Ameliorative Potential of Butea Monosperma on Chronic Constriction Injury of Sciatic Nerve Induced Neuropathic Pain in Rats

The present study was designed to investigate the ameliorative role of ethanolic extract from leaves of Butea monosperma in chronic constriction injury (CCI) of sciatic nerve induced neuropathic pain in rats. Hot plate, acetone drop, paw pressure, Von Frey hair and tail immersion tests were performed to assess the degree of thermal hyperalgesia, cold chemical allodynia, mechanical hyperalgesia & allodynia in the left hind paw and tail thermal hyperalgesia. Further on, thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) and total calcium levels were estimated to assess the biochemical changes in the sciatic nerve tissue. Histopathological changes were also observed in the sciatic nerve tissue. Ethanolic extract of Butea monosperma leaves and pregabalin (serving as positive control) were administered for 14 consecutive days starting from the day of surgery. CCI resulted in significant changes in behavioural and biochemical parameters. Pretreatment of Butea monosperma attenuated CCI induced development of behavioural, biochemical and histopathological alterations in a dose dependent manner, which is comparable to that of pregabalin pretreated group. These findings may be attributed to its potential anti-oxidative, neuroprotective and calcium channel modulatory actions of Butea monosperma.

Conventional medicines such as anti-convulsant (gabapentin, pregabalin and carbamazepine), antidepressants (amitriptyline and duloxetine), topical treatments (lidocaine patch, capsaicin), and opioids are unable to alleviate the neuropathic pain (Dworkin et al. 2010, Lee andNandi 2010).These studies have also reported to exhibit wide spectrum of adverse effects which limit their full clinical exploitation in the management of painful neuropathy (Carol and Jane 2006).Moreover, none of the medication assessed in randomized controlled studies has been found effective in CRPS (Kalita et al. 2006).There is an urgent need of an alternative medicine for the effective management of neuropathy particularly in CRPS.
Various studies have experimentally reported that, herbal medicine can produce beneficial effect on the management of painful neuropathy i.e., Aconiti tuber, Lindera angustifolia, Teucrium polium, Phyllanthus emblica, Vochysia divergens, Cannabis sativa, Nigella sativa, Ocimum sanctum and Ginkgo biloba (Kim et al. 2009, Muthuraman et al. 2008a).Clinical reports have also been documented to provide beneficial effect of herbal drugs in neuropathic pain management (Babbar et al. 2009, Ellis et al. 2009).Butea monosperma (family: Fabaceae), also known as flame of the forest, which is distributed in deciduous forest and in open areas (Gurav et al. 2008) has The leaves, flowers, stem bark and seeds that have been used in traditional medicine as diuretic, anti-diabetic, anthelmintic, anti-microbial, arthritis, wound healing as well as treating burning sensation of the body (Krithikar andBasu 1989, Varier 1995).Experimental reports have also been indicated to possess antiinflammatory, anti-convulsant, and anti-tumour activities (Shahavi and Desai 2008, Kasture et al. 2000, Sehrawat and Sultana 2006).Leaves have also been reported to possess hypoglycemic (Sharma and Garg 2009), ocular anti-inflammatory (Mengi and Deshpande 1995), anti-stress, and anti-anxiety (Soman et al. 2004) actions.Butea monosperma leaves are composed of various bioactive constituents such as euphane triterpenoid, flavonoids, tannins and sterols (Shukla et al. 2002).Fresh decoction of Butea monosperma is commonly used to relieve muscular pain, joint pain and severe headache in some areas of Tamilnadu at Thiruvannamalai, Theni, and Madurai region of India.An ayurvedic formulation namely Mahanarayana taila is used to treat neuralgia being prepared from this plant (Anonymous 2006).However, experimentally its analgesic potential in neuropathic pain remains to be explored.
Hence the present investigation has been undertaken to explore potential of Butea monosperma in CCI induced neuropathic pain in rats.Pregabalin binds to the α2-δ site an auxiliary subunit of voltagegated calcium channels in the CNS, inhibiting excitatory neurotransmitter release.This drug has proven partial effects onset seizures, neuropathic pain associated with diabetic peripheral neuropathy, postherpatic neuralgia, and fibromyalgia (Kumar et al. 2010).Therefore, pregabalin (voltage dependent calcium channel antagonist) serves as a positive control in this investigation.

