Antinociceptive Profile of (-)-Spectaline: A Piperidine Alkaloid from Cassia leptophylla

• Abstract
• Introduction
• Materials and Methods
• Plant material
• Extraction and isolation
• Animals
• Acute toxicity and LD₅₀
• Agents
• Acetic acid-induced abdominal writhing in mice
• Formalin-induced pain in mice
• Capsaicin-induced pain
• Hot plate test
• Tail flick test
• Data analysis
• Results and Discussion
• Acknowledgements
• References

Abstract

The antinociceptive activity of (-)-spectaline (1), a piperidine alkaloid isolated from Cassia leptophylla Vog. (Leguminosae), was investigated. We have also studied the acute oral toxicity of 1 in mice and it did not show any signals of toxicity in doses lower than 400 μmol/kg. The antinociceptive effect of 1 was evaluated on chemical (acetic acid, formalin and capsaicin) and thermal (hot plate and tail flick) pain models in mice, using classical standard drugs. Dipyrone ID₅₀ = 14.68 μmol/kg (4.8 mg/kg), indomethacin ID₅₀ = 0.78 μmol/kg (0.28 mg/kg) and (-)-spectaline ID₅₀ = 48.49 μmol/kg (15.75 mg/kg), all produced a significant inhibition of acetic acid-induced abdominal writhing in mice. (-)-Spectaline was inactive in the hyperalgesic model of formalin and did not show any central analgesic activity (hot plate and tail flick models). In the capsaicin-induced neurogenic pain model, (-)-spectaline presented an important inhibitory effect with an ID₅₀ = 20.81 μg/paw and dipyrone ID₅₀ = 19.89 μg/paw. The ensemble of results permitted us to identify 1 as an antinociceptive compound. The mechanism underlying this antinociceptive effect of 1 remains unknown, but the results suggest that such an effect could be related to pathways associated to vanilloid receptor systems.

Introduction

Phytochemical studies on the leaves, fruits and flowers extracts of Cassia leptophylla Vog. (Leguminosae) have furnished numerous 2,6-alkyl-3-hydroxypiperidine alkaloids [1]. Among these, (-)-spectaline (1) was the major constituent of the alkaloidal mixture and was isolated in 4 % yield from an ethanolic flowers extract [1]. Heterocyclic amines with long aliphatic substituents have been isolated from plants such as Cassia and Prosopis and have received increasing attention due to their variety of pharmacological properties. Ethnopharmacological studies have indicated that this kind of compound may exhibit analgesic, anesthetic, antitumor activities and have effects on the cardiovascular system [2].

Several recent research works have focused on the validation and elucidation of the pharmacological activity of natural products from plants. To date, the biological activity of the major alkaloidal constituent of the ethanolic flower extract of Cassia leptophylla has not been investigated. The purpose of the present study was to evaluate the peripheral antinociceptive activity of the natural product (-)-spectaline, using classical pain models in mice.[]

Materials and Methods

Plant material

The flowers of C. leptophylla Vog. (Leguminosae) were collected in São Paulo State, Brazil (1999) by Dr. Maria Cláudia Marx Young (Botanical Inst. of São Paulo). Botanical material was classified by Dr. Inês Cordeiro (Botanical Inst. of São Paulo). A voucher specimen of C. leptophylla (number Silva 193- SP) was deposited in the Botanic Garden herbarium of São Paulo State, Brazil.

