Abstract
The antinociceptive and anti-inflammatory activities of Nicotiana rustica Linn. (Solanaceae) total extract (NRTE) have been studied using chemical and thermal models in mice. NRTE was obtained by methanol extraction of dried leaves of N. rustica and was administered intraperitoneally at doses of 2.5, 5, 8.5, and 12 mg/kg body wt (bw). It showed significant protective effects against chemical stimuli in the acetic acid and formalin tests. The extract also showed an inhibitory effect in xylene-induced ear edema compared with the reference drug, diclofenac, and produced a significant increase of the latency time of the reaction in the hot-plate test. Furthermore, the antinociceptive effect of NRTE (at a dose of 12 mg/kg bw) was suppressed by naloxone (a non-specific antagonist of opioid receptors) in hot-plate and formalin tests. This is the first report on the analgesic properties of this plant. The extract might act through an opioid-mediated mechanism. These findings suggest that central and peripheral mechanisms are both involved in the analgesic and the anti-inflammatory effects of N. rustica.
Introduction
It is well known that tobacco has dangerous effects on human health. However, some reports have demonstrated that this plant has some beneficial effects such as its action against ulcerative colitis pathogenesis due to its nicotine content and possibly some flavonoid components found in the ethanol smoke extract (Ko & Cho, 2005). In fact, there are constituents in tobacco smoke other than nicotine that have been reported to possess anti-oxidative properties (CitationChen & Loo, 1995; CitationKamisaki et al., 1997; CitationLapenna et al., 1995). Nicotiana rustica Linn. (Solanaceae) is an annual plant originating in Andean South America. Since antiquity, this plant has been used as a sedative by the old civilizations, in particular Maya tribes, to treat convulsions, fever, nervous affections, and pain of the eyes and the skin (CitationTierra, 1986). In ancient China, snuff was considered as a good remedy for many common illnesses such as colds, headaches, and stomach disorders. Therefore, it was preserved in small medicine bottles like other Chinese medicines (CitationDenis, 2007). In Morocco, it is used to treat ear pain, lesions, and cutaneous burns in rural zones. In traditional medicine, N. rustica is used as a vehicle for therapeutic cigarettes: mixed with Origanum vulgare Linn. (Lamiaceae) for pain of the throat, with Eucalyptus for bronchitis, or with leaves of Datura or Brugmansia for asthma and emphysema. This plant is also chewed as a preventive for toothache and decay (CitationBellakhdar, 1997). In addition, nicotine, the psychoactive component of tobacco products, is widely consumed by humans (CitationShields, 2000; CitationSutherland, 2002). The drug exhibits several pharmacological actions in the central and peripheral nervous systems and releases a number of neurotransmitters (CitationMihailescu et al., 2002; CitationMiller et al., 2003). It induces pharmacological effects by acting on nicotinic acetylcholine receptors (nAChRs) (CitationDani, 2001). It has been used as an alternative therapeutic agent for treating ulcerative colitis in some clinical trials (CitationGuslandi & Tittobello, 1998; CitationSandborn et al., 1997). Other animal and clinical studies have suggested the participation of the endogenous opioid system in different behavioral responses to nicotine, mainly concerning its antinociceptive and addictive properties (CitationBerrendero et al., 2005). To date, no scientific work has made reference to the pharmacological properties of N. rustica. The present work is the first report on the analgesic properties of this plant. The antinociceptive and anti-inflammatory capacities of its methanol extract were investigated.
Materials and methods
Animals
Male Swiss mice weighing 20-30 g were housed under standard environmental conditions with free access to food and water ad libitum. They were used for all pharmacological experiments. Throughout the experiments, animals were processed according to the suggested ethical guidelines for the care of laboratory animals and the ethical guidelines for investigation of experimental pain in conscious animals (CitationZimmermann, 1983).
