January 2011

Regular Article

Biol. Pharm. Bull. 34(1) 103107 (2011)

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The Antinociceptive and Anti-inammatory Action of the CHCl3-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; 113 Minatojima, Chuo-ku, Kobe, Hyogo 6508586, Japan: and c Laboratory of Food and Nutritional Sciences, Department of Nutritional Sciences, Kobe Gakuin University; 518 Arise, Ikawadani-cho, Nishi-ku, Kobe, Hyogo 6512180, 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-, CHCl3- and BuOH-soluble phase and its main active component, damnacanthal, isolated from the Noni root, have antinociceptive and anti-inammatory actions in mice. The CHCl3-soluble phase (3 g/kg, per os (p.o.)) signicantly 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 CHCl3-soluble phase (3 g/kg, p.o.) signicantly reduced histamine-induced paw edema. The MeOH- and BuOH-soluble phase had no effect in either test. Furthermore, damnacanthal (10100 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 H1 receptor. These data suggest that the CHCl3-soluble phase of the Noni root has antinociceptive and anti-inammatory effects. Furthermore, these effects of damnacanthal isolated from the Noni root is mediated in part by the histamine H1 receptor.
Key words Morinda citrifolia; damnacanthal; histamine H1 receptor; antinociception; anti-inammatory

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 CHCl3-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. ofcinalis root exhibits antinociceptive and anti-inammatory effects in mice.6) It has been reported that the pharmacological activity of the Noni root was similar to that of the M. ofcinalis root.7) However, the antinociceptive and anti-inammatory activities of the CHCl3-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-inammatory drugs (NSAIDs). The development of alternative analgesic drugs is
To whom correspondence should be addressed.

therefore necessary. The present study was designed to investigate if the CHCl3-soluble phase of the Noni root and damnacanthal has antinociceptive and anti-inammatory effects. MATERIALS AND METHODS Animals Male ddY mice (age, 56 weeks) were obtained from SLC (Hamamatsu, Japan). Mice were housed in cages at 2324 C with a 12-h lightdark 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: A060601-9). 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 identied 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 reux. Extracts was then ltered and evaporated on a rotary evaporator under reduced pressure. These MeOH extracts were then suspended in H2O. They were partitioned with chloroform (CHCl3) and the butanol (BuOH) layer concentrated in vacuo. The obtained extract was freeze-dried to give the CHCl3 extract. The CHCl3 extract (122 g) was chromatographed on Sephadex LH-20 using CHCl3MeOH (1 : 1) to give an anthraquinone-containing fraction. This
2011 Pharmaceutical Society of Japan

e-mail: stoku@pharm.kobegakuin.ac.jp

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fraction was subjected to repeated SiO2 column chromatography using n-hexaneCHCl3 solvent system. It was puried by preparative TLC using a CHCl3MeOH solvent system to afford compounds. HPLC was performed with a Diol column (Inertsil Diol 5 mm, 4.6250 mm, GL Sciences, Tokyo, Japan). The detection wavelength was 254 nm. Elution was carried out with nhexane : CHCl3 (a, 25 : 75), (b, 50 : 50) and (c, 75 : 25) at a ow rate of 1 ml/min. The injection volume was 10 m l (1.0 mg/ml, chloroform-soluble phase in CHCl3). 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 puried damnacanthal were analysed by IR, UV , 1H- and 13C-NMR spectra, and the chemical structures of the isolated damnacanthal are shown in Fig. 1B. Damnacanthal: Yellow 1 amorphous powder; IR n KBr max cm : 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); 1H-NMR (400 MHz, pyridine-d5) d : 7.77 (1H, s, H-4), 8.24 (1H, dd, J7.5, 1.3 Hz, H5), 7.64 (1H, td, J7.5, 1.2 Hz, H-6), 7.70 (1H, td, J7.5, 1.3 Hz, H-7), 8.33 (1H, dd, J7.5, 1.2 Hz, H-8), 10.57 (1H, s, H- 11), 4.14 (3H, s, 1-OCH3); 13C-NMR (100 MHz, pyridine-d5) d : 166.58 (C-1), 119.51 (C-2), 166.41 (C-3), 112.67 (C-4), 141.58 (C- 4a), 126.96 (C-5), 133.67 (C-6), 134.89 (C-7), 127.39 (C-8), 135.41 (C-8a), 180.07 (C-9), 118.21 (C9a), 182.22 (C-10), 132.92 (C-10a), 194.98 (C-11), 64.31 (1OCH3). Sample Preparation and Drugs The MeOH (3 g/kg)-, CHCl3 (3 g/kg)-, and BuOH (3 g/kg)-soluble phase and damnacanthal (10100 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-inammatory 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

