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The present study examined the anti-nociceptive effect of the ethanolic extract of the aerial parts of Hilleria latifolia in chemical (acetic acid-induced abdominal writhing, glutamate, formalin and capsaicin tests) and thermal (tail immersion test) behavioral pain models in rodents. The possible mechanisms of anti-nociceptive action were also assessed with various antagonists in the formalin test.
The Hilleria latifolia extract (HLE) together with morphine and diclofenac (positive controls), showed significant anti-nociceptive activity in all the models used. The anti-nociceptive effect exhibited by HLE in the formalin test was partly or wholly reversed by the systemic administration of naloxone, theophylline and atropine. Glibenclamide, ondansetron, yohimbine, nifedipine and NG-L-nitro-arginine methyl ester /L-NAME, however, did not significantly block the anti-nociceptive effect of the extract. HLE, unlike morphine, did not induce tolerance to its anti-nociceptive effect in the formalin test after chronic administration; morphine tolerance did not also cross-generalize to HLE. Interestingly also, chronic concomitant administration of HLE and morphine significantly suppressed the development of morphine tolerance.
Together, these results indicate that HLE produces dose-related anti-nociception in several models of chemical and thermal pain, without tolerance induction, through mechanisms that involve an interaction with adenosinergic, muscarinic cholinergic and opioid pathways.
KEY WORDS: Hilleria latifolia, formalin, writhing, tail immersion, opioid tolerance
Pain is the most common reason patients seek advice from health professionals. It is one of the most frequent presenting symptoms of different pathologies and represents important medical and economic costs for the community. Current analgesic therapy, despite their proven efficacy in alleviating symptoms and providing pain relief, all have considerable side effects including gastrointestinal, renal damage, respiratory depression, emesis, and tolerance and/or addiction. In addition, many pain sufferers are not satisfied with their pain care and this makes the search for new analgesics that can more effectively treat pain an important challenge to drug research. Medicinal plants are believed to be important sources of new chemical substances with potential therapeutic efficacy. Considering that the most important analgesic prototypes (e.g. salicylic acid and morphine) were originally derived from plant sources, the study of plant species traditionally used as analgesics should still be seen as a useful research strategy in the search of new analgesics.
Hilleria latifolia (Lam.) H. Walt. (Phytolaccaceae) is a perennial herb that is common on cultivated grounds and along forest paths in the forest regions of Ghana. It also occurs in other parts of tropical Africa as well as South America. It is commonly known as Avegboma, Boe or Kukluigbe by the Ewes and Anafranaku by the Akans. In Ghanaian traditional medicine, different parts of the plant are useful in a variety of diseases. The leaves are effective in otalgia,  rheumatism [3-4] and boils  whereas the flowers are used for asthma. The leaves of H. latifolia are also used in Cote d'Ivoire and Congo to treat feverish pains, violent headache and some skin diseases.
In spite of the many uses traditionally, there is little scientific evidence in literature on the effect of this plant on experimental pain. This study therefore examined the anti-nociceptive effect and possible mechanism of action of the ethanolic extract of the aerial parts of H. latifolia in animal models. The current study will help to substantiate the traditional uses of H. latifolia as well as provide an alternative to current analgesics.
MATERIALS AND METHODS
The aerial parts of H. latifolia were collected from the campus of Kwame Nkrumah University of Science and Technology (KNUST), Kumasi near the Botanical Gardens (06°41′12.89″N; 01°33′59.51″W) during the month of July, 2007 and authenticated by Mr. George H. Sam of the Department of Herbal Medicine, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, KNUST, Kumasi, Ghana. A voucher specimen (KNUST/HM1/09/L029) was kept at the herbarium of the Faculty.
Preparation of extract
The plant was room-dried for seven days and pulverized into fine powder. The powder was extracted by cold percolation with 70 % (v/v) ethanol and then concentrated into a green syrupy mass under reduced pressure at 60 °C using a rotary evaporator. It was further dried in a hot air oven at 50 °C for a week and kept in a refrigerator for use. The yield was 19.67 %. This crude extract is subsequently referred to as HLE or extract in this study.
Male ICR mice (15-25 g) and male Sprague-Dawley rats (100-195 g) were purchased from the Noguchi Memorial Institute for Medical Research, Accra, Ghana and kept in the animal house of the Department of Pharmacology, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. They were housed in groups of 5 in stainless steel cages (34ï‚´47ï‚´18 cm3) with soft wood shavings as bedding, fed with normal commercial pellet diet (GAFCO, Tema, Ghana), given water ad libitum and maintained under laboratory conditions (temperature 24±2 °C, relative humidity 60-70%, and 12 hour light-dark cycle). The studies were conducted in accordance with accepted principles for laboratory animal use and care (EEC directive of 1986: 86/609 EEC). All protocols used were approved by the Departmental Ethics Committee.
