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Ligustilide attenuates pain behavior induced by acetic acid or formalin
Journal of Ethnopharmacology 112 (2007) 211–214
Ethnopharmacological communication
Ligustilide attenuates pain behavior induced by acetic acid or formalin Junrong Du a , Yan Yu a , Ya Ke a,b , Chenyuen Wang a , Li Zhu a,c , Zhong Ming Qian a,c,∗ a
Department of Applied Biology & Chemical Technology and National Key Laboratory of Chinese Medicine and Molecular Pharmacology (Shenzhen), Hong Kong Polytechnic University, Kowloon, Hong Kong, PR China b Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, NT, Hong Kong, PR China c Institute for Nautical Medicine and Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, PR China Received 1 November 2006; received in revised form 23 December 2006; accepted 7 February 2007 Available online 11 February 2007
Abstract Danggui is a popular traditional Chinese medicinal (TCM) herb, which has long been used clinically to treat primary dysmenorrhoea. In a recent study, we demonstrated that ligustilide (LIG), one of the main compounds of Danggui essential oil, has multiple effects on uterine smooth muscles and possesses a non-specific antispasmodic function, which suggests that LIG might play a major role in the therapeutic activity of Danggui in primary dysmenorrhoea. Since pain is the main syndrome of dysmenorrhea, the present investigation was carried out to evaluate the analgesic activity of LIG in vivo. LIG was intra-gastrically administered to animals. We demonstrated for the first time that LIG could cause a significant dose-related reduction of acetic acid-induced writhing response and formalin-induced licking time in both the early and late phases. These results showed that LIG possessed antinociceptive and anti-inflammatory activities. These findings plus the data we reported recently implied that LIG not only has an active dilatory effect on myometrium but also an effective role in reducing the neurogenic and inflammatory pain, thus having the potential to be developed into an effective drug for the treatment of various pain syndromes including primary dysmenorrhoea. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Primary dysmenorrhoea; Danggui; Ligustilide (LIG); Acetic acid-induced writhing test; Formalin test; Pain
1. Introduction Danggui (Angelica sinensis (Oliv.) Diels, root), one of the most important traditional Chinese herbs, has been widely used in prescriptions in traditional Chinese medicine. Its pharmacological effects are known for treatment of obstetrical and gynecological disorders (Tsuchida et al., 1987), including primary dysmenorrhoea for a long time (Tsuchida et al., 1987; Mineo et al., 1991; Yim and Wu, 2000; Liu et al., 2001). Primary dysmenorrhoea is painful menstruation with no detectable organ disease and is more common in adolescent women. Its aetiology has not been clearly elucidated (Davis and Westhoff, 2001; Marjoribanks et al., 2003). However, available data show that the pain associated with primary dysmenorrhea is caused
∗ Corresponding author at: Department of Applied Biology & Chemistry Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong, PR China. Tel.: +852 3400 8673; fax: +852 2364 9932. E-mail address: [email protected] (Z.M. Qian).
0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.02.007
by the hypercontractility of uterine muscle, subsequent reduction in blood flow and concomitant uterine ischemia (Alkerlund, 1979). Currently, it is completely unknown what the active ingredients to improve syndromes of primary dysmenorrhoea are. In a recent study (Du et al., 2006), we investigated the effects of ligustilide (LIG), the main compound of the essential oil of Danggui (Gijbels et al., 1982; Takashi, 1982; Naito et al., 1992), on uterine contraction in vitro. Our data demonstrated that LIG inhibits spontaneous periodic uterine contraction in a concentration-dependent manner, suggesting that LIG has a regulatory function in the female reproductive system as a modulator of uterine contractility. We also observed that LIG affected the PGF2␣ - or Ach-induced and high K+ -induced uterine contraction as well as the oxytocin-induced incrase in the contractility of uterine horns that were incubated not only in the Locke solution but also in a Ca2+ -free solution (Du et al., 2006). These results clearly showed that LIG possesses a non-specific antispasmodic function and strongly implied that LIG is one of active ingredients of Danggui to improve syndromes of primary
212
J. Du et al. / Journal of Ethnopharmacology 112 (2007) 211–214
