Journal of Ethnopharmacology 172 (2015) 364–367
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Ethnopharmacological communication
Evaluation of antinociceptive activity of ethanol extract of bark of Polyalthia longifolia Md. Moniruzzaman a,b,n, Afia Ferdous b, Fatama Wahid Bokul b a b
College of Pharmacy, Dongguk University, Goyang 410-820, Republic of Korea 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 17 February 2015 Received in revised form 13 May 2015 Accepted 23 June 2015 Available online 10 July 2015
Ethnopharmacological relevance: Polyalthia longifolia var. pendula is a very popular herb in Bangladesh due to its traditional uses in treatment of rheumatism, bone fracture and gastric ulcer. The present study was conducted to investigate the antinociceptive activity of ethanol extract of P. longifolia (EEPL) bark. Materials and methods: Hot plate and tail immersion tests, acetic acid-induced writhing test, glutamate and formalin-induced paw licking tests in mice were employed in this study. In all the experiments EEPL was administered orally at the doses of 50, 100 and 200 mg/kg body weight. To investigate the possible participation of opioid system in EEPL-mediated effects, naloxone was used to antagonize the action. Results: EEPL showed a significant antinociceptive activity against both heat and chemical-induced nociception. The effects were dose-dependent and significant at the doses of 100 and 200 mg/kg of EEPL. Besides, pretreatment with naloxone caused significant inhibition of the antinociceptive activity induced by EEPL, revealing the possible involvement of the opioid receptors. Conclusion: These results indicate the antinociceptive activity of the bark of P. longifolia and support the ethnomedical use of this plant in treatment of different painful conditions. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Polyalthia longifolia Annonaceae Antinociceptive Medicinal plant
1. Introduction Polyalthia longifolia var. pendula (Family: Annonaceae), also known as debdaru, is a tall evergreen tree grown throughout Asia mainly in Bangladesh, India and Sri Lanka. For a longer period of time, almost all parts of this plant are being used in the traditional medicine system to treat a variety of diseases and disorders (Jothy et al., 2013). Especially the stem bark, which is regarded as the most common serving part of this plant, for its anti-rheumatic, antipyretic and antidiabetic properties (Savithramma et al., 2011). Besides, the bark is also beneficial to treat gastric ulcer, bone fracture (Suneetha et al., 2011), digestive and urinary disorders and so on (Katkar et al., 2010). Preliminary phytochemical screening revealed that the bark is enriched with terpenoids including γ-methoxybutenolide, γ-hydroxybutenolide, 16-hydroxycleroda-4(18), 13-dien-16,15-olide,16-oxocleroda-4(18), 13E-dien-15-oic acid, cleroda-4(18),-13-dien-16,15olide, hydroxycleroda-13-ene-15,16-olide-3-one (Jothy et al., 2013). Other then terpenoids it also contains flavonoids and alkaloids such as polylongine, polyfothine, isooncodine, darienine, penduline and isoursuline etc. (Wu et al., 1990; Jothy et al., 2013). Recently, several researchers reported the scientific validity of the stem bark as a n Corresponding author at: College of Pharmacy, Dongguk University, Goyang 410-820, Republic of Korea. E-mail address: [email protected] (Md. Moniruzzaman).
http://dx.doi.org/10.1016/j.jep.2015.06.053 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
potent antioxidant, antitumor and antibacterial agent (Jothy et al., 2013). However, till now there is no scientific reports investigated its antinociceptive properties in detail. Keeping this in view the present study aims to evaluate the antinociceptive activity of the P. longifolia stem bark in animal models of heat- and chemically-induced acute pain. Furthermore, we also tried to investigate possible mechanism (s) that participated in the EEPL-mediated action.