Drugs and Chemicals
The following drugs and chemicals were used: formalin, acetic acid, theophylline (BDH, Poole, England); diclofenac (KRKA, Slovenia); morphine (PhytoRiker, Accra, Ghana); ondansetron (GlaxoSmithKline, Uxbridge, U.K.); glibenclamide (Daonil®, Sanofi-Aventis, Guildford, UK); nifedipine (Denk Pharma, Germany); yohimbine, atropine, naloxone, NG-L-nitro-arginine methyl ester/ L-NAME, L-glutamic acid, capsaicin (Sigma-Aldrich Inc., St. Louis, MO, USA).
Preliminary phytochemical tests were performed on HLE using methods described by Trease and Evans.
Acetic acid-induced writhing assay
This test was carried out as described previously [6-7] with modifications. Mice were treated with HLE (30, 100 or 300 mg kg-1, p.o.), diclofenac (10, 30 or 100 mg kg-1, i.p.), or vehicle (1 ml 100 g-1, p.o.) 30 min (i.p.) or 1 h (p.o.) before administration of the acetic acid and placed individually in a testing chamber (a Perspex chamber 15 cm - 15 cm - 15 cm). A mirror angled at 45° below the floor of the chamber allowed a complete view of the mice.
Each animal was administered with acetic acid (0.6 %, 10 ml kg-1) intraperitoneally. Injection of acetic acid induced a nociceptive behavior, writhing, an exaggerated extension of the abdomen combined with the outstretching of the hind limbs. Responses were captured (30 min) for analysis by a camcorder (EverioTM, model GZ-MG1300, JVC, Tokyo) placed directly opposite the mirror and attached to a computer. Tracking of the behavior was done using a public domain software JWatcherTM Version 1.0 (University of California, Los Angeles, USA and Macquarie University, Sidney, Australia available at http://www.jwatcher.ucla.edu/.) to obtain the total number of writhes per 5 min, starting 5 min after acetic acid administration. These data expressed in a time course which enabled the observance of changes in the maximal number of writhing induced. A dose-response curve was also plotted to determine the significant anti-nociceptive dose.
Tail immersion test
The tail-immersion test was carried out as described previously  with modifications. This involved immersing the extreme 3.5 cm of the rat's tail in a water bath containing water at a temperature of 48 ± 0.5 °C. The rat reacts by withdrawing the tail. Reaction time was recorded with a stop watch and a cut-off time of 15 s imposed on this measure. Rats were randomly selected to perform one of the study groups (five per group): control, diclofenac (10-100 mg kg-1, i.p.), morphine (1-10 mg kg-1, i.p.) and HLE (30-300 mg kg-1, p.o.). The reaction time (Ta) for the study groups was taken at intervals 0.5, 1, 2, 3, 4 and 5 h after a latency period of 30 min (i.p.) or 1 h (p.o.) following the administration of the drugs or extract. Percentage maximal possible effect (% MPE) was calculated from the reaction times using the formula: [(T2-T1)/(T0-T1) - 100], where T1 and T2 were the pre- and post- drug reaction times respectively, and T0 was the cut-off time.
The test was carried out as described previously [9-10] with a few modifications. Each animal was assigned and acclimatized to one of 20 formalin test chambers (a Perspex chamber 15 cm - 15 cm - 15 cm) for one hour prior to formalin injection. Mice were then pre-treated with the test drugs [HLE (30-300 mg kg-1, p.o.) and morphine (1-10 mg kg-1, i.p.)] 30 min for i.p. route and 1 h for oral route before intraplantar injection of 10 ïl of 5 % formalin. The animals were immediately returned individually into the testing chamber and their nociceptive behaviors captured (1 h) for analysis in the same way as that described previously in the writhing test above. Pain response was scored for 1 h, starting immediately after formalin injection. A nociceptive score was determined for each 5-min time block by measuring the amount of time spent biting/licking of the injected paw . Tracking of the behavior was done using a public domain software JWatcherTM Version 1.0. Average nociceptive score for each time block was calculated by multiplying the frequency and time spent in biting/licking. Data were expressed as the mean ± SEM of scores between 0-10 (first phase) and 10-60 min (second phase) after formalin injection.
