Journal of Ethnopharmacology 154 (2014) 109–115

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Research Paper

Antinociceptive effect of ethanol extract of leaves of Lannea coromandelica Mohammad Zafar Imam n, Md. Moniruzzaman Department of Pharmacy, Stamford University Bangladesh, 51, Siddeswari Road, Dhaka 1217, Bangladesh

art ic l e i nf o

a b s t r a c t

Article history: Received 4 November 2013 Received in revised form 12 February 2014 Accepted 13 March 2014 Available online 21 March 2014

Ethnopharmacological relevance: Lannea coromandelica (Houtt.) Merr. is a plant locally called “Jiga”, found all over Bangladesh. Leaf of the plant is traditionally used in the treatment of local swellings, pains of body, toothache etc. This study evaluated the antinociceptive effect of the ethanol extract of L. coromandelica leaves (EELC). Materials and methods: The antinociceptive activity of the extract (at the doses of 50, 100, and 200 mg/ kg) was evaluated by using chemical- and heat-induced pain models such as acetic acid-induced writhing, hot plate, tail immersion, formalin, and glutamate test. To verify the possible involvement of opioid receptor in the central antinociceptive effect of EELC, naloxone was used to antagonize the effect. Besides, the involvements of ATP-sensitive K þ channel and cGMP pathway were also justified by using glibenclemide and methylene blue. Results: EELC demonstrated significant dose-dependent antinociceptive activity in the chemical- and heat-induced nociception in mice models (po0.05). These findings imply the involvement of both peripheral and central antinociceptive mechanisms. The use of naloxone confirmed the association of opioid receptors in the central antinociceptive effect. EELC also showed the involvements of ATPsensitive K þ channel and cGMP pathway for antinociceptive activity. Conclusions: This study reported the antinociceptive activity of the leaf of L. coromandelica and rationalized the traditional use of the leaf in the treatment of different painful conditions. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Lannea coromandelica Anacardiaceae Medicinal plant Pain Analgesic

1. Introduction Lannea coromandelica (Houtt.) Merr. (Family-Anacardiaceae) is a small deciduous tree, locally known as Jiga in Bangladesh, which is cultivated mainly for living fence posts. Different parts of the plant are used in different ailment in the traditional medicine of the country. Leaf of the plant is traditionally used in injury, hematochezia (Zheng and Xing, 2009). Twigs are used as toothstick, bark for skin diseases, brew, tender leaves and root for stomachache (Franco and Narasimhan, 2009). Boiled leaves are applied to local swellings and body pain (Chopra et al., 1956; Yusuf et al., 2009). The bark is used to treat dyspepsia, general debility, gout, dysentery (Kadir et al., 2013), bruises, wounds, sores, ulcers, sore eyes, gout, swelling and body pain (Singh and Singh, 1994), eruption of skin, ulcers, and toothache (Khan et al., 2009).The sap of the fruit with common salt is used to treat cold and cough (Reddy et al., 2006). Based on the traditional uses researchers have studied the validity of its uses in different diseases. The bark has

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Corresponding author. Tel.: þ 88 01816684184; fax: þ88 02 9355967176. E-mail address: [email protected] (M.Z. Imam).

http://dx.doi.org/10.1016/j.jep.2014.03.032 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

shown anti-inflammatory (Singh and Singh, 1994), hypotensive (Singh and Singh, 1996), antihyperglycemic (Mannan et al., 2010), wound healing, antimicrobial (Sathish et al., 2010), antioxidant and analgesic activity (Alam et al., 2012). Polyflavonoid tannin in the bark with zoosporicidal activity has been reported (Islam et al., 2002). The leaf extract possess antidiarrhoeal (Panda et al., 2012) and anti-inflammatory activity (Gandhidasan et al., 1991). A number of phytochemicals have been isolated form the plant. Leaf and flower contain quercetin-3-arabinoside and ellagic acid (Subramanian and Nair, 1971). Phlobatannin and leucocyanidin have also been isolated from the flower (Nair et al., 1963). Isolated compound from bark includes β-sitosterol, physcion and physcion anthranol B, (Subramanian and Nair, 1971) and dihydroflavonols such as (2R,3S)-( þ)-30 ,5-dihydroxy-40 ,7-dimethoxydihydroflavonol and (2R,3R)-( þ )-40 ,5,7-trimethoxydihydroflavonol and (2R,3R)-( þ)-40 ,7-di-O-methyldihydroquercetin, (2R,3R)-( þ)-40 ,7di-O-methyldihydrokaempferol and (2R,3R)-( þ )-40 -O-methyldihydroquercetin (Islam and Tahara, 2000). The use of L. coromandelica leaves in different painful conditions in folk medicine and lack of scientific study reporting its antinociceptive activity convinced us to design the present study to evaluate the effect of ethanol extract of L. coromandelica leaves

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using different pain models in mice. The present study also further investigated the possible mechanism(s) of action that participate in the EELC-induced antinociception.