EXTRACTION
The fresh leafy part of Butea monosperma was shade dried at room temperature and reduced to coarse powder (sieve n#.10/40).The dried powdered ROLE OF Butea monosperma IN NEUROPATHIC PAIN leaves of Butea monosperma (500 g) were defatted with petroleum ether and then extracted with ethanol (95%) in a Soxhlet apparatus as described in the method of Suzgec-Selcuk and Birteksoz (2011).Ethanolic extract was concentrated under reduced pressure until dryness (yield 14.56 %).CHEMICALS 5, 5'-dithio, bis (2-nitrobenzoic acid) (DTNB), bovine serum albumin (BSA), and reduced glutathione were purchased from Sisco Research Laboratories, Mumbai.Thiobarbituric acid was purchased from Loba Chemie, Mumbai.All other reagents were obtained from SD Fine chemicals, Mumbai, India.

ANIMALS
Wistar rats of both sexes weighing 180-250 g, maintained on standard laboratory diet (Kisan Feeds Ltd., Mumbai, India) and having free access to tap water were employed in the present study.Painful peripheral neuropathy was induced in experimental animals by chronic constriction injury as described in the method of Bennett and Xie (1988), with slight modifications of Sommer and Schafers (1998).In brief, rats were anesthetized with thiopental sodium (35 mg/kg, i.p.).The skin of the lateral surface of the left thigh was incised and a cut was made directly through the biceps femoris muscle to expose the sciatic nerve.Four loose ligatures (silk 4-0), were placed around the nerve proximal part of the trifurcation with a distance of 1 mm between each ligature.The ligatures were loosely tied until a short flick of the ipsilateral hind limb was observed.After performing nerve ligation, muscular and skin layer was immediately sutured with thread, and topical antibiotic was applied at once.Nociceptive threshold was assessed before and after performing surgery on different days i.e. 0, 1, 3, 6, 9, 12, 15, 18, and 21 st day.

EXPERIMENTAL DESIGN
Ten groups, each comprising six Wistar rats were employed in the present study.
Group I (Normal control group): Rats were not subjected to any surgical procedure and were kept for 21 days.
Group II (Sham control group): Rats were subjected to surgical procedure to expose the left sciatic nerve without any nerve ligation process.
Group III (CCI control group): Rats were subjected to surgical procedure to expose the left sciatic nerve with nerve ligation process.

Group IV (CCI + Vehicle treated group):
After subjecting the rats to chronic constriction injury of sciatic nerve, 1 % carboxy methyl cellulose was administered orally for 14 consecutive days from the day of surgery.
Group V (BM per se): Rats were subjected to administration of ethanolic extract of Butea monosperma (400 mg/kg, p.o.) for 14 consecutive days.
Group VI (Pregabalin per se): Rats were subjected to administration of pregabalin (10 mg/kg, p.o.) for 14 consecutive days from the day of surgery.

Group VII to IX (CCI + BM treated group):
After subjecting the rats to CCI, ethanolic extract of Butea monosperma was administered from the day of surgery respectively: 200, 300, and 400 mg/kg, p.o. for 14 consecutive days.VENKATA R.K. THIAGARAJAN et al.

Group X (CCI + Pregabalin treated group):
After subjecting the rats to CCI, pregabalin (10 mg/kg, p.o. for 14 consecutive days) was administered from the day of surgery.
All the groups of animals were employed to assess behavioural tests to determine the degree of nociceptive threshold on certain day intervals, i.e., 0, 1, 3, 6, 9, 12, 15, 18 and 21 st day.All the animals were sacrificed at end of the 21 st day and biochemical analysis was carried out in sciatic nerve tissue homogenate for estimation of total protein content, thiobarbituric reactive substance (TBARS), reduced glutathione and total calcium levels.