Plant material
The leaves of N. rustica were collected in the region of Ketama, northern Morocco, in June 2005. The plant material was botanically identified by Professor Ahmed Ouhammou (A.O) from the Department of Biology, Faculty of Sciences, Semlalia, Cadi Ayyad University. A voucher specimen (5524) was deposited at the herbarium of the aforementioned faculty.
Preparation of the plant extract
The dried leaves were crushed in order to obtain a powder. Then methanol was added to 100 g of this powder to obtain the methanol extract using a Soxhlet apparatus. This extract was evaporated to dryness using a rotary evaporator. The yield was 32% of the starting materials. After, it was dissolved and made up to appropriate volume with 0.9% NaCl just before the start of the experiments. The methanol extract was administered intraperitoneally (i.p.) at different doses (2.5, 5, 8.5, and 12 mg/kg bw) for all tests. Doses of 10, 20, 35, and 50% MTD (Maximum Tolerated Dose) were chosen because they were found to be effective in the different tests used.
Writhing test
This test was performed in mice as previously described by CitationKoster et al. (1959). Acetic acid solution (0.6%) (0.1 mL per 10 g bw) was injected i.p. to separate groups of six mice used for controls and tested animals. Thirty minutes prior to the application of the noxious stimulus, the animals received an intraperitoneal injection of NRTE at doses of 2.5, 5, 8.5, and 12 mg/kg bw. Five min after the i.p. injection of the acetic acid, each animal was isolated in an individual transparent glass box (24 cm × 11 cm × 10 cm) to be observed during 30 min. The number of writhes produced in these animals was counted and recorded. The percentage protection against chemical pain was calculated. For scoring purposes, a writhe is indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb. Control mice received normal saline (10 mL/kg bw, i.p.). Diclofenac (10 mg/kg bw, i.p.) was used as a standard reference drug.
Hot-plate assay
The hot-plate test was performed according to the method of CitationJacob et al. (1974). Mice were placed on an electronically controlled metal plate heated to 55° ± 0.2°C. 30 min prior testing, groups of mice (n = 6) were given NRTE (2.5, 5, 8.5, and 12 mg/kg bw, i.p.), morphine as a reference drug (10 mg/kg bw, i.p.), and saline (negative control). Afterwards the animals were habituated to the non-functioning apparatus for 1 min. Mice that presented baseline reaction times of more than 30 s were not considered in order to avoid tissue damage (the cut-off time). The latency of nociceptive responses such as licking a hind paw or eventually jumping out of the apparatus was recorded in seconds at 0, 30, 60, 90, and 120 min after treatment. In order to determine the involvement of the opioid system in the antinociceptive effect, naloxone (2 mg/kg, i.p.) was administered 15 min before treatment with morphine (10 mg/kg bw, i.p.) or NRTE (12 mg/kg bw, i.p.).
Formalin test
The procedure used was similar to that described previously (CitationHunskaar & Hole, 1987). In this test, the animals were divided into groups of six mice. NRTE (2.5, 5, 8.5, and 12 mg/kg bw), morphine (10 mg/kg bw), and saline (negative control) were intraperitoneally administered. After 30 min, mice were injected subcutaneously with 20 μL of 2% (v/v) solution of formalin into the dorsal right hind paw. Each animal was allowed to adapt to the testing box for 5 min prior to formalin injection. In addition, reversibility by naloxone (an opioidergic antagonist at 2 mg/kg bw) was tested by administering i.p. 15 min before formalin injection as described in CitationAhmadiani et al. (1998). After the different treatments, the animals were observed. The total time (in seconds) spent in licking and biting the injected paw was recorded for quantifying the nociceptive behaviour. On the basis of the response pattern described by CitationTjølsen et al. (1992), two distinct periods are noted and scored separately. The early phase (representing the neurogenic pain) was recorded 0-5 min after formalin injection and the late phase (representing the inflammatory pain) was recorded 20-30 min after the injection. The percentage inhibition of licking for each phase was calculated by the formula:
Xylene-induced ear edema
Mice were divided into groups of six. Thirty min after i.p. injection of NRTE and diclofenac, 0.03 mL xylene was applied to the inner and outer surfaces of the right ear. The left ear remained untreated as a reference. Two hours after xylene application mice were sacrificed by cervical dislocation and both ears were removed. Circular sections were taken using a cork borer with a diameter of 7 mm and weighed. The increase in weight caused by the irritant was measured by subtracting the weight of the untreated left ear section from that of the treated right ear section. Control animals received the irritant and an equal volume of normal saline (10 mL/kg, i.p.), diclofenac (10 mg/kg, i.p.) was used as reference drug (CitationHosseinzadeh & Younesi, 2002).
