Antinociceptive activity of petroleum ether fraction from the MeOH extracts of Paederia scandens in mice.

Abstract

The petroleum ether fraction of MeOH extract from Paederia scandens was evaluated on anti-nociceptive activity in mice using chemical and thermal models of nociception. Given orally, the petroleum ether fraction (PEF) at doses of 20, 40 and 80mg/kg produced significant inhibitions on chemical nociception induced by intraperitoneal acetic acid and subplantar formalin or capsaicin injections and on thermal nociception in the tail-flick test and in the hot plate test. More significant inhibition of nociception was observed at dose of 80mg/kg of the petroleum ether fraction. In the pentobarbital sodium-induced sleeping time test and the open-field test, the petroleum ether fraction neither significantly enhanced the pentobarbital sodium-induced sleeping time nor impaired the motor performance, indicating that the observed anti-nociception in both capsaicin and formalin tests was insensitive to naloxone, but was significantly antagonized by glibenclamide. These results suggested that the petroleum ether fraction produced anti-nociception possibly related to glibenclamide-sensitive [K.sup. +]-ARP channels, which merited further studies regarding the precise site and mechanism of action. The major constituents of the petroleum ether fraction (PEF) determined by GC/MS analysis, are linoleic acid, the sterols and vitamin E. Therefore it can be suggested that they exert synergetic effects and are together responsible for the antinociceptive activity of the PEF-fraction. [c] 2007 Elsevier GmbH. All rights reserved.

Keywords: Paederia scandens; Petroleum ether fraction; Synergetic effect; Anti-nociceptive activity; Glibenclamide; [K.sup. +]-ATP channels.

Introduction

Paederia scandens (Lour.) Merri., a climbing plant, belonging to the family Rubiaceae, is popularly known as the name "Ji Shi Teng" in Chinese which is widely grown in India, China, Japan, Philippines and USA (Dang et al., 2002). It has been traditionally used as folklore medicine and food in Southeast of Asia for thousands of years (Kadota, 2000). The leaves of the plant are used as an ingredient in various foods in Vietnam (Kadota, 2000).

Recently, it has been reported that the iridoid glycosides and the dimeric iridoid glycosides - paederoside, asperuloside, paederosidic acid, deacetylasperuloside, scandoside (Inouye et al., 1969a-c; Kapadia et al., 1979; Dang et al., 2002), were isolated from the MeOH extract from the stems and roots of P. scandens (Zuo et al., 2006; Kim et al., 2004). These chemical constituents of P. scandens have biological activities such as anti-virus, anti-tumor, anti-inflammation and anti-microbial activities (Kapadia et al., 1996; Wang and Huang, 2005). In folklore medicine, the roots, leaves, barks and fruits of P. scandens have been used to treat toothache, chest pain, piles, inflammation of the spleen, diuretic, emetic, rheumatic arthritis and cure bacillary dysentery in China, Japan, Vietnam and other countries in the south-east Asia for thousands of years (Tran, 1987; Kapadia et al., 1996). Although P. scandens is a particularly useful pain-relief in folklore medicine, there has been no report on the anti-nociceptive activity of this plant and its mechanisms of analgesic activity so far.

Based on investigation above, we study the analgesic activity of the MeOH extracts of P. scandens and the anti-nociceptive activity of the petroleum ether, chloroform, n-butanol and water fractions from the MeOH extracts, and found that the petroleum ether fraction (PEF) had a powerful anti-nociceptive activity in preliminary experiment. In present study, we examined further the effects of the PEF on nociception models mice induced by the chemical and the thermal stimuli so as to elucidate the analgesic activity and the possible mechanism of the PEF, and provide scientific basis for the clinical use of P. scandens.

Methods and materials

Plant material

P. scandens was collected at the E' mei Mountain in Sichuan province in July 2005. The plant was identified in the School of Pharmacy, The Second Military Medical University. A voucher specimen of P. scandens (#107) was deposited at the Herbarium of the Department of Pharmacognosy, School of Pharmacy, Second Military Medical University.