The procedure used was similar to that described previously  but with modifications. Before testing, the animals were placed individually in one of 20 transparent Perspex chambers (15 cm - 15 cm - 15 cm). Following an hour adaptation period in the chamber, animals were pre-treated with HLE (10-, 100, 300 mg kg-1, p.o.) and morphine (3 mg kg-1, i.p.) 30 min for i.p. route and 1 h for oral route before intraplantar injection of 20 µl of capsaicin (1.6 µg/paw made in 10 % ethanol, 10 % Tween 80 and 80 % saline). Control animals received vehicle (normal saline, 10 ml kg-1) systemically before intraplantar capsaicin. Pain response (biting/licking of the injected paw) was recorded (10 min) and scored (10 min) in the same way as that described previously in the formalin test above. Data were expressed as the mean ± SEM of scores between 0-10 after capsaicin injection.
The procedure was carried out as described previously [13-14] with modifications. Mice were acclimatized to test chambers and pre-treated with HLE, morphine or vehicle similar to that described for the capsaicin-induced nociception above. Twenty microlitres of glutamate (10 μmol/paw prepared in saline) was injected intraplantarly in the ventral surface to the right hind paw of mice and immediately returned individually into the testing chambers. The nociceptive behavior (biting/licking of the injected paw) of the animals were then captured (15 min) and later scored (15 min) similarly to that described in the formalin test above. Data were expressed as the mean ± SEM of scores between 0-15 min after glutamate injection.
Assessment of some possible mechanisms of action of HLE
To investigate some possible mechanisms by which HLE exerts its anti-nociceptive activity, mice were pre-treated with different drugs in the formalin test. The doses of antagonists, agonists and other drugs were selected based on data from literature and preliminary experiments in our laboratory.
Involvement of the opioid system
Mice were pre-treated with naloxone (a non-selective opioid receptor antagonist; 2 mg kg-1, i.p.) and after 15 min received HLE (30 mg kg-1, p.o.), morphine (3 mg kg-1, i.p.) or vehicle (10 ml kg-1, p.o.). The nociceptive response to formalin injection was recorded 1 h after administration of HLE or vehicle and 30 min after administration of morphine. Another group of mice was pre-treated with vehicle and after 15 min received HLE (30 mg kg-1, p.o.), morphine (3 mg kg-1, i.p.) or vehicle (10 ml kg-1, p.o.), 1, 0.5 and 1 h before formalin injection respectively.
Involvement of the nitric oxide pathway
Mice were pre-treated with L-NAME (NG-L-nitro-arginine methyl ester, a NO synthase inhibitor, 10 mg kg-1, i.p.) and after 15 min received HLE (30 mg kg-1, p.o.), morphine (3 mg kg-1, i.p.) or vehicle. The nociceptive response to formalin injection was recorded 1 h after administration of HLE or vehicle and 30 min after morphine administration.
Involvement of ATP-sensitive K+ channels
Mice were pre-treated with glibenclamide (an ATP-sensitive K+ channel inhibitor, 8 mg kg-1, p.o.) and after 30 min received HLE (30 mg kg-1, p.o.), morphine (3 mg kg-1, i.p.) or vehicle. The nociceptive response to formalin injection was recorded 1 h after administration of HLE or vehicle and 30 min after morphine administration.
Involvement of the adenosinergic system
Mice were pre-treated with theophylline (10 mg kg-1, i.p., a non-selective adenosine receptor antagonist) and after 15 min received HLE (30 mg kg-1, p.o.), morphine (3 mg kg-1, i.p.) or vehicle. The nociceptive response to formalin injection was recorded 1 h after administration of HLE or vehicle and 30 min after morphine administration.
Involvement of 5-HT3-serotonergic receptors
Animals were pre-treated with ondansetron (0.5 mg kg-1, i.p., a 5-HT3 receptor antagonist) and after 15 min received HLE (30 mg kg-1, p.o.), morphine (3 mg kg-1, i.p.) or vehicle ( 10 ml kg-1). The nociceptive response to formalin injection was recorded 1 h after administration of HLE or vehicle and 30 min after morphine administration.
Involvement of α2-adrenoceptors
Mice were pre-treated with yohimbine (3 mg kg-1, p.o., a selective adrenoceptor antagonist) and after 30 min received HLE (30 mg kg-1, p.o.), morphine (3 mg kg-1, i.p.) or vehicle. The nociceptive response to formalin injection was recorded 1 h after administration of HLE or vehicle and 30 min after morphine administration.
Involvement of voltage-gated calcium channels (VGCCs)
Mice were pre-treated with nifedipine (10 mg kg-1, p.o., L-type VGCC blocker) and after 30 min received HLE (30 mg kg-1, p.o.), morphine (3 mg kg-1, i.p.) or vehicle. The nociceptive response to formalin injection was recorded 1 h after administration of HLE or vehicle and 30 min after morphine administration.
