a 10 b

N/Saline ALE 50 ALE 100 ALE 200 Pentazocine 10

Dose (mg/kg) a b Fig. 2: The effect of ALE of C. micranthum on formalin induced licking in rats. p< 0.05; p< 0.01 Student’s t-test, Mean ±SEM for n = 6, ALE= Aqueous Leaf Extract 80 Bajopas Volume 7 Number 2 December, 2014

DISCUSSION reported in the phytochemical constituents. Previous In the present work, the aqueous leaf extract of C. phytochemical screening of C.micranthum revealed micranthum was investigated for acute toxicity, the presence of flavonoids, tannins, alkaloids, analgesic and anti-inflammatory activities. The results phenanthrenes, dihydrophenanthrenes, gums and of acute toxicity studies ( LD 50 ) of aqueous leaf extract glycosides in the plant extract (Pazini, 1993). of C. micranthum in mice and rats showed that the Flavonoids extracted from Elaeagnusangustifolia fruit extract was less-toxic via I.P administration and can be have been reported to have analgesic and anti- used for folkloric medicine, considering the LD 50 by inflammatory effects in rats (Ahmadiani etal., 2000). Lorke (1983); LD 50 < 1.0mg/kg very toxic, LD 50 < Sawant etal (2004) reported that a total alkaloid 10mg/kg toxic, LD 50 up to 100 mg/kg less toxic, LD 50 up fraction of E. alba , may be responsible for both central to 1000 mg/kg slightly toxic and substances with and peripheral analgesic activity in rats. Vougtau LD 50 values greater than 5,000 mg/kg are practically etal (2000) also reported that, flavonoids isolated from non-toxic. The same authors had earlier identified the some medicinal plants have been proven to possess following phytochemical constituents; alkaloids, antinociceptive and anti-inflammatory effect. flavonoids, glycosides, saponins, tannins and Flavonoids have been shown to inhibit prostaglandin phlabotannins in the aqueous leaf extract of C. biosynthesis, a group of powerful pro-inflammatory micranthum (Abdullahi et al., 2014). Some chemical signaling molecules, and also phosphodiesterase constituents found present in the aqueous leaf extract involved in cell activation (Manthey et al. , 2001). The are known to have analgesic and anti-inflammatory aqueous leaf extract of C. Micranthum may have activities. produced its effects by inhibition of cyclo-oxygenase-1 The extract at a higher dose significantly (COX-1) or cyclo –oxygenase-2 (COX-2), but COX-2 reduced the number of acetic acid induced writhes in has been found to be responsible for inflammatory mice which revealed peripheral analgesic property of conditions (Boxtel, 2001). This shows that, the extract the extract in the peripheral tissue. The dose – might have produced analgesic or anti-inflammatory dependent inhibitory activity of the extract on formalin effects by possible inhibition of COX-2, an enzyme induced paw licking in rats showed that the extract responsible for the conversion of arachidonic acid to also possess central analgesic property comparable to prostaglandin. the standard drug. The aqueous leaf extract of C. micranthum may therefore have both central and CONCLUSION peripheral analgesic properties. The inhibitory activity TheCurrent result showed that the aqueous leaf of extract on formalin induced paw licking in rats was extract of C. micranthum possesses antinociceptive found to be greater in the second phase and it is a and anti-inflammatory activities comparable to the measure of the anti-inflammatory property of the standard drugs used in the research and this may extract. The analgesic and anti-inflammatory activities account for use of the plant in the management of of theaqueous leaf extract of C.micranthum may be pain and inflammation in traditional medicine. due to the presence of alkaloids and flavonoids as

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