Evaluation of antinociceptive effect of methanolic leaf and root extracts of Clitoria ternatea Linn. in rats
Abstract
Aim:
Clitoria ternatea Linn. (C. ternatea) is an Ayurvedic herb traditionally used as medicine to relieve inflammatory, rheumatism, ear diseases, fever, arthritis, eye ailments, sore throat and body ache. This study aims to evaluate and elucidate the possible mechanism underlying the antinociceptive action of methanolic extracts of C. ternatea leaf and root using several antinociception models.
Materials and Methods:
The different antinociception models such as hot plate, tail-flick and formalin tests were used along with naloxone (a non-selective opioid antagonist) to establish the antinociceptive activity of both leaf and root extracts.
Results:
Both C. ternatea leaf and root extracts markedly demonstrated antinociceptive action in experimental animals. Results of formalin test showed that the antinociceptive activity of the extracts may be mediated at both central and peripheral level. Moreover, the results of hot plate and tail-flick tests further implies that C. ternatea root extract mediates antinociceptive activity centrally at supraspinal and spinal levels whereas, the C. ternatea leaf extract's antinociceptive activity is mediated centrally at supraspinal level only. It is believed that the opioid receptors are probably involved in antinociceptive activity of both C. ternatea root extract.
Conclusions:
Our studies support the traditional use of C. ternatea leaf and root against pain. The extracts can also be utilised as a new source of central analgesics in treatment of pain.
Introduction
Pain is termed as an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage. This clearly indicates that pain is a multidimensional experience. Pain remains as the primary reason for seeking medical attention as significant medical needs are still unmet.[1] Moreover, throughout the last decade, research analysis has anticipated that analgesics are one of the highest therapeutic categories on which research efforts are concentrated. The current analgesic compounds available in the market still present a wide range of undesired effects leaving an open door for new and better compound. Natural products are believed to be an important source of new chemical substance with potential therapeutic application.[2] With this in view, we further exploited the antinociceptive activity of this plant.
Clitoria ternatea is a very well known Ayurvedic medicine or herb used for centuries as a memory enhancer, nootropic, antistress, anxiolytic, antidepressant, anticonvulsant, tranquilizing and sedative agent. Traditionally, the roots are used for anti-inflammatory and anthelmintic activities and to treat rheumatism, ear-diseases, indigestion, constipation, fever, arthritis, eye ailments and sore throats.[3] The leaves and root are also used in the treatment of a number of ailments including body aches, especially infections, urogenital disorders and as an anthelmintic and antidote to insect stings.[4]
Previous study has reported that methanolic extract of C. ternatea roots markedly reduced the number of writhing in acetic acid-induced writhing test.[5] Also, the ethanolic extract of aerial part of C. ternatea showed significant analgesic activity which was comparable to the reference drug morphine sulphate in radiant heat method. The ethanolic extract also showed a marginal analgesic effect in tail clip test and no effect in acetic acid-induced writhing test.[6] The toxicity of methanolic extracts of C. ternatea root and leaf have been evaluated and found to be safe even at dose of 3.2 g/kg4 and 2 g/kg (p.o.)[7] respectively. Despite all these studies, the precise mechanism underlying the analgesic action of methanolic extracts of C. ternatea leaf and root still remain unclear. Thus the aim of our study is to evaluate and elucidate the possible mechanism underlying the action of methanolic extracts of C. ternatea leaf and root using naloxone (a non-selective opioid antagonist) through several antinociception models such as hot plate, tail-flick and formalin tests.
Materials and Methods
Plant Material
The raw materials of C. ternatea plant samples were collected at Seberang Jaya, Penang, Malaysia. The identification and authenticity work was carried out by a botanist and a voucher specimen (No. 11006) has been deposited at the herbarium of School of Biological Sciences, USM. The plant materials were separated into leaves and roots and washed with water to remove dirt prior to drying at 40°C for 3 days.
Preparation of Plant Extracts and Reference Drugs
Powdered (200 g) samples of leaves and roots were macerated in methanol (2 L) for 72 h under constant stirring conditions for 72 h. The extracts were then filtered through a Buchner funnel fitted with No. 1 Whatman filter paper. The pooled extracts were dried under partial vacuum using rotary evaporator (BUCHI R-110, Switzerland) and lyophilized by freeze drying. The 100, 200 and 400 mg/kg doses of C. ternatea leaves and roots extracts were prepared by suspending the extracts in 2% aqueous Tween 80. Both the extracts and vehicle were administered to the animals orally (p.o.). The reference drugs used in this study were paracetamol (100 mg/kg), morphine sulphate (5 mg/kg) and naloxone (2 mg/kg). All reference drugs were prepared by dissolving in normal saline.