2. Materials and methods 2.1. Plant material and extraction The stem barks of P. longifolia were collected from Savar, Bangladesh and identified in Bangladesh National Herbarium with a voucher specimen number DACB: 37845. The powdered dried barks (250 g) were macerated with 400 ml of ethanol and 9.44 g of extract (yield 3.77% w/w) was obtained. Details are explained in Supplementary materials. 2.2. Phytochemical analyses 2.2.1. Identification of groups of phytochemicals The crude ethanol extract of P. longifolia (EEPL) barks was analyzed in a qualitative way to detect the presence of different
Md. Moniruzzaman et al. / Journal of Ethnopharmacology 172 (2015) 364–367
phytochemicals including alkaloids, glycosides, carbohydrates, saponins, flavonoids, tannins, terpenoids, glucosides, reducing sugars, proteins, gums, and steroids according to the standard procedures described previously (Ghani, 2003). 2.2.2. Total phenolic content determination The total phenolic content was quantified according to the previously described method as explained in Supplementary materials. 2.2.3. Total flavonoid content determination The total flavonoid content was determined according to the previously described method as explained in Supplementary materials. 2.3. Chemicals and drugs The chemicals and drugs used in this study are listed in Supplementary materials. 2.4. Animals Swiss albino mice of 20–25 g body weight were used in this study. Animal handling details are explained in Supplementary materials. All experimental protocols were approved by the Institutional Ethics Committee of Stamford University Bangladesh (No.: SUB/IAEC/12.04). 2.5. Drugs and treatments In both hot plate and tail immersion tests, morphine sulfate (5 mg/ kg) and in writhing and licking tests, diclofenac sodium (10 mg/kg) was used as reference drug. All the drugs were administered intraperitoneally (i.p.) 15 min before the nociceptive stimulation. On the other hand, DMSO (vehicle, 0.1 ml/mouse) or EEPL at the doses of 50, 100, and 200 mg/kg were administered orally before 30 min of the experiments. Naloxone (2 mg/kg), a non-specific opioid receptors antagonist was administered i.p. 15 min prior the treatment with morphine or EEPL. 2.6. Acute toxicity test This test was performed according to Walker et al. (2008) with slight modifications. Animals were divided into desired groups containing 5 in each. EEPL was administered to the animals orally at the doses of 500, 1000, and 2000 mg/kg. The animals were then allowed to take food and water ad libitum and observed for next 72 h to check any abnormal behaviors, allergic symptoms and mortality-induced by EEPL. During the observation period, animals behaviors like scratching and rubbing around the nose and head, irritability or aggression, hypersensitivity to touch and cyanosis around mouth and tail and puffiness around the eyes and mouth were considered as the indications of allergic symptoms. 2.7. Antinociceptive analysis 2.7.1. Hot plate test The hot plate test was performed according to Eddy and Leimbach (1953) as described in Supplementary materials. Then the percentage of the maximal possible effect (%MPE) was calculated using the following equation:
%MPE = [ {(Postdrug latency)−(Pre − drug latency)} /{(Cut off time)−(Pre−drug latency) }] × 100. 2.7.2. Tail immersion test The latency of tail withdrawal from hot water was recorded according to D’Amour and Smith (1941) as described in
365
Supplementary materials. 2.7.3. Acetic acid-induced writhing test The writhing induced by injecting 0.6% acetic was counted for 30 min as described (Moniruzzaman et al., 2015) in Supplementary materials. 2.7.4. Glutamate-induced nociception 10 μM Glutamate was injected into the right hind paw of the mice and the numbers of licking were scored as the degree of nociception as described in Supplementary materials. 2.7.5. Formalin-induced nociception The formalin test was performed according to the previously established method (Imam and Moniruzzaman, 2014) as described in Supplementary materials. 2.7.6. Involvement of opioid system This experiment was performed by administering naloxone as described in Supplementary materials. 2.8. Statistical analysis The results are expressed as mean 7 SEM. The statistical analysis was performed by one way analysis of variance (ANOVA) followed by Dunnett's posthoc test wherever applicable, using SPSS 11.5 software. Differences between groups were considered significant at P o0.05.