Involvement of muscarinic cholinergic system
Mice were pre-treated with atropine (5 mg kg-1, i.p., a non-selective muscarinic receptor antagonist) and after 15 min received HLE (30 mg kg-1, p.o.), morphine (3 mg kg-1, i.p.) or vehicle. The nociceptive response to formalin injection was recorded 1 h after administration of HLE or vehicle and 30 min after morphine administration.
The formalin test was used to ascertain whether, after chronic treatment, tolerance develops to the anti-nociceptive activity of HLE and morphine. The procedure used was similar to that described previously. Mice were divided randomly into five groups (n=5) and treated once daily for 8 days as follows: three groups with saline i.p., one group with HLE 60 mg kg-1, p.o. and one group with morphine 6 mg kg-1, i.p. On day 9, these groups were treated in the following manner: one saline-pre-treated group was treated with saline i.p.; two saline-pre-treated groups were treated with either HLE 30 mg kg-1, p.o. or morphine 3 mg kg-1, i.p.; the group pre-treated with HLE 60 mg kg-1 was treated with HLE 30 mg kg-1, p.o. and the group pre-treated with morphine 6 mg kg-1 was treated with morphine 3 mg kg-1, i.p. HLE and morphine were administered 60 and 30 min before formalin injection, respectively.
In a separate study, HLE was administered to animals chronically treated with morphine to establish whether morphine-induced tolerance cross-generalizes with HLE. This second experiment also investigated whether chronic concurrent treatment of mice with morphine and HLE will abolish the development of morphine tolerance. In the second study, two groups of animals (n = 5) were treated once daily for 8 days with morphine 6 mg kg-1, i.p. Another group (n=5) received both morphine 6 mg kg-1, i.p. and HLE (60 mg kg-1, p.o, 30 min before the morphine) for 8 days. Three other groups of animals (n = 5) received chronic dosing of saline i.p. also for 8 days. On day 9, animals treated with chronic morphine received either morphine (3 mg kg-1, i.p., 30 min before formalin) or HLE (30 mg kg-1, p.o., 60 min before formalin) respectively, whereas three saline-treated groups received either a similar administration of saline, morphine (3 mg kg-1, i.p.), or HLE (30 mg kg-1, p.o.). Additionally, the group that was chronically treated with both morphine and HLE also received morphine (3 mg kg-1, i.p.) 30 min before formalin.
The time-course curves were subjected to two-way (treatment ï‚´ time) repeated measures analysis of variance (ANOVA) with Bonferroni's post hoc test. Total nociceptive score for each treatment was calculated in arbitrary unit as the area under the curve (AUC). To determine the percentage inhibition for each treatment, the following equation was used:
Total nociceptive scores for treatment groups were analyzed using one-way analysis of variance (ANOVA) with drug treatment as a between-subjects factor. Whenever ANOVA was significant, further comparisons between vehicle- and drug- treated groups were performed using the Newman-Keuls test.
ED50 (dose responsible for 50% of the maximal effect) for each drug was determined by using an iterative computer least squares method, with the following nonlinear regression (three-parameter logistic) equation:
Where, X is the logarithm of dose and Y is the response. Y starts at a (the bottom) and goes to b (the top) with a sigmoid shape.
The fitted midpoints (ED50s) of the curves were compared statistically using F test [16-17]. GraphPad Prism for Windows version 4.03 (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses and ED50 determination. P < 0.05 was considered statistically significant in all analysis.
Preliminary phytochemical screening of HLE revealed the presence of saponins, tannins, glycosides, steroids, terpenoids as well as little amounts of flavonoids and alkaloids.
Acetic acid-induced writhing assay
Acetic acid injected intraperitoneally produced the characteristic response described above in control mice pre-treated with physiological saline. Table 1 represents the total number of writhes induced by acetic acid, during 20 min of observation, beginning 10 min after the i.p. injection. HLE (30-300 mg kg-1, p.o. 1 h before) significantly reduced (F6, 25=8.84, P<0.0001, Table 1) the number of abdominal writhes over 20 min with maximal inhibition of 70.60±6.48% at the dose of 300 mg kg-1. Similarly, the NSAID diclofenac (10-100 mg kg-1, i.p., 30 min before) profoundly inhibited (F6,25=8.84, P<0.0001, table 1) the acetic acid-induced writhes by a maximum of 98.10±1.90%.
Figure 6a shows the dose-response curves for the inhibition of acetic acid-induced abdominal writhes in mice. HLE exhibited an inverted U-shaped dose response relationship with ED50 values of approximately 53.21 and 220.80 mg kg-1. Generally, HLE was less potent than diclofenac (ED50=13.81±6.83 mg kg-1).