Animal

Experimental groups consisted of 10 ICR mice (18-22g) per group. They were housed at 21 [+ or -] 1[degrees]C under a 12 h light/12h dark cycle and had free access to standard pellet diet (Purina chow) and tap water. The animals were deprived of food for 15h before the experiment, with free access to drinking water. Each animal was used only once in the experiment. The experimental protocols were approved by the Animal Care and Use Committee of our institute and complied with the recommendations of International Association for the study of pain (Zimmermann, 1983).

Drugs and chemicals

The following reagents and drugs were used: MeOH (AR), petroleum ether (AR), chloroform (AR), n-butanol (AR), formalin (AR) and acetic acid (AR) [Sinopharm chemical Reagent Co., Ltd., China], morphine hydrochloride, acetyl-salicylic acid, pentobarbital sodium (Sihuan Pharmaceutical Factory, Beijing, China), diazepam, naloxone, glibenclamide, capsaicin (Chengdu Pharmaceutical Factory, Chengdu, China).

Morphine hydrochloride, acetyl salicylic acid, glibenclamide, diazepam, pentobarbital sodium and naloxone were dissolved in physiological saline (0.9% NaCl), capsaicin was dissolved in 1% ethanol and 1% Tween 80 in saline (1:1:8). The vehicles used alone had no effects on the nociceptive responses in mice.

The sample preparation

The dried powders of whole plant of P. scandens (2 kg) were extracted with MeOH using Soxhlet apparatus. The MeOH extract was concentrated under reduced pressure to obtain a residue (588.8g). The MeOH extract was subsequently extracted with petroleum ether, chloroform, n-butanol, respectively. The PEF was concentrated under reduced pressure to obtain a residue (688 mg) for bioactivity determination.

Major constituents from the PEF

GC/MS analyses were performed using the Finnigan Vayager equipped with a VF-5 ms fused-silica capillary column (30 x 0.25mm i.d., film thickness 0.25 [micro]m, VARIAN, USA). The column temperature was programmed at 100 [degrees]C (2min), increasing to 300 [degrees]C at rate of 15[degrees]C/min, injector temperature was 250 [degrees]C; carrier gas (helium) was set at a flow rate of 1.0ml/min; ionization energy 70 eV, and scan mode EI. 1 [micro]l of sample was injected with split ratio of 1:30 (v/v) and the compounds were identified by matching their mass spectra and retention times with standard obtained from NIST (National Institute of Standards and Technologies) whenever possible.

Protocol

The anti-nociceptive activity of the PEF of P. scandens was evaluated on the chemical nociception in the test models of acetic acid-induced writhing, capsaicin- and formalin-induced hind paw licking, and on the thermal nociception in the hot-plate test and the tail-flick test. In all of the nocifensive tests, conscious (un-anesthetized) mice were used. The doses of the positive drugs were determined on the basis of the principle of their pharmacokinetics and clinical use. The PEF of P. scandens was administered orally. The doses selection-20, 40, 80 mg/kg - for the fraction was based on the results of preliminary experiments. Control groups were treated with a similar volume of vehicle that had been used to dilute the fraction.

Abdominal constriction induced by acetic acid

In the acetic acid-induced writhing test (Garcia et al., 2004), groups of overnight fasted mice (n = 10) were treated with the PEF of P. scandens, vehicle or acetylsalicylic acid, 1 h before the administration of acetic acid (0.7%, 10 ml/kg, i.p.). The number of writhing was counted for each animal, starting 3 min after acetic acid injection over the period of 12 min.

Formalin test

In formalin test (Santos and Calixto, 1997), groups of mice were treated as above with the PEF of P. scandens or vehicle and after 60 min, each mouse was given 20 [micro] l of 5% formalin (in 0.9% saline, subplantar) into the right hind-paw. The duration of paw licking (s) as an index of painful response was determined at 0-5 min (early phase, neurogenic) and 20-25 min (late phase, inflammatory) after formalin injection. Morphine was used as a positive control drug, which was administrated at the dose of 10 mg/kg, s.c., 30 min before the test. In order to verify the possible mechanism of the PEF of P. scandens, anti-nociception (80 mg/kg) animal groups pretreated with naloxone or glibenclamide were used. Naloxone or glibenclamide was administered 15 min before the PEF of P. scandens or morphine.