3. Results and discussion The present study demonstrated the central and peripheral antinociceptive effects of EEPL when assessed in thermal and chemical models of nociception as measured by hot-plate and tailimmersion tests, acetic acid-induced writhing test, glutamate and formalin-induced licking tests respectively. Furthermore, the oral administration of EEPL at 500–2000 mg/kg doses did not show any adverse effects like abnormal behavior or allergic manifestation or mortality during 72 h of observation period. The absence of toxicity presented by EEPL enabled us to establish the doses used in this study. Our starting experiments, the hot plate and tail immersion tests which are extensively used methods for investigating the central antinociceptive activities of a drug candidate where hot plate indicates the supraspinally organized response of pain and tail immersion represents the spinal mechanism. It has been demonstrated that the opioid agents exhibit their antinociceptive activity through the interaction with supraspinal (m1, ĸ3, δ1, s2) and spinal (m2, ĸ3, δ2) nociceptors (Morales and Perez-Garcia, 2001; Jinsmaa et al., 2005). In our study, EEPL exhibited antinociceptive effects both in hot plate and tail immersion tests by increasing hot plate latency and tail withdrawal time, respectively. The significant (P o0.01) effect was found from the higher doses of EEPL (100 and 200 mg/kg). As expected, morphine also produced similar pattern of effect in these tests (P o0.001) (Table 1 and S1). Besides, pretreatment with naloxone (2 mg/kg) caused a marked reduction (P o0.05) of the antinociceptive activity produced by EEPL especially with the doses of 100 and 200 mg/kg. Therefore, these findings suggest that the opioid receptors may contribute at least in part to the EEPL-mediated action. The acetic acid-induced writhing in mice, a widely used model of visceral pain, is highly sensitive and useful to screen new analgesic drugs. It has been reported that acetic acid induces writhing syndromes by increasing the production of different proinflammatory mediators like prostaglandin, prostacyclin and other
366
Md. Moniruzzaman et al. / Journal of Ethnopharmacology 172 (2015) 364–367
Table 1 Antinociceptive effect of EEPL, morphine and reversal effect of naloxone in hot plate test. Treatment (mg/Kg)
Response time (s) % MPE
Control (0.1 ml/mouse) Morphine (5) EEPL (50) EEPL (100) EEPL (200) NLX (2) NLX (2) þMorphine (5) NLX (2) þEEPL (50) NLX (2) þEEPL (100) NLX (2) þEEPL (200)
Pretreatment
30 min
60 min
90 min
120 min
5.89 7 0.26 5.87 7 0.20 5.727 0.74 5.42 7 0.15 5.067 0.49 5.69 7 0.22 5.767 0.09 5.417 0.37 5.25 7 0.13 5.52 7 0.31
6.047 0.13 11.90 7 0.57n (42.68) 7.157 0.54 (10.07) 8.22 70.41 (19.23) 9.59 70.38n (30.32) 5.89 70.23 8.03 70.54a (15.90) 6.177 0.26 (5.22) 6.737 0.26 (10.04) 7.85 7 0.34 (16.04)
6.247 0.22 15.02 7 0.82n (64.76) 8.20 7 0.52 (17.42) 10.54 7 0.51n (35.09) 11.94 7 1.00n (46.04) 5.95 7 0.27 10.51 70.82a (33.33) 7.08 7 0.33 (11.46) 8.077 0.32 (19.07) 9.03 7 0.70 (24.21)
6.62 7 0.16 16.177 0.60n (72.90) 8.89 7 0.60 (22.19) 11.45 7 1.00n (41.36) 13.187 0.32n (54.32) 5.87 7 0.19 11.39 7 0.78a (39.54) 7.53 7 0.33 (14.51) 8.65 7 0.29b (23.04) 9.54 7 0.50c (27.76)
6.707 0.22 16.38 7 0.90n (74.38) 9.80 70.52 (28.58) 11.477 0.49n (41.52) 13.96 7 0.34n (59.57) 6.18 70.16 12.017 0.22a (43.91) 8.60 70.43 (21.88) 9.80 70.55 (30.82) 11.377 0.26c (40.40)
Each value is presented as the mean7 SEM (n ¼5). EEPL ¼ Ethanol extract of bark of Polyalthia longifolia; NLX ¼ Naloxone. n
a b c
Po 0.001 compared with the control group (Dunnett's test). P o0.001 compared with the morphine group (Bonferroni's test). Po 0.05 compared with the EEPL 100 mg/kg group (Bonferroni's test). Po 0.05 compared with the EEPL 200 mg/kg group (Bonferroni's test).
80 160 140 120
** **
40
**
**
20
Number of licking
Number of writhing
60
100
**
80 60
**
**
40 20
0
Co
o ntr
clo
fen
S ac
o
g g/k
g g/k
m diu
0 L5
m
P
EE
PL
0 10
g g/k
m
PL
EE
0 20
m
Co
EE
140
160
120
140
100
* 80
60
40
ntr Di
**
20
Number of licking (late phase)
Number of licking (early phase)
Di
0
l
ol
clo
fen
S ac
od
0 L5
mg
P
EE
EE
PL
0 10
mg
/kg EE
PL
0 20
mg
/kg
120 100
**
80
**
60
**
40 20
0
/kg
ium
**
0
Co
ntr
ol
cl Di
ofe
na
o cS
diu
m EE
PL
m 50
g/k
g
EE
PL
1
m 00
g/k
g
EE
PL
2
m 00
g/k
g Co
ntr
ol
cl Di
ofe
na
o cS
diu
g
m
g/k
PL
EE
m 50
EE
PL
1
m 00
g/k
g
EE
PL
2
m 00
g/k
g
Fig. 1. Antinociceptive effects of EEPL and diclofenac sodium on the nociception induced by (A) 0.6% acetic acid (10 ml/kg), (B) glutamate (10 μMol/paw) and (C–D) 2.5% formalin (20 μl/paw). Acetic acid and glutamate were given after 30 min and formalin after 60 min of EEPL administration. Statistical analysis was performed using one-way ANOVA followed by the Dunnett's posthoc test. **P o 0.001; *Po 0.05 compared to the respective control.