Experimental Animals
Sprague-Dawley male rats (180-220g) were obtained from the animal house of University Sains Malaysia for antinociceptive studies. The animals were kept in a ventilated departmental animal room under standard environmentally controlled conditions of 24 ± 1°C with 12 h light and 12 h dark cycle. Animals were provided with standard rodent pellet diet and food was with drawn 24 h before the experiment began. Water was allowed ad libitium. All studies were approved by Animal Ethics Committees, USM.
Formalin test
The study was carried out according to method described by Hunskaar and Hole.[8] The control group received 2% aqueous Tween 80 (10 mL/kg, p.o.) and the reference group were given paracetamol (100 mg/kg, p.o.) and morphine sulphate (5 mg/kg, s.c.) respectively. Extract at doses of 100, 200 and 400mg/kg were administered orally to the animals. After 1 h oral administration of extracts, control and paracetamol (except only 15 min for morphine), 100 μL of 3% formalin in saline was injected into the subcutaneous tissue on the plantar surface of the left hind paw of rats. The time spent in licking the injected paw by each rat was observed as soon (0-5 min, post injection) as the formalin was injected and later (late phase 15-30 min, post injection). These phases represent neurogenic and inflammatory pain responses, respectively. The mean of the time spent on licking the injected paw in each group was determined.
Hot plate test
The hot plate test was used as previously described.[9] The heated surface of a hot plate analgesia meter (model PE34, series 3, IITC Life Science Series, Woodland hills, USA) was maintained at 55 ± 1°C. Each animal was placed on the heated surface of the plate. Prior to treatment, the thermal reaction time of each rat (licking of the forepaws or jumping response) was performed at 0 and 10 min interval. The average of the two readings was obtained as the initial reaction time. Only rats that showed nociceptive responses within 15 s were used in the experiments.
The animals in the control group received 2% aqueous Tween 80 (10 mL/kg, p.o.) while the reference groups were treated with morphine sulphate (5 mg/kg, s.c.), paracetamol (100 mg/kg, p.o.) and naloxone (2 mg/kg, i.p.) respectively. The rats in the test groups were treated with different doses (100, 200 and 400 mg/kg, p.o.) of leaf and root extracts respectively. In order to determine the involvement of opioid system on the antinociceptive effect, naloxone (2 mg/kg, i.p.) was administered 10 min before morphine (5 mg/kg, s.c.) or C. ternatea root extract (400 mg/kg, p.o.) respectively. After 30 min of treatment with all various doses of extracts (except only 15 min for morphine and 10 min for naloxone), rat were placed on a hot plate maintained at 55 ± 1°C. The latency of nociceptive response (reaction time) of each rat that was identified by the time for licking and flicking of a hind limb or jumping was recorded. The reaction time was measured every 15 min in a 60-min period at intervals of 30, 45, 60, 75 and 90 min. In order to minimize damage to the animal paw the cut-off time for latency of response was taken as 45 s.
Tail flick test
The method of D’Amour and Smith[10] as modified by Rahman et al.[11] was adopted for this test. An analgesiometer model 33T, series 8, IITC Life Sciences Series, Woodland hills, USA) was used to record tail flick latencies. Prior to treatment, the thermal reaction time of each rat (flicking or removing the tail) was performed at 0 and 10 min interval. The average of the two readings was obtained as the initial reaction time. Only rats that showed nociceptive responses within 4 s were used in the experiments. The rats were treated with the similar treatment groups as in hot plate test. After 30 min of treatment with various doses of extracts (except only 15 min for morphine and 10 min for naloxone), the tail-flick response of the rat was measured by gently placing the rat tail at a central position of a focused light beam from a 45-W projection bulb. The time taken by the animals to withdraw (flick) its tail from heat induced by the light beam was recorded as the reaction time. The reaction time was measured every 15 min in a 60-min period at intervals of 30, 45, 60, 75 and 90 min. The cut-off time was 10 s to prevent injury to the rat tail.
Statistical Analysis
The data obtained were analysed using SPSS software program version 15.0 and expressed as a mean ± standard deviation (SD). Statistically significant differences between groups was calculated by the application of one way analysis of variance (ANOVA) followed by Dunnett's post hoc analysis. P Values less than 0.05 (P < 0.05) were used as the significant level.