2. Materials and methods 2.1. Plant material and extraction The leaves of L. coromandelica were collected from the Shitalakkha barrage side of Demra, Dhaka in May 2012. The samples were then identified by Bushra Khan, Principal Scientific Officer, Bangladesh National Herbarium, Mirpur, Dhaka, Bangladesh. A voucher specimen (DACB: 37549) has been deposited in the Herbarium for further reference. Powdered dried leaves (250 g) were macerated with 420 ml of ethanol with occasional stirring at 257 2 1C for 3 days. The extract was then filtered using a Buchner funnel and cotton filter. The solvent was completely removed by rotary evaporator and 9.44 g extract (Yield 3.78%) was obtained. This crude extract was used for the acute toxicity and antinociceptive activity studies. 2.2. Chemicals and drugs The following drugs and chemicals were used in the current study: morphine sulfate, diclofenac sodium (Square Pharmaceuticals Ltd., Bangladesh), naloxone (Hamein Pharmaceuticals GmbH), acetic acid (Merck, Germany), ethanol (Merck, Germany), formalin (Merck, Germany), methylene blue (Merck, Germany), L-glutamic acid (Merck, Germany), glibenclemide (Square Pharmaceuticals Ltd., Bangladesh), dimethylsulfoxide (DMSO) (Merck, Germany). 2.3. Animals Swiss albino mice (20–25 g) of both sex were collected from the Animal Resources Branch of the International Center for Diarrheal Disease Research, Bangladesh (ICDDR,B). The animals were kept in standard laboratory conditions (relative humidity 55–60%; room temperature 25 72 1C; 12 h light/dark cycle) and were provided with standard diet (ICDDR,B formulated) and clean water ad libitum. The animals were acclimatized to the laboratory environment for a period of 14 days before performing experiments. All the experimental animals were treated following the Ethical Principles and Guidelines for Scientific Experiments on Animals (1995) formulated by The Swiss Academy of Medical Sciences and the Swiss Academy of Sciences. The experimental processes were approved by the Institutional Ethics Committee (SUB/IAEC/12.01). AVMA Guidelines for the Euthanasia of Animals: 2013 Edition was followed in euthanasia of mice using Pentobarbital. 2.4. Drugs and treatments EELC was dissolved in dimethylsulfoxide (DMSO) and orally administered to the test animals 30 min before the experiments at the doses of 50, 100, and 200 mg/kg body weight in both the chemical-induced pain and heat-induced pain models. The standard drug morphine sulfate (5 mg/kg) used in hot plate and tail immersion tests and diclofenac sodium (10 mg/kg) in writhing and licking tests were prepared with saline water and administered intraperitoneally 15 min before the experiments while the animals in control group received vehicle (DMSO) orally at the dose of 10 ml/kg body weight 30 min before the experiments. Naloxone, a non-selective opioid receptor antagonist, was injected intraperitoneally at 2 mg/kg dose 15 min before the administration of morphine sulfate or EELC (50, 100, and 200 mg/kg) to investigate