Md. Moniruzzaman et al. / Journal of Ethnopharmacology 172 (2015) 364–367
cytokines in the peripheral tissue fluid, which in turn excite the nerve endings of the peripheral nociceptors (Ikeda et al., 2001; Ferdous et al., 2008). Our results demonstrated that EEPL can significantly (P o0.001) inhibit the number of writhing episodes induced by acetic acid (Fig. 1A). The highest percentage of writhing inhibition (73.49%) was found with the dose of 200 mg/kg of EEPL. Furthermore, the oral administration of EEPL produced a dose dependant inhibition of licking in the glutamate test. The significant (Po 0.001) effect was found at the dose of 100 and 200 mg/kg of EEPL with percent inhibition of 50.35 and 68.09 respectively (Fig. 1B). It is well established that glutamate produce nociceptive responses through the interaction with peripheral, spinal and supraspinal site of action with both NMDA and nonNMDA receptors (Beirith et al., 2002). So it is conceivable that the antinociceptive action exhibited by EEPL may be associated with its interaction with the glutamatergic system. In our next experiment, we observed that EEPL can also inhibit number of licking induced by 2.5% of formalin, in a dose-dependent fashion. However, EEPL at 200 mg/kg dose only could produce significant (Po0.05) effect (29.3% of licking inhibition) in the early phase of formalin test (Fig. 1C). On the other hand, in late phase, all doses of EEPL significantly (Po0.001) diminished number of licking with highest percent inhibition of 65.55% (Fig. 1D). It is well established that, in the first phase of formalin test, formalin causes direct activation of type C nociceptive nerve endings, releasing neuropeptides such as substance P, among others. In contrast, the second phase is related to the release of chemical mediators such as histamine, serotonin, bradykinin, prostaglandins and excitatory amino acids which are responsible to cause inflammation (Tjølsen et al., 1992; Santos and Calixto, 1997). Therefore, it is conceivable that the extract could produce antinociceptive effect, more prominently in the late phase, through down regulation of synthesis or action of the above mentioned nociceptive mediators. Although, our findings in thermal models revealed the central antinociceptive action of EEPL through possible involvement of opioid receptors, it is showing a quite different result of expectation in the early phase of formalin test where the nerve endings are directly involved in production of nociceptive responses. Thus, it is possible that the less efficacy produced by EEPL in early phase of formalin test is due to the differences in the mechanisms with thermal models where the antinociceptive action of EEPL is more prominent. Our phytochemical studies revealed that EEPL contains alkaloids, flavonoids, glycosides, terpenoids and tannins. Besides, total phenolic and flavonoid contents were quantified as 9.3172.57 mg/g and 13.1773.13 mg/g respectively. It has been reported that flavonoids can inhibit the increased level of Ca2 þ as well as release of different pro-inflammatory mediators like tumor necrosis factor-α. Besides, tannins also attributed for their antinociceptive activity (Starec et al., 1988; Kempuraj et al., 2005). Therefore it is conceivable that these groups of compounds may be participated in the observed antinociceptive effects produced by EEPL.
4. Conclusions Taken together, the results presented in current study provide the biological evidence for the antinociceptive activity of P. longifolia stem bark and support the traditional use of this plant as an analgesic agent in folk medicine. Moreover, the alteration of antinociceptive activity by treatment with naloxone suggests the possible involvement of the opioid receptors in the EEPL-induced antinociception in thermally-induced pain. In spite of that, more higher doses of EEPL may produce higher efficacy, which could be a query of the readers. Therefore, further phytopharmacological investigations leading to isolation and characterization of pure compound(s) is required to elucidate the exact mechanism(s) of the observed bioactivities.
367
Conflict of interest Authors declare that they have not any conflict of interest.
Acknowledgments Author's wishing to express profound heartfelt gratitude to Professor Dr. Bidyut Kanti Datta, Chairman, Department of Pharmacy, Stamford University Bangladesh, for his permission to use the facilities of the pharmacology laboratory.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jep.2015.06.053.
References Beirith, A., Santos, A.R.S., Calixto, J.B., 2002. Mechanisms underlying the nociception and paw oedema caused by injection of glutamate in to the mouse paw. Brain Res. 924, 219–228. D’Amour, F.E., Smith, D.L., 1941. A method for determining loss of pain sensation. J. Pharmacol. Exp. Ther. 72, 74–79. Eddy, N.B., Leimbach, D., 1953. Synthetic analgesics: II. Dithienylbutinyl and dithienylbutylamines. J. Pharmacol. Exp. Ther. 107, 385–393. Ferdous, M., Rouf, R., Shilpi, J.A., Uddin, S.J., 2008. Antinociceptive activity of the ethanolic extract of Ficus racemosa Lin. (Moraceae). Orient. Pharm. Exp. Med. 8, 93–96. Ghani, A., 2003. Medicinal Plants of Bangladesh (Chemical Constituents and Uses), Second ed. Asiatic Society of Bangladesh, Dhaka-1000, Bangladesh, pp. 331–332 274. Ikeda, Y., Ueno, A., Naraba, H., Oh-ishi, S., 2001. Involvement of vanilloid receptor VR1 and prostanoids in the acetic acid-induced writhing responses of mice. Life Sci. 69, 2911–2919. Imam, M.Z., Moniruzzaman, M., 2014. Antinociceptive effect of ethanol extract of leaves of Lannea coromandelica. J. Ethnopharmacol. 