dysmenorrhoea. Since pain is the main syndrome of dysmenorrhea, we therefore examined the analgesic effect of LIG in mice in this study. 2. Materials and methods 2.1. Animal The Department of Health of the Hong Kong Government and the Animal Ethics Committee of The Hong Kong Polytechnic University approved the use of animals for this study. Female ICR mice (8 weeks old, 20–24 g) were supplied by the animal Center, National Key Laboratory of Chinese Medicine and Molecular Pharmacology (Shenzhen) of The Hong Kong Polytechnic University. All animals were housed in pairs in stainless steel cages at 21 ± 2 ◦ C and provided free access to food and water. Rooms were in a cycle of 12 h of light (7:00–19:00) and 12 h of darkness (from 19:00 to 7:00). 2.2. Preparation of ligustilide Danggui was purchased from the Danggui Cultivating Base of Good Agricultural Practice in Min Xian County, Gansu Province, PR China. Its identity was confirmed by comparison with descriptions of characteristics and appropriate monograph in the Chinese Pharmacopoeia (The State Pharmacopoeia Commission of People’s Republic of China, 2000). Ligustilide (LIG) was prepared by a well-established procedure in our laboratory (Du et al., 2006). Briefly, essential oil of Angelica sinensis was extracted using supercritical-CO2 fluid. LIG was isolated from the oil by silica-gel column chromatography and identified by electron impact ionisation MS, 1 H NMR and 13 C NMR spectrometric techniques. The purity was found to be >98% based on the percentage of total peak area by the HPLC analysis in this study. 2.3. Acetic acid-induced writhing test The writhing test was basically conducted as described by Fukawa et al. (1980) and Ribeiro et al. (2000b). Female ICR mice were pre-treated with LIG (2.5, 5, 10 mg/kg) or aspirin (200 mg/kg) (i.g.), 1 h before the administration of 0.6% aqueous solution of acetic acid (10 ml/kg, i.p.). Each mouse was placed in a transparent observation box and the number of writhes was counted for 30 min after the acetic acid administration. The number of writhes in each treated group was compared to that of a control group (3% Tween 80-treatment). The inhibition of writhes in percentage [(control mean − test mean)/control mean] × 100 was calculated.
into the dorsal surface of the right hind paw. Immediately after the formalin injection, the mouse was returned to the observation box, and the amount of time spent licking and biting the injected paw was measured with a hand-stop watch from 0 to 10 min (the first phase that is thought to be due to a direct chemonociceptive effect of formalin) and from 10 to 30 min (the second phase that is mainly mediated by inflammatory reactions) (Tjolsen et al., 1992) after the formalin injection. One hour before the injection of formalin, the animals were treated with LIG (2.5, 5, 10 mg/kg, i.g.), morphine (5 mg/kg, i.p.) or the same volume of vehicle (3% Tween 80-treatment the control). The effect of the drugs at different doses was compared with that of the control. The inhibition of writhes in percentage [(control mean − test mean)/control mean] × 100 was also calculated. 2.5. Statistical analysis All the data were expressed as the mean ± S.D. of eight experiments. Analysis of variance (ANOVA) was used to find out the statistical difference if more than two groups were compared. SPSS software (version 11.0) was used for all statistical calculations. The p values less than 0.05 were considered significantly different. 3. Results 3.1. Effect of LIG on acetic acid-induced writhing movements in mice Treatment of LIG led to a dose-dependent reduction in the number of acetic acid-induced writhing movements in mice (Fig. 1). The inhibition of writhes in percentage [(control mean − test mean)/control mean] × 100 ranged from 23.6 to 66.1% after oral administration of LIG from 2.5 to 10 mg/kg (Fig. 1). The number of writhing movements/30 min at 10 and 5 mg/kg of LIG groups were 9.4 ± 3.3 and 18.5 ± 7.1, respectively, significantly lower than that of the control (27.1 ± 9.2)
2.4. Formalin test The procedure used for the formalin test was the same as reported by Nakano et al. (2000). Approximately 20 min before testing, the female mice were individually placed in a transparent observation box for adaptation. Then, the animal was taken out, and 20 l of 2% formalin in 0.9% saline was injected s.c.
Fig. 1. Effects of ligustilide on acetic acid-induced writhing movements in mice. Mice were treated with aspirin (200 mg/kg, i.g.), ligustilide (LIG: 2.5, 5 or 10 mg/kg, i.g.) or the same volume of vehicle (the control) 1 h before injection of 0.6% aqueous solution of acetic acid (10 ml/kg, i.p.). The number of writhes was counted for 30 min after acetic acid administration. Data were presented as the mean ± S.E.M. (n = 8). * p < 0.05, ** p < 0.01 vs. the control.
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213
(p < 0.01 or 0.05). The inhibitory effects of LIG at 10 mg/kg and aspirin at 200 mg/kg on the number of acetic acid-induced writhing movements showed no significant difference (p > 0.05) (Fig. 1).
the 1st and 2nd phases of the formalin-induced pain were less extent compared to that of morphine (5 mg/kg, i.p.).
