Abstract
In this work we describe the analgesic, anti-inflammatory and toxic activities as well as the phytochemical profile of the ethanol extract from Tabernaemontana catharinensis A. DC. (Apocynaceae) stem bark. Analgesic evaluation was carried out against chemical and thermal stimuli. Anti-inflammatory activity was investigated on carrageenan-induced edema in rats and toxicological studies (LD50) were conducted in mice. Phytochemical analyses were performed by standardized methodology. In an analgesic assay, acetic acid-induced writhings were significantly inhibited by extract doses of 37.5 mg/kg (40.97%), 75 mg/kg (77.70%) and 150 mg/kg (88.98%). A central analgesia was also observed using T. catharinensis extract at all doses tested, particularly noticed at 60 and 90 min following administration. The extract significantly reduced edema development by 30.35% (37.5 mg/kg), 34.46% (75 mg/kg), and 56.42% (150 mg/kg) when assessed 180 min following carrageenan intraplantar injection, demonstrating an effective anti-inflammatory action. The LD50 value was 2200 mg/kg. Phytochemical analyses of ethanol extract from Tabernaemontana catharinensis stem bark showed the presence of alkaloids and terpenoids, which may be responsible for the observed pharmacological activities described in this work.
Introduction
Tabernaemontana catharinensis A. DC. (syn, Tabernaemontana affinis Müll. Arg., Tabernaemontana australis Müll. Arg., and Tabernaemontana hilariana Müll. Arg.) (Apocynaceae) is a plant species known in Brazil as “leiteiro de vaca” (Cow’s udder), due to an intensive latex production by its aerial parts (CitationLeeuwenberg, 1994). T. catharinenis stem bark and roots have been used against snakebite envenomation in Brazil, Uruguay, Paraguay, and Argentina. Indeed, previous results have shown that T. catharinenis root bark extract neutralizes the lethality and myotoxicity induced by Crotalus durissus terrificus (South American rattlesnake) venom in rats, which were subsequently attributed to the action of the alkaloid 12-methoxy-4-methylvoachalotine (CitationBatina et al., 2000; CitationDe Almeida et al., 2004). Moreover, a partial neutralization of the myotoxic effects of Bothrops jararacussu snake venom and its myotoxins BthTX-I and BthTX-II by the aqueous extract of Tabernaemontana catharinensis was shown in rat isolated muscle preparations (CitationVeronese et al., 2005).
Genus Tabernaemontana encompasses several species that are traditionally used for the treatment of fever, acute or chronic pain, and dysentery (CitationLeeuwenberg, 1994). Phytochemical analysis of these plants has shown the presence of alkaloids and terpenoids possessing anti-inflammatory and analgesic properties, such as in T. pandacaqui and T. pachysiphon (CitationAchenbach et al., 1994; CitationNielsen et al., 1994; CitationIngkaninan et al., 1999; CitationTaesotikul et al., 2003). Moreover, several other alkaloids and terpenoids from the Apocynaceae family have been described as potential analgesic and anti-inflammatory compounds, as demonstrated by triterpenoids, lupeol acetate, α-amyrin and lupeol cinnamates, from Himatanthus sucuuba (CitationDe Miranda et al., 2000).
The aim of this work was to evaluate the analgesic and anti-inflammatory effects of T. catharinenis ethanol extract and to correlate these data with those obtained from phytochemical analysis.
Materials and methods
Materials
The following drugs were used. Acetic acid (Chemco, Campinas, Brazil), aspirin (Bayer, São Paulo, Brazil), carrageenan (Sigma, St. Louis, MO), indomethacin (Indocid®, Merck Sharp-Dolme, Cramlington, UK), morphine sulphate (Dimorf ®, São Paulo). Solvents were from Synth (São Paulo) and Polyoxyethylene Sorbitan Monolaurate (Tween® 20) from USB (Cleveland, Ohio).
Animals
Swiss albino male 18–25 g mice and Wistar albino 150–250 g male rats, bred at the University of São Paulo (Ribeirão Preto) or at ANILAB (Paulínia, Brazil), were used for pharmacological assays. Animals were housed in standard cages under a 12 h light/12 h dark cycle at a controlled temperature (21° ± 3°C) with free access to rat platelet food or water and acclimatized to the laboratory environment for at least 2 h prior to experiments. Animals were used only once. Pharmacological assays were performed in a sound-attenuated room at controlled temperature (20–22°C).
Efforts were made to avoid unnecessary stress and pain to the experimental animals. Experiments were performed according to the rules of the Brazilian Society of Neurosciences, which follow the guidelines for animal care prepared by the Committee on Care and Use of Laboratory Animal Resources, National Research Council, USA.
