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Spinal serotonergic system is partially involved in antinociception induced by Trigonella foenum-graecum (TFG) leaf extract
Journal of Ethnopharmacology 95 (2004) 13–17
Spinal serotonergic system is partially involved in antinociception induced by Trigonella foenum-graecum (TFG) leaf extract Alireza Parvizpur, Abolhassan Ahmadiani∗ , Mohammad Kamalinejad Department of Pharmacology, Neuroscience Research Center, School of Medicine, Shaheed Beheshti University of Medical Sciences, P.O. Box 19835-355, Tehran, IR, Iran Received 19 May 2002; received in revised form 24 May 2004; accepted 27 May 2004 Available online 17 August 2004
Abstract It has been reported that Trigonella foenum-graecum (TFG) extract exerts analgesic, anti-inflammatory and anti-pyretic effects in different experimental models. The major objective of this paper was to investigate the site and mechanism of the analgesia induced by Trigonella foenum-graecum extract. We studied the analgesic effects of different doses of Trigonella foenum-graecum extract after i.p., i.t. and i.c.v. administration in formalin test, using male NMRI rats (200–250 g). Trigonella foenum-graecum extract showed analgesic effects in i.p. (1 g/kg) and i.t. (0.5, 1, and 2 mg/rat) (P < 0.05 in all groups) but not in i.c.v. (1 and 3 mg/rat) administrations. Based on the similarities between the effects of Trigonella foenum-graecum extract with those of nonsteroidal anti-inflammatory drugs (NSAIDs) and the role of 5-HT system in analgesic effects of NSAIDs, we tried to investigate the role of spinal 5-HT system in analgesic effects of Trigonella foenum-graecum extract. After lesioning of spinal 5-HT system by 5,7-dihydroxytryptamine (5,7-DHT), it was shown that the analgesic effect of Trigonella foenum-graecum extract (0.5 and 3 mg/rat) in the second phase of formalin test, was abolished completely and reduced relatively after using a low-dose (0.5 mg/rat) and a high-dose (3 mg/rat), respectively (P < 0.05). So, the antinociception partially remained (P < 0.05) after using the latter dose. Meanwhile, administration of naloxone (2 mg/kg, i.p.) had no effect on the Trigonella foenum-graecum extract (1 g/kg, i.p.) analgesia. In conclusion, this study confirms the central action of Trigonella foenum-graecum extract and that spinal 5-HT system is partially involved in the analgesia induced by it in the second phase of formalin test and also indicates for co-existence of other analgesic mechanism(s). © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Trigonella foenum-graecum; Serotonergic system; Analgesia; Rat
1. Introduction Most protocols for the control of pain rely on using nonsteroidal anti-inflammatory drugs (NSAIDs) and opioid analgesics; however, both of them produce several side effects: NSAIDs produce gastrointestinal (GI) disturbances and ulceration, renal damage and hypersensitivity reactions; while opioids induce nausea, constipation, confusion, respiratory depression, and possibly dependence (Dray and Urban, 1996). Therefore, searching for less harmful compounds is still an outstanding domain of investigation. Traditional medicine ∗
Corresponding author. Tel.: +98 21 2403154; fax: +98 21 2403154. E-mail address: [email protected] (A. Ahmadiani).
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.05.020
and medical plants are known as good sources for natural analgesic agents. It has been shown that the plant Trigonella foenum-graecum (TFG, Fenugreek), from the family of Papilionaceae, produces antinociceptive, antiinflammatory and anti-pyretic effects in different laboratory models (Ahmadiani et al., 2001; Javan et al., 1997), which are similar to those of NSAIDs. The plant is known to contain flavonoids, nicotinic acid and salicylate (Javan et al., 1997). The involvement of serotonin in the modulation of nociceptive impulse in the spinal cord has been widely studied. There are at least three families of 5-HT receptors in the spinal cord (5-HT1 , 5-HT2 , and 5-HT3 ) with different affinities for 5-HT (Bardin et al., 1997). Moreover, some reports suggest that serotonergic system involves in the antinocicep-
14
A. Parvizpur et al. / Journal of Ethnopharmacology 95 (2004) 13–17
tive action of NSAIDs. Parachloro phenylalanine (5-HT synthesis inhibitor) prevents the antinociceptive activity of aspirin in hot plate test (Pini et al., 1995), and also abolishes the antinociceptive effect of aspirin in formalin test (Vitale et al., 1998). Pini et al. (1995, 1997) reported that aspirin displayed antinociceptive activity in both hot plate and formalin tests where serotonergic pathways involved in this antinociceptive activity. In dental pain, aspirin induced analgesia was antagonized by pre-treatment with either cyproheptadine, parachloro phenylalanine or 5,7-dihydroxytryptamine (Shyu et al., 1984). Opioids produce spinal analgesia by presynaptic inhibition of the release of transmitters, postsynaptic inhibition of evoked activity and postsynaptic disinhibition (Dickenson, 1994). Based on the similarity between the effects of Trigonella foenum-graecum extract and NSAIDs, the present study was designed to clarify the mechanism of action of the extract by determining its site of action and to study the probable interference of serotonergic and opioid systems. 2. Materials and methods 2.1. Plant material Trigonella foenum-graecum L. (Fenugreek) from the family of Papilionaceae, is extensively cultivated in most regions of the world. The leaves of the plant were obtained from the local market. The plant was authenticated by M. Kamalinejad (Department of Pharmacognosy, Faculty of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran) and voucher specimen coded 417 has been deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran. 2.2. Preparation of the plant extract Fresh green leaves were separated and cleaned, then dried in shade at room temperature. Dried leaves (100 g) were decocted in water for 30 min. Thereafter the extract was filtered and concentrated with a rotary evaporator apparatus (Heidolph, Heizbad WB, Germany). The final weight of the crude extract was 21 g. The extract was maintained at 4 ◦ C throughout the experiments. When it was administered, the extract was dissolved in appropriate volume of saline according to the route of administration. 2.3. Experimental animals Male NMRI rats (200–250 g, Pasteur Institute, Iran) were used in all experiments. The animals were housed in plexiglass cages in groups of four under a temperature range of 21–25 ◦ C and 12:12 h light/dark cycle. Food and water were available ad libitum.