Tail immersion test
As shown by the time course curves in figure 1, all test drugs caused an increase in the tail withdrawal latency, calculated as a percentage of the maximum possible effect (% MPE). Two-way ANOVA (treatment ´ time) revealed a significant effect of drug treatments on the tail withdrawal latencies (HLE: F3,112=9.90; P<0.0001; diclofenac: F3,112=26.47; P<0.0001 and morphine: F3,112=25.09; P<0.0001; Fig. 1a, c, e). HLE (30-300 mg kg-1, p.o, 1 h before) increased the tail withdrawal latencies (F3,15=3.918, P=0.030; Fig.1b) with a significant effect at the dose of 30 mg kg-1 (P<0.05). Diclofenac (10-100 mg kg-1, i.p.) elicited a significant anti-nociceptive activity by dose-dependently increasing the tail withdrawal latencies of animals pre-treated with it (F3,16= 6.804, P=0.0036; Fig. 1d). Morphine (1-10 mg kg-1, i.p., fig 1f) also showed similar effects (F3, 16 =9.43, P= 0.0008).
Dose-response curves for the anti-nociceptive effects of HLE, diclofenac and morphine in the tail immersion test are shown in Fig 6b. HLE displayed a U-shaped dose response relationship with ED50 values of 56.86 and 156.68 mg kg-1. By comparing the ED50 values from the curves, HLE was significantly less potent than diclofenac (ED50 19.18±24.11 mg kg-1) and morphine (ED50 2.24±2.35 mg kg-1).
Injection of formalin (5 %, 10 µl) into the ventral surface of the right hind paw evoked a characteristic biphasic licking response in the mice as previously reported [18-19]. This consisted of an initial intense response to pain beginning immediately after formalin injection and rapidly decaying within 10 min after formalin injection (first/neurogenic phase) and then followed by a slowly rising but longer-lasting response (second/inflammatory phase) from10-60 min after formalin injection with maximum effect at approximately 20-30 min after formalin injection. [20-21]
Figure 2 shows the effect of pre-treatment of HLE and morphine on formalin-induced pain in mice. All drug-treated groups displayed (Figure 2a, c) significant reduction in formalin-induced nociceptive behavior when compared with the vehicle-treated group [(HLE: F3,192=3.92; P<0.05; morphine: F3,192=15.29; P<0.0001; Two-way ANOVA (treatment ï‚´ time)]. Oral administration of HLE (30-300 mg kg-1) 1 h before the injection of formalin inhibited both neurogenic (F3,16=2.71; P=0.0797, fig 2b) and inflammatory (F3,16=6.648; P=0.0051, fig 2b) phases of formalin-induced licking, though inhibition did not reach statistical significance in the neurogenic phase. Morphine (1-10 mg kg-1, i.p.), the positive analgesic control, produced marked dose-related inhibition of both the neurogenic (F3,16= 3.531, P=0.039, fig 2d) and inflammatory (F3,16= 15.54, P<0.0001, fig 2d) pain phases.
HLE (30-300 mg kg-1) displayed an inverted U-shaped dose response relationship as shown in figure 6d. The ED50 values were approximately 35.80 and 310.46 mg kg-1 for the first phase and 37.15 and 123.03 mg kg-1 for the second phase. Comparison of ED50s obtained by F-test (Fig.6d) revealed that the extract was more potent in the second phase than the first.
Capsaicin induced a clear nociceptive response exhibited by biting and licking of the injected paw. Oral administration of HLE (30-300 mg kg-1) 60 min before the intraplantar injection of capsaicin produced dose-dependent attenuation of capsaicin-induced neurogenic pain (F5,24=10.21; P<0.0001, fig 3a) with a maximal inhibition of 59.49±7.89 % at the dose of 300 mg kg-1. Similarly, morphine (3 mg kg-1, i.p.30 min before) profoundly inhibited (F5,24=10.21; P<0.0001, fig 3a) the neurogenic pain by 84.07±4.88%. Figure 6c shows the dose-response curves for the inhibition of capsaicin-induced neurogenic pain by HLE in mice. The ED50 from the non-linear regression was 90±116.76 mg kg-1.
Figure 3b shows the effects of HLE and morphine on glutamate-induced nociception. HLE (30-300 mg kg-1, p.o., 1 h before i.pl. glutamate) produced dose-dependent inhibition of glutamate-induced pain (F5, 22=8.00; P<0.0002, fig 3b) with a maximal inhibition of 53.41±8.25% at the dose of 100 mg kg-1. Similarly, morphine (3 mg kg-1, i.p.30 min before) profoundly inhibited (F5, 22=8.00; P<0.0002, fig 3b) the glutamate-evoked nocifensive behaviors by 92.22±4.66 %. HLE exhibited an inverted U-shaped dose-response relationship (fig 6c) with ED50 values of 188.36 mg kg-1and 28.58 mg kg-1from the non-linear regressional analysis.
