January 2011 Regular Article. Biol. Pharm. Bull. 34(1) (2011)

Transcription

1 January 2011 Regular Article Biol. Pharm. Bull. 34(1) (2011) 103 The Antinociceptive and Anti-inflammatory Action of the CHCl 3 -Soluble Phase and Its Main Active Component, Damnacanthal, Isolated from the Root of Morinda citrifolia Kanako OKUSADA, a Kazuo NAKAMOTO, a Mikako NISHIDA, a Wakako FUJITA-HAMABE, a Kohei KAMIYA, b Yoshiyuki MIZUSHINA, c Toshiko SATAKE, b and Shogo TOKUYAMA*,a a Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Kobe Gakuin University; b Department of Pharmacognosy and Natural Product Chemistry, School of Pharmaceutical Sciences, Kobe Gakuin University; Minatojima, Chuo-ku, Kobe, Hyogo , Japan: and c Laboratory of Food and Nutritional Sciences, Department of Nutritional Sciences, Kobe Gakuin University; 518 Arise, Ikawadani-cho, Nishi-ku, Kobe, Hyogo , Japan. Received August 28, 2010; accepted October 16, 2010; published online October 21, 2010 Morinda citrifolia (Rubiaceae, Noni) is a traditional medicine with various pharmacological activities. We investigated if the MeOH-, CHCl 3 - and BuOH-soluble phase and its main active component, damnacanthal, isolated from the Noni root, have antinociceptive and anti-inflammatory actions in mice. The CHCl 3 -soluble phase (3 g/kg, per os (p.o.)) significantly reduced pain-related behavior observed in the formalin test. These effects were not suppressed by pretreatment with naloxone (1 mg/kg, intraperitoneally (i.p.)), an opioid receptor antagonist. The CHCl 3 -soluble phase (3 g/kg, p.o.) significantly reduced histamine-induced paw edema. The MeOH- and BuOH-soluble phase had no effect in either test. Furthermore, damnacanthal ( mg/kg, p.o.)) exerted an antinociceptive effect on chemical nociceptive stimuli, and decreased histamine-induced paw edema. Damnacanthal was weakly bound to the histamine H 1 receptor. These data suggest that the CHCl 3 -soluble phase of the Noni root has antinociceptive and anti-inflammatory effects. Furthermore, these effects of damnacanthal isolated from the Noni root is mediated in part by the histamine H 1 receptor. Key words Morinda citrifolia; damnacanthal; histamine H 1 receptor; antinociception; anti-inflammatory Morinda citrifolia (M. citrifolia, Rubiaceae), known as Noni, is a tropical plant that grows widely across Polynesia. In Japan, it is called Yaeyama-aok and grows in Okinawa Prefecture. Noni has traditionally been used for the prevention and treatment of various diseases, 1) and is widely known as a health food and supplement. We have reported that the Noni fruit inhibits copperinduced low-density lipoprotein oxidation, 2) and prevents ischemic neuronal damage through suppression of the development of post-ischemic glucose intolerance. 3) Noni, which contains many biologically active compounds, could therefore be an important source of new drugs. There are few pharmacological studies of the dried root of the Noni plant. We recently reported that two anthraquinone glycosides from the BuOH-soluble phase of the Noni root decreased blood glucose levels in streptozotocin-induced type-1 diabetes in mice. 4) Furthermore, we isolated 10 anthraquinones in the CHCl 3 -soluble phase of the Noni root, and found that damnacanthal was the main component. 