- Email: [email protected]
Evaluation of antinociceptive effect of ethanol extract of Hedyotis corymbosa Linn. whole plant in mice
Journal of Ethnopharmacology 161 (2015) 82–85
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Ethnopharmacological communication
Evaluation of antinociceptive effect of ethanol extract of Hedyotis corymbosa Linn. whole plant in mice Md Moniruzzaman a,n, Afia Ferdous b, Shamima Irin 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 1 August 2014 Received in revised form 10 November 2014 Accepted 5 December 2014 Available online 13 December 2014
Ethnopharmacological relevance: Hedyotis corymbosa (Linn.) Lam. is a small herb commonly called as khetpapra, traditionally used to treat a wide range of diseases including abdominal pain, arthritis and inflammation. This study was conducted to evaluate the antinociceptive effect of ethanol extract of Hedyotis corymbosa (EEHC) whole plant. Materials and methods: The antinociceptive activity of EEHC was evaluated in mice using both chemicaland heat-induced pain models such as acetic acid-induced writhing, hot plate, tail immersion, formalin, and glutamate tests at 50, 100, and 200 mg/kg doses. In order to verify the possible involvement of opioid receptors in the central antinociceptive effect of EEHC, the effects found in hot plate and tail immersion tests were antagonized with naloxone. Results: EEHC produced a dose-dependent antinociceptive effect against the chemical- and heat-induced pain in mice, significantly at 100 and 200 mg/kg doses. These findings suggest that the action of EEHC involves both peripheral and central antinociceptive mechanisms. The antinociceptive activity of EEHC was significantly attenuated by pretreatment with naloxone, indicating the influence of opioid receptors on the exertion of EEHC action centrally. Conclusions: This study reports the antinociceptive activity of Hedyotis corymbosa and possible underlying mechanism(s) that supports the traditional use of this plant in the treatment of different painful conditions. & 2014 Published by Elsevier Ireland Ltd.
Keywords: Hedyotis corymbosa Rubiaceae Medicinal plant Pain Analgesic
1. Introduction Hedyotis corymbosa (Linn.) Lam. (Family—Rubiaceae), a small weedy flowering herb in Bangladesh commonly known as khetpapra, grows in the fallow lands (Walter, 1964). This plant has wide therapeutic applications in the practice of traditional medicine in Bangladesh. More specifically, this plant possesses stomachic, febrifuge and anthelmintic activities and thereby has been extensively used to treat abdominal pain and toothache (Yusuf et al., 2009; Denni et al., 2012). This plant is also used as an important ingredient of “nilavimbu kudineer choornam”, a classical preparation used to treat inflammation, arthralgia, arthritis, fever and jaundice in Tamil Nadu, India (Anbarasu et al., 2011). Preliminary phytochemical screening has revealed that Hedyotis corymbosa contains geniposide, iridoid glycosides, 6α-hydroxygeniposide, scandoside methyl ester (6βhydroxygeniposide), 10-o-benzoylscandoside-methyl-ester, asperulosidic acid, asperuloside, deacetyl asperuloside, 10-o-p-hydroxy
n
Corresponding author. Mobile: þ 82 1037404442. E-mail address: [email protected] (M. Moniruzzaman).
http://dx.doi.org/10.1016/j.jep.2014.12.011 0378-8741/& 2014 Published by Elsevier Ireland Ltd.
benzoylscandoside-methyl-ester and cinnamic acid (Otuska et al., 1991; Noiarsa et al., 2008) as well as ursolic acid that possesses hepatoprotective and antibacterial activities (Sultana et al., 2010). Based on the traditional uses, researchers provided substantial scientific evidences revealing the beneficial impact of this plant highlighting its anticancer, hepatoprotective, antiulcer, antioxidant, anti-malarial, antibacterial and antifungal activities, but not the anti-inflammatory activities in detail. This prompted us to conduct the present study where we evaluated the impact of the ethanol extract of Hedyotis corymbosa whole plant in different nociceptive models of mice. Moreover, we also tried to understand the possible mechanism underlying the antinociceptive effect of this plant.
2. Materials and methods 2.1. Plant material and extraction Hedyotis corymbosa were collected from Comilla, Bangladesh in August 2012. 5.11% w/w extract was obtained through cold maceration process. Details are explained in Supplementary materials.