the involvement of opioid receptor system. Methylene blue, a non specific inhibitor of NO/guanylyl cyclase (20 mg/kg) and glibenclamide, an ATP-sensitive K þ channel inhibitor (10 mg/kg) were also injected intraperitoneally to verify the involvement of cGMP and ATP-sensitive K þ channel pathway respectively. 2.5. Acute toxicity test Swiss albino mice were divided into control and three test groups which contain five animals each. EELC was administered orally at the doses of 1000, 2000, and 3000 mg/kg. The mice were allowed food and water ad libitum and all animals were observed for abnormal behaviors, allergic symptoms and mortality for the next 72 h (Walker et al., 2008). 2.6. Phytochemical screening The crude ethanol extract of L. coromandelica (EELC) leaves were qualitatively tested for the detection of carbohydrates, saponins, flavonoids, tannins, alkaloids, glycosides, glucosides, reducing sugars, proteins, gums, and steroids following standard procedures (Ghani, 2003). 2.7. Antinociceptive activity test 2.7.1. Hot plate test The mice that showed forepaw licking, withdrawal of the paw (s) or jumping response within 15 s on hot plate kept at a temperature of 50 70.5 1C were selected for this study 24 h prior to the experiment. Mice were fasted overnight with water given ad libitum. The animals were treated with morphine or EELC and were placed on Eddy's hot plate (Kshitij Innovations, Haryana, India) kept at a temperature of 507 0.5 1C. A cut off period of 20 s was maintained to avoid paw tissue damage (Eddy and Leimbach, 1953). The response in the form of forepaw licking, withdrawal of the paw(s) or jumping was recorded at 30, 60, 90, and 120 min following treatment. Then the percentage of the maximal possible effect (% MPE) was calculated using the following formula: % MPE ¼[(Postdrug latency) (Predrug latency)/(Cut off time)  (Predrug latency)]  100. 2.7.2. Tail immersion test To evaluate the central analgesic property the tail immersion test was performed. This procedure is based on the observation that morphine like drugs prolongs the tail withdrawal time from hot water in mice (D0 Amour and Smith, 1941). One to two cm of tail of the mice pretreated with morphine or EELC were immersed in warm water kept constant at 5470.5 1C. The latency between tail sub-mersion and deflection of tail was recorded. Mice that showed a latency period between 1.5 and 3.5 s were selected for this experiment and the pre-treatment latency was recorded. A latency period of 20 s was maintained to avoid tail tissue damage in mice. The latency period of the tail-withdrawal response was taken as the index of antinociception and was determined at 30, 60, 90, and 120 min after the administration of the drug and extract. Then the % MPE was calculated from using the same formula used in hot plate test. 2.7.3. Acetic acid-induced writhing test The mice were treated with drug or EELC and then the writhing was induced by injecting 0.6% acetic acid after 15 and 30 min, respectively, at the dose 10 ml/kg body weight. Five minutes after the injection of acetic acid, the mice were observed and the number of writhing was counted for 30 min (Sulaiman et al., 2010). The contractions of the abdomen, elongation of the body,

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twisting of the trunk and/or pelvis ending with the extension of the limbs were considered as complete writhing. 2.7.4. Formalin-induced nociception Mice were injected with 20 μl of a 2.5% formalin solution (0.92% formaldehyde) made up in saline, injected into the sub-plantar region of the right hind paw 60 min after EELC treatment and 15 min after injection of Diclofenac sodium. Licking or biting of the injected paw was recorded as nociceptive response at 0–5 min (neurogenic phase) and 15–30 min (inflammatory phase) after formalin injection (Santos, Calixto, 1997; Santos et al., 1999). 2.7.5. Glutamate-induced nociception 20 μL of glutamate (10 μmol/paw) was injected into the ventral surface of the right hind paw of the mice 30 min after EELC treatment and 15 min after injection of diclofenac sodium. The mice were observed for 15 min following glutamate injection. The number of licking of its injected paw was indicative of nociception (Beirith et al., 2002).

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2.8.3. Involvement of ATP-sensitive K þ channel pathway Possible contribution of K þ channel in the antinociceptive effect of EELC was evaluated by using the method described in Mohamad et al. (2011) and Perimal et al. (2011). The mice were pretreated with glibenclamide (10 mg/kg), an ATP-sensitive K þ channel inhibitor, intraperitonially 15 min before administration of either vehicle or EELC. The mice were challenged with i.p. injection of 0.6% acetic acid, 30 min post-treatment. Following the injection of acetic acid, the animals were immediately placed in a chamber and the number of writhing was recorded for 30 min, starting from 5 min post injection. 2.9. Statistical analysis The results are presented as Mean7 SEM. The statistical analysis of the results was performed using one way analysis of variance (ANOVA) followed by Dunnett's post hoc test or Bonferroni's test as appropriate using SPSS 11.5 software. Differences between groups were considered significant at the level of po 0.05.

2.8. Analysis of the possible mechanism of action of EELC 3. Results 2.8.1. Involvement of opioid system The possible participation of the opioid system in the antinociceptive effect of EELC was examined by injecting naloxone hydrochloride (2 mg/kg, i.p.), a non-selective opioid receptor antagonist, 15 min prior to the administration of either morphine or EELC. Then the hot plate and tail immersion latencies were sequentially measured at pretreatment, 30, 60, 90 and 120 min with the same cut off time of 20 s for the safety of animals (Khan et al., 2011). 2.8.2. Involvement of cyclic guanosine monophosphate (cGMP) pathway To verify the possible involvement of cGMP in the antinociceptive action caused by EELC, the mice were pre-treated with methylene blue (20 mg/kg), a non specific inhibitor of NO/guanylyl cyclase, intraperitonially 15 min before administration of EELC. Then the nociceptive responses against 0.6% acetic acid injection were observed for 30 min, starting from 5 min post injection. The numbers of abdominal writhing were counted as indication of pain behavior (Abacioğlu et al., 2000; Perimal et al., 2011).