Capsaicin test

In the capsaicin test (Goncales et al., 2005), mice were pretreated with the PEF of P. scandens or vehicle 60 min before the subplantar injection of capsaicin (1.6 [micro] g, 20 [micro]l) into the right hind paws. A morphine (10 mg/kg, s.c. 30 min before the test)-treated animal group was included as a positive control. The amount of time each mouse spent on licking the injected paw was recorded over the first 5-min period. In order to verify the possible involvement of endogenous opioids and/or the [K.sup.+] - ATP channels in the anti-nociceptive activity, the high dose of the fraction was examined in the groups of mice pretreated with naloxone or glibenclamide. Naloxone or glibenclamide was administered 15 min before the fraction or morphine.

Tail-flick test

In the tail-flick test (S'anchez-Mateo et al., 2006), the apparatus consisting of a circulating immersion water heater was used. The thermostat was adjusted so that a constant temperature of 54 [+ or -] 1 [degrees] C was maintained in the water bath. Before treatment, the rear 3.5 cm of each mouse's tail was immersed into the water bath and the time taken to flick the tail was recorded in seconds. In this thermal test, pre-treatment latencies were determined three times with intervals of 15 min. Only mice showing a pretreatment reaction for less or equal to 3s were selected for the study. Immediately after the basal latency assessment, the mice were pre-treated with the fraction or vehicle 1 h before the measurement. A morphine (10 mg/kg, s.c. 30 min before the test)-treated animal group was included as a positive control. The cut-off time was 7 s in the tail-flick measurements in order to minimize tissue injuries.

Hot-plate test

The hot-plate test (Franzotti et al., 2000) was carried out on groups of female mice using a hot-plate apparatus (model YLS-6B, China), maintained at 55 [+ or -] 1 [degrees] C. Only mice that showed initial nociceptive responses between 5 and 30s were selected for the experiment. The latency to first sign of hind paw licking or jumping to avoid heat nociception was taken as an index of nociceptive threshold. In this test, pretreatment latencies were determined three times with intervals of 20 min. The groups of mice were pre-treated with the fraction or vehicle and 1 h later the measurement started. A morphine (10 mg/kg, s.c. 30 min before the test)-treated animal group was included as a positive control. The cut-off time was 60s in the hot-plate test in order to minimize skin damages.

Open-field test

The effect of the chemical compounds on spontaneous locomotor activity and exploratory behavior was assessed by the open-field test (Tsuda et al., 1996). The photoelectrical spontaneous locomotor activity apparatus (model ZZ-6, China) was a round arena (34 cm in diameter) with the floor divided into 21 equal areas. Immediately after evaluation, each animal was transferred to the apparatus and observed for 5 min. The number of rearing responses, the number of areas crossed by all paws, and the total time spent on being immobilized (immobility) were recorded. One hour before the test, the groups of mice were pre-treated with the fraction or vehicle. A diazepam (1.0 mg/kg, i.p.)-treated animal group was included as a positive control.

Pentobarbital sodium-induced sleeping time

In this test (F.A. Santos et al., 2005), groups of mice (n = 10) were treated orally with the fraction, and vehicle 60 min before the injection of sodium pentobarbitone. Diazepam was used as the reference drug. The time between losing and regaining righting reflex was considered as the duration of sleep time in seconds.

[FIGURE 1 OMITTED]

Statistical analysis

All data were expressed as the mean [+ or -] S.E.M. Data was subjected to ANOVA followed by Dunnett's multiple comparison test. p [less than or equal to] 0.05 was considered significant.