M. Moniruzzaman et al. / Journal of Ethnopharmacology 161 (2015) 82–85
2.2. Phytochemical analyses The crude ethanol extract of Hedyotis corymbosa (EEHC) plant was qualitatively tested to confirm the presence of carbohydrates, saponins, flavonoids, tannins, alkaloids, glycosides, glucosides, reducing sugars, proteins, gums, and steroids following standard procedures (Ghani, 2003). 2.3. Chemicals and drugs The following drugs and chemicals were used in the current study: morphine sulfate (Gonoshasthaya Pharmaceuticals Ltd., Savar, Bangladesh), diclofenac sodium (Square Pharmaceuticals Ltd., Dhaka, Bangladesh), naloxone (Hameln Pharmaceuticals GmbH, Hameln, Germany), acetic acid, L-glutamic acid, formalin, ethanol and 99% dimethylsulfoxide (DMSO) (Merck, Darmstadt, Germany). 2.4. Animals Swiss albino mice (20–25 g) of both sex were used in all experiments. Animal handling details are described in Supplementary materials. All experimental protocols were approved by the Institutional Ethics Committee of Stamford University Bangladesh (No: SUB/IAEC/12.01). 2.5. Drugs and treatments The animals were divided into five groups containing 5–7 animals each for every experiment. The standard drug morphine sulfate (5 mg/kg) used in both hot plate and tail immersion tests and diclofenac sodium (10 mg/kg) employed in writhing and licking tests were administered intraperitoneally (i.p.) 15 min before the experiments while the animals in control group received vehicle (DMSO) orally at a dose of 0.1 ml/mouse 30 min before the experiments. EEHC was dissolved in DMSO and orally administered to the test animals at the doses of 50, 100, and 200 mg/kg body weight. 2.6. Acute toxicity test The mice were divided into control and three test groups each containing five animals. EEHC was administered to the animals orally at doses of 1000, 2000, and 3000 mg/kg. The mice were allowed to 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). 2.7. Antinociceptive analysis 2.7.1. Hot plate test The hot plate test was used to measure the central analgesic activity according to Eddy and Leimbach (1953) as described in Supplementary materials. 2.7.2. Tail immersion test This experiment is based on the previous observation revealing that morphine like drugs prolong the time of tail withdrawal from hot water in mice. This test was performed according to D’Amour and Smith (1941) as described in Supplementary materials. 2.7.3. Acetic acid-induced writhing test The mice were injected with 0.6% acetic acid and then the number of writhing was counted for 30 min as described in Supplementary materials.
83
2.7.4. Formalin-induced nociception The formalin test was performed according to the previously established method (Santos and calixto, 1997) as described in Supplementary materials. 2.7.5. Glutamate-induced nociception 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. 2.7.6. Involvement of the opioid system To evaluate the possible participation of the opioid system in the antinociceptive effect of EEHC, we reversed the hot plate and tail immersion latencies as described in Supplementary materials. 2.8. Statistical analysis The results are expressed as Mean 7SEM. The statistical analysis was performed by one way ANOVA followed by Dunnett's or Bonferroni's post-hoc test using SPSS 11.5 software. Differences between groups were considered significant at p o0.05. 3. Results and discussion The present study demonstrates that oral administration of EEHC can elicit dose-dependent antinociceptive effect in different models of nociception and provides some evidence on the mechanism implicated in these effects. Besides, EEHC at the doses of 1000–3000 mg/kg p.o. did not produce any abnormal behavior or allergic manifestation or mortality during the observation period of 72 h. Therefore, it is conceivable that EEHC may not be toxic at our experimental doses up to 3000 mg/kg. We investigated the antinociceptive activity of Hedyotis corymbosa using hot plate and tail immersion tests on mice that are widely used to evaluate the impact of centrally acting analgesics which have been shown to elevate the pain threshold of mice towards heat (Hiruma-Lima et al., 2000). These tests also scrutinize the involvement of narcotic drugs that produce antinociceptive effects through interaction with opioid receptors, specifically with μ, δ and the κ-subtypes (Turner, 1965; Dietis et al., 2011). In our study, EEHC at both 100 and 200 mg/kg doses significantly increased the time latency in hot plate test suggesting the central antinociceptive activity of EEHC. This effect was parallel to the impact of EEHC in the tail immersion test. On the other hand, in both hot plate and tail immersion tests, naloxone reversed the antinociceptive effect of EEHC to some extent (Tables 1 and S1). Therefore, it is conceivable that the central antinociceptive effect of EEHC may be influenced by opioid receptors in the spinal and supraspinal level. Next, we evaluated the antinociceptive activity of EEHC using acetic acid-induced writhing test