3.1. Phytochemical screening Preliminary phytochemical screening tests of the crude extract of L. coromandelica showed the presence of alkaloid, glycoside, carbohydrate, saponin, and tannin. 3.2. Acute toxicity test Administration of EELC at the doses of 1000–3000 mg/kg orally had no effect on their behavioral responses or caused no allergic manifestation and no mortality during the observation period of 72 h after administration. Therefore, it can be indicated that EELC has low toxicity profile and the LD50 is more than 3000 mg/kg. 3.3. Hot plate test In the hot plate test there were no significant differences in the antinociceptive effect of EELC at 50, and 100 mg/kg doses. It showed the antinociceptive effect only at 200 mg/kg dose

Table 1 Antinociceptive effect of ethanol extract of L. coromandelica leaf, morphine, and reversal effect of naloxone in hot plate test. Treatment (mg/kg)

Control (0.1 ml/mouse) Morphine (5) EELC (50) EELC (100) EELC (200) NLX (2) Control (0.1 ml/mouse) þNaloxone (2) NLX (2) þMorphine (5) NLX (2) þEELC (50) NLX (2) þEELC (100) Naloxone (2) þEELC (200)

Response time (s) (% MPE) Pretreatment

30 min

60 min

90 min

120 min

6.417 0.37 5.87 7 0.20 5.65 7 0.14 4.46 7 0.44 5.36 7 0.12 7.22 7 0.40 6.87 7 0.26 5.93 7 0.27 5.36 7 0.13 4.357 0.16 5.34 7 0.60

7.137 0.35 13.107 1.17nn (51.17) 7.497 0.55 (12.84) 8.337 0.44 (24.90) 9.357 0.51 (27.24) 7.05 7 0.18 7.03 7 0.16 7.46 7 0.57a (10.87) 7.077 0.27 (11.68) 7.34 7 0.31 (19.10) 7.36 7 0.28 (13.74)

9.32 70.42 15.45 71.46nn (67.83) 8.10 70.91 (17.05) 9.59 70.38 (33.01) 10.54 70.51 (35.36) 6.65 70.30 7.94 70.12 7.85 70.16a (13.65) 7.37 70.35 (13.72) 8.36 70.21 (25.63) 8.68 70.26 (22.75)

9.62 7 0.24 15.707 0.86nn (69.60) 10.047 0.79 (30.62) 11.047 0.45 (42.36) 12.667 0.36n (49.85) 6.08 7 0.35 8.81 7 0.13 9.25 7 0.19a (23.59) 8.39 7 0.32 (20.70) 8.53 7 0.21b (26.66) 9.487 0.38c (28.24)

9.747 0.47 15.84 7 1.38 (70.59) 13.32 7 0.99 (53.45) 13.46 7 1.73 (57.90) 14.88 7 1.09n (65.05) 4.25 7 0.63 8.58 7 0.85 9.22 7 0.32 (23.41) 11.75 7 0.88 (43.66) 12.047 0.83 (49.14) 12.20 7 0.85 (46.80)

Each value is presented as the Mean 7 SEM (n¼ 5). EELC¼ Ethanol extract of. Lannea coromandelica leaves; NLX¼ Naloxone; p o 0.05 compared with the EELC 50 group (Bonferroni's test). n

po 0.05 compared with the control group (Dunnett's test). p o0.01 compared with the control group (Dunnett's test). a p o 0.001 compared with the morphine group (Bonferroni's test). b p o0.05 compared with the EELC 100 group (Bonferroni's test). c po 0.05 compared with the EELC 200 group (Bonferroni's test). nn