5) Other pharmacological studies of the Noni root reported that monotropein isolated from the BuOH-soluble phase of the M. officinalis root exhibits antinociceptive and anti-inflammatory effects in mice. 6) It has been reported that the pharmacological activity of the Noni root was similar to that of the M. officinalis root. 7) However, the antinociceptive and anti-inflammatory activities of the CHCl 3 -soluble phase and damnacanthal isolated from the Noni root are unknown. A considerable number of analgesic drugs are currently available for the treatment of pain. However, their use is limited due to their side effects, such as vomiting after opioid treatment and serious cardiovascular effects associated with the long-term use of non-steroidal anti-inflammatory drugs (NSAIDs). The development of alternative analgesic drugs is therefore necessary. The present study was designed to investigate if the CHCl 3 -soluble phase of the Noni root and damnacanthal has antinociceptive and anti-inflammatory effects. MATERIALS AND METHODS Animals Male ddy mice (age, 5 6 weeks) were obtained from SLC (Hamamatsu, Japan). Mice were housed in cages at C with a 12-h light dark cycle (light on at 8 a.m. to 8 p.m.). Food and water were available ad libitum. The present study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, which has been adopted by the Japanese Pharmacological Society. Experiments were approved by Ethical Committee for Animal Experimentation at Kobe Gakuin University (approved number: A ). Plant Materials The dried roots of the tropical plant Noni (M. citrifolia, Rubiaceae) were collected in August 2004 at Okinawa in Japan and the plant was identified by Dr. K. Kamiya. A voucher specimen has been deposited in Department of Pharmacognosy and Natural Product Chemistry, Kobe Gakuin University. Extraction, Fractionation and Isolation Dried powdered roots were extracted with MeOH ten under reflux. Extracts was then filtered and evaporated on a rotary evaporator under reduced pressure. These MeOH extracts were then suspended in H 2 O. They were partitioned with chloroform (CHCl 3 ) and the butanol (BuOH) layer concentrated in vacuo. The obtained extract was freeze-dried to give the CHCl 3 extract. The CHCl 3 extract (122 g) was chromatographed on Sephadex LH-20 using CHCl 3 MeOH (1 : 1) to give an anthraquinone-containing fraction. This To whom correspondence should be addressed. stoku@pharm.kobegakuin.ac.jp 2011 Pharmaceutical Society of Japan

2 104 Vol. 34, No. 1 fraction was subjected to repeated SiO 2 column chromatography using n-hexane CHCl 3 solvent system. It was purified by preparative TLC using a CHCl 3 MeOH solvent system to afford compounds. HPLC was performed with a Diol column (Inertsil Diol 5 mm, mm, GL Sciences, Tokyo, Japan). The detection wavelength was 254 nm. Elution was carried out with n- hexane : CHCl 3 (a, 25 : 75), (b, 50 : 50) and (c, 75 : 25) at a flow rate of 1 ml/min. The injection volume was 10 ml (1.0 mg/ml, chloroform-soluble phase in CHCl 3 ). Damnacanthal contained in the chloroform-soluble phase could be separated by HPLC using three solvent systems (Fig. 1A). The yields of damnacanthal from the dried root 0.434%. The purified damnacanthal were analysed by IR, UV, 1 H- and 13 C-NMR spectra, and the chemical structures of the isolated damnacanthal are shown in Fig. 1B. Damnacanthal: Yellow amorphous powder; IR n KBr max cm 1 : 3070, 1668, 1647, 1565, 1344, 1329, 1261, 1132, 980; UV l max (MeOH) nm (log e): 283 (4.23), 247 (4.26), 203 (4.12); 1 H-NMR (400 MHz, pyridine-d 5 ) d: 7.77 (1H, s, H-4), 8.24 (1H, dd, J 7.5, 1.3 Hz, H- 5), 7.64 (1H, td, J 7.5, 1.2 Hz, H-6), 7.70 (1H, td, J 7.5, 1.3 Hz, H-7), 8.33 (1H, dd, J 7.5, 1.2 Hz, H-8), (1H, s, H- 11), 4.14 (3H, s, 1-OCH 3 ); 13 C-NMR (100 MHz, pyridine-d 5 ) d: (C-1), (C-2), (C-3), (C-4), (C- 4a), (C-5), (C-6), (C-7), (C-8), (C-8a), (C-9), (C- 9a), (C-10), (C-10a), (C-11), (1- OCH 