in mouse model. Acetic acid, a potent inducer of writhing syndrome (abdominal contractions, twisting of dorsoabdominal muscles, and reduction of motor activity), causes algesia by increasing the level of proinflammatory endogenous agents and cytokines in the peripheral tissue fluid, which then excite the peripheral nociceptive nerve endings (Ikeda et al., 2001). In our study, EEHC at the doses of 100 and 200 mg/kg significantly reduced the number of writhing episodes in mice, indicating the inhibition of acetic acid-induced visceral nociception (Fig. 1A). Besides, the oral administration of EEHC (50, 100 and 200 mg/ kg) also caused inhibition of the glutamate-induced nociception in a dose dependent manner (ED50 ¼63.6 74.14 mg/kg), significantly at the latter two doses (Fig. 1B). As expected, the reference drug diclofenac sodium also produced significant antinociceptive effect
84
M. Moniruzzaman et al. / Journal of Ethnopharmacology 161 (2015) 82–85
Table 1 Antinociceptive effect of EEHC, morphine, and reversal effect of naloxone in tail immersion test. Treatment (mg/kg)
Response time (s) (% MPE)
Control (0.1 ml/mouse) Morphine (5) EEHC (50) EEHC (100) EEHC (200) NLX (2) Control (0.1 ml/mouse) þNLX (2) NLX (2) þMorphine NLX (2) þEEHC (50) NLX (2) þEEHC (100) NLX (2) þEEHC (200)
Pretreatment
30 min
60 min
90 min
120 min
1.84 7 0.14 1.687 0.04 2.08 7 0.13 2.03 7 0.08 2.047 0.18 2.067 0.10 1.727 0.16 1.96 7 0.18 1.93 7 0.11 2.107 0.23 2.05 7 0.06
2.377 0.06 3.08 7 0.32 (7.66) 2.34 7 0.11 (1.44) 2.727 0.11 (3.84) 2.917 0.24 (4.85) 2.157 0.16 1.977 0.08 2.30 7 0.18 (1.88) 2.177 0.114 (1.30) 2.28 7 0.12 (1.03) 2.34 7 0.06 (1.62)
2.56 7 0.18 3.99 7 0.26n (12.60) 3.067 0.25 (5.45) 3.517 0.14n (8.26) 3.81 7 0.11n (9.87) 2.767 0.14 2.167 0.07 2.65 7 0.09a (3.84) 2.43 7 0.09 (2.76) 2.79 7 0.10 (3.89) 3.007 0.05c (5.31)
2.80 7 0.24 4.39 7 0.28n (14.82) 3.30 7 0.07 (6.83) 3.717 0.07 (9.36) 4.22 7 0.18n (12.15) 2.50 7 0.13 2.177 0.11 2.99 7 0.14a (5.71) 2.98 7 0.10 (5.78) 2.917 0.13b (4.54) 3.217 0.12c (6.46)
2.93 7 0.22 4.447 0.07n (15.07) 3.517 0.17 (7.96) 3.767 0.14 (9.60) 4.177 0.18n (11.85) 2.32 7 0.28 2.39 7 0.16 3.157 0.12a (6.62) 3.277 0.08 (7.38) 3.447 0.17 (7.50) 3.487 0.17 (7.98)
Each value is presented as the Mean 7SEM (n¼5). EEHC ¼ Ethanol extract of Hedyotis corymbosa; NLX ¼ Naloxone. 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 EEHC 100 group (Bonferroni's test). c po 0.05 compared with the EEHC 200 group (Bonferroni's test). a
100
200 180 160
60
* 40
20
*
*
Number of licking
Number of writhing
80
140 120 *
100 80 60 40
*
* *
20 0
0
160
160 140 120 100 80 60 40
*
Number of Licking (Late phase)
Number of Licking (Early phase)
180
140 120 100 *
80 60
*
40
20
20
0
0
*
Fig. 1. Effects of oral administration of EEHC on the nociception induced by (A) 0.6% acetic acid (10 ml/kg), (B) glutamate (10 μMol/paw) and (C and D) 2.5% formalin (20 μl/ paw). Treatments were given 30 min before i.p. injection of acetic acid and intraplanter injection of glutamate and 1 h before intraplanter injection of formalin. npo 0.001 significantly different compared to respective control.
both in acetic acid-induced writhing, formalin and glutamate tests (p o0.001). To further evaluate the antinociceptive activity of EEHC, we performed the formalin test which revealed EEHC-mediated
inhibition of the number of formalin-induced lickings in a dosedependent manner. However, the inhibitory effect of EEHC was much prominent in the inflammatory phase (15–30 min) rather than in the early phase of the pain model. In the prevailed
M. Moniruzzaman et al. / Journal of Ethnopharmacology 161 (2015) 82–85
experimental condition, EEHC at the doses of 100 and 200 mg/kg significantly (p o0.001) reduced the nociception in the late phase (ED50 ¼1177 5.85 mg/kg) where diclofenac sodium significantly (p o0.001) reduced the pain in both phases (Fig. 1C and D). The corresponding ED50 values for all parameters are depicted in Supplementary materials (S2). Our findings in writhing and formalin tests can be related with a previous work (Fatema and Hossain, 2014) revealing that 250 and 500 mg/kg of EEHC produced significant dose dependent analgesic effect in mice. We found discrepancy in between the results of this report and our study, which in our opinion is mainly due to the differences in the experimental conditions and methods. Our phytochemical analysis has revealed that the crude ethanolic extract of Hedyotis corymbosa whole plant contains alkaloids, saponins, flavonoids, carbohydrates, glycosides, steroids and tannins. The total phenolic and flavonoid contents of this plant extract were found to be considerably higher (11.6 and 4.4 mg/g, respectively) (Yadav and Agarwala, 2011). It has been reported that these phytochemicals can interact directly with the cyclooxygenase pathway and elicit analgesic and anti-inflammatory activities (Ramprasath et al., 2006). Taken together, it is conceivable that these natural constituents might play an important role in the observed antinociceptive effects of EEHC.