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(p o0.05) (Table 1). The antinociceptive effect of EELC is dosedependent. The effect of naloxone was not noticeable at any of the doses. Administration of morphine at the doses of 5 mg/kg demonstrated a significant antinociceptive effect when compared with control group (p o0.01). 3.4. Tail immersion test The tail-immersion test results showed significant antinociceptive activity (po 0.001) compared with control, at the doses of 100 and 200 mg/kg. The antinociceptive effect of 50, 100, and 200 mg/ kg of EELC were comparable to that of the reference drug (Table 2). The antinociceptive activity of morphine used in this test was antagonized by naloxone. Naloxone significantly decreased the antinociceptive effect produced by morphine (p o0.001) and EELC at 100 and 200 mg/kg group (p o0.05). 3.5. Acetic acid-induced writhing test The administration of EELC at 50, 100, and 200 mg/kg doses caused a significant reduction in the number of writhing episodes induced by acetic acid compared to the control (p o0.001). The percentage inhibition of constrictions was calculated as 72.78% (diclofenac sodium), 18.78% (EELC 50 mg/kg), 34.63% (EELC 100 mg/kg), and 71.52% (EELC 200 mg/kg) (Table 3). 3.6. Formalin-induced nociception The results showed that EELC at 50, 100, and 200 mg/kg dose caused a significant dose-dependent inhibition of both neurogenic (0–5 min) and inflammatory (15–30 min) phases of formalin induced licking (Table 4). However, its antinociceptive effect was more pronounced in the second phase of this model of pain. Here, EELC 100 and 200 mg/kg significantly (p o0.001) reduced the nociception in late phase. Diclofenac sodium (10 mg/kg, i.p.) significantly (p o0.001) reduced formalin-induced nociception in both phases. 3.7. Glutamate-induced nociception The result of glutamate-induced nociception test showed that administration of EELC (50, 100, and 200 mg/kg) produced significant inhibition of the glutamate-induced nociception (Table 5). Diclofenac sodium (10 mg/kg) was used as a positive control drug

which showed 68.63% inhibition of licking as compared to the control group. All treatments showed significant antinociceptive activity compared with the control group (p o0.001). 3.8. Involvement of cyclic guanosine monophosphate (cGMP) pathway The present study looked at the effects of 50, 100, and 200 mg/ kg EELC and methylene blue (20 mg/kg) treatments. EELC and Table 3 Antinociceptive effect of ethanol extract of L. coromandelica leaf in acetic acidinduced abdominal writhing test in mice. Treatment (mg/kg)

Writhing (Mean 7SEM)

% Inhibition

Control (0.1 ml/mouse) Diclofenac sodium (10) EELC (50) EELC (100) EELC (200)

68.87 4.024 18.727 2.51n 55.88 7 5.05 44.98 7 2.37n 19.60 7 3.43n

– 72.78 18.78 34.63 71.52

Values are expressed as Mean 7 SEM (n¼ 5); EELC ¼Ethanol extract of Lannea coromandelica leaves. n

Denotes p o 0.001 compared with control group (Dunnett's test).

methylene blue administration alone significantly inhibited acetic

Table 4 Antinociceptive effect of ethanol extract of L. coromandelica leaf in formalin test in mice. Treatment (mg/kg)

Number of licking or biting (Mean 7SEM)

% Inhibition

Early phase (0–5 min)

Early phase Late phase (0–5 min) (15–30 min)

Late phase (15–30 min)

Control 1167 12.84 (0.1 ml/mouse) Diclofenac 26.2 7 1.46n sodium (10) EELC (50) 1107 6.04 EELC (100) 102.40 7 4.28 EELC (200) 95.40 7 5.30

135 73.39

–

–

6.20 70.86n

77.41

95.41

128.60 74.70 92.40 73.49n 36.4 73.85n

5.17 11.72 17.76

4.74 31.56 73.04

Values are expressed as Mean 7 SEM (n¼ 5); EELC ¼Ethanol extract of Lannea coromandelica leaves. n

Denotes p o 0.001 compared with control group (Dunnett's test).

Table 2 Antinociceptive effect of ethanol extract of L. coromandelica leaf, morphine, and reversal effect of naloxone in tail immersion test. Treatment (mg/kg)

Control (0.1 ml/mouse) Morphine (5) EELC (50) EELC (100) EELC (200) Naloxone (2) Control (0.1 ml/mouse) þNaloxone (2) NLX (2) þMorphine (5) NLX (2) þEELC (50) NLX (2) þEELC (100) Naloxone (2) þEELC (200)

Response times (s) (% MPE) Pretreatment

30 min

60 min

90 min

120 min

1.98 7 0.17 1.54 7 0.12 2.167 0.15 2.137 0.09 2.22 7 0.18 1.87 7 0.12 1.747 0.18 2.02 7 0.18 2.077 0.11 2.217 0.29 2.05 7 0.06