Assessment of possible mechanism of action of HLE
The results presented in figure 4a show that the pre-treatment of mice with naloxone (2 mg kg-1, i.p.) significantly reversed (P<0.05) the anti-nociception by HLE (30 mg kg-1, p.o.) in the inflammatory phase and had no effect on phase 1. Naloxone also significantly reversed the anti-nociception caused by morphine (3 mg kg-1 i.p.) in both phases of formalin-induced pain (P<0.05 and P<0.01 respectively; fig. 4c).
Previous treatment of the animals with theophylline (10 mg kg-1, i.p.) significantly abolished the anti-nociception caused by HLE (30 mg kg-1, p.o.) in the second phase of the formalin test (P<0.05; fig. 4a). Theophylline also completely reversed the anti-nociception caused by morphine (3 mg kg-1, i.p.) in both phases of the formalin test (P<0.05 and P<0.01 respectively; fig. 4c).
Systemic pre-treatment of mice with L-NAME (10 mg kg-1, i.p.) or glibenclamide (8 mg kg-1, p.o.) did not significantly prevent the anti-nociception caused by HLE (30 mg kg-1, p.o.) in both phases of the formalin test (fig 4a). However, both L-NAME and glibenclamide blocked morphine (3 mg kg-1 i.p.) anti-nociception in the first phase (both P<0.05; fig 4c).
Ondansetron (0.5 mg kg-1, i.p.) did not significantly block anti-nociception caused by HLE (30 mg kg-1, p.o.) in both phases of the formalin test (fig. 4d). In contrast, ondansetron significantly reversed the anti-nociception caused by morphine (3 mg kg-1 i.p.) in both phases (P<0.001 and P<0.05 respectively; fig. 4b).
Systemic pre-treatment of mice with atropine (5 mg kg-1, i.p.) completely reversed the anti-nociception caused by HLE (30 mg kg-1, p.o.) in both phases of the formalin test (P<0.01 and P<0.05 respectively; fig. 4b). Atropine significantly abolished the anti-nociception caused by morphine (3 mg kg-1 i.p.) in the second phase (P<0.05) but caused no significant change in the first (fig. 4d).
Yohimbine (3 mg kg-1, p.o.) and nifedipine (10 mg kg-1, p.o.) did not significantly inhibit the anti-nociception caused by either HLE (30 mg kg-1, p.o.) or morphine (3 mg kg-1, i.p.) in both phases of the formalin test (fig. 4b, d).
Morphine (3 mg kg-1, i.p.) significantly attenuated nociceptive responses in both phases (F3,16= 27.87, P<0.0001 phase 1; F3,16= 5.41, P<0.01 phase 2; fig 5b) of formalin test in chronic vehicle-treated animals. However, the same dose of morphine administered at day 9 in animals chronically treated with morphine (6 mg kg-1, i.p.) failed to show such effect indicating development of tolerance (Fig.5b). In contrast, oral administration of 30 mg kg-1 HLE showed a comparable anti-nociceptive activity in mice given chronic treatment of either HLE 60 mg kg-1, p.o or vehicle, indicating lack of tolerance development (Fig. 5a). Furthermore, 30 mg kg-1, p.o. HLE still demonstrated anti-nociceptive activity in mice chronically treated with morphine(6 mg kg-1, i.p.), indicating that no cross-tolerance exists with morphine (Fig. 5a). Additionally, the repeated administration of HLE (60 mg kg-1, p.o) 30 min prior to each morphine (3 mg kg-1, i.p.) injection to mice during the 8-day protocol significantly attenuated the development of tolerance to morphine (Fig 5b).
This study has demonstrated that oral administration of the ethanolic extract of the aerial parts of Hilleria latifolia exerts significant anti-nociceptive activity against thermal- (tail immersion) as well as chemical- (acetic acid, glutamate, capsaicin and formalin) induced nociception in mice. This anti-nociceptive effect was partly or wholly reversed by the systemic administration of the naloxone, theophylline and atropine. Glibenclamide, ondansetron, yohimbine, nifedipine and L-NAME, however, did not significantly alter the anti-nociceptive effect of the extract.
In order to obtain a full picture of the analgesic property of HLE, several behavioral animal models of nociception which differ with respect to stimulus quality, intensity and duration were employed. The nociceptive tests were selected such that both peripherally- and centrally-mediated effects were investigated; in all, the extract showed peripheral and central anti-nociceptive activity.