Extraction and isolation

The ethanolic extracts of fruits and flowers (500 g) were concentrated, redissolved in MeOH-H₂O (8 : 2) and partitioned with hexanes, CH₂Cl₂, AcOEt and n-BuOH, successively. The CH₂Cl₂ soluble fraction was concentrated yielding a CH₂Cl₂ portion (39 g) which was extracted with aqueous HCl (40 %; 3 × 50 mL) and the combined aqueous fractions were alkalinized to pH 11 with concentrated NH₄OH. The resulting aqueous basic solution was exhaustively extracted with dichloromethane, dried over anhydrous MgSO₄ and concentrated, furnishing 9 g of a crude alkaloidal fraction. The crude alkaloidal portion was chromatographed on a neutral Al₂O₃ column with a gradient mixture of CHCl₃/hexane (9 : 1 to 9.5 : 0.5) and CHCl₃/MeOH/hexane (8 : 0.5 : 1 to 9 : 1 : 0) as eluent, affording the piperidine alkaloids (-)-spectaline (1) (4.82 g), purity 95 %, [α]_D ²⁰: -12° (c 0,14, CH₂Cl₂) and 3-O-acetylspectaline (151 mg) as well as another three unpurified complex alkaloidal mixtures. The NMR data were in according with the literature values [1].

Animals

All the experiments were performed on male Swiss mice from LASSBio of Universidade Federal do Rio de Janeiro (UFRJ), weighing 25 ± 5 g. Animals were maintained only with access to water for 8 hour before the experiments. Experiments reported in this study were performed in according to current guidelines of laboratory animal care and ethical guidelines for investigation (CAUAP-IBCCF, Brazil) and the ethical guidelines for investigations of experimental pain in conscious animals [3].

Acute toxicity and LD₅₀

Acute toxicity studies of 1 were performed in male Swiss mice. In this assay, increasing doses of the test substance were orally administered to groups of five animals for each dose (10 - 1000 μmol/kg, p. o.). The animals were observed for 14 days. At the end of this period the number of survivors was recorded. The acute toxicology effect was estimated by a method described previously [4] and was expressed as LD₅₀ according to Litchfield and Wilcoxon [5].

Agents

Acetic acid and indomethacin (Merck), arabic gum and dipyrone (Sigma Chemical), capsaicin (Calbiochem) and morphine sulfate (Dimorf-Cristalia-BR). Capsaicin was dissolved in absolute DMSO and the final concentration of DMSO did not exceed 10 %, which had no effect per se. A solution of formalin 2.5 % was prepared with formaldehyde (Merck) in saline (NaCl 0.9 %)

Acetic acid-induced abdominal writhing in mice

The response to intraperitoneal 0.6 % acetic acid solution injection, a contraction of the abdominal muscle and stretching of the hind limbs, was induced as previously reported [6]. Animals were pre-treated with (-)-spectaline (10 - 300 μmol/kg, p. o.) and negative control animals received a similar volume of arabic gum used as vehicle (10 mL/kg, p. o.). Positive control mice groups received dipyrone (1 - 300 μmol/kg, p. o.) and indomethacin (0.3 - 30 μmol/kg, p. o.). The substances were administered 60 min before 0.6 % acetic acid injection. The animals were placed in separate transparent boxes and after 10 min the number of abdominal writhings was counted over a period of 20 min. The antinociceptive activity was expressed as the reduction in the number of abdominal writhings.

Formalin-induced pain in mice

The formalin test was carried out as described by Tjolsen et al. [7]. Animals were injected subplantarly with 20 μL of 2.5 % formalin in the hind paw. (-)-Spectaline (300 μmol/kg, p. o.) or vehicle (10 mL/kg) was administered p. o. 60 min before formalin injection. The time that mice spent licking or biting the injected paw or leg was recorded. On the basis of the response pattern described by Tjolsen et al. [7] two distinct periods of intensive licking activity were identified and scored separately unless otherwise stated. The first period (earlier or neurogenic phase) was recorded 0 - 5 min after the formalin injection and the second period (later or inflammatory phase) was recorded 15 - 30 min after the injection.

Capsaicin-induced pain

Mice were injected with 20 μL of capsaicin (1.6 μg/paw, DMSO 10 %) into the plantar region of the right hind paw. The pain response was scored during a period of 20 min as the number of episodes of paw linking [8]. The test [dipyrone or (-)-spectaline] substance was administered by the subplantar route 30 min before capsaicin injection.