4. Conclusion The present findings provide the evidence for the ethnopharmacological use of the plant products in different painful conditions. Our results indicate that the EEHC possesses antinociceptive activity that is prominent in both heat- and chemical-induced nociception models. The present study also suggests that the antinociceptive activity of EEHC could involve both central and peripheral mechanisms as well as the opioid receptors. Therefore, it may be worth further investigation to isolate potential bioactive compound(s) that may act as a lead(s) in new drug development.
Acknowledgments The authors are thankful to Professor Dr. Bidyut Kanti Datta, Chairman, Department of Pharmacy, Stamford University Bangladesh for providing necessary support for the study.
85
Appendix A. Supplementary materials Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2014.12.011. References Anbarasu, K., Kumar, M.K.T., Ramachandran, S., 2011. Antipyretic, antiinflammatory and analgesic properties of nilavembu kudineer choornam: a classical preparation used in the treatment of chikungunya fever. Asian Pacific Journal of Tropical Medicine 4, 819–823. D’Amour, F.E., Smith, D.L., 1941. A method for determining loss of pain sensation. Journal of Pharmacology and Experimental Therapeutics 72, 74–79. Denni, M., Mammen, D., Ramesh, S., 2012. Pharmacognosy of Hedyotis corymbosa (L.) Lam. The Global Journal of Pharmaceutical Research 1, 584–589. Dietis, N., Rowbotham, D.J., Lambert, D.G., 2011. Opioid receptor subtypes: fact or artifact. British Journal of Anaesthesia 107, 8–18. Eddy, N.B., Leimbach, D., 1953. Synthetic analgesics: II. Dithienylbutinyl and dithienylbutylamines. The Journal of Pharmacology and Experimental Therapeutics 107, 385–393. Fatema, U.K., Hossain, M.S., 2014. Analgesic effect of ethanol extract of Hedyotis corymbosa L. whole plant. International Research Journal of Pharmacy 5, 21–24. Ghani, A., 2003. Medicinal Plants of Bangladesh with Chemical Constituents and Uses, second ed. Asiatic Society of Bangladesh, Dhaka p. 274. Hiruma-Lima, C.A., Gracioso, J.S., Bighetti, E.J.B., Germonsen Robineou, L., Souza Brito, A.R.M., 2000. The juice of the fresh leaves of Boerhaavia diffusa L. (Nyctaginaceae) markedly reduces pain in mice. Journal of Ethnopharmacology 71, 267–274. Ikeda, Y., Ueno, A., Naraba, H., Oh-ishi, S., 2001. Involvement of vanilloid receptor VR1 and prostanoids in the acid-induced writhing responses of mice. Life Sciences 69, 2911–2919. Noiarsa, P., Ruhirawat, S., Otsuka, H., Kanchanapoom, T., 2008. Chemical constituents from Oldenlandia Corymbosa L. of Thai origin. Journal of Natural Medicine 62, 249–250. Otuska, H., Yoshoimura, K., Yamasaki, K., Cantoria, M.C., 1991. Isolation of 10-O-acyl iridoid glycosides from Philippine medicinal plant, Oldenlandia Corymbosa L. (Rubiaceae). Chemical and Pharmaceutical Bulletin 39, 2049–2052. Ramprasath, V.R., Shanthi, P., Sachdanandam, P., 2006. Immunomodulatory and anti-inflammatory effects of Semecarpus anacardium Linn. nut milk extract in experimental inflammatory conditions. Biological & Pharmaceutical Bulletin 29, 693–700. 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. Sultana, T., Rashid, M.A., Ali, M.A., Mahmood, S.F., 2010. Hepatoprotective and antibacterial activity of ursolic acid extracted from Hedyotis corymbosa L. Bangladesh Journal of Scientific and Industrial Research 45, 27–34. Turner, R.A., 1965. Screening Methods in Pharmacology. Academic Press, New York p. 158. 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. Journal of Ethnopharmacology 120, 169–175. Walter, H.L., 1964. Oldenlandia corymbosa (Rubiaceae). Grana Palynologica 5, 330–341. Yadav, R.N.S., Agarwala, M., 2011. Phytochemical analysis of some medicinal plants. Journal of Phytology 3, 10–14. Yusuf, M., Begum, J., Hoque, M.N., Chowdhury, J.U., 2009. Medicinal Plants of Bangldesh, Second ed. Bangladesh Council of Scientific and Industrial Research Laboratories Chittagong, Chittagong, Bangladesh p. 794.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Ethnopharmacological communication