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 m l 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 CHCl3-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 m mol, 10 m l) was injected intraplantarly (i.pl.) into the right hind paw to induce acute inammation.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 H1 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 H1 Receptor Binding Assay The histamine H1 receptor binding afnity assay was conducted at Cerep Incorporated (Celle lEvescault, France), according to an established method.10) The inuence of damnacanthal (109 103 M) on histamine H1 receptors was tested in a radioligand binding assay with HEK-293 cells transfected with the human recombinant histamine H1 receptor using [3H]-pyrilamine (1 nmol/l; incubation time, 60 min at 22 C). Bound radioactivity was measured with a scintillation counter. The histamine H1 binding assay was done in duplicate. Non-specic binding was dened using 1 m M unlabeled pyrilamine. Statistical Analyses Data are meanS.E.M. The statistical signicance of differences between the control and CHCl3 soluble phase-treated group and damnacanthal-treated group were analyzed using one-way ANOVA followed by Scheffes multiple-comparison test for the acetic acid writhing test and formalin test. p0.05 was considered signicant. RESULTS Effect of the MeOH-, CHCl3- and BuOH-Soluble Phase of the Noni Root on the Chemical Nociceptive Stimuli in the Formalin Test The CHCl3-soluble phase (3 g/kg) did not reduce pain response time in the early phase, but signicantly suppressed pain-related behavior in the late phase (p0.01) of the formalin test. Similarly, indomethacin (50 mg/kg) as a positive control signicantly 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 CHCl3 Soluble Phase-Induced Antinociception The antinociceptive effect in the late phase of the CHCl3-soluble phase (3 g/kg) was not affected by pretreatment with NLX (1 mg/kg). On the other hand, morphine (5 mg/kg) signicantly attenuated pain-related behavior in the early and late phase in a NLX-reversible manner (Fig. 3). Effect of the CHCl3-Soluble Phase of the Noni Root on Histamine-Induced Paw Edema The saline-injected hind

Fig. 1. HPLC Proles (A) and Structural Formula (B) of Damnacanthal, Isolated from the CHCl3-Soluble Phase of the Noni Root
Elution was carried out with n-hexane : chloroform25 : 75 (a), 50 : 50 (b) and 75 : 25 (c).

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Fig. 2. Effect of the MeOH-, CHCl3- and BuOH-Soluble Phase of the Noni Root on Chemical Nociceptive Stimuli in the Formalin Test
The cumulative nociceptive response time of biting, licking and shaking of the injected hind paw was measured during 30 min after injection (i.pl.) of formalin at 5% (V/V). Responses were measured at 05 min (early phase) and 530 min (late phase) after formalin injection. Indomethacin was used as a positive control. Each column indicates meanS.E.M. (n515) ** p0.01, * p0.05 vs. Vehicle, Scheffe test.

Fig. 4. Effect of the CHCl3-Soluble Phase of the Noni Root on HistamineInduced 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 m mol 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 meanS.E.M. (n56). ** p0.01, * p0.05 vs. HistamineVehicle, ## p0.01 vs. SalineVehicle, Scheffe test.

Fig. 3. Effect of Naloxone on CHCl3 Soluble Phase-Induced Antinociception in the Formalin Test
The cumulative nociceptive response time of biting, licking and shaking of the injected hind paw was measured during 30 min after injection (i.pl.) of formalin at 5% (V/V). (a) Responses were measured at 05 min (early phase) and 530 min (late phase) after formalin injection. Morphine was used as a positive control. Each column indicates meanS.E.M. (n911) ** p0.01, * p0.05 vs. Vehicle, ## p0.01 vs. Morphine. Scheffe test. NLX: naloxone, n.s.: not signicant.