Hot plate test

The hot plate test was used to measure response latency according to the method described by Eddy and Leimback [9], with minor modifications. In these experiments, the hot plate apparatus (Ugo Basile, Model-DS 37) was maintained at 55.5 ± 1 °C. Animals were placed on the heated surface and the time between placement and licking of the paws or jumping was recorded as latency. Latency was recorded for vehicle control groups (10 mL/kg) or pre-treated groups with 1 (100 and 300 μmol/kg). The test compounds were administered after animal selection on a time of 30 min. The selection was made on the basis of the reactivity to the test. Pre-treatment times 0 and 30 min were used for assay adaptation and selection of the animals, respectively. Animals showing a reaction time within the range of 4 - 7 sec were selected. Compounds were administered at time 30 min and treatment latencies were recorded at times 60, 90, 120 and 150 min

Tail flick test

The antinociceptive response was determined as describe previously by the modified tail-flick test [10]. Briefly, the tail flick latencies to thermal stimulation (radiant heat) were determined at 0, 30, 60, 90, 120 min, after administration of 1 (300 μmol/kg) or vehicle (10 mL/kg). The basal latencies were found to be 5 - 7 sec. A cut-off time of 20 sec was followed to prevent any injury to the tail.

Data analysis

Results are presented as mean ± standard error of the mean (SEM). Significance was tested by means of Mann-Whitney test at a *P value less than 0.05. When appropriate, the mean ID₅₀ values accompanied by their respective 95 % confidence limits (i. e., the dose or concentration which reduce response by 50 % relative to the control values) were determined by linear regression from individual experiments with linear regression GraphPad software.

Results and Discussion

The antinociceptive profile effect of (-)-spectaline, a new piperidine alkaloid, was investigated. This compound was isolated from the flowers of Cassia leptophylla Vog. (Leguminosae) by Bolzani at al. [1]. As part of a pharmacological study of the possible antinociceptive activity of this compound, we first determined the acute toxicity of 1. A single administration of the test substance by oral route, lowered to the dose of 400 μmol/kg did not produce any sign of toxicity in mice. No significant changes in body or organ weight were observed. Thus, this result indicates that 1 has no significant toxicological effects. For this reason, a maximal dose of 300 μmol/kg [97.5 mg/kg] was used to determine the general profile of the antinociceptive effect of 1.

The first indication of the antinociceptive property of 1 was observed in the acetic acid-induced abdominal writhing model in mice. Fig. [1] shows the dose-response of 1 ID₅₀ = 48.49 (15 - 49) μmol/kg [15.75 mg/kg] and the positive controls (reference standards) dipyrone ID₅₀ = 14.68 (7 - 28) μmol/kg and indomethacin ID₅₀ = 0.78 (0.03 - 16) μmol/kg that were able to inhibit significantly the abdominal writhing in mice, when compared with the control animals (vehicle) that present constrictor response 100 % (data not show).

The abdominal writhing elicited by acetic acid has been reported to be a poor specific antinociceptive model and is used for evaluation of peripheral analgesic effects. Acetic acid acts indirectly by releasing endogenous mediators that stimulate the nociceptive effect but also activates neurons that are sensitive to other substances such as narcotics and centrally acting agents [11]. Based on these observations and on the antinociceptive activity found for 1 in this model, we decided to use other pharmacological models to investigate the antinociceptive profile of this new natural product.

These assays were performed in order to determine whether the antinociceptive effect of 1 was caused by central or peripheral mechanisms. In the hot plate and tail flick tests no significant difference between pre-treatment and treatment latency values were observed (Figs. [2] and [3]). The results show that 1 did not induce a pain latency increase on hot plate (Fig. [2]) and tail flick (Fig. [3]). By the way, morphine 14.9 μmol/kg (10 mg/kg, i. p.), an analgesic narcotic drug, was used as positive control. As expected, morphine produced analgesia and induced an increase in time latency of pain.

Using a classical pain model, 1 and indomethacin were evaluated in the formalin-induced pain model. Fig. [4] shows the effects of systemic injections of the 1 and indomethacin in both phases of the formalin-induced pain model. Indomethacin had no effect on the first phase, but it produced a reduction (40.3 %) of the second phase at 100 μmol/kg. Interestingly, 1, given by the oral route at a high dose (300 μmol/kg) [97.5 mg/kg], had no effect in both phases.