2.4. Drugs Trigonella foenum-graecum extract was prepared as described before (Javan et al., 1997). 5,7-Dihydroxytriptamine (5,7-DHT), pentobarbital, naloxone and desipramine were purchased from Sigma. 2.5. Surgical procedures Intrathecal cannula was implanted under pentobarbital (50 mg/kg, i.p.) anaesthesia as described previously (Yaksh and Rudy, 1976). Briefly, a polyethylene tube (PE-10) filled with sterile saline, was inserted through a small incision into the atlanto-occipital membrane and extended 8.5 cm caudally to the lumbar enlargement of the spinal cord. A period of at least 2 days allowed the animals to recover from the surgery before the analgesia testing. Any animal that showed signs of motor deficit following cannula implantation, was excluded from the study. For i.c.v. administration of Trigonella foenum-graecum extract a 23-ga stainless steel guide cannula, containing a 27-ga stainless steel wire as a stylet, was stereotaxically implanted into the lateral cerebroventricle (i.c.v.) of rats (AP, 0.7 mm from bregma; L, 1.4 mm from the midline and H, 3.3 mm from the surface of dura) (Paxinos and Watson, 1986). The cannula was fixed to the skull with acrylic dental cement. Animals were allowed to recover from the surgery for at least 2 days before being used for the experiments. For i.c.v. injection of the drugs, the guide cannula was opened and a 27-ga injection needle, which was connected to a 25 l—Hamilton microsyring, was inserted into the cannula and after injecting the extract, it was replaced by a stylet. Three minutes later, formalin test was started. In the end of the experiments, pontamine sky blue was injected into the i.c.v. The brain was removed and after being fixed with 10% formalin, the correct injection into i.c.v. was confirmed by evaluating the spreading of the dye into the i.c.v. All experiments were performed between 9:00 a.m. and 3:00 p.m. The adaptation period before formalin test was 30 min. 2.6. Antinociceptive tests The method of formalin test used was the same as described previously (Dubuisson and Dennis, 1977). Briefly, 50 l of 2.5% formaldehyde was injected subcutaneously into the plantar surface of the animal hind paw. Rats were placed in a plexiglass chamber (30 cm×30 cm×30 cm) and a mirror was placed under its floor with a 45◦ angle in order to allow an unobstructed view of the paw. All animals were brought to the test chamber 30 min prior to the experiments, for adaptation. Behavioral responses were detected and recorded for 1 h after the formalin injection. The first 5 min after the administration of formalin was considered as the first phase, and the period between the 16 and 60 min as the second phase. The Trigonella foenum-graecum extract was dissolved in saline and administered i.p. (1 g/kg/ml), i.t. (0.5, 1, 2, and
A. Parvizpur et al. / Journal of Ethnopharmacology 95 (2004) 13–17
3 mg/rat/10 l) and i.c.v. (1, and 3 mg/rat/10 l). Formalin test was carried out 45 min after i.p. injection of the extract and 3 min after i.t. or i.c.v. administrations. Control groups received saline. 5,7-Dihydroxytryptamine was used to destroy the spinal serotonergic system. Desipramine (10 mg/kg i.p. in 0.9% saline) was also administrated to prevent the uptake of neurotoxin by chatecholaminergic neurons and to destroy them. 5,7-dihydroxytryptamine, 200 g/10 l (in 0.9% saline containing ascorbic acid, 0.2 mg/ml) was administered i.t. 45 min after the administration of desipramine. Five days after i.t. administration of neurotoxin, Trigonella foenum-graecum extract (0.5 or 3 mg/rat, i.t.) was administered and 3 min later formalin test was performed. Two control groups of animals received vehicle of neurotoxin and after 5 days, saline or Trigonella foenum-graecum extract (0.5 or 3 mg/rat, i.t.) was administered before formalin test. In the other experiments, two groups of animals received Trigonella foenum-graecum extract (1 g/kg i.p.), 30 min later saline or naloxone (2 mg/kg i.p.) was administered and 10 min later formalin test was performed.