Results

Chemical compounds in the PEF of P. scandens

The results of GC-MS analysis on the PEF showed predominance of fatty acids, sterol and alkanes (Fig. 1) (Table 1). Twenty-six constituents were identified. The levels of the 9 major components identified (91.61%) were n-hexadecanoic acid (6.8%); cis-9, cis-12-octadecadienoic acid (46.72%); ethyl (9E, 12E)-9, 12-octadecadienoate (4.21%); linolenic acid ethyl ester (2.37%); squalene (2.02%); vitamin E (10.72%); campesterol (3.44%); stigmasterol (2.92%) and beta-sitosterol (12.41%). Among the remaining constituents (8.39%) widdrol hydroxyether, phytol and 2-octadecoxyethanol were detected in percentages ranging from 1.06% to 1.69%.

Effect of abdominal constriction induced by acetic acid

In the acetic acid-induced writhing test, the treatment with fraction decreased significant amount of inhibition on the fraction decreased significant amount of inhibition on the mean number of writhes (Fig. 2). These were in the order of 44.22 [+ or -] 4.74, 32.18 [+ or -] 3.55, 29.09 [+ or -] 3.24, and 25 [+ or -] 3.82, respectively, for the controls and the fraction at the tested doses of 20, 40 and 80 mg/kg. The positive drug, acetylsalicylic acid (200 mg/kg), also manifested the significantly diminished number of writhes (23.9 [+ or -] 5.07). Therefore, the data showed that the fraction had a dose-dependent inhibition of writhing on nociception.

Effect of the formalin test

In the formalin test, the vehicle-treated animals showed the mean licking times (s) of 83.83 [+ or -] 13.56 in the first-phase and 45.17[+ or -]12.86 in the second-phase (Fig. 3A). Pretreatment with the fraction caused significant diminutions of both the first-phase (49.5 [+ or -] 6.76, 39.83 [+ or -] 5.14, and 25.67 [+ or -] 4.39s) and the second-phase (13.17 [+ or -] 5.66, 12.17 [+ or -] 4.4, and 1.33 [+ or -] 0.71s) pain responses, at the tested doses of 20, 40 and 80mg/kg, respectively. Morphine (10 mg/kg), the reference drug also significantly suppressed the formalin-response in both phases (first-phase, 6.67 [+ or -] 1.26 and second-phase, 0.17 [+ or -] 0.17s). When used alone, naloxone (1 mg/kg, s.c.) and glibenclamide (2 mg/kg, i.p.), the respective opioid-receptor and [[KAPPA].sup.+] -ATP channel antagonists failed to modify the formalin-induced nociceptive responses in a significant manner (Fig. 3B) (naloxone: first-phase, 85.83 [+ or -] 9.17 and second-phase, 47.5 [+ or -] 6.57; glibenclamide: first-phase, 89.33 [+ or -] 18.81 and second-phase, 46.5 [+ or -] 8.06s). In the combination studies, naloxone notably antagonized only the morphine anti-nociception but not the fraction [naloxone + morphine: first-phase, 74.33 [+ or -] 11.01 and second-phase, 47 [+ or -] 8.25s; naloxone + dose (80 mg/kg): first-phase, 31.17 [+ or -] 4.31 and second-phase, 2 [+ or -] 1.06s]. In contrast, glibenclamide exhibited significant antagonism to the fraction but not to morphine (glibenclamide + the high dose: first-phase, 54.5 [+ or -] 7.31 and second-phase, 40.17 [+ or -] 6.71s; glibenclamide + morphine: first-phase, 10.83 [+ or -] 1.94 and second-phase, 0.83 [+ or -] 0.48s) (Fig. 3B).