154, 109–115. Jinsmaa, Y., Fujitab, Y., Shiotanib, K., Miyazakic, A., Lib, T., Tsuda, Y., Okada, Y., Amboe, A., Sasakie, Y., Bryanta, S.D., Lawrence, H., Lazarus, L.H., 2005. Differentiation of opioid receptor preference by [Dmt1]endomorphin- 2-mediated antinociception in the mouse. Eur. J. Pharmacol. 509, 37–42. Jothy, S.L., Choong, Y.S., Saravanam, D., Deivanai, S., Latha, L.Y., Vijayarathna, S., Sasidharan, S., 2013. Polyalthia longifolia sonn: an ancient remedy to explore for novel therapeutic agents. Res. J. Pharm. Biol. Chem. Sci. 4, 714–730. Katkar, K.V., Suthat, A.C., Chauhan, V.S., 2010. The chemistry, pharmacologic, and therapeutic applications of Polyalthia longifolia. Pharmacogn. Rev. 4, 62–68. Kempuraj, D., Madhappan, B., Christodoulou, S., Boucher, W., Cao, J., Papadopoulou, N., 2005. Flavonols inhibit pro-inflammatory mediator release, intracellular calcium ion levels and protein kinase C theta phosphorylation in human must cells. Br. J. Pharmacol. 145, 934–944. Moniruzzaman, M., Ferdous, A., Irin, S., 2015. Evaluation of antinociceptive effect of ethanol extract of Hedyotis corymbosa Linn. whole plant in mice. J. Ethnopharmacol. 161, 82–85. Morales, L., Perez-Garcia, C., Alguacil, L.F., 2001. Effects of yohimbine on the antinociceptive and place conditioning effects of opioid agonists in rodents. Br. J. Pharmacol. 133, 172–178. Santos, A.R.S., Calixto, J.B., 1997. Further evidence for the involvement of tachykinin receptor subtypes in formalin and capsaicin models of pain in mice. Neuropeptides 31, 381–389. Savithramma, N., Rao, M.L., Suhrulatha, D., 2011. Screening of medicinal plants for secondary metabolites. Middle-East J. Sci. Res. 8, 579–584. Starec, M., Waitzová, D., Elis, J., 1988. Evaluation of the analgesic effect of RG-tannin using the “hotplate” and “tailflick” method in mice. Cesk Farm 37, 319–321. Suneetha, J., Prasanthi, S., Naidu, B.V.A.R., Reddi, T.V.V.S., 2011. Indigenous phytotherapy for bone fractures from Eastern Ghats. Indian J. Tradit. Knowl. 10, 550–553. Tjølsen, A., Berge, O.G., Hunskaar, S., Rosland, J.H., Hole, K., 1992. The formalin test: an evaluation of the method. Pain 51, 5–17. Walker, C.I.B., Trevisan, G., Rossato, M.F., Franciscato, C., Pereirac, M.E., Ferreira, J., Manfron, M.P., 2008. Antinociceptive activity of Mirabilis jalapa in mice. J. Ethnopharmacol. 120, 169–175. Wu, Y.C., Duh, C.Y., Wang, S.K., Chen, K.S., Yang, T.H., 1990. Two new natural azafluorene alkaloids and a cytotoxic aporphine alkaloid from Polyalthia longifolia. J. Nat. Prod. 53, 1327–1331.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Ethnopharmacological communication
Evaluation of antinociceptive activity of ethanol extract of bark of Polyalthia longifolia Md. Moniruzzaman a,b,n, Afia Ferdous b, Fatama Wahid Bokul b a b
College of Pharmacy, Dongguk University, Goyang 410-820, Republic of Korea 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 17 February 2015 Received in revised form 13 May 2015 Accepted 23 June 2015 Available online 10 July 2015
Ethnopharmacological relevance: Polyalthia longifolia var. pendula is a very popular herb in Bangladesh due to its traditional uses in treatment of rheumatism, bone fracture and gastric ulcer. The present study was conducted to investigate the antinociceptive activity of ethanol extract of P. longifolia (EEPL) bark. Materials and methods: Hot plate and tail immersion tests, acetic acid-induced writhing test, glutamate and formalin-induced paw licking tests in mice were employed in this study. In all the experiments EEPL was administered orally at the doses of 50, 100 and 200 mg/kg body weight. To investigate the possible participation of opioid system in EEPL-mediated effects, naloxone was used to antagonize the action. Results: EEPL showed a significant antinociceptive activity against both heat and chemical-induced nociception. The effects were dose-dependent and significant at the doses of 100 and 200 mg/kg of EEPL. Besides, pretreatment with naloxone caused significant inhibition of the antinociceptive activity induced by EEPL, revealing the possible involvement of the opioid receptors. Conclusion: These results indicate the antinociceptive activity of the bark of P. longifolia and support the ethnomedical use of this plant in treatment of different painful conditions. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Polyalthia longifolia Annonaceae Antinociceptive Medicinal plant
1. Introduction Polyalthia longifolia var. pendula (Family: Annonaceae), also known as debdaru, is a tall evergreen tree grown throughout Asia mainly in Bangladesh, India and Sri Lanka. For a longer period of time, almost all parts of this plant are being used in the traditional medicine system to treat a variety of diseases and disorders (Jothy et al., 2013). Especially the stem bark, which is regarded as the most common serving part of this plant, for its anti-rheumatic, antipyretic and antidiabetic properties (Savithramma et al., 2011). Besides, the bark is also beneficial to treat gastric ulcer, bone fracture (Suneetha et al., 2011), digestive and urinary disorders and so on (Katkar et al., 2010). Preliminary phytochemical screening revealed that the bark is enriched with terpenoids including γ-methoxybutenolide, γ-hydroxybutenolide, 16-hydroxycleroda-4(18), 13-dien-16,15-olide,16-oxocleroda-4(18), 13E-dien-15-oic acid, cleroda-4(18),-13-dien-16,15olide, hydroxycleroda-13-ene-15,16-olide-3-one (Jothy et al., 2013). Other then terpenoids it also contains flavonoids and alkaloids such as polylongine, polyfothine, isooncodine, darienine, penduline and isoursuline etc. (Wu et al., 1990; Jothy et al., 2013). Recently, several researchers reported the scientific validity of the stem bark as a n Corresponding author at: College of Pharmacy, Dongguk University, Goyang 410-820, Republic of Korea. E-mail address: [email protected] (Md. Moniruzzaman).