The abdominal writhing test, a peritoneovisceral inflammatory pain model, is a very sensitive and convenient method for screening anti-nociceptive effect of compounds. Although in terms of specificity this method may have some insufficiencies (i.e. writhing may be suppressed by muscle relaxants and other non-analgesic drugs, leaving scope for the misinterpretation of results), it generally has a good correlation between the ED50 values obtained in animals using this test and analgesic doses administered in man. The nociceptive effect induced in this model is easily prevented by non-steroidal anti-inflammatory drugs, as well as by opioids and analgesics with central actions. HLE significantly inhibited the abdominal constriction induced by acetic acid in mice. The actions of acetic acid are known to be the indirect cause of the release of nociceptive endogenous mediators such as bradykinin, substance P, serotonin, histamine, sympathomimetic amines, prostaglandins (PGE2 and PGF2α) and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-8.[24-28] The inhibitory effects of HLE on inflammatory pain and abdominal constrictions produced after administration of acetic acid in this study might therefore be said to be due to interference with activation of nociceptors by one of these endogenous mediators.
The anti-nociceptive effects of HLE, morphine and diclofenac were confirmed by the use of a thermal nociceptive stimulation (tail immersion in a 48°C water bath). The tail immersion test, a variant of the tail-flick pain model, is a sensitive and particularly useful test for demonstrating dose-related activity. The effectiveness of analgesics in this model is also highly correlated with relief of human pain. HLE significantly attenuated thermal nociception in rats in this model, though not as effectively as morphine and diclofenac. The tail immersion test gives a response that is believed to be a spinally-mediated reflex  but the mechanism of response could also involve higher neural structures. It is therefore suggested that HLE exerts its anti-nociceptive effects, at least in part, by spinally-mediated central mechanisms.
The formalin-induced paw pain, an in vivo model of persistent pain, is a valid model for analgesic study. It is undoubtedly the most predictive of acute pain  and very popular for the rapid and easy screening of pharmacological targets in drug evaluation.[32-33] HLE showed significant anti-nociceptive effects in this model. The formalin test produces a distinct biphasic nociceptive response. A first phase (neurogenic pain), occurring within seconds of formalin injection, is elicited by direct chemical activation of nociceptive primary afferent fibers. A second, later phase (inflammatory pain), occurs as a result of ongoing activity in primary afferents and increased sensitivity of dorsal horn neurons.[10,34-35] Therefore, the test can be used to clarify the possible mechanism of anti-nociceptive effect of a proposed analgesic. Centrally acting drugs, such as opioids, inhibit both phases equally, however, many NSAIDs and corticosteroids inhibit only the late phase. HLE inhibited both phases of the formalin test but more effectively the second than the first. This implies that HLE is effective against both neurogenic and inflammatory pain. The inhibitory effect in the second phase also suggests anti-inflammatory action of HLE.
Hilleria latifolia extract, given orally, elicited a dose-dependent anti-nociceptive effect on the capsaicin-induced neurogenic paw licking response. Capsaicin (8-methyl-N-vanillyl-6-nonenamide), the pungent algesic substance obtained from hot red chilli peppers, is regarded a valuable pharmacological tool for studying a subset of mammalian primary sensory C-fibers and Aδ afferent neurons including polymodal nociceptors and warm thermoceptors. It has been proposed that the capsaicin-induced nociception occurs as a result of the activation of the capsaicin (vanilloid) receptor, TRPV1, a ligand-gated non-selective cation channel present in primary sensory neurons.[38-40] The effect of HLE in this pain model suggests that HLE is effective against neurogenic pain in mice and its action may be due to an interaction with the capsaicin receptor (TPRV1).
Results obtained in this study also show that oral administration of HLE produced a significant inhibition of the nociceptive response caused by intraplantar injection of glutamate into the mouse hind paw. Glutamate, acting through a variety of receptors, plays an important role in peripheral and central pain transmission. Its intraplantar injection evokes thermal and mechanical hyperalgesia [42-44] as well as spontaneous lifting and licking behaviors in mice. The nociceptive response induced by glutamate appears to involve peripheral, spinal and supraspinal sites of action and is largely mediated by both NMDA and non-NMDA receptors as well as by the release of nitric oxide or by some nitric oxide-related substance. The inhibitory capabilities of HLE by interference with the nociceptive response induced by glutamate, demonstrates, at least in part, an interaction of HLE with the glutamatergic system.