Statistical analysis
The results are presented as mean values ± SEM (Standard error of the mean). Statis tical significance between treated groups and control was performed by the application of one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls as the post hoc test. Values with P < 0.05−0.001 were considered significant.
Results and discussion
The results outlined above showed that Nicotiana rust ica total extract exhibited a significant effect against pain in the current antinociceptive and anti- inflammatory models in mice: writhing, hot plate, formalin, and xylene-induced ear edema tests, since it provided comparable pharmacological effects with the reference drugs. The methanol extract was more active at lower concentrations in all tests. Therefore, this effect might be due to solubility problems of the extract.
In this paper we studied the peripheral analgesic effect of the methanol extract of N. rustica using the acetic acid writhing test. shows that NRTE produced significant effect in this viscero-somatic model of pain. The intraperitoneal administration of the methanol extract of N. rustica leaves significantly reduced in a dose-dependent manner the number of writhes. The most potent protection exhibited by the extract was 73.7% at a dose of 12 mg/kg bw. Importantly, the antinociceptive effect of NRTE was found to be more potent than diclofenac, which is one of the standard non-steroidal anti-inflammatory drugs acting peripherally by inhibiting the cyclooxygenase enzymes (COXs) and consequent inhibition of prostaglandin (PG) synthesis. Diclofenac induced only 47.4% inhibition of writhes. The noxious agent induces indirectly the release of endogenous mediators which stimulate the nociceptive neurons sensitive to non-steroidal anti-inflammatory drugs (NSAIDs) and opioids (CitationCollier et al., 1968). Hence, the antinociceptive effect of NRTE is probably mediated by its peripheral effect by inhibiting the PG synthesis via the blockade of the COX.
Table 1. Effect of N. rustica total extract (NRTE) on acid acetic-induced visceral pain.
Secondly, the central analgesic effect of the extract was examined by the use of the hot-plate test, which is a specific central antinociceptive test (CitationParkhouse & Pleuvry, 1979). Data in show that the methanol extract of N. rustica significantly and dose- dependently delayed the reaction times of mice in this thermal model of pain. Morphine increased the reaction times of the animals. The duration of the analgesic effect was also measured using this thermal model. The most important analgesic effect was obtained at 30 min after intraperitoneal administration of extract and persisted until the following second hour. The increase in latency time induced by NRTE was smaller than that induced by morphine. These effects were inhibited by naloxone, an opioid antagonist. Opioid agents exert their analgesic effects via supraspinal and spinal receptors (CitationReisine & Pasternack, 1996). Therefore, it is apparent that the extract exerts its effect via the central opioid system.
Table 2. Effect of the N. rustica total extract (NRTE) and morphine on pain threshold in the hot-plate test.