Evaluation of antinociceptive effect of ethanol extract of Hedyotis corymbosa Linn. whole plant in mice Md Moniruzzaman a,n, Afia Ferdous b, Shamima Irin 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 1 August 2014 Received in revised form 10 November 2014 Accepted 5 December 2014 Available online 13 December 2014
Ethnopharmacological relevance: Hedyotis corymbosa (Linn.) Lam. is a small herb commonly called as khetpapra, traditionally used to treat a wide range of diseases including abdominal pain, arthritis and inflammation. This study was conducted to evaluate the antinociceptive effect of ethanol extract of Hedyotis corymbosa (EEHC) whole plant. Materials and methods: The antinociceptive activity of EEHC was evaluated in mice using both chemicaland heat-induced pain models such as acetic acid-induced writhing, hot plate, tail immersion, formalin, and glutamate tests at 50, 100, and 200 mg/kg doses. In order to verify the possible involvement of opioid receptors in the central antinociceptive effect of EEHC, the effects found in hot plate and tail immersion tests were antagonized with naloxone. Results: EEHC produced a dose-dependent antinociceptive effect against the chemical- and heat-induced pain in mice, significantly at 100 and 200 mg/kg doses. These findings suggest that the action of EEHC involves both peripheral and central antinociceptive mechanisms. The antinociceptive activity of EEHC was significantly attenuated by pretreatment with naloxone, indicating the influence of opioid receptors on the exertion of EEHC action centrally. Conclusions: This study reports the antinociceptive activity of Hedyotis corymbosa and possible underlying mechanism(s) that supports the traditional use of this plant in the treatment of different painful conditions. & 2014 Published by Elsevier Ireland Ltd.
Keywords: Hedyotis corymbosa Rubiaceae Medicinal plant Pain Analgesic
1. Introduction Hedyotis corymbosa (Linn.) Lam. (Family—Rubiaceae), a small weedy flowering herb in Bangladesh commonly known as khetpapra, grows in the fallow lands (Walter, 1964). This plant has wide therapeutic applications in the practice of traditional medicine in Bangladesh. More specifically, this plant possesses stomachic, febrifuge and anthelmintic activities and thereby has been extensively used to treat abdominal pain and toothache (Yusuf et al., 2009; Denni et al., 2012). This plant is also used as an important ingredient of “nilavimbu kudineer choornam”, a classical preparation used to treat inflammation, arthralgia, arthritis, fever and jaundice in Tamil Nadu, India (Anbarasu et al., 2011). Preliminary phytochemical screening has revealed that Hedyotis corymbosa contains geniposide, iridoid glycosides, 6α-hydroxygeniposide, scandoside methyl ester (6βhydroxygeniposide), 10-o-benzoylscandoside-methyl-ester, asperulosidic acid, asperuloside, deacetyl asperuloside, 10-o-p-hydroxy
n
Corresponding author. Mobile: þ 82 1037404442. E-mail address: [email protected] (M. Moniruzzaman).
http://dx.doi.org/10.1016/j.jep.2014.12.011 0378-8741/& 2014 Published by Elsevier Ireland Ltd.
benzoylscandoside-methyl-ester and cinnamic acid (Otuska et al., 1991; Noiarsa et al., 2008) as well as ursolic acid that possesses hepatoprotective and antibacterial activities (Sultana et al., 2010). Based on the traditional uses, researchers provided substantial scientific evidences revealing the beneficial impact of this plant highlighting its anticancer, hepatoprotective, antiulcer, antioxidant, anti-malarial, antibacterial and antifungal activities, but not the anti-inflammatory activities in detail. This prompted us to conduct the present study where we evaluated the impact of the ethanol extract of Hedyotis corymbosa whole plant in different nociceptive models of mice. Moreover, we also tried to understand the possible mechanism underlying the antinociceptive effect of this plant.
2. Materials and methods 2.1. Plant material and extraction Hedyotis corymbosa were collected from Comilla, Bangladesh in August 2012. 5.11% w/w extract was obtained through cold maceration process. Details are explained in Supplementary materials.