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Fig. 1. Trigonella foenum-graecum extract (1 g/kg, i.p.) induced analgesia in the first and second phases of formalin test. Bars indicate mean ± S.E.M.( n = 7), TFG: Trigonella foenum-graecum extract, ∗∗∗ P < 0.001 vs. control group.
2.7. Preliminary phytochemical tests Preliminary phytochemical properties of the extract were studied for alkaloids, terpenoids, flavonoids and tannin (Wagner and Bladt, 1996). 2.8. Statistical analysis The results of the experiments were expressed as mean ± S.E.M. The differences were estimated by means of Student’s unpaired t-test or ANOVA followed by Tukey’s test. When the probability was less than 0.05 (P < 0.05), the difference was considered to be significant.
Fig. 2. The effect of Trigonella foenum-graecum extract (0.5, 1, 2, and 3 mg/rat, i.t.) in the first phase of formalin test. Bars indicate mean ± S.E.M. (n = 7), TFG: Trigonella foenum-graecum extract, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs. control group.
3. Results 1. Phytochemical studies showed that the applied extract contained alkaloid and steroids, while flavonoids and tannins were absent. 2. Administration of Trigonella foenum-graecum extract via i.p. (Fig. 1) and i.t. (Figs. 2 and 3) routes produced significant analgesia in both the first and second phases of formalin test, but its i.c.v. administration did not produce analgesia in this test (Table 1). 3. Pre-treatment with 5,7-DHT did not affect the first phase analgesia (Table 2) induced by Trigonella foenum-graecum extract (0.5 mg/rat). However, although the second phase analgesia of Trigonella foenum-graecum extract, 3 mg/rat but not 0.5 mg/rat, was significantly reduced by pre-treatment with 5,7DHT, the extract still had significant analgesic effect (Fig. 4).
Fig. 3. Trigonella foenum-graecum extract (0.5, 1, 2, and 3 mg/rat, i.t) induced analgesia in the second phase of formalin test. Bars indicate mean ± S.E.M. (n = 6–8), TFG: Trigonella foenum-graecum extract, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs. control group.
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A. Parvizpur et al. / Journal of Ethnopharmacology 95 (2004) 13–17
Table 1 The effect of i.c.v. administration of Trigonella foenum-graecum extract on formalin induced pain Treatment
Dose
Formalin test
10 l 1 mg/10 l 3 mg/10 l
Saline Trigonella foenum-graecum extract Trigonella foenum-graecum extract
First phase
Second phase
2.30 ± 0.08 2.04 ± 0.06 2.10 ± 0.06
1.88 ± 0.06 2.02 ± 0.10 1.90 ± 0.05
Pain score: mean ± S.E.M. (n = 6–8), TFG: Trigonella foenum graecum extract.
Table 2 Pre-treatment with 5,7-DHT did not affect the antinociception induced by Trigonella foenum-graecum extract (0.5 mg/rat, i.t.) in the first phase of formalin test Treatment
N
Pain score
Vehicle + saline Vehicle + Trigonella foenum-graecum 5,7-DHT + Trigonella foenum-graecum
7 7 8
2.23 ± 0.03 1.94 ± 0.10∗ 1.95 ± 0.07∗
Vehicle = 0.9% saline containing ascorbic acid 0.2 mg/ml. TFG: Trigonella foenum graecum. ∗ P < 0.05 vs. vehicle + saline group.
Fig. 4. The effect of spinal 5-HTergic system disruption, using 5,7-DHT, on the antinociception induced by 0.5 and 3 mg/rat (i.t.) of Trigonella foenumgraecum extract, in the second phase of formalin test. Bars indicate mean ± S.E.M. (n = 6–8), TFG: Trigonella foenum-graecum extract, V: vehicle of neurotoxin, N: neurotoxin 5,7-DHT, S: saline, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
Table 3 Naloxone (2 mg/kg, i.p.) did not affect the antinociception induced by Trigonella foenum-graecum extract (1 g/kg, i.p.) in formalin test Treatment
Saline + saline Trigonella foenum-graecum extract + naloxone Trigonella foenum-graecum extract + saline
Formalin test First phase
Second phase
2.29 ± 0.08 1.70 ± 0.04∗∗∗
1.88 ± 0.06 1.20 ± 0.13∗∗∗
1.75 ± 0.07∗∗∗
1.04 ± 0.09∗∗∗
Pain score: mean ± S.E.M., n = 6–8. TFG: Trigonella foenum-graecum. ∗∗∗ P < 0.001 vs. saline + saline group.
4. Administration of naloxone (2 mg/kg, i.p.) did not induce any significant change in Trigonella foenum-graecum extract analgesia (1 g/kg, i.p.) (Table 3).