3.2. Effect of LIG on formalin-induced nociception in mice
The present investigation was carried out to evaluate the analgesic activity of LIG, the main compound of the essential oil of Danggui. The study was carried out in vivo. LIG was administered intra-gastrically to animals. We demonstrated for the first time that LIG (2.5–10 mg/kg) could cause a significant doserelated reduction of acetic acid-induced writhing response and formalin-induced licking time in both the early and late phases. These results showed that LIG possessed antinociceptive and anti-inflammatory activities. We (Du et al., 2006) had recently reported that LIG inhibited spontaneous periodic uterine contraction in a concentration-dependent manner and affected the PGF2␣ - or Ach-induced and high K+ -induced uterine contraction as well as the oxytocin-induced increase in the contractility of uterine horns that were incubated not only in the Locke solution but also in a Ca2+ -free solution. These results plus the findings obtained from the present study implied that LIG not only has an active dilatory effect on myometrium but also an effective role in reducing neurogenic and inflammatory pain. The data also suggested that LIG might play a major role in the therapeutic activity of Danggui and supported at least partly the validity of the use of Danggui in traditional medicine. The acetic acid-induced writhing is a visceral pain model and widely used for detecting both central and peripheral analgesia (Fukawa et al., 1980). It was reported that prostaglandin biosynthesis plays an important role in the nociceptive mechanism in this pain model (Franzotti et al., 2002). In the current study, acetic acid injection was demonstrated to induce a characteristic writhing response in the mice. LIG at higher doses (5 and 10 mg/kg) produced a significantly analgesic effect on the number of writhes induced by acetic acid, suggesting that LIG might have a role to inhibit the synthesis of prostaglandins. Further study on this possibility is needed. In addition to prostaglandins, several other inflammatory mediators, including sympathomimetic amines, tumour necrosis factor-␣, interleukin-1 and interleukin-8, have been reported to be associated with the nociceptive response to acetic acid in mice (Duarte et al., 1988; Ferreira et al., 1988, 1993a,b; Ribeiro et al., 2000a). Recently, Ribeiro et al. (2000b) showed that writhing response induced by acetic acid is highly dependent on both peritoneal macrophages and mast cells. In order to understand the mechanisms involved in the role of LIG, additional investigations are needed to find out the relationship between the treatment of LIG and the changes in inflammatory mediators or the functions of peritoneal macrophages and mast cells in mice. Formalin is known to produce biphasic pain behaviors (Wheeler-Aceto and Cowan, 1991). The first transient phase is ascribed to the direct effect of formalin on sensory C fibers, and the second prolonged phase is associated to the development of an inflammatory response and the release of algesic mediators (Hunskaar and Hole, 1987; Shibata et al., 1989). It was reported that substance P and bradykinin participate in the manifestation of the first-phase responses, and histamine, serotonin,
An s.c. injection of 2% formalin into the dorsal surface of the hind paw produced biphasic pain behaviors consisting of licking and biting of the injected paw: the first transient phase lasted for the first 10 min post-injection and was followed by the second prolonged phase beginning from 10 min post-injection. In the control group, the licking time induced by 2% formalin were 74.7 ± 18.7 and 87.1 ± 20.1 (s), respectively, during the first and second phases (Fig. 2A and B). LIG at doses of 5 and 10 mg/kg had inhibitory effects on the first phase of the formalin-induced pain behavior, a very significant decrease in the licking and biting time was produced by 5 and 10 mg/kg of LIG (p < 0.01; Fig. 2A). Similar inhibitory effects of LIG on the second phase were also found (Fig. 2B), however, a significant decrease in the licking and biting time was produced only by 10 mg/kg of LIG (p < 0.05). The effects of LIG (2.5, 5.0 and 10 mg/kg) on both of
Fig. 2. Effects of ligustilide on the formalin-induced nociception in mice. Animals were treated with morphine (5 mg/kg, i.p.), ligustilide (LIG: 2.5, 5, 10 mg/kg, i.g.) or the same volume of vehicle (the control) 1 h before 20 l of 2% formalin in 0.9% saline was injected s.c. into the dorsal surface of the right hind paw. The time spent licking and biting the injected paw was measured with a hand-stop watch from 0 to 10 min (the first phase—A) and from 10 to 30 min (the second phase—B) after formalin injection. Data were presented as the mean ± S.E.M. (n = 8). * p < 0.05, ** p < 0.01 vs. control.