http://dx.doi.org/10.1016/j.jep.2015.06.053 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
potent antioxidant, antitumor and antibacterial agent (Jothy et al., 2013). However, till now there is no scientific reports investigated its antinociceptive properties in detail. Keeping this in view the present study aims to evaluate the antinociceptive activity of the P. longifolia stem bark in animal models of heat- and chemically-induced acute pain. Furthermore, we also tried to investigate possible mechanism (s) that participated in the EEPL-mediated action.
2. Materials and methods 2.1. Plant material and extraction The stem barks of P. longifolia were collected from Savar, Bangladesh and identified in Bangladesh National Herbarium with a voucher specimen number DACB: 37845. The powdered dried barks (250 g) were macerated with 400 ml of ethanol and 9.44 g of extract (yield 3.77% w/w) was obtained. Details are explained in Supplementary materials. 2.2. Phytochemical analyses 2.2.1. Identification of groups of phytochemicals The crude ethanol extract of P. longifolia (EEPL) barks was analyzed in a qualitative way to detect the presence of different
Md. Moniruzzaman et al. / Journal of Ethnopharmacology 172 (2015) 364–367
phytochemicals including alkaloids, glycosides, carbohydrates, saponins, flavonoids, tannins, terpenoids, glucosides, reducing sugars, proteins, gums, and steroids according to the standard procedures described previously (Ghani, 2003). 2.2.2. Total phenolic content determination The total phenolic content was quantified according to the previously described method as explained in Supplementary materials. 2.2.3. Total flavonoid content determination The total flavonoid content was determined according to the previously described method as explained in Supplementary materials. 2.3. Chemicals and drugs The chemicals and drugs used in this study are listed in Supplementary materials. 2.4. Animals Swiss albino mice of 20–25 g body weight were used in this study. Animal handling details are explained in Supplementary materials. All experimental protocols were approved by the Institutional Ethics Committee of Stamford University Bangladesh (No.: SUB/IAEC/12.04). 2.5. Drugs and treatments In both hot plate and tail immersion tests, morphine sulfate (5 mg/ kg) and in writhing and licking tests, diclofenac sodium (10 mg/kg) was used as reference drug. All the drugs were administered intraperitoneally (i.p.) 15 min before the nociceptive stimulation. On the other hand, DMSO (vehicle, 0.1 ml/mouse) or EEPL at the doses of 50, 100, and 200 mg/kg were administered orally before 30 min of the experiments. Naloxone (2 mg/kg), a non-specific opioid receptors antagonist was administered i.p. 15 min prior the treatment with morphine or EEPL. 2.6. Acute toxicity test This test was performed according to Walker et al. (2008) with slight modifications. Animals were divided into desired groups containing 5 in each. EEPL was administered to the animals orally at the doses of 500, 1000, and 2000 mg/kg. The animals were then allowed to take food and water ad libitum and observed for next 72 h to check any abnormal behaviors, allergic symptoms and mortality-induced by EEPL. During the observation period, animals behaviors like scratching and rubbing around the nose and head, irritability or aggression, hypersensitivity to touch and cyanosis around mouth and tail and puffiness around the eyes and mouth were considered as the indications of allergic symptoms. 2.7. Antinociceptive analysis 2.7.1. Hot plate test The hot plate test was performed according to Eddy and Leimbach (1953) as described in Supplementary materials. Then the percentage of the maximal possible effect (%MPE) was calculated using the following equation:
%MPE = [ {(Postdrug latency)−(Pre − drug latency)} /{(Cut off time)−(Pre−drug latency) }] × 100. 2.7.2. Tail immersion test The latency of tail withdrawal from hot water was recorded according to D’Amour and Smith (1941) as described in
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Supplementary materials. 2.7.3. Acetic acid-induced writhing test The writhing induced by injecting 0.6% acetic was counted for 30 min as described (Moniruzzaman et al., 2015) in Supplementary materials. 2.7.4. Glutamate-induced nociception 10 μM Glutamate was injected into the right hind paw of the mice and the numbers of licking were scored as the degree of nociception as described in Supplementary materials. 2.7.5. Formalin-induced nociception The formalin test was performed according to the previously established method (Imam and Moniruzzaman, 2014) as described in Supplementary materials. 2.7.6. Involvement of opioid system This experiment was performed by administering naloxone as described in Supplementary materials. 2.8. Statistical analysis The results are expressed as mean 7 SEM. The statistical analysis was performed by one way analysis of variance (ANOVA) followed by Dunnett's posthoc test wherever applicable, using SPSS 11.5 software. Differences between groups were considered significant at P o0.05.