4. Discussion It is well known that formalin is useful to cause neurogenic and inflammatory pain and its subcutaneous injection produces a biphasic pain response in rats. We used tail-flick test to measure the antinociception effect of Trigonella foenum-graecum extract. Tail flick test is very sensitive to centrally acting drugs (Carlisson and Jurna, 1987). Centrally acting analgesic drugs inhibit the pain response in both phases equally, while the peripherally acting drugs inhibit only the second phase. I.p. administration of Trigonella foenum-graecum extract exerts antinociceptive effects in tail flick test (Javan et al., 1997) and in both phases of formalin test (Fig. 1) that propose its activity, at least partially, results from its central action. Furthermore, i.t. administration of Trigonella foenum-graecum extract caused analgesia in formalin test which confirms the central action of the extract (Figs. 2 and 3). However, i.c.v. administration of Trigonella foenum-graecum extract did not exert any antinociceptive effect (Table 1) while i.p. administration of it exerted anti-inflammatory effect. Since algesia in the second phase of formalin test is related to inflammation, it seems that the anti-inflammatory action of Trigonella foenum-graecum extract potentiates its analgesic effect (Ahmadiani et al., 2001). It is well known that 5-HTergic system is involved in the modulation of nociceptive transmission in the spinal cord. High-densities of 5-HT1A and 5-HT3 receptors have been found in superficial lamina of the rat spinal dorsal horn, where nociceptive messages are relayed from primary afferent to spinal neurons. 5-HT1A and 5-HT3 receptors may contribute to the serotonergic regulation of spinal nociceptive transmission (Oyama et al., 1996). Analgesia induced by intrathcal administration of Trigonella foenum-graecum extract (0.5 mg/rat) in the first phase of formalin test was not abolished by pre-treatment with i.t. 5,7-DHT (Table 2). Therefore, there might be another mechanism of action for the first phase analgesia exerted by the extract. Pre-treatment with 5,7-DHT reduced the analgesic effect of Trigonella foenum-graecum extract (3 mg/rat) in the second phase of formalin test significantly (Fig. 4), although it was still able to induce antinociception. Therefore, it is proposed
A. Parvizpur et al. / Journal of Ethnopharmacology 95 (2004) 13–17
that another mechanism of action for the second phase of analgesia co-exists and the spinal serotonergic system is at least partially involved in the Trigonella foenum-graecum extract analgesia in the second phase of formalin test. Trigonella foenum-graecum extract has antinociceptive, anti-inflammatory and anti-pyretic activity, therefore an apparent similarity exists between the effects of NSAIDs and Trigonella foenum-graecum extract. The antinociceptive activity of aspirin was prevented by pre-treatment with parachloro phenylalanine, 100 mg/kg/day, administered for 4 days. It has been reported that the monoaminergic pathways in the central nervous system, are involved in the antinociceptive effect of aspirin and other NSAIDs (Pini et al., 1995). Therefore, the possible role of 5-HT system in Trigonella foenum-graecum analgesia produces another similarity between Trigonella foenum-graecum extract and NSAIDs. Naloxone did not affect (Table 3) the analgesia induced by Trigonella foenum-graecum extract in both phases of the formalin test. So, it seems that the antinociceptive effects of Trigonella foenum-graecum extract are not dependent on the opioid system. Since it has been reported that NSAIDs can inhibit COX isozymes and there are also many reports indicating the role of purinergic system in pain and platelet aggregation, and concerning the relationship between NSAIDs and purinergic system (Puri and Colman, 1997), these two pathways should also be taken into consideration in clarifying the mechanism of action of the extract. In conclusion, Trigonella foenum-graecum extract exerted analgesia in both phases of formalin test in i.p. and i.t. but not i.c.v. administrations. The analgesia induced by Trigonella foenum-graecum extract in the second phase of formalin test was at least partially dependent on intact spinal serotonergic system but there was (were) probably other mechanism(s) for the analgesia induced by the extract in both phases of formalin test. Therefore, it is necessary to determine the effect of Trigonella foenum-graecum extract on COX isozymes and gastric ulceration, platelet aggregation and purinergic system in order to understand more about the analgesic mechanism of this extract.