2.217 0.17 3.09 7 0.38 (8.42) 2.69 7 0.15 (2.99) 3.20 7 0.18 (5.98) 3.38 7 0.16n (6.52) 2.25 7 0.18 2.017 0.10 2.067 0.09 (0.24) 2.217 0.13 (0.78) 2.247 0.14b (0.19) 2.39 7 0.06c (1.92)

2.647 0.26 3.977 0.27n (13.15) 3.02 7 0.19 (4.84) 3.43 7 0.10 (7.30) 3.63 7 0.08n (7.92) 2.69 7 0.20 2.117 0.10 2.60 7 0.10a (3.27) 2.53 7 0.06 (2.59) 2.88 7 0.13 (3.75) 3.047 0.06 (5.50)

2.78 7 0.26 4.377 0.29n (15.35) 3.337 0.18 (6.54) 3.96 7 0.10n (10.22) 4.20 7 0.17n (11.13) 2.30 7 0.17 2.147 0.13 2.977 0.14a (5.33) 2.96 7 0.12 (4.96) 3.09 7 0.08b (4.96) 3.34 7 0.09 (7.22)

3.417 0.44 4.077 0.30 (13.71) 3.75 7 0.13 (8.90) 4.077 0.21 (10.86) 4.22 7 0.19 (11.23) 2.22 7 0.32 2.337 0.19 3.357 0.23 (7.42) 3.357 0.08 (7.14) 3.647 0.19 (8.05) 3.747 0.19 (9.44)

Each value is presented as the Mean 7 SEM (n¼ 5). EELC¼ Ethanol extract of. Lannea coromandelica leaves; NLX ¼ Naloxone; p o0.05 compared with the EELC 50 group (Bonferroni's test). n

po 0.001 compared with the control group (Dunnett's test). p o 0.001 compared with the morphine group (Bonferroni's test). b p o0.05 compared with the EELC 100 group (Bonferroni's test). c po 0.05 compared with the EELC 200 group (Bonferroni's test). a

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Table 5 Effect of ethanol extract of L. coromandelica leaf in glutamate-induced nociception in mice. Treatment (mg/kg)

Licking number (Mean 7 SEM)

% Inhibition

Control (0.1 ml/mouse) Diclofenac sodium (10) EELC (50) EELC (100) EELC (200)

183.0 7 10.64 57.40 7 3.04n 120.0 7 8.26n 79.007 2.00n 59.007 2.51n

– 68.63 34.43 56.83 67.76

Values are expressed as Mean 7 SEM (n ¼5); EELC¼ Ethanol extract of Lannea coromandelica leaves. n

Denotes po 0.001 compared with control group (Dunnett's test).

Table 6 Effect of ethanol extract of L. coromandelica leaf on involvement of cyclic guanosine monophosphate (cGMP) pathway. Treatment (mg/kg)

Writhing (Mean 7 SEM)

% Inhibition

Control (0.1 ml/mouse) Methylene blue (MB) (20) EELC (50) EELC (100) EELC (200) MB (20)þ EELC (50) MB (20)þ EELC (100) MB (20)þ EELC (200)

74.30 7 1.97 52.30 7 3.30 50.30 7 2.50n 41.007 1.28n 23.80 7 2.48n 48.107 0.80 29.107 1.83a 21.30 7 0.75

– 29.61 32.30 44.82 67.97 35.26 60.83 71.33

Values are expressed as Mean 7 SEM (n¼ 5); MB¼Methylene blue, EELC¼ Ethanol extract of Lannea coromandelica leaves; n

a

Denotes po 0.001 compared with control group (Dunnett's test). p o 0.01 compared with the EELC 200 group (Bonferroni's test).

Table 7 Effect of ethanol extract of L. coromandelica leaf on involvement of ATP-sensitive K þ channel pathway. Treatment (mg/kg)

Writhing (Mean 7 SEM)

% Inhibition

Control (0.1 ml/mouse) Glibenclamide (G) (10) EELC (50) EELC (100) EELC (200) G (10) þEELC (50) G (10) þEELC (100) G (10) þEELC (200)

76.7071.42 77.10 71.85 53.90 73.99n 42.90 71.73n 20.50 72.04n 60.30 71.95 51.60 75.44 41.30 71.77

– – 29.73 44.07 73.27 21.38 32.72 46.15

Values are expressed as Mean 7 SEM (n¼ 5); G ¼Glibenclamide, EELC¼ Ethanol extract of Lannea coromandelica leaves; n

Denotes po 0.001 compared with control group (Dunnett's test).

acid induced abdominal writhing (Table 6). Given together, methylene blue significantly (p o0.001) enhanced EELC (100 and 200 mg/kg) induced antinociception compared to the treatment of both EELC and methylene blue alone.