Table 1. The chemical compounds in the PEF (GC-MS analysis)

Start RT  End RT                 Compound                     % Area
(min)     (min)

 7.78      7.81   2.4[alpha],5,6,7,8-Hexahydro-3,5,5,9-        0.13
                  tertramethyl-1H-benzocycloheptene

 7.97      8.02   [alpha]-Longipinene                          0.07

 8.02      8.07   [beta]-Guaiene                               0.18

 8.12      8.17   -                                            0.1

 8.73      8.77   8,9-Dehydro-9- formyl-cyclosolongifolene     0.11

 9.94      9.99   Widdrol hydroxyether                         1.06

10.13     10.19   Octahydro-4,4,8,8-tetramethy 1-[4.sub.x],    0.2
                  7-methano- [4.sub.x]H- naphth[1,8,sub.a.-b]

10.33     10.38   3,7,11,15-Tetramethly-2- hexadecen 1-ol      0.24

10.39     10.42   Hexahydrofarnesyl acetone                    0.1

10.52     10.57   2-cis-9-Octadecenyloxyethanol                0.2

11.08     11.15   Z-11-Hexadecenoice acid                      0.58

11.20     11.28   Hexadecenoice acid                           6.8

11.39     11.44   Palmitic acid ethyl ester                    0.87

12.17     12.21   Phytol                                       1.08

12.31     12.47   Cis-9, cis-12-octadecadienoate acid         46.72

12.48     12.51   Ethyl (9E, 12E)-9, 12-octadecedienoate       4.21

12.52     12.56   Linolenic acid ethyl ester                   2.37

14.36     14.41   n-Octacosane                                 0.4

14.45     14.5    Methyl betadecyl ketone                      0.73

15.32     15.47   2-Octadecoxycthanol                          1.69

16.02     16.09   Sqyalcne                                     2.02

18.51     18.65   Vitamin E                                   10.72

19.84     19.99   Campesterol                                  3.44

20.15     20.3    Stimasterol                                  2.92

20.95     21.16   [beta]-Sitosterol                           12.41

-, not detected.

Effect of the capsaicin test

The effects of the PEF and morphine against capsaicin-induced nociception in mice were shown in Fig. 4A. When compared to vehicle-treated controls (43.5 [+ or -] 5.3 s), the dose-dependent reduction in the duration of paw licking was observed in mice pretreated with the fraction (28.17 [+ or -], 24.83 [+ or -] 4.45, and 19.67 [+ or -] 3.49 s, respectively, for the doses of 20, 40 and 80 mg/kg). Morphine, the positive control used in the study also caused significant anti-nociception (0.33 [+ or -] 0.21 s). The effects of naloxone (1 mg/kg, s.c.) and glibenclamide (2 mg/kg, i.p.) on the anti-nociceptive activities of morphine (10 mg/kg, s.c.) and the fraction (80 mg/kg) were shown in Fig. 4B. Employed alone, naloxone and glibenclamide produced no effect on capsaicin-induced paw licking response (naloxone: 49.5 [+ or -] 5.17; glibenclamide: 45.5 [+ or -] 6.13 s). In the combination studies, while the naloxone pretreatment selectively antagonized the anti-nociceptive effect of morphine [morphine + naloxone: 38.83 [+ or -]6.85; the dose (80 mg/kg) + naloxone: 23.83 [+ or -] 5.1s]. Interestingly, glibenclamide exhibited significant antagonism to the fraction but not to morphine [glibenclamide + morphine: 0.33 [+ or -] 0.21; glibenclamide + dose (80 mg/kg): 40.17 [+ or -] 5.22s].

[FIGURE 2 OMITTED]

Effect of the tail-flick test

In the tail-flick test, the fraction showed a significant effect on the duration in the hot water, when compared to vehicle-treated control group (1.52 [+ or -] 0.15 s) (Fig. 5). The mean of the duration in the group of the dose (20, 40 and 80 mg/kg) was 1.69 [+ or -] 0.13, 2.1 [+ or -] 0.15, 2.25 [+ or -] 0.09s, respectively. Whereas the positive control group treated with morphine (10mg/kg) exhibited the powerful activity (4.95 [+ or - ] 0.35 s).

[FIGURE 3 OMITTED]

Effect of the hot-plate test

In the hot-plate test, the mean of the durations in the group of the dose (20, 40 and 80 mg/kg), the positive control group (morphine, 10mg/kg) and control group were as follows: the group of the dose (20, 40 and 80 mg/kg) 15.13 [+ or -] 0.82, 19.85 [+ or -] 0.02, 27.87 [+ or -] 4.08 s; the positive control group 44.4 [+ or -] 3.5 s;

control group 12.92 [+ or -] 1.47 s, respectively. The result showed that the group treated with the fraction and morphine had the powerful anti-nociceptive effect (Fig. 6).