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References Ahmadiani, A., Javan, M., Semnanian, S., Barat, E., Kamalinejad, M., 2001. Anti-inflammatory and anti-pyretic effect of Trigonella foenumgraecum leaves extract in the rat. Journal of Ethnopharmacology 75, 283–286. Bardin, L., Bardin, M., Lavarenne, J., Eschlier, A., 1997. Effect of intrathecal serotonin on nociception in rats: influence of the test used. Experimental Brain Research 113, 81–87. Carlisson, K.H., Jurna, I., 1987. Depression by flupirtine, a novel analgesic agent of motor and sensory response of nociceptive system in the rat spinal cord. European Journal of Pharmacology 143, 89–99. Dickenson, A., 1994. In: Gebhart, G.F., Hammond, D.L., Jensen, T.S. (Eds.), Progress in Pain Research and Management, vol. 2. IASP Press, Seattle, pp. 525–550. Dray, A., Urban, L., 1996. New pharmacological strategies for pain relief. Annual Reviews of Pharmacology and Toxicology 36, 253–260. Dubuisson, D., Dennis, S.G., 1977. The formalin test: a quantitative study of the analgesic effects of morphine, meperidine and the brain stem stimulation in rats and cats. Pain 4, 161–174. Javan, M., Ahmadiani, A., Semnanian, S., Kamalinejad, M., 1997. Antinociceptive effects of Trigonella foenum-graecum leaves extract. Journal of Ethnopharmacology 58, 125–129. Oyama, T., Ueda, M., Kuraishi, Y., Akaike, A., Satoh, M., 1996. Dual effect of serotonin on formalin-induced nociception in the rat spinal cord. Neuroscience Research 25, 129–135. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. Pini, L.A., Sandrini, M., Vitale, G., 1995. Involvement of brain serotonergic system in the antinociceptive action of acetylsalicylic acid in the rat. Inflammation Research 44, 30–35. Pini, L.A., Vitale, G., Ottani, A., Sandrini, M., 1997. Serotonin and opiate involvement in the antinociceptive effect of acetylsalicylic acid. Pharmacology 54, 84–91. Puri, R.N., Colman, R.W., 1997. ADP-induced platelet activation. Critical Reviews in Biochemistry and Molecular Biology 3, 437–502. Shyu, K.W., Lin, M.T., Wu, T.C., 1984. Possible role of central serotonergic neurons in the development of dental pain and aspirin-induced analgesia in the monkey. Experimental Neurology 84, 179–187. Vitale, G., Pini, L.A., Ottani, A., Sandrini, M., 1998. Effect of acetylsalicylic acid on formalin test and on serotonin system in the rat brain. General Pharmacology 31, 753–758. Wagner, H., Bladt, S., 1996. Plant Drug Analysis. Springer, Berlin, pp. 359–364.. Yaksh, T.L., Rudy, T.S., 1976. Chronic catheterization of the spinal subarachnoid space. Physiology and Behavior 17, 1031–1036.
Spinal serotonergic system is partially involved in antinociception induced by Trigonella foenum-graecum (TFG) leaf extract Alireza Parvizpur, Abolhassan Ahmadiani∗ , Mohammad Kamalinejad Department of Pharmacology, Neuroscience Research Center, School of Medicine, Shaheed Beheshti University of Medical Sciences, P.O. Box 19835-355, Tehran, IR, Iran Received 19 May 2002; received in revised form 24 May 2004; accepted 27 May 2004 Available online 17 August 2004
Abstract It has been reported that Trigonella foenum-graecum (TFG) extract exerts analgesic, anti-inflammatory and anti-pyretic effects in different experimental models. The major objective of this paper was to investigate the site and mechanism of the analgesia induced by Trigonella foenum-graecum extract. We studied the analgesic effects of different doses of Trigonella foenum-graecum extract after i.p., i.t. and i.c.v. administration in formalin test, using male NMRI rats (200–250 g). Trigonella foenum-graecum extract showed analgesic effects in i.p. (1 g/kg) and i.t. (0.5, 1, and 2 mg/rat) (P < 0.05 in all groups) but not in i.c.v. (1 and 3 mg/rat) administrations. Based on the similarities between the effects of Trigonella foenum-graecum extract with those of nonsteroidal anti-inflammatory drugs (NSAIDs) and the role of 5-HT system in analgesic effects of NSAIDs, we tried to investigate the role of spinal 5-HT system in analgesic effects of Trigonella foenum-graecum extract. After lesioning of spinal 5-HT system by 5,7-dihydroxytryptamine (5,7-DHT), it was shown that the analgesic effect of Trigonella foenum-graecum extract (0.5 and 3 mg/rat) in the second phase of formalin test, was abolished completely and reduced relatively after using a low-dose (0.5 mg/rat) and a high-dose (3 mg/rat), respectively (P < 0.05). So, the antinociception partially remained (P < 0.05) after using the latter dose. Meanwhile, administration of naloxone (2 mg/kg, i.p.) had no effect on the Trigonella foenum-graecum extract (1 g/kg, i.p.) analgesia. In conclusion, this study confirms the central action of Trigonella foenum-graecum extract and that spinal 5-HT system is partially involved in the analgesia induced by it in the second phase of formalin test and also indicates for co-existence of other analgesic mechanism(s). © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Trigonella foenum-graecum; Serotonergic system; Analgesia; Rat
1. Introduction Most protocols for the control of pain rely on using nonsteroidal anti-inflammatory drugs (NSAIDs) and opioid analgesics; however, both of them produce several side effects: NSAIDs produce gastrointestinal (GI) disturbances and ulceration, renal damage and hypersensitivity reactions; while opioids induce nausea, constipation, confusion, respiratory depression, and possibly dependence (Dray and Urban, 1996). Therefore, searching for less harmful compounds is still an outstanding domain of investigation. Traditional medicine ∗
Corresponding author. Tel.: +98 21 2403154; fax: +98 21 2403154. E-mail address: [email protected] (A. Ahmadiani).