Heat hyperalgesic test
Heat thermal sensitivity of the hind paw was assessed by using Eddy's hot plate as described method of Eddy et al. (1950) with slight modification for assessing the degree of noxious thermal sensation.The rats were placed on the top of a preheated (52.5° ± 0.5°C) hot plate surface, allowing access to the left hind paw withdrawal response to degree of the nociceptive threshold.The cut-off time of 20s was maintained.

Cold chemical allodynic test
Cold chemical thermal sensitivity of the hind paw was assessed using acetone drop method as described by Choi et al. (1994) with slight modification for assessing the reactivity to nonnoxious cold chemical stimuli.The rats were placed on the top of a wire mesh grid, allowing access to the hind paws.Acetone (100 µL) was sprayed on the plantar surface of rat's left hind paw.Cold chemical sensitive reaction with respect to either paw licking, shaking or rubbing the left hind paw was observed and recorded as paw withdrawal threshold.The cutoff time of 20s was maintained.

Mechanical hyperalgesic test
Mechanical sensation of the hind paw as an index of mechano-hyperalgesic test was assessed by pressure stimulation method as described by Randall and Selitto (1957).Briefly, mechanical nociceptive threshold, expressed in grams, as measured by applying increasing pressure to the left hind paw.Withdrawal of left hind paw was used to assess the mechanical nociceptive threshold.The cut-off pressure 450 g was maintained.

Mechanical allodynia test
Mechanical sensation of the hind paw as an index of mechano-allodynia was assessed as described method of Chaplan et al. (1994).Briefly, calibrated nylon filaments, in terms of different bending forces, were applied to the mid plantar surface of left hind paw.The filaments were applied ten times, starting with the softest and continuing in ascending order of stiffness.A brisk withdrawal of the left hind limb was considered as a positive response.The criterion for the threshold value, in grams, was equal to the filament evoking a withdrawal of the paw 5 times out of 10 trials i.e., 50% response.The cut-off pressure 30 g was maintained.

Tail heat hyperalgesic test
Spinal thermal sensitivity was assessed by the tail immersion test as described method of Necker and Hellon (1978).Briefly, the terminal part of the rat's tail (1 cm) was immersed in heat-noxious temperature (52° ± 0.5°C), until the tail was withdrawn.The duration of the tail withdrawal reflex was used to assess the thermal heat hyperalgesia.The cut-off time of 10s was maintained.

OXIDATIVE STRESS
After 21 days of surgery, animals were sacrificed by cervical dislocation and sciatic nerve was ROLE OF Butea monosperma IN NEUROPATHIC PAIN immediately isolated from the body.The proximal part of sciatic nerve tissue homogenate (10%, w/v) was prepared with 0.1 M Tris-HCl buffer (pH 7.4) and supernatant of homogenate was employed to estimate total protein content, TBARS, reduced glutathione and total calcium content.

Estimation of tissue protein
Protein concentration was estimated according to the method of Lowry et al. (1951), using bovine serum albumin (BSA) as a standard.The absorbance was determined spectrophotometrically at 750 nm.

Estimation of lipid peroxidation
Estimation of lipid peroxidation was done by measuring the levels of malondialdehyde [MDA: thiobarbituric acid reactive substances (TBARS)] as the described method of Okhawa et al. (1979).The concentration of TBARS in tissue homogenate was expressed in terms of nmol of malondialdehyde per mg of protein.1,1,3,3-Tetramethoxypropane (1-10 nmol) was used as the standard.