3 ). Sample Preparation and Drugs The MeOH (3 g/kg)-, CHCl 3 (3 g/kg)-, and BuOH (3 g/kg)-soluble phase and damnacanthal ( mg/kg) were orally administrated to mice 30 min before each test. Samples were suspended in 1% CMC-Na. Indomethacin (50 mg/kg), a non-steroidal anti-inflammatory drug (NSAIDs), was orally administrated to mice 30 min before administration of each soluble phase and damnacanthal. Morphine (5 mg/kg), a m opioid receptor agonist, was subcutaneously administrated to mice 30 min before administration of each soluble phase and damnacanthal. A Fig. 1. HPLC Profiles (A) and Structural Formula (B) of Damnacanthal, Isolated from the CHCl 3 -Soluble Phase of the Noni Root Elution was carried out with n-hexane : chloroform 25 : 75 (a), 50 : 50 (b) and 75 : 25 (c). B Naloxone (NLX; 1 mg/kg, intraperitoneally (i.p.)), a non-selective opioid receptor antagonist, was administered 10 min before administration of each soluble phase. Formalin Test The formalin test was carried out as described. 8) Mice were injected with 10 ml of 5% formalin [formaldehyde solution 37% (w/w) diluted in saline] into the subplantar space of the right hind paw 30 min after the treatment of the CHCl 3 -soluble phase (per os (p.o.)) and damnacanthal (p.o.). The times spent in licking, biting and shaking behaviors were measured during 0 to 5 min (early phase) and from 5 to 30 min (late phase) after formalin injection. Histamine-Induced Paw Edema Histamine (0.5 mmol, 10 ml) was injected intraplantarly (i.pl.) into the right hind paw to induce acute inflammation. 9) The thickness of the injected hind paw was measured every 10 min or 60 min for 300 min after histamine injection (i.pl.). The intraperitoneal administration of diphenhydramine (30 mg/kg), a histamine H 1 receptor antagonist, was used as a positive control in the test. The area under the curve (AUC) value for the effect of restraining paw edema on each mouse was then calculated. Histamine H 1 Receptor Binding Assay The histamine H 1 receptor binding affinity assay was conducted at Cerep Incorporated (Celle l Evescault, France), according to an established method. 10) The influence of damnacanthal ( M) on histamine H 1 receptors was tested in a radioligand binding assay with HEK-293 cells transfected with the human recombinant histamine H 1 receptor using [ 3 H]-pyrilamine (1 nmol/l; incubation time, 60 min at 22 C). Bound radioactivity was measured with a scintillation counter. The histamine H 1 binding assay was done in duplicate. Non-specific binding was defined using 1 m M unlabeled pyrilamine. Statistical Analyses Data are mean S.E.M. The statistical significance of differences between the control and CHCl 3 soluble phase-treated group and damnacanthal-treated group were analyzed using one-way ANOVA followed by Scheffe s multiple-comparison test for the acetic acid writhing test and formalin test. p 0.05 was considered significant. RESULTS Effect of the MeOH-, CHCl 3 - and BuOH-Soluble Phase of the Noni Root on the Chemical Nociceptive Stimuli in the Formalin Test The CHCl 3 -soluble phase (3 g/kg) did not reduce pain response time in the early phase, but significantly suppressed pain-related behavior in the late phase (p 0.01) of the formalin test. Similarly, indomethacin (50 mg/kg) as a positive control significantly attenuated painrelated behavior in the late phase of the formalin test, but not the early phase. However, the MeOH (3 g/kg)- and BuOH (3 g/kg)-soluble phase had no effect in the formalin tests (Fig. 2). Effect of NLX on CHCl 3 Soluble Phase-Induced Antinociception The antinociceptive effect in the late phase of the CHCl 3 -soluble phase (3 g/kg) was not affected by pretreatment with NLX (1 mg/kg). On the other hand, morphine (5 mg/kg) significantly attenuated pain-related behavior in the early and late phase in a NLX-reversible manner (Fig. 3). Effect of the CHCl 3 -Soluble Phase of the Noni Root on Histamine-Induced Paw Edema The saline-injected hind