3.9. Involvement of ATP-sensitive K þ channel pathway The present study looked at the effects of 50, 100, and 200 mg/ kg EELC and glibenclemide (10 mg/kg) treatments. It was found that glibenclamide (10 mg/kg) administration alone did not alter abdominal writhing count when assessed through the injection of 0.6% acetic acid (Table 7). When given together, antinociceptive activity of EELC (100 mg/kg, 200 mg/kg) was markedly decreased by glibenclamide.

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4. Discussion The present study demonstrates that oral administration of EELC elicits dose-dependent antinociceptive effect in both chemical- and heat-induced animal models. The significant increase in latency time in hot plate by EELC, at 200 mg/kg dose, suggests central antinociceptive activity of EELC. The effect is further supported by the results observed in the tail immersion test as the tail withdrawal response in hot water-induced pain is selective only for centrally acting analgesics, while the peripherally acting agents are inactive (Srinivasan et al., 2003). To approach possible antinociceptive mechanism(s) for EELC, we examined the effect of naloxone, a non-selective opioid receptor antagonist, against the antinociceptive effect of EELC. The results of the hot plate and tail immersion test indicated that antinociceptive effect of EELC was reversed to some extent by naloxone. These data suggest that the antinociceptive effect of EELC may be partly exerted through opioid receptors at the spinal and supraspinal level. Both tailimmersion and hot-plate tests are based on measuring the response of the animal to thermal stimuli where the tailimmersion monitor a spinal reflex, and the hotplate is used for supraspinal reflex (Arslan and Bektas, 2010). In agreement with this suggestion, it has been demonstrated that μ2- and δ-opioid receptors are involved in spinal mechanism, while μ1/μ2-opioid receptors may mediate mainly supraspinal analgesia (Jinsmaa et al., 2004, 2005). Therefore, there is a high possibility that the central antinociceptive effect of EELC may be prominent on μ-opioid receptors. The acetic acid-induced writhing test has been used for the evaluation of peripheral antinociceptive activity (Trongsakul et al., 2003). In addition, writhing response is widely accepted as a model visceral pain due to the release of endogen mediators of pain, such as prostaglandins, kinins, etc. (Deraedt et al., 1980; Ahmed et al., 2007; Sulaiman et al., 2008; Zheng et al., 2009). Oral administration of EELC produced significant antinociceptive effect in acetic acid-induced writhing test. Intraperitoneal administration of acetic acid causes an increase in cyclooxygenase (COX), lipoxygenase (LOX), prostaglandins (PGs), histamine, serotonin, bradykinin, substance P, IL-1β, IL-8,TNF-α in the peripheral tissue fluid. Increased level of these mediators causes the excitation of primary afferent nociceptors entering dorsal horn of the central nervous system (Ikeda et al., 2001). The release of these inflammatory mediators is also thought to contribute to increased blood brain barrier (BBB) permeabilization or disruption (Radu et al., 2013). Besides, injection of pain inducing agent like acetic acid is also reported to increase vasodilation and vascular fluid permeability and these events were reversed by plant extracts (Anosike et al., 2012). The inhibition of writhing response in our study clearly indicated the peripheral antinociceptive effect of EELC in addition to its central effect. The potent antinociceptive effect of EELC in the acetic acid-induced model, therefore, suggests that EELC may be involved in the inhibition of the release of endogenous nociceptive mediators and may involve the permeability reversal effect at both BBB and vascular level. Formalin test results show that EELC caused significant and dose-dependent antinociception at both neurogenic (early phase) and inflammatory (late phase) pain responses caused by formalin in mice. Formalin is a well-described model of nociception and can be consistently inhibited by typical analgesic and anti-inflammatory drugs, including diclofenac sodium, morphine, indomethacin and dexamethasone (Hunskaar and Hole, 1987; Tjølsen et al., 1992). Considering the inhibitory property of EELC on the second phase of formalin, we might suggest an anti-inflammatory action of the plant extract. In addition, recent studies have shown that formalin activates primary afferent sensory neurons through a specific and direct action on Transient Receptor Potential Vanilloid-1 (TRPA-1), a