M. Moniruzzaman et al. / Journal of Ethnopharmacology 161 (2015) 82–85
2.2. Phytochemical analyses The crude ethanol extract of Hedyotis corymbosa (EEHC) plant was qualitatively tested to confirm the presence of carbohydrates, saponins, flavonoids, tannins, alkaloids, glycosides, glucosides, reducing sugars, proteins, gums, and steroids following standard procedures (Ghani, 2003). 2.3. Chemicals and drugs The following drugs and chemicals were used in the current study: morphine sulfate (Gonoshasthaya Pharmaceuticals Ltd., Savar, Bangladesh), diclofenac sodium (Square Pharmaceuticals Ltd., Dhaka, Bangladesh), naloxone (Hameln Pharmaceuticals GmbH, Hameln, Germany), acetic acid, L-glutamic acid, formalin, ethanol and 99% dimethylsulfoxide (DMSO) (Merck, Darmstadt, Germany). 2.4. Animals Swiss albino mice (20–25 g) of both sex were used in all experiments. Animal handling details are described in Supplementary materials. All experimental protocols were approved by the Institutional Ethics Committee of Stamford University Bangladesh (No: SUB/IAEC/12.01). 2.5. Drugs and treatments The animals were divided into five groups containing 5–7 animals each for every experiment. The standard drug morphine sulfate (5 mg/kg) used in both hot plate and tail immersion tests and diclofenac sodium (10 mg/kg) employed in writhing and licking tests were administered intraperitoneally (i.p.) 15 min before the experiments while the animals in control group received vehicle (DMSO) orally at a dose of 0.1 ml/mouse 30 min before the experiments. EEHC was dissolved in DMSO and orally administered to the test animals at the doses of 50, 100, and 200 mg/kg body weight. 2.6. Acute toxicity test The mice were divided into control and three test groups each containing five animals. EEHC was administered to the animals orally at doses of 1000, 2000, and 3000 mg/kg. The mice were allowed to 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). 2.7. Antinociceptive analysis 2.7.1. Hot plate test The hot plate test was used to measure the central analgesic activity according to Eddy and Leimbach (1953) as described in Supplementary materials. 2.7.2. Tail immersion test This experiment is based on the previous observation revealing that morphine like drugs prolong the time of tail withdrawal from hot water in mice. This test was performed according to D’Amour and Smith (1941) as described in Supplementary materials. 2.7.3. Acetic acid-induced writhing test The mice were injected with 0.6% acetic acid and then the number of writhing was counted for 30 min as described in Supplementary materials.
83
2.7.4. Formalin-induced nociception The formalin test was performed according to the previously established method (Santos and calixto, 1997) as described in Supplementary materials. 2.7.5. Glutamate-induced nociception 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. 2.7.6. Involvement of the opioid system To evaluate the possible participation of the opioid system in the antinociceptive effect of EEHC, we reversed the hot plate and tail immersion latencies as described in Supplementary materials. 2.8. Statistical analysis The results are expressed as Mean 7SEM. The statistical analysis was performed by one way ANOVA followed by Dunnett's or Bonferroni's post-hoc test using SPSS 11.5 software. Differences between groups were considered significant at p o0.05. 3. Results and discussion The present study demonstrates that oral administration of EEHC can elicit dose-dependent antinociceptive effect in different models of nociception and provides some evidence on the mechanism implicated in these effects. Besides, EEHC at the doses of 1000–3000 mg/kg p.o. did not produce any abnormal behavior or allergic manifestation or mortality during the observation period of 72 h. Therefore, it is conceivable that EEHC may not be toxic at our experimental doses up to 3000 mg/kg. We investigated the antinociceptive activity of Hedyotis corymbosa using hot plate and tail immersion tests on mice that are widely used to evaluate the impact of centrally acting analgesics which have been shown to elevate the pain threshold of mice towards heat (Hiruma-Lima et al., 2000). These tests also scrutinize the involvement of narcotic drugs that produce antinociceptive effects through interaction with opioid receptors, specifically with μ, δ and the κ-subtypes (Turner, 1965; Dietis et al., 2011). In our study, EEHC at both 100 and 200 mg/kg doses significantly increased the time latency in hot plate test suggesting the central antinociceptive activity of EEHC. This effect was parallel to the impact of EEHC in the tail immersion test. On the other hand, in both hot plate and tail immersion tests, naloxone reversed the antinociceptive effect of EEHC to some extent (Tables 1 and S1). Therefore, it is conceivable that the central antinociceptive effect of EEHC may be influenced by opioid receptors in the spinal and supraspinal level. Next, we evaluated the antinociceptive activity of EEHC using acetic acid-induced writhing test