Moreover, NRTE exhibited a considerable antinociceptive effect in the formalin test (). This method is also very useful for elucidating the mechanism of pain and analgesia (CitationTjølsen et al., 1992). The injection of formalin into the mouse paw produces two distinct phases of pain-like behaviour. The early phase of intensive pain begins immediately after formalin injection (0-5 min), after which nociception appears to diminish. The late phase of moderate pain starts about 20 min after formalin administration. Methanol extract of N. rustica and morphine significantly inhibited the two phases. Drugs which act mainly centrally, such as narcotic analgesics, inhibit both phases of formalin-induced pain, while drugs, such as aspirin, hydrocortisone, and dexamethasone, which are primarily peripherally acting, only inhibit the late phase (CitationChen et al., 1995; CitationElisabetsky et al., 1995; CitationSantos et al., 1995). Naloxone totally reversed the antinociceptive effect of the extract and morphine in the early phase. However, in the anti-inflammatory phase, naloxone partially inhibited the analgesic activity of NRTE. Thus, the extract appears to act centrally on opioid receptors and peripherally by inhibition of cyclooxygenase.
Table 3. Effect of N. rustica total extract (NRTE) and morphine on formalin-induced nociception.
The delay of the onset of the response is probably due to the viscosity of the extract which affects its absorption and not to a metabolic action on neurotransmitter synthesis. As the antinociceptive activity of the extract was inhibited by naloxone, the active compounds of the extract are likely to act specifically on opioid receptors.
In xylene-induced mouse ear edema, the results show that NRTE exerted a potent anti-inflammatory effect. This test has certain advantages for natural product testing and has a good predictive value for screening anti-inflammatory agents (CitationJacobs et al., 1985). Intraperitoneal administration of the extract at lower doses, 30 min before topical application of xylene, caused a potent and a dose-dependent inhibition of increase in ear edema. The most important inhibition produced by the extract was 53% at a dose of 12 mg/kg bw. Diclofenac showed an inhibition of 59% in ear plug weight. The effect of NRTE was similar to the reference drug (). The extract may have inhibited the release of pro-inflammatory mediators of the acute inflammation such as histamine and prostaglandin via the inhibition of the phospholipase A2 (PLA2). It may also have reduced the release of substance P, which is one of the causes of the neurogenic inflammation. Of course, this effect needs validation by other researches. Consideration of NRTE as a complicated mixture suggests that its active principles would have strong anti-inflammatory effect.
Table 4. Effect of N. rustica total extract (NRTE) on xylene induced ear edema in mice.
Experimental evidence obtained in this study indicated that the peripheral analgesic effect of the extract may be mediated via inhibition of cyclooxygenases and/or lipoxygenases (and other inflammatory mediators). The results obtained tend to suggest that NRTE probably exerts its anti-inflammatory and peripheral antinociceptive effects by inhibiting the release, synthesis, and production of inflammatory mediators, including: prostaglandins, histamine, polypeptide, kinins, etc. While the central analgesic action of the extract, which was completely suppressed by naloxone, an opiate antagonist, may be mediated through inhibition of central pain receptors. The delay of the reaction times of the mice increased significantly and dose-dependently in the thermal model used in this study and showed that the extract might have some opioidergic agonists acting like opiates. At this stage of experience, it is too early to know if there is a direct action on receptors or indirect action via the release and/or the synthesis of the mediators of the nociception.
In conclusion, the data obtained in the animal models used in this study demonstrate that some compounds of Nicotiana rustica methanol extract have central and/or peripheral antinociceptive and anti-inflammatory activities and confirm the traditional use of this plant in the ancient cultures in painful diseases like headaches, pain of skin, and sore throat. These activities may be mediated by the opioid receptors and the inhibition of cyclooxygenase and phospholipase A2 enzymes. Agents of natural origin with very little side effects are required as substitute chemical therapeutics (CitationVerpoorte, 1999). Therefore, the isolation of the precise active constituents may increase the potency of the extract. Since NRTE is a complicated mixture, experiences concerning the specification of the active compounds which act on the opioid transmission are under investigation. The findings obtained allow us to speculate that these chemical compounds might have contributed, at least in part, to the antinociceptive, anti-inflammatory effects in this study.
Acknowledgements
The authors are thankful to A. Regragui, Department of Biology, Laboratory of Animal Physiology, Unit of Ecopharmacology for his technical assistance.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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