Earlier reports showed that indomethacin, a cyclooxygenase inhibitor, attenuated the pain response in the second phase, but not in the first phase, of the formalin test in the mouse [12]. Indeed, these studies have been cited as evidence that the second phase of the formalin response is a valid model of inflammatory pain [7] and the mediators such as prostaglandin E₂ are not necessary in the first phase [13].

It has been documented that inflammatory mediators, such as kinins [14], [15], prostaglandins [12], serotonin [16] but not tachykinins [17] account for the edema formation caused by subplantar formalin injection. Thus, (-)-spectaline could not interfere with the release and/or action of such inflammatory mediators.

Injection of inflammatory agents into the paw can produce a formalin-like behavioral pain response. The results of Fig. [5] show that 1 and dipyrone (1 - 100 μmol/kg) given by the subplantar route, dose-dependently inhibited capsaicin-induced licking. The calculated mean ID₅₀ values were: 1 was 20.81 (18 - 24) μg/paw and dipyrone 19.89 (13 - 54) μg/paw. The final concentration of DMSO (10 %) per se did not have any effect and does not interfere with the pain threshold.

Capsaicin (8-methyl-N-vanillylnon-6-enamide) the active principle in Chile pepper is thought to interact to produce pain by selectively activating VR1-type vanilloid receptors on peripheral sensory fibers [18]. It has also been shown that a capsaicin-operated cation-specific ion channel exists, which is closely associated with the capsaicin system in the membrane of sensory neurones and induces pain by depleting tachykinins, like substance P and neurokinin, from stored pools [19]. The fact that 1 affects capsaicin-induced pain suggests that this compound may directly interfere with primary afferent-mediated transmission of pain signals to the central nervous system [20]. For the first time, we identified 1 as an antinociceptive compound, an abundant natural product that could represent an important molecular prototype for new analgesic agents. A discrepancy finding is the absence of an antinociceptive effect of 1 when it was tested in formalin-induced hyperalgesia in the mouse. This suggests that, depending on the agent or the model of nociception, 1 may present with different effects. In summary, we have reported that the 1 elicited dose-related antinociception when assessed against abdominal constriction acetic acid-induced pain and capsaicin-induced neurogenic pain in the mouse. The precise mechanism underlying the antinociceptive action of 1 has yet to be determined. Finally, these results suggest a direct interaction with the binding of the vanilloid system or excitatory amino acid on its receptors might also account, at least in part, for its antinociceptive action. We are engaged, at the moment, in the determination of its mechanism of action.

Acknowledgements

The authors would like to thank Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro - FAPERJ (MSAM), Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP (VSB, CVJr), Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (ALPM, EJB and VSB) for financial support and fellowships.

References

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Dr. Eliezer J. Barreiro

LASSBio

UFRJ

C.P. 68006.

CEP 21944-910- Rio de Janeiro

RJ

Brazil

Phone: +55-21-22609192 ext. 220/223/238.