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member of the Transient Receptor Potential family (TRP) of cation channels that is highly expressed by a subset of C-Fibre nociceptors (McNamara et al., 2007). Preliminary phytochemical screening revealed that tannins, triterpinoids and flavonoids are present in L. coromandelica. The total phenolic content in the methanolic extract of the leaves of L. coromandelica estimated by gallic acid and tannic acid equivalents was found to be 7.43 and 11.864 respectively (Reddy et al., 2011). These compounds may be involved in its noticeable antinociceptive effect in these models. Flavonoids, for example, have been found to suppress the intracellular Ca2 þ ion elevation in a dose dependent manner, as well as the release of proinflammatory mediators such as TNF-α (Kempuraj et al., 2005). Flavonoids may increase the amount of endogenous serotonin or may interact with 5-HT2 and 5-HT3 receptors which may be involved in the mechanism of central analgesic activity (Annegowda et al., 2010). There are also reports on the role of flavonoid, a powerful antioxidant (Vinson et al., 1995; Brown and Rice-Evans, 1998), in analgesic activity primarily by targeting prostaglandins (Ramesh et al., 1998). Moreover, EELC showed significant analgesic activity in the entire experimental model which may be due to its high flavonoid. Moreover, flavonoids have the ability to inhibit ecosanoid biosynthesis. Ecosanoids, such as prostaglandins are involved in various immunological responses and are the end products of the cyclooxyginase and lypoxygenase pathways (Jothimanivannan et al., 2010). Tannins are also found to have a contribution in antinociceptive activity (Ramprasath et al., 2006). So it can be assumed that cyclooxygenase (COX) inhibitory activity along with antioxidant activity may reduce the production of arachidonic acid from phospholipid or may inhibit the enzyme system responsible for the synthesis of prostaglandins and ultimately relieve pain-sensation. The oral administration of EELC also produced a dosedependent inhibition of the nociceptive response caused by glutamate into hind paw of mice. The nociceptive response caused by glutamate involves peripheral, spinal and supraspinal sites of action with glutamate receptors such as AMPA, Kainate and NMDA receptors. This interaction contributes in modulating this nociceptive response (Neugebauer, 2001a,b; Beirith et al., 2002). So the antinociceptive activity by EELC may be associated with its interaction with the glutamatergic system. The current study also investigated the possible participation of cGMP pathway on the antinociceptive activity of EELC. Physiological functions such as pain and analgesia are influenced by the cellular level of cGMP regulated by the action of sGC mediated by nitric oxide (NO) (Abacioğlu et al., 2000). It has been reported that the activity of cGMP on the ion channels may be direct or through the activation of protein kinases and phosphodiesterases (Xu et al., 1995). The availability of cGMP regulates the activation or deactivation of nociresponsive neurons while concentration of intracellular cGMP is regulated by the action of soluble guanylyl cyclase (sGC) and as well as by the rate of degradation by cGMP-specific phosphodiesterases. To detect the possible involvement of cGMP in EELCinduced antinociception, methylene blue (MB), a guanylyl cyclase and/or NO inhibitor, was administered prior to inducing nociception with intraperitoneal injection of acetic acid. The result presents that the pretreatment with methylene blue significantly reduced the nociception caused by acetic acid and also enhanced the antinociceptive effect exerted by EELC. It has been suggested that MB promotes antinociceptive activity by inhibiting peripheral NO and sGC that resulted in NO interference (Abacioğlu et al., 2000). As pretreatment with MB and subsequent administration of EELC at all doses increased antinociceptive effect in acetic acidinduced writhing model in mice than the groups where only EELC were administered, it is quite clear that the antinociceptive effect of EELC involves the NO–cGMP pathway.

The results of the present study also demonstrate that glibenclamide, an ATP-sensitive K þ channel antagonist, partly reversed the antinociceptive activity shown by EELC. Previous studies reports specific blockade of ATP-sensitive K þ channel by glibenclamide while not affecting other types of K þ channel like Ca2 þ activated and voltage-gated K þ channels (Alves and Duarte, 2002; Jesse et al., 2007). The results of this study, therefore, might indicate the involvement of ATP sensitive K þ channel opening and subsequent efflux of K þ ion and membrane repolarization and/or hyperpolarization by EELC which reduces the membrane excitability (Lawson, 1996). In conclusion, the results of the present study indicate that EELC possesses significant antinociceptive activity that is prominent in chemical-induced pain models than heat-induced models. However, the central effect shown in the study was to some extent reversed by the antagonist naloxone that suggests the involvement of opioid receptor in the mechanism. The antinociceptive effect of EELC also involved cGMP pathway and ATP sensitive K þ channel pathway. Further studies are required to fully understand the mechanisms of action of EELC and testing of potential isolated compounds to find the bioactive compound that may act as a lead for drug development.

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