3. Results and discussion The present study demonstrated the central and peripheral antinociceptive effects of EEPL when assessed in thermal and chemical models of nociception as measured by hot-plate and tailimmersion tests, acetic acid-induced writhing test, glutamate and formalin-induced licking tests respectively. Furthermore, the oral administration of EEPL at 500–2000 mg/kg doses did not show any adverse effects like abnormal behavior or allergic manifestation or mortality during 72 h of observation period. The absence of toxicity presented by EEPL enabled us to establish the doses used in this study. Our starting experiments, the hot plate and tail immersion tests which are extensively used methods for investigating the central antinociceptive activities of a drug candidate where hot plate indicates the supraspinally organized response of pain and tail immersion represents the spinal mechanism. It has been demonstrated that the opioid agents exhibit their antinociceptive activity through the interaction with supraspinal (m1, ĸ3, δ1, s2) and spinal (m2, ĸ3, δ2) nociceptors (Morales and Perez-Garcia, 2001; Jinsmaa et al., 2005). In our study, EEPL exhibited antinociceptive effects both in hot plate and tail immersion tests by increasing hot plate latency and tail withdrawal time, respectively. The significant (P o0.01) effect was found from the higher doses of EEPL (100 and 200 mg/kg). As expected, morphine also produced similar pattern of effect in these tests (P o0.001) (Table 1 and S1). Besides, pretreatment with naloxone (2 mg/kg) caused a marked reduction (P o0.05) of the antinociceptive activity produced by EEPL especially with the doses of 100 and 200 mg/kg. Therefore, these findings suggest that the opioid receptors may contribute at least in part to the EEPL-mediated action. The acetic acid-induced writhing in mice, a widely used model of visceral pain, is highly sensitive and useful to screen new analgesic drugs. It has been reported that acetic acid induces writhing syndromes by increasing the production of different proinflammatory mediators like prostaglandin, prostacyclin and other
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Table 1 Antinociceptive effect of EEPL, morphine and reversal effect of naloxone in hot plate test. Treatment (mg/Kg)
Response time (s) % MPE
Control (0.1 ml/mouse) Morphine (5) EEPL (50) EEPL (100) EEPL (200) NLX (2) NLX (2) þMorphine (5) NLX (2) þEEPL (50) NLX (2) þEEPL (100) NLX (2) þEEPL (200)
Pretreatment
30 min
60 min
90 min
120 min
5.89 7 0.26 5.87 7 0.20 5.727 0.74 5.42 7 0.15 5.067 0.49 5.69 7 0.22 5.767 0.09 5.417 0.37 5.25 7 0.13 5.52 7 0.31
6.047 0.13 11.90 7 0.57n (42.68) 7.157 0.54 (10.07) 8.22 70.41 (19.23) 9.59 70.38n (30.32) 5.89 70.23 8.03 70.54a (15.90) 6.177 0.26 (5.22) 6.737 0.26 (10.04) 7.85 7 0.34 (16.04)
6.247 0.22 15.02 7 0.82n (64.76) 8.20 7 0.52 (17.42) 10.54 7 0.51n (35.09) 11.94 7 1.00n (46.04) 5.95 7 0.27 10.51 70.82a (33.33) 7.08 7 0.33 (11.46) 8.077 0.32 (19.07) 9.03 7 0.70 (24.21)
6.62 7 0.16 16.177 0.60n (72.90) 8.89 7 0.60 (22.19) 11.45 7 1.00n (41.36) 13.187 0.32n (54.32) 5.87 7 0.19 11.39 7 0.78a (39.54) 7.53 7 0.33 (14.51) 8.65 7 0.29b (23.04) 9.54 7 0.50c (27.76)
6.707 0.22 16.38 7 0.90n (74.38) 9.80 70.52 (28.58) 11.477 0.49n (41.52) 13.96 7 0.34n (59.57) 6.18 70.16 12.017 0.22a (43.91) 8.60 70.43 (21.88) 9.80 70.55 (30.82) 11.377 0.26c (40.40)
Each value is presented as the mean7 SEM (n ¼5). EEPL ¼ Ethanol extract of bark of Polyalthia longifolia; NLX ¼ Naloxone. n
a b c
Po 0.001 compared with the control group (Dunnett's test). P o0.001 compared with the morphine group (Bonferroni's test). Po 0.05 compared with the EEPL 100 mg/kg group (Bonferroni's test). Po 0.05 compared with the EEPL 200 mg/kg group (Bonferroni's test).