4. Discussion
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prostaglandin and bradykinin are involved in the second-phase responses (Santos and Calixto, 1997a,b; Otuki et al., 2001). Studies also indicated that the formalin test is a useful method for examining nociception and its modulation by pharmacological or natural products. In this study, it was found that treatment of mice with LIG at higher doses (5 and 10 mg/kg in the 1st, and 10 mg/kg in the 2nd phases) resulted in a statistically significant antinociceptive effect on the formalin-induced paw-licking. This implied that LIG is effective at reducing not only the neurogenic but also the inflammatory pain. However, it is currently unknown whether LIG has a role to inhibit the release of substance P and bradykinin in the first-phase responses and histamine and other mediators in the second-phase responses. Taken together, the present data indicated that LIG might contribute to the analgesic activity, both central and peripheral, of Danggui. It could also be concluded that LIG has a potential anti-inflammatory activity against acute and chronic phases of inflammation. The findings of this study plus the data we reported recently about the effects of LIG on uterine contraction suggested that LIG has a potential to be developed into an effective drug for the prevention and treatment of primary dysmenorrhoea and various pain syndromes. Acknowledgements The studies in this laboratory were supported by Research Grants of National Key laboratory of Chinese Medicine and Molecular Pharmacology in Shenzhen, The Hong Kong Polytechnic University Research Grants (A-256, G-YE98, G-YX14, G-YD78, G-T616, and G-T856), Natural Science Project of University in Jiangsu Province (04KJB310113), and Research Grants of Nantong University. References Alkerlund, M., 1979. Pathophysiology of dysmenorrhea. Acta Obstetricia et Gynecologica Scandinavica 87, 27–32. Davis, A.R., Westhoff, C.L., 2001. Primary dysmenorrhea in adolescent girls and treatment with oral contraceptives. Journal of Pediatrics Adolescent and Gynecology 14, 3–8. Du, J.R., Bai, B., Kuang, X., Yu, Y., Wang, C.Y., Ke, Y., Xu, Y.J., Tzang, H.C., Qian, Z.M., 2006. Ligustilide inhibits spontaneous and agonists- or K+ depolarization-induced contraction of rat uterus. Journal of Ethnopharmacology 108, 54–58. Duarte, I.D., Nakamura, M., Ferreira, S.H., 1988. Participation of the sympathetic system in acetic aid-induced writhing in mice. Brazilian Journal of Medical and Biological Research 21, 341–433. Ferreira, S.H., Lorenzetti,.B.B., Bristow, A.F., Poole, S., 1988. Interleukin-1 beta as a potent hyperalgesic agent antagonized by a tripeptide analogue. Nature 334, 698–700. Ferreira, S.H., Lorenzetti, B.B., Poole, S., 1993a. Bradykinin initiates cytokinemediated inflammatory hyperalgesia. British Journal of Pharmacology 110, 1227–1231. Ferreira, S.H., Lorenzetti, B.B., Cunha, F.Q., Poole, S., 1993b. Bradykinin release of TNF-alpha plays a key role in the development of inflammatory hyperalgesia. Agents and Actions 38 (Spec No: C7-9). Franzotti, E.M., Santos, C.V., Rodrigues, H.M., Mourao, R.H., Andrade, M.R., Antoniolli, A.R., 2002. Anti-inflammatory, analgesic and acute toxicity of Sida cardiafolia L. Journal of Ethnopharmacology 72, 273–278.
Fukawa, K., Kawano, O., Hibi, M., Misaki, N., Ohba, S., Hatanaka, Y., 1980. A method for evaluating analgesic agents in rats. Journal of Pharmacological Methods 4, 251–259. Gijbels, M.J.M., Scheffer, J.J.C., Svendsen, A.B., 1982. Phthalides in the essential oil from roots of LeVisticum officinale. Planta Medica 44, 207– 211. Hunskaar, S., Hole, K., 1987. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 30, 103–114. Liu, J., Burdette, J.E., Xu, H., Gu, C., Breemen, R.B., Bhat, K.P., Booth, N., Constantinou, A.I., Pezzuto, J.M., Fong, H.H., Farnsworth, N.R., Bolton, J.L., 2001. Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms. Journal of Agriculture and Food Chemistry 49, 2472–2479. Marjoribanks, J., Proctor, M.L., Farquhar, C., 2003. Non-steroidal antiinflammatory drugs for primary dysmenorrhoea. Cochrane Database of Systematic Reviews 4 (CD001751). Mineo, S., Takayasu, M., Syoichi, S., Masao, Y., Naokata, M., 1991. Evaluation of Angelicae radix (Touki) by the inhibitory effect on platelet aggregation. Chemical Pharmaceutical Bulletin 39, 2046–2048. Naito, T., Katsuhara, T., Niitsu, K., Ikeya, Y., Okada, M., Mitsuhashi, H., 1992. Two phthalides from Ligusticum Chuangxiong. Phytochemistry 31, 639–642. Nakano, H., Minami, T., Abe, K., Arai, T., Tokumura, M., Ibii, N., OkudaAshitaka, E., Mori, H., Ito, S., 2000. Effect of intrathecal nocistatin on the formalin-induced pain in mice versus that of nociceptin/orphanin FQ. Journal of Pharmacology and Experimental Therapeutics 292, 331–336. Otuki, M.F., Lima, F.V., Malheiros, A., Cechinel-Filho, V., Delle, M.F., Yunes, R.A., Calixto, J.B., 2001. Evaluation of the antinociceptive action caused by ether fraction and a triterpene isolated from resin of Protium kleinii. Life Science 69, 2225–2236. Ribeiro, R.A., Vale, M.L., Ferreira, S.H., Cunha, F.Q., 2000a. Analgesic effect of thalidomide on