Plant material and extraction
T. catharinensis stem bark was collected in Assis (São Paulo, Brazil), and authenticated in the Botanical Institute of UNICAMP (Campinas). Voucher specimens (02940, 02944, and 03224) were deposited at the Herbarium of FFCL of Ribeirão Preto, University of São Paulo. Dried and pulverized stem bark sample was macerated in 95% ethanol for 24 h at 25°C. The filtrate was concentrated in a rotory evaporator, lyophilized, and resuspended in distilled water prior use.
Phytochemical analysis
Phytochemical properties of T. catharinensis aqueous extract were assessed by thin-layer chromatography on silica gel PF254 plates (5 × 10 cm) (Macherey-Nagel, Germany). Solvent systems consisting of hexane/ethyl acetate (7:3, w/v) or chloroform/methanol (8:2, w/v) were used as developer. Detection was performed using UV light exposition (254 and 366 nm) or spraying with vanillin-sulfuric acid solution followed by heating.
Antinociceptive activity on acetic acid-induced writhing
Male Swiss mice were distributed in control and experimental groups of 7 animals each. The T. catharinensis extract (37.5, 75 and 150 mg/kg, intraperitoneally), aspirin (100 mg/kg, per ora) and vehicle (saline 100 μL, intraperitoneally) were administered 60 min prior to the intraperitoneal (i.p.) injection of 100 μL of 1% acetic acid. After a latency period of 5 min, the numbers of writhing and stretching responses was followed during the succeeding 30 min, and expressed as a percentage of analgesic activity by the equation:
where N is the averaged number of abdominal constrictions of animals treated with saline (controls) and N1 the number of abdominal constrictions of animals treated with T. catharinensis extract or aspirin.
Nociceptive response induced by a thermal stimulus
Rats were divided into control and experimental groups of 9 animals each, and individually placed in an acrylic box under a mobile infrared heating lamp (55° ± 1°C) (Ugo Basile, Varese, Italy). The time interval elapsed until the animal reacted with shaking and/or lifting its right hind-paw was recorded during 1 h prior to each treatment (basal time reaction). The experimental protocol, performed as described by CitationHargreaves et al. (1988), with modifications, consisted of the administration of 37.5, 75, and 150 mg/kg (i.p.) of the T. catharinensis extract, morphine (5 mg/kg, i.p.), or vehicle (saline 100 μL, i.p.), respectively, given 60 min before starting to record the latency for the withdrawal response at 30, 60, 120, and 180 min. The same conditions used to assess the basal time reaction were followed. Cut-off time was always 30 s to avoid tissue damage to the animal.
Anti-inflammatory activity on carrageenan-induced rat paw edema
The T. catharinensis anti-inflammatory effect was analyzed by measuring the inhibition of carrageenan- induced hind paw edema, according to CitationWinter et al. (1962). Briefly, mice were divided into control and experimental groups of 7 animals each. The right hind paw volume of each animal was measured prior to treatments with a Plethysmometer 7140 (Ugo Basile). The extract (37.5, 75, and 150 mg/kg, i.p.), indomethacin (10 mg/kg, i.p.), and vehicle (saline 100 μL, i.p.) were administered to each animal 60 min prior to the injection of the edema-inducing agent (50 μL of an aqueous solution of carrageenan; 1%, w/v) into the right hind paw plantar surface. Paw volumes were recorded after 30, 60, 120, and 180 min following carrageenan injection. Results were expressed as percentages of edema inhibition using the following equation:
where Vt is the average volume for each group and V0 average volume for each group prior to indomethacin or extract treatment (CitationLanhers et al., 1991).
Toxicological assays
Male Swiss mice (n = 6) were treated with T. catharinesis extract in 2.5%, v/v Tween 20, following a geometric dose sequence (150, 300, 600, 1200, and 2400 mg/kg, p.o.). Controls received 0.1 mL of saline containing 2.5% Tween 20 (v/v) (p.o.). Signs of toxicity were recorded during the succeeding 24 h. The LD50 was calculated from plotted data.
Statistical analysis
Statistical significance was determined by ANOVA following Student’s t-test. A value of p < 0.05 was considered statistically significant.
Results and discussion
summarizes the results of the phytochemical analysis of the ethanol extract of T. catharinensis stem bark, showing the presence of alkaloids and terpenoids. Previous results have shown the presence of the alkaloid 12-methoxy-4-methylvoachalotine, as well as five other indole alkaloids, in the root bark of this plant (CitationPereira et al., 1999; CitationBatina et al., 2000). Alkaloids and terpenoids are commonly present in plants showing analgesic and anti-inflammatory properties (CitationIngkaninan et al., 1999; CitationDahanukar et al., 2000; CitationTaesotikul et al., 2003). Flavonols and tannins were not found in the analyzed extract. These results should be of relevance as a guideline for future T. catharinensis extract purification.