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.05.020
and medical plants are known as good sources for natural analgesic agents. It has been shown that the plant Trigonella foenum-graecum (TFG, Fenugreek), from the family of Papilionaceae, produces antinociceptive, antiinflammatory and anti-pyretic effects in different laboratory models (Ahmadiani et al., 2001; Javan et al., 1997), which are similar to those of NSAIDs. The plant is known to contain flavonoids, nicotinic acid and salicylate (Javan et al., 1997). The involvement of serotonin in the modulation of nociceptive impulse in the spinal cord has been widely studied. There are at least three families of 5-HT receptors in the spinal cord (5-HT1 , 5-HT2 , and 5-HT3 ) with different affinities for 5-HT (Bardin et al., 1997). Moreover, some reports suggest that serotonergic system involves in the antinocicep-
14
A. Parvizpur et al. / Journal of Ethnopharmacology 95 (2004) 13–17
tive action of NSAIDs. Parachloro phenylalanine (5-HT synthesis inhibitor) prevents the antinociceptive activity of aspirin in hot plate test (Pini et al., 1995), and also abolishes the antinociceptive effect of aspirin in formalin test (Vitale et al., 1998). Pini et al. (1995, 1997) reported that aspirin displayed antinociceptive activity in both hot plate and formalin tests where serotonergic pathways involved in this antinociceptive activity. In dental pain, aspirin induced analgesia was antagonized by pre-treatment with either cyproheptadine, parachloro phenylalanine or 5,7-dihydroxytryptamine (Shyu et al., 1984). Opioids produce spinal analgesia by presynaptic inhibition of the release of transmitters, postsynaptic inhibition of evoked activity and postsynaptic disinhibition (Dickenson, 1994). Based on the similarity between the effects of Trigonella foenum-graecum extract and NSAIDs, the present study was designed to clarify the mechanism of action of the extract by determining its site of action and to study the probable interference of serotonergic and opioid systems. 2. Materials and methods 2.1. Plant material Trigonella foenum-graecum L. (Fenugreek) from the family of Papilionaceae, is extensively cultivated in most regions of the world. The leaves of the plant were obtained from the local market. The plant was authenticated by M. Kamalinejad (Department of Pharmacognosy, Faculty of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran) and voucher specimen coded 417 has been deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran. 2.2. Preparation of the plant extract Fresh green leaves were separated and cleaned, then dried in shade at room temperature. Dried leaves (100 g) were decocted in water for 30 min. Thereafter the extract was filtered and concentrated with a rotary evaporator apparatus (Heidolph, Heizbad WB, Germany). The final weight of the crude extract was 21 g. The extract was maintained at 4 ◦ C throughout the experiments. When it was administered, the extract was dissolved in appropriate volume of saline according to the route of administration. 2.3. Experimental animals Male NMRI rats (200–250 g, Pasteur Institute, Iran) were used in all experiments. The animals were housed in plexiglass cages in groups of four under a temperature range of 21–25 ◦ C and 12:12 h light/dark cycle. Food and water were available ad libitum.
2.4. Drugs Trigonella foenum-graecum extract was prepared as described before (Javan et al., 1997). 5,7-Dihydroxytriptamine (5,7-DHT), pentobarbital, naloxone and desipramine were purchased from Sigma. 2.5. Surgical procedures Intrathecal cannula was implanted under pentobarbital (50 mg/kg, i.p.) anaesthesia as described previously (Yaksh and Rudy, 1976). Briefly, a polyethylene tube (PE-10) filled with sterile saline, was inserted through a small incision into the atlanto-occipital membrane and extended 8.5 cm caudally to the lumbar enlargement of the spinal cord. A period of at least 2 days allowed the animals to recover from the surgery before the analgesia testing. Any animal that showed signs of motor deficit following cannula implantation, was excluded from the study. For i.c.v. administration of Trigonella foenum-graecum extract a 23-ga stainless steel guide cannula, containing a 27-ga stainless steel wire as a stylet, was stereotaxically implanted into the lateral cerebroventricle (i.c.v.) of rats (AP, 0.7 mm from bregma; L, 1.4 mm from the midline and H, 3.3 mm from the surface of dura) (Paxinos and Watson, 1986). The cannula was fixed to the skull with acrylic dental cement. Animals were allowed to recover from the surgery for at least 2 days before being used for the experiments. For i.c.v. injection of the drugs, the guide cannula was opened and a 27-ga injection needle, which was connected to a 25 l—Hamilton microsyring, was inserted into the cannula and after injecting the extract, it was replaced by a stylet. Three minutes later, formalin test was started. In the end of the experiments, pontamine sky blue was injected into the i.c.v. The brain was removed and after being fixed with 10% formalin, the correct injection into i.c.v. was confirmed by evaluating the spreading of the dye into the i.c.v. All experiments were performed between 9:00 a.m. and 3:00 p.m. The adaptation period before formalin test was 30 min. 2.6. Antinociceptive tests The method of formalin test used was the same as described previously (Dubuisson and Dennis, 1977). Briefly, 50 l of 2.5% formaldehyde was injected subcutaneously into the plantar surface of the animal hind paw. Rats were placed in a plexiglass chamber (30 cm×30 cm×30 cm) and a mirror was placed under its floor with a 45◦ angle in order to allow an unobstructed view of the paw. All animals were brought to the test chamber 30 min prior to the experiments, for adaptation. Behavioral responses were detected and recorded for 1 h after the formalin injection. The first 5 min after the administration of formalin was considered as the first phase, and the period between the 16 and 60 min as the second phase. The Trigonella foenum-graecum extract was dissolved in saline and administered i.p. (1 g/kg/ml), i.t. (0.5, 1, 2, and
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3 mg/rat/10 l) and i.c.v. (1, and 3 mg/rat/10 l). Formalin test was carried out 45 min after i.p. injection of the extract and 3 min after i.t. or i.c.v. administrations. Control groups received saline. 5,7-Dihydroxytryptamine was used to destroy the spinal serotonergic system. Desipramine (10 mg/kg i.p. in 0.9% saline) was also administrated to prevent the uptake of neurotoxin by chatecholaminergic neurons and to destroy them. 5,7-dihydroxytryptamine, 200 g/10 l (in 0.9% saline containing ascorbic acid, 0.2 mg/ml) was administered i.t. 45 min after the administration of desipramine. Five days after i.t. administration of neurotoxin, Trigonella foenum-graecum extract (0.5 or 3 mg/rat, i.t.) was administered and 3 min later formalin test was performed. Two control groups of animals received vehicle of neurotoxin and after 5 days, saline or Trigonella foenum-graecum extract (0.5 or 3 mg/rat, i.t.) was administered before formalin test. In the other experiments, two groups of animals received Trigonella foenum-graecum extract (1 g/kg i.p.), 30 min later saline or naloxone (2 mg/kg i.p.) was administered and 10 min later formalin test was performed.