inflammatory pain. European Journal of Pharmacology 391, 97–103. Ribeiro, R.A., Vale, M.L., Thomazzi, S.M., Paschoalato, A.B., Poole, S., Ferreira, S.H., Cunha, F.Q., 2000b. Involvement of resident macrophages and mast cells in the writhing nociceptive response induced by zymosan and acetic acid in mice. European Journal of Pharmacology 387, 111– 118. Santos, A.R., Calixto, J.B., 1997a. Ruthenium red and capsazepine antinociceptive effect in formalin and capsaicin models of pain in mice. Neuroscience Letter 235, 73–76. Santos, A.R., Calixto, J.B., 1997b. Further evidence for the involvement of tachykinin receptor subtypes in formalin and capsaicin models of pain in mice. Neuropeptides 31, 381–389. Shibata, M., Ohkubo, T., Takahashi, H., Inuki, R., 1989. Modified formalin test: characteristic biphasic pain response. Pain 38, 347–352. Takashi, Y., 1982. Quantitative determination of ligustilide and butylidenephthalide in touki (Angelica radix) and senkyu (Cnidii rhizome) by high-performance liquid chromatography. Hokkaidoritsu Eisei Kenkyusyoho 32, 12–16. Tjolsen, A., Berge, O.G., Hunskaar, S., Rosland, J.H., Hole, K., 1992. The formalin test: an evaluation of the method. Pain 51, 5–17. The State Pharmacopoeia Commission of People’s Republic of China, 2000. Pharmacopoeia of the People’s Republic of China, vol. 1. Chemical Industry Press, Beijing, p. 101. Tsuchida, T., Kobayashi, M., Kaneko, K., Mitsuhashi, H., 1987. Studies on the constituents of Umbelliferae plants. XVI. Isolation and structures of three new ligustilide derivatives from Angelica acutiloba. Chemical Pharmaceutical Bulletin 35, 4460–4464. Wheeler-Aceto, H., Cowan, A., 1991. Neurogenic and tissue-mediated components of formalin-induced oedema: evidence for supraspinal regulation. Agents and Actions 34, 264–269. Yim, T.K., Wu, W.K., 2000. Myocardial protection against ischaemiareperfusior injury by a Polygonum multiflorum extract supplemented “Dang-Gui decoction for enriching blood”, a compound formulation, ex vivo. Phytotherapy Research 14, 195–199.
Ethnopharmacological communication
Ligustilide attenuates pain behavior induced by acetic acid or formalin Junrong Du a , Yan Yu a , Ya Ke a,b , Chenyuen Wang a , Li Zhu a,c , Zhong Ming Qian a,c,∗ a
Department of Applied Biology & Chemical Technology and National Key Laboratory of Chinese Medicine and Molecular Pharmacology (Shenzhen), Hong Kong Polytechnic University, Kowloon, Hong Kong, PR China b Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, NT, Hong Kong, PR China c Institute for Nautical Medicine and Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, PR China Received 1 November 2006; received in revised form 23 December 2006; accepted 7 February 2007 Available online 11 February 2007
Abstract Danggui is a popular traditional Chinese medicinal (TCM) herb, which has long been used clinically to treat primary dysmenorrhoea. In a recent study, we demonstrated that ligustilide (LIG), one of the main compounds of Danggui essential oil, has multiple effects on uterine smooth muscles and possesses a non-specific antispasmodic function, which suggests that LIG might play a major role in the therapeutic activity of Danggui in primary dysmenorrhoea. Since pain is the main syndrome of dysmenorrhea, the present investigation was carried out to evaluate the analgesic activity of LIG in vivo. LIG was intra-gastrically administered to animals. We demonstrated for the first time that LIG could cause a significant dose-related reduction of acetic acid-induced writhing response and formalin-induced licking time in both the early and late phases. These results showed that LIG possessed antinociceptive and anti-inflammatory activities. These findings plus the data we reported recently implied that LIG not only has an active dilatory effect on myometrium but also an effective role in reducing the neurogenic and inflammatory pain, thus having the potential to be developed into an effective drug for the treatment of various pain syndromes including primary dysmenorrhoea. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Primary dysmenorrhoea; Danggui; Ligustilide (LIG); Acetic acid-induced writhing test; Formalin test; Pain
1. Introduction Danggui (Angelica sinensis (Oliv.) Diels, root), one of the most important traditional Chinese herbs, has been widely used in prescriptions in traditional Chinese medicine. Its pharmacological effects are known for treatment of obstetrical and gynecological disorders (Tsuchida et al., 1987), including primary dysmenorrhoea for a long time (Tsuchida et al., 1987; Mineo et al., 1991; Yim and Wu, 2000; Liu et al., 2001). Primary dysmenorrhoea is painful menstruation with no detectable organ disease and is more common in adolescent women. Its aetiology has not been clearly elucidated (Davis and Westhoff, 2001; Marjoribanks et al., 2003). However, available data show that the pain associated with primary dysmenorrhea is caused
∗ Corresponding author at: Department of Applied Biology & Chemistry Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong, PR China. Tel.: +852 3400 8673; fax: +852 2364 9932. E-mail address: [email protected] (Z.M. Qian).