Fax: +55-21-22602299

Email: eliezer@pharma.ufrj.br

References

• 1 Bolzani V S, Gunatilaka A AL, Kingston D GI. Bioactive and other piperidine alkaloids from Cassia leptophylla .  Tetrahedron. 1995;  51 5929-34
  CrossrefPubMedSearch in Google Scholar
• 2 Banba Y, Abe C, Nemoto H, Kato A, Adachi I, Takahata H. Asymmetric synthesis of fagomine and Its analogues.  Tetrahedron Asymmetry. 2001;  12 817-9
  CrossrefPubMedSearch in Google Scholar
• 3 Zimmerman M. Ethical guidelines for investigation of experimental pain in conscious animals.  Pain. 1983;  16 109-10
  CrossrefPubMedSearch in Google Scholar
• 4 Souza Brito A RM. Manual de ensaios toxicológicos in vivo . Unicamp Campinas, Brasil; 1995
  Search in Google Scholar
• 5 Litchfield J T, Wilcoxon F A. A simplified method of evaluating dose effect experiments.  J Pharmacol Exp Ther. 1949;  95 99-113
  PubMedSearch in Google Scholar
• 6 Koster R, Anderson M, De Beer E J. Acetic acid for analgesic screening.  Fed Proc. 1959;  18 418-20
  PubMedSearch in Google Scholar
• 7 Tjolsen A, Berge O G, Hunskaar S, Rosland J N, Hole K. The formalin test: an evaluation of the method.  Pain. 1992;  51 5-17
  CrossrefPubMedSearch in Google Scholar
• 8 Sakurada T, Katsumata K, Tan-No K, Sakurada S, Kisara K. The capsaicin test in mice for evaluating tachykinin antagonists in the spinal cord.  Neurophamacology. 1992;  31 1279-85
  CrossrefPubMedSearch in Google Scholar
• 9 Eddy N B, Leimback D. Synthetic analgesics. II. Dithienylbutenyl and diethylenylbutylamines.  J Pharmacol Exp Ther. 1953;  107 385-93
  PubMedSearch in Google Scholar
• 10 D’Amour F E, Smith D L. Method for determining loss of pain sensation.  J Pharmacol Exp Ther. 1941;  72 74-9
  PubMedSearch in Google Scholar
• 11 Collier H OJ, Dinneen L C, Johnson C A, Schhneider C. The abdominal constriction response and its supression by analgesic drugs in the mouse.  Br J Pharmacol Chemoth. 1968;  32 295-310
  PubMedSearch in Google Scholar
• 12 Hunskaar S, Berger O -G, Hole K. Dissociation between antinociceptive and anti-inflammatory effects of acetylsalicylic acid and indomethacin in the formalin test.  Pain. 1986;  25 125-32
  CrossrefPubMedSearch in Google Scholar
• 13 Hong Y, Abbott F V. Behavioural effects of intra-plantar injection of inflammatory mediators in the rat.  Neuroscience. 1994 ;  63 827-36
  CrossrefPubMedSearch in Google Scholar
• 14 Chapman V, Dickenson A H. The spinal and peripheral roles of bradykinin and prostaglandins in nociceptive processing in the rat.  Eur J Pharmacol. 1992;  219 427-33
  CrossrefPubMedSearch in Google Scholar
• 15 Corrêa C R, Kyle D J, Chakravarty S, Calixto J B. Antinociceptive profile of the pseudopeptide B₂ bradykinin receptor antagonist NPC 18 688 in mice.  Br J Pharmacol. 1996;  117 552-8
  PubMedSearch in Google Scholar
• 16 Sugishita E, Amagaya S, Ogihara Y. Anti-inflammatory testing methods: comparative evaluation of mice and rats.  J Pharmacobiod. 1981;  4 565-75
  PubMedSearch in Google Scholar
• 17 Santos A RS, Calixto J B. Further evidence for the involvement of tachykinin receptor subtypes in formalin and capsaicin models of pain in mice.  Neuropeptides. 1997;  31 381-9
  CrossrefPubMedSearch in Google Scholar
• 18 Piper A S, Yeats J C, Bevan S, Docherty R J. A study of the voltage dependence of capsaicin-activated membrane currents in rat sensory neurones before and after acute desensitization.  J Physiol. 1999;  518 721-33
  CrossrefPubMedSearch in Google Scholar
• 19 Caterina M j, Schumacher M A, Tominaga M, Rosen T A, Levine J D, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway.  Nature. 2000;  389 816-24
  PubMedSearch in Google Scholar
• 20 Davis J B, Gray J, Gunthorpe M J, Hatcher J P, Davey P T, Hughes A S. et al . Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia.  Nature. 2000;  405 183-7
  CrossrefPubMedSearch in Google Scholar

Dr. Eliezer J. Barreiro

LASSBio

UFRJ

C.P. 68006.

CEP 21944-910- Rio de Janeiro

RJ

Brazil

Phone: +55-21-22609192 ext. 220/223/238.

Fax: +55-21-22602299

Email: eliezer@pharma.ufrj.br