Table 1. Phytochemical analysis of the extract from T. catharinenis stem bark.
As shown in , the number of acid acetic-induced writhes was significantly reduced by T. catharinensis extract at doses of 37.5 mg/kg (40.97%), 75 mg/kg (77.70%), and 150 mg/kg (88.98%), in a dose-dependent manner (r = −0.88, p < 0.01). An inhibitory effect of 54.93% was also promoted by a 100 mg/kg dose of aspirin (positive control). However, this effect was lower than that obtained by extract doses of 75 and 150 mg/kg. Abdominal injection of acetic acid in mice has been shown to induce release of arachidonic acid via the cyclooxygenase pathway, suggesting an increase of prostaglandin production in the nociceptive mechanism in visceral pain (CitationFranzotti et al., 2002). As shown, all doses of the T. catharinensis extract assayed exhibited dose-dependent peripheral analgesic activity, which is possibly related to the inhibition of prostaglandin production.
Table 2. Antinociceptive effect of T. catharinensis on writhing induced by acetic acid in mice.
The use of thermal stimuli is highly suitable for testing the efficiency of opioid analgesic compounds. These assays have also been proven for animals showing an impairment of motor activity. However, these assays are not sensitive for the assessment of non-steroid analgesic drugs (CitationPlummer et al., 1996). In we can observe the central analgesic effect induced by T. catharinensis extract. Morphine showed its highest analgesic effect after 60 min (). The latency time for the animal writhing/stretching response was significantly increased by extract doses of 37.5, 75, and 150 mg/kg, especially when observed 60 and 90 min following treatment. Indeed, at 150 mg/kg, the highest extract dose administrated, the period of latency was almost twice as high compared to the control group when observed 60 min following treatment. After 90–120 min, a progressive decrease of analgesic activity of both extract and morphine was observed. This last observation has also been demonstrated in other reports using this assay (CitationDar et al., 2005; CitationElhabazi et al., 2006). Thus, T. catharinensis extract seems to present a mechanism of central analgesia, besides its involvement in inhibiting the production of prostaglandins and other autacoids linked to peripheral analgesic activity.
Table 3. Antinociceptive effect of T. catharinensis extract against a thermal stimulus.
The carrageenan-induced rat paw edema model is generally used for the screening of anti-inflammatory drugs. It is characterized by a primary event involving the release of high concentrations of histamine and serotonin (CitationVinegar et al., 1969). After the first 60 min, however, the edema is marked by the presence of prostaglandins (PGI2) and bradykinin, when its volume is maximal (CitationDi Rosa et al., 1971; CitationGilman, 1985). The anti-inflammatory effect induced by T. catharinensis extract on carrageenan-induced rat paw edema is shown in . At a dose of 150 mg/kg, the extract reduced by 42.38% edema development when the assessment was performed 120 min after carrageenan injection. After 180 min, extract doses of 37.5, 75, and 150 mg/kg promoted a significant edema inhibition of 30.35, 34.46, and 56.42%, respectively. At lower doses or time intervals, no anti-inflammatory effect was observed in any group. Indomethacin induced an effective anti-inflammatory action, inhibiting the paw edema by 47.40 and 65.89% after 120 and 180 min, respectively. T. catharinensis extract exhibited its most marked anti-inflammatory effect during the later phase of edema (120–180 min). A similar effect is described to indomethacin (CitationDi Rosa et al., 1971). Taking the peripheral analgesic results and anti-inflammatory response together, we strongly suggest that T. catharinensis extract acts as a bradykinin/protaglandin synthesis inhibitor.
Table 4. Anti-inflammatory effect of T. catharinensis extract on rat paw edema-induced by the intraplantar injection of carrageenan.
No sign of toxicity was observed in the control or experimental animals, just a slight sedation in the last group up to 60 min after extract administration. Neither diarrhea, nor piloerection, motor impairment, hyper-excitability, alterations to respiratory frequency or ataxia were noted. The value of LD50 (24 h) was 2200 mg/kg, i.e., about 14 times higher than the most effective dose of extract (150 mg/kg), demonstrating the relative safety of the extract.
Although it is difficult to establish a definitive relation, it is logical to assume that the analgesic and anti-inflammatory activities of T. catharinensis extract associated with its inhibitory activities against snake venom lethality and myotoxicity might be one reason for its popular use against snake bite envenomations in some South American countries.
In conclusion, this work shows that T. catharinensis extracts had pronounced central and peripheral analgesic actions, an effective anti-inflammatory effect, and a high LD50 value. The presence of alkaloids and terpenoids may be responsible for the pharmacological activities observed. It should be pointed out that an intensive effort is therefore underway to purify the active compound(s).
Acknowledgements
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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