Fig. 5. Effect of Damnacanthal on Chemical Nociceptive Stimuli in the Formalin Test

The cumulative nociceptive response time of biting, licking and shaking of the injected hind paw was measured during 30 min after injection (i.pl.) of formalin at 5% (V/V). Responses were measured at 05 min (early phase) and 530 min (late phase) after formalin injection. Indomethacin was used as a positive control. Each column indicates meanS.E.M. (n411). ** p0.01, * p0.05 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 CHCl3-soluble phase showed signicant inhibition of the edema. Diphenhydramine (30 mg/kg) signicantly inhibited the paw edema (Fig. 4). Effect of Damnacanthal on Chemical Nociception in the Formalin Test Damnacanthal (10100 mg/kg) did not reduce pain response time in the rst phase, but signicantly suppressed pain-related behavior in the second phase (p0.01). Damnacanthal (30100 mg/kg) exhibited a signicant antinociceptive effect in a dose-dependent manner. Indomethacin (50 mg/kg) signicantly 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 signicant inhibition of histamine-induced paw edema (Fig. 6). Effects of Damnacanthal in the Displacement of [3H]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 m mol 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 meanS.E.M. (n56). ** p0.01 vs. Vehicle, Scheffe test.

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Fig. 7. Displacement of [3H]-Pyrilamine Binding by Damnacanthal or Histamine

Histamine H1 radioligand ([3H]pyrilamine) displacement curves for drugs using HEK293 cells. Displacement of [3H]pyrilamine binding to the histamine H1 receptor by different concentrations of [3H]pyrilamine. The data, shown as percentage of specic binding, t according to a one-site ligandreceptor model.

placed [3H]-pyrilamine binding to human HEK293 cells in a concentration-dependent manner. Similarly, histamine also displaced [3H]-pyrilamine binding to human HEK293 cells in a concentration-dependent manner. Specic binding of [3H]pyrilamine was displaced with increasing concentrations of unlabeled pyrilamine (Fig. 7). DISCUSSION We examined the antinociceptive effect of the MeOH-, CHCl3- and BuOH-soluble phase in a model of acute inammatory pain induced by formalin. In general, the early phase (010 min) of formalin-induced pain behavior is produced by direct stimulation of primary afferent nerves, whereas pain behaviors associated with the late phase (1030 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 inammatory reaction.8) In the present study, we obtained evidence that the CHCl3-soluble phase exhibited a signicant antinociceptive effect on chemical nociceptive stimuli. In particular, the CHCl3-soluble phase reduced pain-related behavior in the late phase. The antinociceptive effect of the CHCl3-soluble phase may therefore be (at least in part) due to attenuation of the central sensitization induced by certain inammatory 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 CHCl3-soluble phase. The CHCl3-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 CHCl3-soluble phase may have an anti-inammatory effect because administration of indomethacin attenuated pain-related behavior in the late phase of the formalin test. To examine the anti-inammatory effect of the CHCl3-soluble phase, we evaluated histamine-induced paw edema in mice. Histamine is a potent mediator of acute inamma-

tion.13) It is produced in the early phase of acute inammation to increase vascular permeability. The CHCl3-soluble phase signicantly 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 H1 receptor. Therefore, we suggest that the CHCl3-soluble phase exerts anti-inammatory 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 H1 and H2 receptors are involved in the modulation of pain sensations.1517) In the peripheral nervous system, it is reported that pyrilamine (histamine H1 receptor antagonist) and cimetidine (histamine H2 receptor antagonist) show analgesic effects in the formalin test.18) In addition, anti-histaminic drugs show antinociceptive effects on formalin-induced stimuli.1921) Thus, histamine seems to be involved in the stimulation of nociceptive bers, 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 CHCl3-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 immunodeciency virus (HIV).27) However, there is no evidence that damnacanthal has an antinociceptive and anti-inammatory effect on chemical nociceptive stimuli. Here, we found that damnacanthal possesses antinociceptive and anti-inammatory effects. Therefore, in the present study, damnacanthal may have been one of the active components of the CHCl3-soluble phase of the Noni root. Interestingly, damnacanthal was weakly bound to the histamine H1 receptor. Damnacanthal may therefore exert its antinociceptive and anti-inammatory effects through binding to in part the histamine H1 receptor. In conclusion, we demonstrated that the CHCl3-soluble phase of the Noni root has antinociceptive and anti-inammatory effects. In addition, damnacanthal was shown to be the main active component through in part a histamine H1 receptor-mediated system in the production of these effects in the CHCl3-soluble phase. Acknowledgments Parts of this study was Academic Frontier Project, Cooperative Research Center of Life Sciences. REFERENCES
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