80 160 140 120
** **
40
**
**
20
Number of licking
Number of writhing
60
100
**
80 60
**
**
40 20
0
Co
o ntr
clo
fen
S ac
o
g g/k
g g/k
m diu
0 L5
m
P
EE
PL
0 10
g g/k
m
PL
EE
0 20
m
Co
EE
140
160
120
140
100
* 80
60
40
ntr Di
**
20
Number of licking (late phase)
Number of licking (early phase)
Di
0
l
ol
clo
fen
S ac
od
0 L5
mg
P
EE
EE
PL
0 10
mg
/kg EE
PL
0 20
mg
/kg
120 100
**
80
**
60
**
40 20
0
/kg
ium
**
0
Co
ntr
ol
cl Di
ofe
na
o cS
diu
m EE
PL
m 50
g/k
g
EE
PL
1
m 00
g/k
g
EE
PL
2
m 00
g/k
g Co
ntr
ol
cl Di
ofe
na
o cS
diu
g
m
g/k
PL
EE
m 50
EE
PL
1
m 00
g/k
g
EE
PL
2
m 00
g/k
g
Fig. 1. Antinociceptive effects of EEPL and diclofenac sodium on the nociception induced by (A) 0.6% acetic acid (10 ml/kg), (B) glutamate (10 μMol/paw) and (C–D) 2.5% formalin (20 μl/paw). Acetic acid and glutamate were given after 30 min and formalin after 60 min of EEPL administration. Statistical analysis was performed using one-way ANOVA followed by the Dunnett's posthoc test. **P o 0.001; *Po 0.05 compared to the respective control.
Md. Moniruzzaman et al. / Journal of Ethnopharmacology 172 (2015) 364–367
cytokines in the peripheral tissue fluid, which in turn excite the nerve endings of the peripheral nociceptors (Ikeda et al., 2001; Ferdous et al., 2008). Our results demonstrated that EEPL can significantly (P o0.001) inhibit the number of writhing episodes induced by acetic acid (Fig. 1A). The highest percentage of writhing inhibition (73.49%) was found with the dose of 200 mg/kg of EEPL. Furthermore, the oral administration of EEPL produced a dose dependant inhibition of licking in the glutamate test. The significant (Po 0.001) effect was found at the dose of 100 and 200 mg/kg of EEPL with percent inhibition of 50.35 and 68.09 respectively (Fig. 1B). It is well established that glutamate produce nociceptive responses through the interaction with peripheral, spinal and supraspinal site of action with both NMDA and nonNMDA receptors (Beirith et al., 2002). So it is conceivable that the antinociceptive action exhibited by EEPL may be associated with its interaction with the glutamatergic system. In our next experiment, we observed that EEPL can also inhibit number of licking induced by 2.5% of formalin, in a dose-dependent fashion. However, EEPL at 200 mg/kg dose only could produce significant (Po0.05) effect (29.3% of licking inhibition) in the early phase of formalin test (Fig. 1C). On the other hand, in late phase, all doses of EEPL significantly (Po0.001) diminished number of licking with highest percent inhibition of 65.55% (Fig. 1D). It is well established that, in the first phase of formalin test, formalin causes direct activation of type C nociceptive nerve endings, releasing neuropeptides such as substance P, among others. In contrast, the second phase is related to the release of chemical mediators such as histamine, serotonin, bradykinin, prostaglandins and excitatory amino acids which are responsible to cause inflammation (Tjølsen et al., 1992; Santos and Calixto, 1997). Therefore, it is conceivable that the extract could produce antinociceptive effect, more prominently in the late phase, through down regulation of synthesis or action of the above mentioned nociceptive mediators. Although, our findings in thermal models revealed the central antinociceptive action of EEPL through possible involvement of opioid receptors, it is showing a quite different result of expectation in the early phase of formalin test where the nerve endings are directly involved in production of nociceptive responses. Thus, it is possible that the less efficacy produced by EEPL in early phase of formalin test is due to the differences in the mechanisms with thermal models where the antinociceptive action of EEPL is more prominent. Our phytochemical studies revealed that EEPL contains alkaloids, flavonoids, glycosides, terpenoids and tannins. Besides, total phenolic and flavonoid contents were quantified as 9.3172.57 mg/g and 13.1773.13 mg/g respectively. It has been reported that flavonoids can inhibit the increased level of Ca2 þ as well as release of different pro-inflammatory mediators like tumor necrosis factor-α. Besides, tannins also attributed for their antinociceptive activity (Starec et al., 1988; Kempuraj et al., 2005). Therefore it is conceivable that these groups of compounds may be participated in the observed antinociceptive effects produced by EEPL.
4. Conclusions Taken together, the results presented in current study provide the biological evidence for the antinociceptive activity of P. longifolia stem bark and support the traditional use of this plant as an analgesic agent in folk medicine. Moreover, the alteration of antinociceptive activity by treatment with naloxone suggests the possible involvement of the opioid receptors in the EEPL-induced antinociception in thermally-induced pain. In spite of that, more higher doses of EEPL may produce higher efficacy, which could be a query of the readers. Therefore, further phytopharmacological investigations leading to isolation and characterization of pure compound(s) is required to elucidate the exact mechanism(s) of the observed bioactivities.
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Conflict of interest Authors declare that they have not any conflict of interest.
Acknowledgments Author's wishing to express profound heartfelt gratitude to Professor Dr. Bidyut Kanti Datta, Chairman, Department of Pharmacy, Stamford University Bangladesh, for his permission to use the facilities of the pharmacology laboratory.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jep.2015.06.053.
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