Effect on the open-field test

The fraction (20, 40 and 80 mg/kg) did not affect the motor coordination in mice. The mean permanence time of animals and the length of the route in the apparatus, obtained, in the fraction-treated groups, weren't statistically different from those of vehicle-treated control group over 5 min period. Only diazepam (1 mg/kg, i.p.) significantly (p < 0.01) affected the mobile performance in comparison with the control group.

Effect on pentobarbital sodium-induced sleeping time

The effects of the fraction and diazepam on pentobarbital sodium-induced sleeping time were as follows: vehicle-treated controls: 42.25 [+ or -] 1.85 s; the dose (20, 40 and 80 mg/kg): 39.5 [+ or -] 4.94, 39.87 [+ or -] 6.32 and 40.14 [+ or-] 4.88s, respectively; diazepam: 166.12 [+ or -] 7.67s. Only diazepam but not the fraction prolonged the sleeping time significantly produced (p < 0.001) (Fig. 7).

Discussion

In the presented experiments, the PEF of Paederia scandens demonstrated significant both peripheral and central analgesic activities against chemical nociception in mice induced by intraperitoneal acetic acid, sub-plantar capsaicin or formalin and thermal nociception.

[FIGURE 4 OMITTED]

The PEF is a complex mixture of fatty acids and sterols, which comprises a great part of the fraction. In the previous study, it was well known that stigmasterol and [beta]-sitosterol could, significantly and dose-dependently, reduce formalin-induced pain, acetic acid induced abdominal constrictions and increase the reaction time in the hot-plate test (Al-Yousuf et al., 2002; Erazo et al., 2006). Moreover, a previous study reported the positional and geometric isomers of octadecadienoic acid had the property of anti-inflammation and anti-nociception (Bassaganya-Riera et al., 2002). Due to complexity of the chemical compounds from the PEF, it would be difficult to attribute the observed activity to any single chemical component present in it. So it might equally be true that the antinociceptive activity of the PEF was related to the combination of three major chemical compounds - cis-9, cis-12-octadecadienoic acid, stigmasterol and beta-sitosterol.

The acetic acid-induced writhing method was widely used for the evaluation of peripheral anti-nociceptive activity, which was able to determine the anti-nociceptive effect of compounds or dose levels that might appear inactive in other methods like tail-flick test (Bentley et al., 1983; Gene et al., 1998). However, it was known that constriction induced by acetic acid was considered to be a non-selective anti-nociceptive model, since acetic acid indirectly induced the release of endogenous mediators stimulated the nociceptive neurons that were sensitive to non-steroidal antiinflammatory drugs (NSAIDs) (Sanchez-Mateo et al., 2006). Our results indicated that the PEF of P. scandens could reduce the number of writhing on animal model, showed powerful anti-nociceptive effects. However, the results of this writhing test alone didn't ascertain whether the anti-nociceptive effect was central or peripheral.

[FIGURE 5 OMITTED]

In order to confirm it, the formalin test was carried out. The advantage of the formalin model of nociception was that it could discriminate pain in its central and/or peripheral components. The test consists of two different phases which can be separated in time: the first one is generated in the periphery through the activation of nociceptive neurons by the direct action of formalin and the second phase occurs through the activation of the ventral horn neurons at the spinal chord level (Tjolsen et al., 1992). Central analgesic drugs, such as narcotics, inhibited equally in both phases, while peripherally acting drugs, such as steroids (hydrocortisone, dexamethasone) and NSAIDs (aspirin) suppressed mainly in the later phase (Trongsakul et al., 2003). In this test, the PEF of P. scandens at more effective doses in the writhing test could reduce the duration of the paw licking (s) obviously in both the first phase (neurogenic) and the second phase (inflammatory) of the formalin test. It was reasonable that the PEF of P. scandens had the same anti-nociceptive activity as central analgesic morphine. Furthermore, the significant effect of the PEF of P. scandens on the tail-fick response and on the hot plate response, in the thermal tests, provided a further confirmation of their central effect, since the tail-flick test and the hot plate test were predominantly a spinal reflex, and were considered to be selective for centrally acting analgesic compounds, like pethidine, while peripheral analgesic was known to be inactive on this kind of painful stimulus (Srinivasan et al., 2003).