3 January Fig. 2. Effect of the MeOH-, CHCl 3 - and BuOH-Soluble Phase of the Noni Root on Chemical Nociceptive Stimuli in the Formalin Test (V/V). Responses were measured at 0 5 min (early phase) and 5 30 min (late phase) after formalin injection. Indomethacin was used as a positive control. Each column indicates mean S.E.M. (n 5 15) ** p 0.01, * p 0.05 vs. Vehicle, Scheffe test. Fig. 4. Effect of the CHCl 3 -Soluble Phase of the Noni Root on Histamine- Induced Paw Edema The thickness of the injected hind paw was measured between 0 min and 300 min. The thickness measured every 10 min or 60 min for 300 min after the histamine injection (i.pl.) at 0.5 mmol is described in the Materials and Methods. The area under the curve (AUC) was calculated. Diphenhydramine was used as a positive control. Each column indicates mean S.E.M. (n 5 6). ** p 0.01, * p 0.05 vs. Histamine Vehicle, ## p 0.01 vs. Saline Vehicle, Scheffe test. Fig. 3. Effect of Naloxone on CHCl 3 Soluble Phase-Induced Antinociception in the Formalin Test (V/V). (a) Responses were measured at 0 5 min (early phase) and 5 30 min (late phase) after formalin injection. Morphine was used as a positive control. Each column indicates mean S.E.M. (n 9 11) ** p 0.01, * p 0.05 vs. Vehicle, ## p 0.01 vs. Morphine. Scheffe test. NLX: naloxone, n.s.: not significant. Fig. 5. Effect of Damnacanthal on Chemical Nociceptive Stimuli in the Formalin Test (V/V). Responses were measured at 0 5 min (early phase) and 5 30 min (late phase) after formalin injection. Indomethacin was used as a positive control. Each column indicates mean S.E.M. (n 4 11). ** p 0.01, * p 0.05 vs. Vehicle, Scheffe test. paws used as control showed no increase of paw volume during the entire experiment; on the other hand, hind paw edema was successfully induced by intraplantar injection of histamine. Administration of the CHCl 3 -soluble phase showed significant inhibition of the edema. Diphenhydramine (30 mg/kg) significantly inhibited the paw edema (Fig. 4). Effect of Damnacanthal on Chemical Nociception in the Formalin Test Damnacanthal ( mg/kg) did not reduce pain response time in the first phase, but significantly suppressed pain-related behavior in the second phase (p 0.01). Damnacanthal ( mg/kg) exhibited a significant antinociceptive effect in a dose-dependent manner. Indomethacin (50 mg/kg) significantly reduced pain-related behavior in the second phase (Fig. 5). Effect of Damnacnthal on Histamine-Induced Paw Edema Administration of damnacanthal (100 mg/kg) and diphenhydramine (30 mg/kg) showed significant inhibition of histamine-induced paw edema (Fig. 6). Effects of Damnacanthal in the Displacement of [ 3 H]- Pyrilamine in Human HEK293 Cells Damnacanthal dis- Fig. 6. Effect of Damnacanthal on Histamine-Induced Paw Edema The thickness of the injected hind paw was measured between 0 min and 300 min. The thickness measured every 10 min or 60 min for 300 min after the histamine injection (i.pl.) at 0.5 mmol is described in the Materials and Methods. The area under the curve (AUC) was calculated. Diphenhydramine used as a positive control in the test. Each column indicates mean S.E.M. (n 5 6). ** p 0.01 vs. Vehicle, Scheffe test.