Results
Formalin test
In this study, the C. ternatea leaf and root extracts demonstrated antinociceptive activity against both neurogenic and inflammatory phase of formalin assay. Extracts at all dose levels significantly (<0.05) reduced the paw licking time when compared to control group for both phases [Table 1]. The morphine inhibited the licking of the paw at both phases which supports its centrally acting analgesic activity.[12] Paracetamol, a weak analgesic and anti-inflammatory drug reduced the paw licking time during both phases when compared to vehicle. The C. ternatea root (400 mg/kg) reduced the paw licking time in next after morphine during both phases. The antinociceptive activity of C. ternatea leaf (400 mg/kg) and root (200 mg/kg and 100 mg/kg) was better than paracetamol during the first phase. Interestingly, both C. ternatea leaf and root extracts at all dose levels reduced the paw licking time significantly better than paracetamol during the inflammatory phase.
Effect of C. ternatea leaves and root extracts on the reaction time of rats in the formalin test
Hot plate test
The results of hot plate test are reported in Table 2. Morphine (5 mg/kg, s.c.) markedly increased pain latency (P < 0.01) throughout the observation period. Both C. ternatea leaf (200 and 400 mg/kg) and root (100, 200 and 400 mg/kg) extracts prolonged the latency time significantly (P < 0.01) when compared to control (vehicle) group.
Effect of C. ternatea leaf and root extracts on the reaction time of rats in the hot plate test
Tail-flick test
The results of tail flick experiment are reported in Table 3. The tail-flick latency response was significantly prolonged from 30–75 min after administration of C. ternatea root (400 mg/kg, p.o.) extract compared to control (P < 0.01). Conversely, the C. ternatea root (100 and 200 mg/kg) and leaf extract at all doses (100, 200 and 400 mg/kg) did not prolong the tail-flick latency time throughout the observation period. Morphine (5 mg/kg, s.c.) significantly increased the tail-flick latency response throughout the 60 min observation period when compared to control (vehicle).
Effect of C. ternatea leaf and root extracts on the reaction time of rats in the tail flick test
Involvement of opioid receptors
As expected, morphine (5 mg/kg, s.c.) markedly increased pain latency (P < 0.01) throughout the reaction time in both hot plate and tail-flick tests. After naloxone (2 mg/kg, i.p.) pre-treatment, the morphine (5 mg/kg, s.c.) was antagonized by naloxone. Interestingly, the C. ternatea root (400 mg/kg) extracts was antagonized by naloxone in both hot plate and tail-flick tests [Tables 4 and 5].
Effect of C. ternatea root extracts on the reaction time of rats in the hot plate after pre-treatment with naloxone
Effect of C. ternatea root extract on the reaction time of rats in the tail flick after pre-treatment with naloxone
Discussion
The toxicity of methanolic extracts of C. ternatea root and leaf have been evaluated and found to be safe even at dose of 3.2 g/kg and 2 g/kg (p.o.) respectively.[37] It is important to note that the highest dose used in the present study (400 mg/kg, p.o.) was 5 and 8 fold lower than the LD50 following an acute toxicity study of both C. ternatea leaf (LD50 > 2000mg/kg)[7] and root (LD50 = 3200 mg/kg)[3] extracts respectively. On the whole, these findings substantiate the safe traditional use of C. ternatea leaf in the treatment of pain, inflammatory conditions and other disorders in Ayurvedic medicine.[4]
The formalin test in rodents has an important feature whereby the animals show two phases of nociceptive behaviors which involves two distinctive different stimuli.[13] Hunskaar and Hole[8] have clearly differentiated that the early phase may be direct action of formalin on nociceptors and this phase can be inhibited by centrally acting analgesics. On the contrary, the late phase occurs due to inflammatory response mediated by prostaglandins, serotonin, bradykinin and histamine and can be inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs), steroids and as well as centrally acting analgesics. As both the C. ternatea leaf and root extracts reduced the paw licking time significantly (P < 0.05) at both phases when compared to the control group, hence suggest the centrally acting antinociceptive activity of both the extracts. In addition, the antinociceptive activity of the extracts was better than paracetamol which suggest the potential medicinal use of the extracts in treatment of pain in substitute of paracetamol. The antinociceptive activity of C. ternatea root (400 mg/kg) may be mediated like morphine as the reduction of paw licking time was comparable with morphine at both phases. Considering the antinociceptive of C. ternatea of both leaf and root extracts on the inflammatory phase of formalin test, is indicative of an anti-inflammatory action of the extracts. The C. ternatea leaf and root extracts could have possibly block or reduce prostaglandin, serotonin, bradykinin and histamine synthesis which contribute to the reduction of nociception at the late (inflammatory) phase.