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Fig. 1. Trigonella foenum-graecum extract (1 g/kg, i.p.) induced analgesia in the first and second phases of formalin test. Bars indicate mean ± S.E.M.( n = 7), TFG: Trigonella foenum-graecum extract, ∗∗∗ P < 0.001 vs. control group.
2.7. Preliminary phytochemical tests Preliminary phytochemical properties of the extract were studied for alkaloids, terpenoids, flavonoids and tannin (Wagner and Bladt, 1996). 2.8. Statistical analysis The results of the experiments were expressed as mean ± S.E.M. The differences were estimated by means of Student’s unpaired t-test or ANOVA followed by Tukey’s test. When the probability was less than 0.05 (P < 0.05), the difference was considered to be significant.
Fig. 2. The effect of Trigonella foenum-graecum extract (0.5, 1, 2, and 3 mg/rat, i.t.) in the first phase of formalin test. Bars indicate mean ± S.E.M. (n = 7), TFG: Trigonella foenum-graecum extract, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs. control group.
3. Results 1. Phytochemical studies showed that the applied extract contained alkaloid and steroids, while flavonoids and tannins were absent. 2. Administration of Trigonella foenum-graecum extract via i.p. (Fig. 1) and i.t. (Figs. 2 and 3) routes produced significant analgesia in both the first and second phases of formalin test, but its i.c.v. administration did not produce analgesia in this test (Table 1). 3. Pre-treatment with 5,7-DHT did not affect the first phase analgesia (Table 2) induced by Trigonella foenum-graecum extract (0.5 mg/rat). However, although the second phase analgesia of Trigonella foenum-graecum extract, 3 mg/rat but not 0.5 mg/rat, was significantly reduced by pre-treatment with 5,7DHT, the extract still had significant analgesic effect (Fig. 4).
Fig. 3. Trigonella foenum-graecum extract (0.5, 1, 2, and 3 mg/rat, i.t) induced analgesia in the second phase of formalin test. Bars indicate mean ± S.E.M. (n = 6–8), TFG: Trigonella foenum-graecum extract, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs. control group.
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Table 1 The effect of i.c.v. administration of Trigonella foenum-graecum extract on formalin induced pain Treatment
Dose
Formalin test
10 l 1 mg/10 l 3 mg/10 l
Saline Trigonella foenum-graecum extract Trigonella foenum-graecum extract
First phase
Second phase
2.30 ± 0.08 2.04 ± 0.06 2.10 ± 0.06
1.88 ± 0.06 2.02 ± 0.10 1.90 ± 0.05
Pain score: mean ± S.E.M. (n = 6–8), TFG: Trigonella foenum graecum extract.
Table 2 Pre-treatment with 5,7-DHT did not affect the antinociception induced by Trigonella foenum-graecum extract (0.5 mg/rat, i.t.) in the first phase of formalin test Treatment
N
Pain score
Vehicle + saline Vehicle + Trigonella foenum-graecum 5,7-DHT + Trigonella foenum-graecum
7 7 8
2.23 ± 0.03 1.94 ± 0.10∗ 1.95 ± 0.07∗
Vehicle = 0.9% saline containing ascorbic acid 0.2 mg/ml. TFG: Trigonella foenum graecum. ∗ P < 0.05 vs. vehicle + saline group.