0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.02.007
by the hypercontractility of uterine muscle, subsequent reduction in blood flow and concomitant uterine ischemia (Alkerlund, 1979). Currently, it is completely unknown what the active ingredients to improve syndromes of primary dysmenorrhoea are. In a recent study (Du et al., 2006), we investigated the effects of ligustilide (LIG), the main compound of the essential oil of Danggui (Gijbels et al., 1982; Takashi, 1982; Naito et al., 1992), on uterine contraction in vitro. Our data demonstrated that LIG inhibits spontaneous periodic uterine contraction in a concentration-dependent manner, suggesting that LIG has a regulatory function in the female reproductive system as a modulator of uterine contractility. We also observed that LIG affected the PGF2␣ - or Ach-induced and high K+ -induced uterine contraction as well as the oxytocin-induced incrase in the contractility of uterine horns that were incubated not only in the Locke solution but also in a Ca2+ -free solution (Du et al., 2006). These results clearly showed that LIG possesses a non-specific antispasmodic function and strongly implied that LIG is one of active ingredients of Danggui to improve syndromes of primary
212
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dysmenorrhoea. Since pain is the main syndrome of dysmenorrhea, we therefore examined the analgesic effect of LIG in mice in this study. 2. Materials and methods 2.1. Animal The Department of Health of the Hong Kong Government and the Animal Ethics Committee of The Hong Kong Polytechnic University approved the use of animals for this study. Female ICR mice (8 weeks old, 20–24 g) were supplied by the animal Center, National Key Laboratory of Chinese Medicine and Molecular Pharmacology (Shenzhen) of The Hong Kong Polytechnic University. All animals were housed in pairs in stainless steel cages at 21 ± 2 ◦ C and provided free access to food and water. Rooms were in a cycle of 12 h of light (7:00–19:00) and 12 h of darkness (from 19:00 to 7:00). 2.2. Preparation of ligustilide Danggui was purchased from the Danggui Cultivating Base of Good Agricultural Practice in Min Xian County, Gansu Province, PR China. Its identity was confirmed by comparison with descriptions of characteristics and appropriate monograph in the Chinese Pharmacopoeia (The State Pharmacopoeia Commission of People’s Republic of China, 2000). Ligustilide (LIG) was prepared by a well-established procedure in our laboratory (Du et al., 2006). Briefly, essential oil of Angelica sinensis was extracted using supercritical-CO2 fluid. LIG was isolated from the oil by silica-gel column chromatography and identified by electron impact ionisation MS, 1 H NMR and 13 C NMR spectrometric techniques. The purity was found to be >98% based on the percentage of total peak area by the HPLC analysis in this study. 2.3. Acetic acid-induced writhing test The writhing test was basically conducted as described by Fukawa et al. (1980) and Ribeiro et al. (2000b). Female ICR mice were pre-treated with LIG (2.5, 5, 10 mg/kg) or aspirin (200 mg/kg) (i.g.), 1 h before the administration of 0.6% aqueous solution of acetic acid (10 ml/kg, i.p.). Each mouse was placed in a transparent observation box and the number of writhes was counted for 30 min after the acetic acid administration. The number of writhes in each treated group was compared to that of a control group (3% Tween 80-treatment). The inhibition of writhes in percentage [(control mean − test mean)/control mean] × 100 was calculated.
into the dorsal surface of the right hind paw. Immediately after the formalin injection, the mouse was returned to the observation box, and the amount of time spent licking and biting the injected paw was measured with a hand-stop watch from 0 to 10 min (the first phase that is thought to be due to a direct chemonociceptive effect of formalin) and from 10 to 30 min (the second phase that is mainly mediated by inflammatory reactions) (Tjolsen et al., 1992) after the formalin injection. One hour before the injection of formalin, the animals were treated with LIG (2.5, 5, 10 mg/kg, i.g.), morphine (5 mg/kg, i.p.) or the same volume of vehicle (3% Tween 80-treatment the control). The effect of the drugs at different doses was compared with that of the control. The inhibition of writhes in percentage [(control mean − test mean)/control mean] × 100 was also calculated. 2.5. Statistical analysis All the data were expressed as the mean ± S.D. of eight experiments. Analysis of variance (ANOVA) was used to find out the statistical difference if more than two groups were compared. SPSS software (version 11.0) was used for all statistical calculations. The p values less than 0.05 were considered significantly different. 3. Results 3.1. Effect of LIG on acetic acid-induced writhing movements in mice Treatment of LIG led to a dose-dependent reduction in the number of acetic acid-induced writhing movements in mice (Fig. 1). The inhibition of writhes in percentage [(control mean − test mean)/control mean] × 100 ranged from 23.6 to 66.1% after oral administration of LIG from 2.5 to 10 mg/kg (Fig. 1). The number of writhing movements/30 min at 10 and 5 mg/kg of LIG groups were 9.4 ± 3.3 and 18.5 ± 7.1, respectively, significantly lower than that of the control (27.1 ± 9.2)
2.4. Formalin test The procedure used for the formalin test was the same as reported by Nakano et al. (2000). Approximately 20 min before testing, the female mice were individually placed in a transparent observation box for adaptation. Then, the animal was taken out, and 20 l of 2% formalin in 0.9% saline was injected s.c.