in mouse model. Acetic acid, a potent inducer of writhing syndrome (abdominal contractions, twisting of dorsoabdominal muscles, and reduction of motor activity), causes algesia by increasing the level of proinflammatory endogenous agents and cytokines in the peripheral tissue fluid, which then excite the peripheral nociceptive nerve endings (Ikeda et al., 2001). In our study, EEHC at the doses of 100 and 200 mg/kg significantly reduced the number of writhing episodes in mice, indicating the inhibition of acetic acid-induced visceral nociception (Fig. 1A). Besides, the oral administration of EEHC (50, 100 and 200 mg/ kg) also caused inhibition of the glutamate-induced nociception in a dose dependent manner (ED50 ¼63.6 74.14 mg/kg), significantly at the latter two doses (Fig. 1B). As expected, the reference drug diclofenac sodium also produced significant antinociceptive effect
84
M. Moniruzzaman et al. / Journal of Ethnopharmacology 161 (2015) 82–85
Table 1 Antinociceptive effect of EEHC, morphine, and reversal effect of naloxone in tail immersion test. Treatment (mg/kg)
Response time (s) (% MPE)
Control (0.1 ml/mouse) Morphine (5) EEHC (50) EEHC (100) EEHC (200) NLX (2) Control (0.1 ml/mouse) þNLX (2) NLX (2) þMorphine NLX (2) þEEHC (50) NLX (2) þEEHC (100) NLX (2) þEEHC (200)
Pretreatment
30 min
60 min
90 min
120 min
1.84 7 0.14 1.687 0.04 2.08 7 0.13 2.03 7 0.08 2.047 0.18 2.067 0.10 1.727 0.16 1.96 7 0.18 1.93 7 0.11 2.107 0.23 2.05 7 0.06
2.377 0.06 3.08 7 0.32 (7.66) 2.34 7 0.11 (1.44) 2.727 0.11 (3.84) 2.917 0.24 (4.85) 2.157 0.16 1.977 0.08 2.30 7 0.18 (1.88) 2.177 0.114 (1.30) 2.28 7 0.12 (1.03) 2.34 7 0.06 (1.62)
2.56 7 0.18 3.99 7 0.26n (12.60) 3.067 0.25 (5.45) 3.517 0.14n (8.26) 3.81 7 0.11n (9.87) 2.767 0.14 2.167 0.07 2.65 7 0.09a (3.84) 2.43 7 0.09 (2.76) 2.79 7 0.10 (3.89) 3.007 0.05c (5.31)
2.80 7 0.24 4.39 7 0.28n (14.82) 3.30 7 0.07 (6.83) 3.717 0.07 (9.36) 4.22 7 0.18n (12.15) 2.50 7 0.13 2.177 0.11 2.99 7 0.14a (5.71) 2.98 7 0.10 (5.78) 2.917 0.13b (4.54) 3.217 0.12c (6.46)
2.93 7 0.22 4.447 0.07n (15.07) 3.517 0.17 (7.96) 3.767 0.14 (9.60) 4.177 0.18n (11.85) 2.32 7 0.28 2.39 7 0.16 3.157 0.12a (6.62) 3.277 0.08 (7.38) 3.447 0.17 (7.50) 3.487 0.17 (7.98)
Each value is presented as the Mean 7SEM (n¼5). EEHC ¼ Ethanol extract of Hedyotis corymbosa; NLX ¼ Naloxone. 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 EEHC 100 group (Bonferroni's test). c po 0.05 compared with the EEHC 200 group (Bonferroni's test). a
100
200 180 160
60
* 40
20
*
*
Number of licking
Number of writhing
80
140 120 *
100 80 60 40
*
* *
20 0
0
160
160 140 120 100 80 60 40
*
Number of Licking (Late phase)
Number of Licking (Early phase)
180
140 120 100 *
80 60
*
40
20
20
0
0
*
Fig. 1. Effects of oral administration of EEHC on the nociception induced by (A) 0.6% acetic acid (10 ml/kg), (B) glutamate (10 μMol/paw) and (C and D) 2.5% formalin (20 μl/ paw). Treatments were given 30 min before i.p. injection of acetic acid and intraplanter injection of glutamate and 1 h before intraplanter injection of formalin. npo 0.001 significantly different compared to respective control.
both in acetic acid-induced writhing, formalin and glutamate tests (p o0.001). To further evaluate the antinociceptive activity of EEHC, we performed the formalin test which revealed EEHC-mediated
inhibition of the number of formalin-induced lickings in a dosedependent manner. However, the inhibitory effect of EEHC was much prominent in the inflammatory phase (15–30 min) rather than in the early phase of the pain model. In the prevailed
M. Moniruzzaman et al. / Journal of Ethnopharmacology 161 (2015) 82–85
experimental condition, EEHC at the doses of 100 and 200 mg/kg significantly (p o0.001) reduced the nociception in the late phase (ED50 ¼1177 5.85 mg/kg) where diclofenac sodium significantly (p o0.001) reduced the pain in both phases (Fig. 1C and D). The corresponding ED50 values for all parameters are depicted in Supplementary materials (S2). Our findings in writhing and formalin tests can be related with a previous work (Fatema and Hossain, 2014) revealing that 250 and 500 mg/kg of EEHC produced significant dose dependent analgesic effect in mice. We found discrepancy in between the results of this report and our study, which in our opinion is mainly due to the differences in the experimental conditions and methods. Our phytochemical analysis has revealed that the crude ethanolic extract of Hedyotis corymbosa whole plant contains alkaloids, saponins, flavonoids, carbohydrates, glycosides, steroids and tannins. The total phenolic and flavonoid contents of this plant extract were found to be considerably higher (11.6 and 4.4 mg/g, respectively) (Yadav and Agarwala, 2011). It has been reported that these phytochemicals can interact directly with the cyclooxygenase pathway and elicit analgesic and anti-inflammatory activities (Ramprasath et al., 2006). Taken together, it is conceivable that these natural constituents might play an important role in the observed antinociceptive effects of EEHC.