The intraplantar injection of formalin, capsaicin or glutamate is known to release endogenous chemical mediators such as neuropeptides, excitatory neurotransmitters, PGE2, NO and kinins in periphery and spinal cord that contribute to the nociceptive process.[13,34,45-47] Therefore, the suppression of the capsaicin-, formalin- and glutamate-induced licking response caused by treatment with HLE, are complementary indications that the anti-nociceptive action of this extract could be associated with its ability to inhibit the production or action of some of these mediators.
With the exception of the capsaicin test, HLE showed a typical biphasic dose-response pattern in all nociceptive tests used (fig.6). The exact biochemical mechanism underlying this pharmacological inversion is not yet clear and requires further studies to establish it. Nonetheless, the activation of various pathways at different doses of HLE may be responsible for the bell-shape.
In an attempt to further characterize some of the mechanisms through which HLE exerts its activity, the anti-nociceptive effect of HLE was assessed in the presence of various antagonists of notable mediators of the nociceptive pathway including naloxone, theophylline, L-NAME, glibenclamide, atropine, ondansetron, yohimbine and nifedipine. The formalin test was selected for this study, since it is more specific and with its biphasic control of pain, reflects different pathological processes and allows the elucidation of the possible mechanism involved in analgesia. Naloxone, a non-selective opioid antagonist significantly reversed the anti-nociceptive effect of HLE only in the second phase suggesting a possible peripheral opioidergic involvement in the actions of HLE.
The anti-nociceptive effects of HLE and morphine were reversed by theophylline implicating the involvement of adenosinergic pathway in their actions. Adenosine acts at several P1 receptors (A1, A2A, A2B, and A3) all of which are coupled to G proteins. Activation of A1 receptors produce anti-nociception while activation of A2 and A3 receptors produce pro-nociception. Since theophylline blocks adenosine A1 and A2 receptors, the anti-nociceptive effects may be due to activation of A1 receptors and/or an increment in endogenous adenosine either centrally or peripherally. The involvement of adenosine in morphine anti-nociception is well known [49-51] and has been confirmed in this study.
The reversal of the anti-nociceptive effects of HLE by the non-selective muscarinic receptor antagonist, atropine implicates the muscarinic cholinergic system in the actions of the extract. It is well reported that the activation of muscarinic receptors (M1-M4) induces anti-nociception in various pain paradigms including thermal, inflammatory and neuropathic pain.[52-54] Therefore, the anti-nociceptive effects of HLE may be due to activation of one or more of the muscarinic receptors and/or an increment in endogenous acetylcholine either centrally or peripherally.
Since glibenclamide (ATP-sensitive K+ channel blocker), ondansetron (a 5-HT3 receptor antagonist), yohimbine (a selective α2-adrenoceptor antagonist), nifedipine (L-type VGCC blocker) and L-NAME (a selective inhibitor of NO biosynthesis) did not significantly alter the anti-nociceptive effect of the HLE, it is speculated that the extract's anti-nociceptive mechanism may not significantly involve ATP-sensitive K+ channels, 5-HT3 serotonergic receptors, α2-adrenoceptors, L-type voltage-gated calcium channels or the nitric oxide pathway. However, further pharmacological and chemical studies are necessary to characterize the precise mechanism(s) responsible for the anti-nociceptive action of HLE.
Opioids, such as morphine, are clinically used primarily as analgesics. But the development of tolerance that necessitates dose escalation regardless of disease progression, greatly limit their effectiveness and usage.[55-56] Since the present study revealed the possible involvement of the opioidergic pathway in the anti-nociceptive activity of HLE, a study was carried out to determine if repeated administration of HLE could lead to the development of analgesic tolerance. The study further determined if morphine tolerance could cross-generalize to HLE and whether concurrent administration of morphine and HLE could abolish morphine tolerance. The results suggest that, unlike morphine, HLE does not induce tolerance to its anti-nociceptive effect after chronic administration in the formalin test. In fact, HLE was even more effective after chronic administration. The lack of tolerance development after HLE treatment cannot be attributed to the use of a low dose, because HLE was chronically administered at the dose maximally active in the late phase of formalin-induced pain. In view of the opioidergic activity of HLE without tolerance development, it is suggested that HLE might have components acting via pathways that interfere with the mechanism of opioid tolerance development. This view is supported by the fact that HLE attenuated the development of morphine tolerance in this current study. Another interesting finding is that morphine tolerance does not cross-generalize to HLE, implying the extract can be used to treat pain in opioid tolerant individuals.
In conclusion, the current study demonstrates that the ethanolic extract of the aerial parts of Hilleria latifolia has anti-nociceptive activity in chemical and thermal models of nociception without inducing tolerance. The anti-nociceptive effect involves an interaction with adenosinergic, muscarinic cholinergic and opioid pathways.