Fig. 1. Effects of ligustilide on acetic acid-induced writhing movements in mice. Mice were treated with aspirin (200 mg/kg, i.g.), ligustilide (LIG: 2.5, 5 or 10 mg/kg, i.g.) or the same volume of vehicle (the control) 1 h before injection of 0.6% aqueous solution of acetic acid (10 ml/kg, i.p.). The number of writhes was counted for 30 min after acetic acid administration. Data were presented as the mean ± S.E.M. (n = 8). * p < 0.05, ** p < 0.01 vs. the control.
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(p < 0.01 or 0.05). The inhibitory effects of LIG at 10 mg/kg and aspirin at 200 mg/kg on the number of acetic acid-induced writhing movements showed no significant difference (p > 0.05) (Fig. 1).
the 1st and 2nd phases of the formalin-induced pain were less extent compared to that of morphine (5 mg/kg, i.p.).
3.2. Effect of LIG on formalin-induced nociception in mice
The present investigation was carried out to evaluate the analgesic activity of LIG, the main compound of the essential oil of Danggui. The study was carried out in vivo. LIG was administered intra-gastrically to animals. We demonstrated for the first time that LIG (2.5–10 mg/kg) could cause a significant doserelated reduction of acetic acid-induced writhing response and formalin-induced licking time in both the early and late phases. These results showed that LIG possessed antinociceptive and anti-inflammatory activities. We (Du et al., 2006) had recently reported that LIG inhibited spontaneous periodic uterine contraction in a concentration-dependent manner and affected the PGF2␣ - or Ach-induced and high K+ -induced uterine contraction as well as the oxytocin-induced increase in the contractility of uterine horns that were incubated not only in the Locke solution but also in a Ca2+ -free solution. These results plus the findings obtained from the present study implied that LIG not only has an active dilatory effect on myometrium but also an effective role in reducing neurogenic and inflammatory pain. The data also suggested that LIG might play a major role in the therapeutic activity of Danggui and supported at least partly the validity of the use of Danggui in traditional medicine. The acetic acid-induced writhing is a visceral pain model and widely used for detecting both central and peripheral analgesia (Fukawa et al., 1980). It was reported that prostaglandin biosynthesis plays an important role in the nociceptive mechanism in this pain model (Franzotti et al., 2002). In the current study, acetic acid injection was demonstrated to induce a characteristic writhing response in the mice. LIG at higher doses (5 and 10 mg/kg) produced a significantly analgesic effect on the number of writhes induced by acetic acid, suggesting that LIG might have a role to inhibit the synthesis of prostaglandins. Further study on this possibility is needed. In addition to prostaglandins, several other inflammatory mediators, including sympathomimetic amines, tumour necrosis factor-␣, interleukin-1 and interleukin-8, have been reported to be associated with the nociceptive response to acetic acid in mice (Duarte et al., 1988; Ferreira et al., 1988, 1993a,b; Ribeiro et al., 2000a). Recently, Ribeiro et al. (2000b) showed that writhing response induced by acetic acid is highly dependent on both peritoneal macrophages and mast cells. In order to understand the mechanisms involved in the role of LIG, additional investigations are needed to find out the relationship between the treatment of LIG and the changes in inflammatory mediators or the functions of peritoneal macrophages and mast cells in mice. Formalin is known to produce biphasic pain behaviors (Wheeler-Aceto and Cowan, 1991). The first transient phase is ascribed to the direct effect of formalin on sensory C fibers, and the second prolonged phase is associated to the development of an inflammatory response and the release of algesic mediators (Hunskaar and Hole, 1987; Shibata et al., 1989). It was reported that substance P and bradykinin participate in the manifestation of the first-phase responses, and histamine, serotonin,
An s.c. injection of 2% formalin into the dorsal surface of the hind paw produced biphasic pain behaviors consisting of licking and biting of the injected paw: the first transient phase lasted for the first 10 min post-injection and was followed by the second prolonged phase beginning from 10 min post-injection. In the control group, the licking time induced by 2% formalin were 74.7 ± 18.7 and 87.1 ± 20.1 (s), respectively, during the first and second phases (Fig. 2A and B). LIG at doses of 5 and 10 mg/kg had inhibitory effects on the first phase of the formalin-induced pain behavior, a very significant decrease in the licking and biting time was produced by 5 and 10 mg/kg of LIG (p < 0.01; Fig. 2A). Similar inhibitory effects of LIG on the second phase were also found (Fig. 2B), however, a significant decrease in the licking and biting time was produced only by 10 mg/kg of LIG (p < 0.05). The effects of LIG (2.5, 5.0 and 10 mg/kg) on both of
Fig. 2. Effects of ligustilide on the formalin-induced nociception in mice. Animals were treated with morphine (5 mg/kg, i.p.), ligustilide (LIG: 2.5, 5, 10 mg/kg, i.g.) or the same volume of vehicle (the control) 1 h before 20 l of 2% formalin in 0.9% saline was injected s.c. into the dorsal surface of the right hind paw. The time spent licking and biting the injected paw was measured with a hand-stop watch from 0 to 10 min (the first phase—A) and from 10 to 30 min (the second phase—B) after formalin injection. Data were presented as the mean ± S.E.M. (n = 8). * p < 0.05, ** p < 0.01 vs. control.