4. Conclusion The present findings provide the evidence for the ethnopharmacological use of the plant products in different painful conditions. Our results indicate that the EEHC possesses antinociceptive activity that is prominent in both heat- and chemical-induced nociception models. The present study also suggests that the antinociceptive activity of EEHC could involve both central and peripheral mechanisms as well as the opioid receptors. Therefore, it may be worth further investigation to isolate potential bioactive compound(s) that may act as a lead(s) in new drug development.
Acknowledgments The authors are thankful to Professor Dr. Bidyut Kanti Datta, Chairman, Department of Pharmacy, Stamford University Bangladesh for providing necessary support for the study.
85
Appendix A. Supplementary materials Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2014.12.011. References Anbarasu, K., Kumar, M.K.T., Ramachandran, S., 2011. Antipyretic, antiinflammatory and analgesic properties of nilavembu kudineer choornam: a classical preparation used in the treatment of chikungunya fever. Asian Pacific Journal of Tropical Medicine 4, 819–823. D’Amour, F.E., Smith, D.L., 1941. A method for determining loss of pain sensation. Journal of Pharmacology and Experimental Therapeutics 72, 74–79. Denni, M., Mammen, D., Ramesh, S., 2012. Pharmacognosy of Hedyotis corymbosa (L.) Lam. The Global Journal of Pharmaceutical Research 1, 584–589. Dietis, N., Rowbotham, D.J., Lambert, D.G., 2011. Opioid receptor subtypes: fact or artifact. British Journal of Anaesthesia 107, 8–18. Eddy, N.B., Leimbach, D., 1953. Synthetic analgesics: II. Dithienylbutinyl and dithienylbutylamines. The Journal of Pharmacology and Experimental Therapeutics 107, 385–393. Fatema, U.K., Hossain, M.S., 2014. Analgesic effect of ethanol extract of Hedyotis corymbosa L. whole plant. International Research Journal of Pharmacy 5, 21–24. Ghani, A., 2003. Medicinal Plants of Bangladesh with Chemical Constituents and Uses, second ed. Asiatic Society of Bangladesh, Dhaka p. 274. Hiruma-Lima, C.A., Gracioso, J.S., Bighetti, E.J.B., Germonsen Robineou, L., Souza Brito, A.R.M., 2000. The juice of the fresh leaves of Boerhaavia diffusa L. (Nyctaginaceae) markedly reduces pain in mice. Journal of Ethnopharmacology 71, 267–274. Ikeda, Y., Ueno, A., Naraba, H., Oh-ishi, S., 2001. Involvement of vanilloid receptor VR1 and prostanoids in the acid-induced writhing responses of mice. Life Sciences 69, 2911–2919. Noiarsa, P., Ruhirawat, S., Otsuka, H., Kanchanapoom, T., 2008. Chemical constituents from Oldenlandia Corymbosa L. of Thai origin. Journal of Natural Medicine 62, 249–250. Otuska, H., Yoshoimura, K., Yamasaki, K., Cantoria, M.C., 1991. Isolation of 10-O-acyl iridoid glycosides from Philippine medicinal plant, Oldenlandia Corymbosa L. (Rubiaceae). Chemical and Pharmaceutical Bulletin 39, 2049–2052. Ramprasath, V.R., Shanthi, P., Sachdanandam, P., 2006. Immunomodulatory and anti-inflammatory effects of Semecarpus anacardium Linn. nut milk extract in experimental inflammatory conditions. Biological & Pharmaceutical Bulletin 29, 693–700. 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. Sultana, T., Rashid, M.A., Ali, M.A., Mahmood, S.F., 2010. Hepatoprotective and antibacterial activity of ursolic acid extracted from Hedyotis corymbosa L. Bangladesh Journal of Scientific and Industrial Research 45, 27–34. Turner, R.A., 1965. Screening Methods in Pharmacology. Academic Press, New York p. 158. 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. Journal of Ethnopharmacology 120, 169–175. Walter, H.L., 1964. Oldenlandia corymbosa (Rubiaceae). Grana Palynologica 5, 330–341. Yadav, R.N.S., Agarwala, M., 2011. Phytochemical analysis of some medicinal plants. Journal of Phytology 3, 10–14. Yusuf, M., Begum, J., Hoque, M.N., Chowdhury, J.U., 2009. Medicinal Plants of Bangldesh, Second ed. Bangladesh Council of Scientific and Industrial Research Laboratories Chittagong, Chittagong, Bangladesh p. 794.