The hot plate test is suitable for differentiating central analgesics from peripheral acting analgesics. Centrally acting analgesics prolongs the response whereas the peripheral analgesics of the acetylsalicylic acid (e.g. aspirin) or phenyl-acetic acid type (e.g. diclofenac) do not affect these responses.[14] In this study, C. ternatea leaf and root extracts prolongs the latency time significantly (P < 0.01) when compared to control (vehicle) group, hence supportive of the centrally mediated antinociceptive activity of C. ternatea leaf and root extracts. The hot plate responses (paw-licking) is considered to be mediated by complex supraspinally organized behavior.[915] Seeing that both the C. ternatea extracts prolongs the hot plate latency time significantly, it can be further implied that the antinociceptive activity of C. ternatea leaf and root extracts could be mediated centrally at supraspinal level.
In tail flick test, the responses are tail flick or try to escape.[14] The tail flick response is regarded as spinal reflex and does not involve a high degree of sensory-motor coordination and is normally under physiologic control of higher centers.[1617] The escape reaction meanwhile is regarded as a complex phenomenon mediated by the brain. The tail flick can discriminate between non-opioid analgesics and centrally acting morphine-like analgesics.[14] As the tail flick latency response was significantly prolonged from 30-75 min after administration of C. ternatea root (400 mg/kg, p.o.), these findings suggest that C. ternatea root extract mediates antinociceptive activity centrally at supraspinal and spinal levels. Moreover, the findings suggest a centrally acting morphine-like antinociceptive activity of the C. ternatea root extracts which may bind with opioid receptors. Whereas, the C. ternatea leaf extract's antinociceptive activity is mediated centrally at supraspinal level only as the leaf extract did not prolong the tail-flick latency response. The C. ternatea leaf extract may acts on opiate receptors but not acting as morphine-like analgesics.
In order to determine the involvement of opioid receptors, the C. ternatea root extract was pre-treated with opioid receptor antagonist nalaxone. Naloxone is a non-selective antagonist, potent at μ, κ and δ opioid receptors.[18] It reverses the opioid effects by competitively binding with the opioid receptors but does not change the cell function.[1819] Therefore by taking all these together, it is believed that the antinociceptive activity of C. ternatea root extract is most likely to be mediated through opioid receptor sub-types. However, the precise opioid receptor involved in the antinociceptive activity of the extracts could not be determined as the naloxone a non-selective antagonist blocks all μ, κ and δ opioid receptors. Therefore, it is of important to further investigate the possible receptors involved in the analgesic activity of C. ternatea root extract by conducting competitive receptor binding assay which determines the binding affinity of the extract with opioid receptors.[20]
Conclusion
The data obtained from the present study indicates that in formalin test the antinociceptive activity of C. ternatea leaf and root extracts at all tested dose levels may be mediated through both central and peripheral level. Both the extracts exhibit anti-inflammatory activity. Further evaluation of hot plate test, confirms the centrally acting analgesic effects of C. ternatea leaf and root extracts and indicates both the extracts could be mediated centrally at supraspinal level. The tail-flick test suggest that C. ternatea root extract's antinociceptive activity is not only mediated centrally at supraspinal level but also at spinal level the extract may be a centrally acting morphine-like analgesic which bind with opioid receptors. Whereas, the C. ternatea leaf extract's antinociceptive activity is mediated centrally at supraspinal level only but not acting as morphine-like analgesics but may acts on opiate receptors. The analgesic activity of root extract was antagonised by naloxone and thus indicative of opioid receptor activity. These findings thus justify traditional use of this plant in the treatment of pain, inflammatory conditions and other central nervous system disorders and validate its claim of being used for said purpose in Ayurvedic medicine. The present study is, to our knowledge the first to demonstrate that C. ternatea root extract acts on opioid receptor sub-types. Further studies are in progress to evaluate the type of opioid-like receptors involved in the antinociceptive activity of C. ternatea root extract and identify the active components for further pre-clinical evaluations before clinical study can be initiated in humans.
Footnotes
Source of Support: Nil
Conflict of Interest: No
Acknowledgements
This research project was supported by Center for Drug Research, Universiti Sains Malaysia under the Research University Grant Scheme (1001/CDADAH/813041). The author (L. Kamilla) would like to acknowledge the scholarship provided by MyPhD, Ministry of Higher Education, Malaysia.
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