4. Discussion
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prostaglandin and bradykinin are involved in the second-phase responses (Santos and Calixto, 1997a,b; Otuki et al., 2001). Studies also indicated that the formalin test is a useful method for examining nociception and its modulation by pharmacological or natural products. In this study, it was found that treatment of mice with LIG at higher doses (5 and 10 mg/kg in the 1st, and 10 mg/kg in the 2nd phases) resulted in a statistically significant antinociceptive effect on the formalin-induced paw-licking. This implied that LIG is effective at reducing not only the neurogenic but also the inflammatory pain. However, it is currently unknown whether LIG has a role to inhibit the release of substance P and bradykinin in the first-phase responses and histamine and other mediators in the second-phase responses. Taken together, the present data indicated that LIG might contribute to the analgesic activity, both central and peripheral, of Danggui. It could also be concluded that LIG has a potential anti-inflammatory activity against acute and chronic phases of inflammation. The findings of this study plus the data we reported recently about the effects of LIG on uterine contraction suggested that LIG has a potential to be developed into an effective drug for the prevention and treatment of primary dysmenorrhoea and various pain syndromes. Acknowledgements The studies in this laboratory were supported by Research Grants of National Key laboratory of Chinese Medicine and Molecular Pharmacology in Shenzhen, The Hong Kong Polytechnic University Research Grants (A-256, G-YE98, G-YX14, G-YD78, G-T616, and G-T856), Natural Science Project of University in Jiangsu Province (04KJB310113), and Research Grants of Nantong University. References Alkerlund, M., 1979. Pathophysiology of dysmenorrhea. Acta Obstetricia et Gynecologica Scandinavica 87, 27–32. Davis, A.R., Westhoff, C.L., 2001. Primary dysmenorrhea in adolescent girls and treatment with oral contraceptives. Journal of Pediatrics Adolescent and Gynecology 14, 3–8. Du, J.R., Bai, B., Kuang, X., Yu, Y., Wang, C.Y., Ke, Y., Xu, Y.J., Tzang, H.C., Qian, Z.M., 2006. Ligustilide inhibits spontaneous and agonists- or K+ depolarization-induced contraction of rat uterus. Journal of Ethnopharmacology 108, 54–58. Duarte, I.D., Nakamura, M., Ferreira, S.H., 1988. Participation of the sympathetic system in acetic aid-induced writhing in mice. Brazilian Journal of Medical and Biological Research 21, 341–433. Ferreira, S.H., Lorenzetti,.B.B., Bristow, A.F., Poole, S., 1988. Interleukin-1 beta as a potent hyperalgesic agent antagonized by a tripeptide analogue. Nature 334, 698–700. Ferreira, S.H., Lorenzetti, B.B., Poole, S., 1993a. Bradykinin initiates cytokinemediated inflammatory hyperalgesia. British Journal of Pharmacology 110, 1227–1231. Ferreira, S.H., Lorenzetti, B.B., Cunha, F.Q., Poole, S., 1993b. Bradykinin release of TNF-alpha plays a key role in the development of inflammatory hyperalgesia. Agents and Actions 38 (Spec No: C7-9). Franzotti, E.M., Santos, C.V., Rodrigues, H.M., Mourao, R.H., Andrade, M.R., Antoniolli, A.R., 2002. Anti-inflammatory, analgesic and acute toxicity of Sida cardiafolia L. Journal of Ethnopharmacology 72, 273–278.
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