Fig. 4. The effect of spinal 5-HTergic system disruption, using 5,7-DHT, on the antinociception induced by 0.5 and 3 mg/rat (i.t.) of Trigonella foenumgraecum extract, in the second phase of formalin test. Bars indicate mean ± S.E.M. (n = 6–8), TFG: Trigonella foenum-graecum extract, V: vehicle of neurotoxin, N: neurotoxin 5,7-DHT, S: saline, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
Table 3 Naloxone (2 mg/kg, i.p.) did not affect the antinociception induced by Trigonella foenum-graecum extract (1 g/kg, i.p.) in formalin test Treatment
Saline + saline Trigonella foenum-graecum extract + naloxone Trigonella foenum-graecum extract + saline
Formalin test First phase
Second phase
2.29 ± 0.08 1.70 ± 0.04∗∗∗
1.88 ± 0.06 1.20 ± 0.13∗∗∗
1.75 ± 0.07∗∗∗
1.04 ± 0.09∗∗∗
Pain score: mean ± S.E.M., n = 6–8. TFG: Trigonella foenum-graecum. ∗∗∗ P < 0.001 vs. saline + saline group.
4. Administration of naloxone (2 mg/kg, i.p.) did not induce any significant change in Trigonella foenum-graecum extract analgesia (1 g/kg, i.p.) (Table 3).
4. Discussion It is well known that formalin is useful to cause neurogenic and inflammatory pain and its subcutaneous injection produces a biphasic pain response in rats. We used tail-flick test to measure the antinociception effect of Trigonella foenum-graecum extract. Tail flick test is very sensitive to centrally acting drugs (Carlisson and Jurna, 1987). Centrally acting analgesic drugs inhibit the pain response in both phases equally, while the peripherally acting drugs inhibit only the second phase. I.p. administration of Trigonella foenum-graecum extract exerts antinociceptive effects in tail flick test (Javan et al., 1997) and in both phases of formalin test (Fig. 1) that propose its activity, at least partially, results from its central action. Furthermore, i.t. administration of Trigonella foenum-graecum extract caused analgesia in formalin test which confirms the central action of the extract (Figs. 2 and 3). However, i.c.v. administration of Trigonella foenum-graecum extract did not exert any antinociceptive effect (Table 1) while i.p. administration of it exerted anti-inflammatory effect. Since algesia in the second phase of formalin test is related to inflammation, it seems that the anti-inflammatory action of Trigonella foenum-graecum extract potentiates its analgesic effect (Ahmadiani et al., 2001). It is well known that 5-HTergic system is involved in the modulation of nociceptive transmission in the spinal cord. High-densities of 5-HT1A and 5-HT3 receptors have been found in superficial lamina of the rat spinal dorsal horn, where nociceptive messages are relayed from primary afferent to spinal neurons. 5-HT1A and 5-HT3 receptors may contribute to the serotonergic regulation of spinal nociceptive transmission (Oyama et al., 1996). Analgesia induced by intrathcal administration of Trigonella foenum-graecum extract (0.5 mg/rat) in the first phase of formalin test was not abolished by pre-treatment with i.t. 5,7-DHT (Table 2). Therefore, there might be another mechanism of action for the first phase analgesia exerted by the extract. Pre-treatment with 5,7-DHT reduced the analgesic effect of Trigonella foenum-graecum extract (3 mg/rat) in the second phase of formalin test significantly (Fig. 4), although it was still able to induce antinociception. Therefore, it is proposed
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that another mechanism of action for the second phase of analgesia co-exists and the spinal serotonergic system is at least partially involved in the Trigonella foenum-graecum extract analgesia in the second phase of formalin test. Trigonella foenum-graecum extract has antinociceptive, anti-inflammatory and anti-pyretic activity, therefore an apparent similarity exists between the effects of NSAIDs and Trigonella foenum-graecum extract. The antinociceptive activity of aspirin was prevented by pre-treatment with parachloro phenylalanine, 100 mg/kg/day, administered for 4 days. It has been reported that the monoaminergic pathways in the central nervous system, are involved in the antinociceptive effect of aspirin and other NSAIDs (Pini et al., 1995). Therefore, the possible role of 5-HT system in Trigonella foenum-graecum analgesia produces another similarity between Trigonella foenum-graecum extract and NSAIDs. Naloxone did not affect (Table 3) the analgesia induced by Trigonella foenum-graecum extract in both phases of the formalin test. So, it seems that the antinociceptive effects of Trigonella foenum-graecum extract are not dependent on the opioid system. Since it has been reported that NSAIDs can inhibit COX isozymes and there are also many reports indicating the role of purinergic system in pain and platelet aggregation, and concerning the relationship between NSAIDs and purinergic system (Puri and Colman, 1997), these two pathways should also be taken into consideration in clarifying the mechanism of action of the extract. In conclusion, Trigonella foenum-graecum extract exerted analgesia in both phases of formalin test in i.p. and i.t. but not i.c.v. administrations. The analgesia induced by Trigonella foenum-graecum extract in the second phase of formalin test was at least partially dependent on intact spinal serotonergic system but there was (were) probably other mechanism(s) for the analgesia induced by the extract in both phases of formalin test. Therefore, it is necessary to determine the effect of Trigonella foenum-graecum extract on COX isozymes and gastric ulceration, platelet aggregation and purinergic system in order to understand more about the analgesic mechanism of this extract.
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