[FIGURE 6 OMITTED]

[FIGURE 7 OMITTED]

The capsaicin-induced paw-licking responses were mediated by release of the excitatory amino acid glutamate and by sensory neuropeptides like substance P released from sensory neurons at the spinal cord, which was correspondent with the mechanism of the late phase of the formalin test (Santos and Calixto, 1997; Otuki et al., 2001). So the data obtained in the capsaicin test and the late phase of the formalin test indicated that this fraction had anti-nociceptive activity. The anti-nociception caused by the PEF of P. scandens seemed to be unrelated to motor impairment or sedation since the mice tested in open-field and pentobarbital sodium-induced sleeping time tests showed no significant effect on these behaviors.

To verify possible anti-nociceptive mechanisms, we had examined the effect of naloxone, a non-selected opioid receptor antagonist and glibenclamide, a blocker of K + -ATP channels on the anti-nociceptive activity of the PEF of P. scandens. Interestingly, the data obtained in both formalin and capsaicin tests showed that the anti-nociception produced by the PEF of P. scandens was naloxone-sensitive, but sensitive to glibenclamide. Since naloxone failed to antagonize the anti-nociception in formalin and capsaicin tests, so the anti-nociceptive activity of the fraction might be the involvement of K + -ATP channels. It is well known that the nociception in the formalin test and hot plate test, as well as capsaicin test results from the accumulation of intracellular Ca2 + concentration that, in turn, initiates a number of second/third messenger-mediated intracellular cascades (Mayer and Miller, 1990; Collingridge and Singer, 1990; Sommer and Seeburg, 1992; Schoepp and Conn, 1993). In addition, several reports had indicated that intra-plantar injections of formalin as well as capsaicin evoke increases in primary afferent activity (Peterson et al., 1997; Ren et al., 2005). In primary afferent neurons, capsaicin activates vanilloid receptor (TRPV1) and causes accumulation of intracellular [Ca.sup.2+], which is necessary for capsaicin-evoked transmitter release (Sakurada et al., 2003). The anti-nociception of the PEF may thus involve opening of glibenclamide-sensitive [K.sup.+] channels, hyper-polarization of capsaicin-sensitive sensory afferent neurons and decreased release of neurotransmitters. Future studies using neonatal capsaicin-treated animals and immunohistochemical studies that show Fos-like immunoreactivity in spinal cord may help in elucidating the mechanism of the anti-nociception.

In conclusion, the study demonstrated the antinociceptive activity of the PEF of P. scandens in the test models of nociception induced by chemical and thermal stimuli, and further suggested that anti-nociceptive activity might be related to the involvement of [K.sup.+]-ATP channels, which merited further studies regarding the precise site and the mechanism of action.

Acknowledgements

The authors are grateful to the Kai-run pharmaceutical factory for financial support.

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Yu-Feng Chen (a), Nan Li (a), Yu-Liang Jiao (b), Peng Wei (a), Qiao-Yan Zhang (a), Khalid Rahman, Han-Chen Zheng (a), Lu-Ping Qin (a), *

(a) Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, PR China

(b) Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, 800 Xiang-yin Road, Shanghai 200433, PR China

(c) Faculty of Science, School of Biomolecular Science, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK

* Corresponding author. Tel./fax: + 8621 25070394.

E-mail address: [e-mail] (L.-p. Qin).

0944-7113/$-see front matter [C] 2007 Elsevier GmbH. All rights reserved. doi: 10.1016/j.phymed.2007.09.023