4 106 Vol. 34, No. 1 Fig. 7. Displacement of [ 3 H]-Pyrilamine Binding by Damnacanthal or Histamine Histamine H 1 radioligand ([ 3 H]pyrilamine) displacement curves for drugs using HEK293 cells. Displacement of [ 3 H]pyrilamine binding to the histamine H 1 receptor by different concentrations of [ 3 H]pyrilamine. The data, shown as percentage of specific binding, fit according to a one-site ligand receptor model. placed [ 3 H]-pyrilamine binding to human HEK293 cells in a concentration-dependent manner. Similarly, histamine also displaced [ 3 H]-pyrilamine binding to human HEK293 cells in a concentration-dependent manner. Specific binding of [ 3 H]- pyrilamine was displaced with increasing concentrations of unlabeled pyrilamine (Fig. 7). DISCUSSION We examined the antinociceptive effect of the MeOH-, CHCl 3 - and BuOH-soluble phase in a model of acute inflammatory pain induced by formalin. In general, the early phase (0 10 min) of formalin-induced pain behavior is produced by direct stimulation of primary afferent nerves, whereas pain behaviors associated with the late phase (10 30 min) are related to the sensitization of dorsal horn neurons due to the initial barrage of primary afferent input during the early phase or the formalin-induced inflammatory reaction. 8) In the present study, we obtained evidence that the CHCl 3 -soluble phase exhibited a significant antinociceptive effect on chemical nociceptive stimuli. In particular, the CHCl 3 -soluble phase reduced pain-related behavior in the late phase. The antinociceptive effect of the CHCl 3 -soluble phase may therefore be (at least in part) due to attenuation of the central sensitization induced by certain inflammatory substances. It is well known that opioidergic nervous systems regulate the various types of pain. The activation of opioid receptors has a pivotal role in the production of analgesic effects in animal and human. Some researchers have shown that the aqueous extracts of the fruit and roots of the Noni plant have an antinociceptive effect via opioid receptors. 11,12) In the present study, these effects were not inhibited by pretreatment of NLX on the antinociceptive effect of the CHCl 3 -soluble phase. The CHCl 3 -soluble phase may therefore produce an antinociceptive effect without acting on opioid receptors. This discrepancy may be dependent upon the main ingredients of the Noni root. The CHCl 3 -soluble phase may have an anti-inflammatory effect because administration of indomethacin attenuated pain-related behavior in the late phase of the formalin test. To examine the anti-inflammatory effect of the CHCl 3 -soluble phase, we evaluated histamine-induced paw edema in mice. Histamine is a potent mediator of acute inflammation. 13) It is produced in the early phase of acute inflammation to increase vascular permeability. The CHCl 3 -soluble phase significantly suppressed the paw edema induced by histamine. This test was inhibited by treatment with diphenhydramine, so this response seems to be partly mediated by the histamine H 1 receptor. Therefore, we suggest that the CHCl 3 -soluble phase exerts anti-inflammatory effects by mechanisms similar to those of histamine. Histamine activates polymodal nociceptors and produces pain. 14) The role of histamine in pain is different in the central nervous system and peripheral nervous system. In the former, histamine H 1 and H 2 receptors are involved in the modulation of pain sensations ) In the peripheral nervous system, it is reported that pyrilamine (histamine H 1 receptor antagonist) and cimetidine (histamine H 2 receptor antagonist) show analgesic effects in the formalin test. 18) In addition, anti-histaminic drugs show antinociceptive effects on formalin-induced stimuli ) Thus, histamine seems to be involved in the stimulation of nociceptive fibers, and its antagonists show antinociceptive effects, considering histaminergic neurons are important for pain modulation. 22) We demonstrated that damnacanthal is the major component in the CHCl 3 -soluble phase of the Noni root. 5) Damnacanthal has been reported to inhibit: selective tyrosine kinase activity, 23) activation of nuclear factor kappa-b, 24) progression of cancer, 25,26) and the human immunodeficiency virus (HIV). 27) However, there is no evidence that damnacanthal has an antinociceptive and anti-inflammatory effect on chemical nociceptive stimuli. 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