Estimation of reduced glutathione
Reduced glutathione was measured as described method of Ellman (1959).Equal quantity of sciatic nerve homogenate was mixed with 10% trichloroacetic acid and centrifuged to separate proteins.To 10 µL of this supernatant, 2 mL of phosphate buffer (pH 8.4), 500 µL of 5, 5'-dithio, bis (2-nitrobenzoic acid) and 400 µL double distilled water were added.Mixture was vortexed and the absorbance was taken at 412 nm within 15 min.The concentration of reduced glutathione was expressed as µg per mg of protein in sciatic nerve tissue.

Estimation of total calcium
Total calcium levels were estimated in sciatic nerve tissue as described method of Severinghaus and Ferrebee (1950) with slight modification of Muthuraman et al. (2008b).Briefly, sciatic nerve tissue homogenate was mixed with 1 mL of trichloroacetic acid (4%) in ice cold conditions and centrifuged at 2,000 r.p.m. for 10 minutes.The clear supernatant was used for the estimation of total calcium ion by atomic emission spectroscopy at 556 nm.
HISTOPATHOLOGICAL ASSESSMENT Samples of sciatic nerve were stored in the fixative solution (10 % formalin) and cut into 4 µm thickness.
Staining was done by using hematoxylin and eosin (H & E) as described method of Sudoh et al. (2004).Nerve sections were analyzed qualitatively under light microscope (450 X) for axonal degeneration.

Statistical analysis
All the results were expressed as standard error of mean (SEM).Data obtained from behavioural tests were statistically analyzed by using twoway repeated ANOVA, while data of biochemical parameters was analyzed using one way ANOVA.In both cases, Tukey's multiple range test were applied for post-hoc analysis.A value of p<0.05 was considered to be statistically significant.

RESULTS
EFFECT OF BUTEA MONOSPErMA ON HEAT HYPERALGESIC TEST Chronic constriction injury (CCI) of sciatic nerve resulted in significant development of noxious thermal hyperalgesia, indicated by decrease in left hind paw withdrawal threshold, after the 3 rd day of surgery as compared to sham control.Administration of ethanolic extract of Butea monosperma (BM 200, 300, and 400 mg/kg, p.o.) attenuated CCI induced decrease in the nociceptive threshold for thermal hyperalgesia in a dose dependent manner.Treatment of pregabalin also produced similar effects.However, statistically significant attenuation was recorded only with medium and high dose of Butea monosperma.Further, vehicle, BM per se and pregabalin did not show any significant effect on heat hyperalgesic test (Fig. 1).VENKATA R.K. THIAGARAJAN et al.
EFFECT OF BUTEA MONOSPErMA ON COLD CHEMICAL ALLODYNIC TEST Chronic constriction injury of sciatic nerve resulted in significant development of non-noxious cold chemical allodynia, indicated by decrease in left hind paw withdrawal threshold, after the 3 rd day of surgery as compared to sham control.Administration of ethanolic extract of Butea monosperma (BM 200, 300, and 400 mg/kg, p.o.) attenuated CCI induced decrease in the nociceptive threshold for thermal allodynia in a dose dependent manner.Treatment of pregabalin also produced similar effects.However, statistically significant attenuation was recorded only with medium and high dose of Butea monosperma.Further, vehicle, Butea monosperma per se and pregabalin did not show any significant effect on cold chemical allodynic test (Fig. 2).
EFFECT OF BUTEA MONOSPErMA ON MECHANICAL HYPERALGESIC TEST Chronic constriction injury of sciatic nerve resulted in significant development of noxious static mechanical hyperalgesia, indicated by decrease in left hind paw withdrawal threshold, after the 3 rd day of surgery as compared to sham control.Administration of ethanolic extract of Butea monosperma (BM 200, 300, and 400 mg/kg, p.o.) attenuated CCI induced decrease in the nociceptive threshold for mechanical hyperalgesia in a dose dependent manner.Treatment of pregabalin also produced similar effects.However, statistically significant attenuation was recorded only with medium and high dose of Butea monosperma.Further, vehicle, Butea monosperma per se and pregabalin did not show any significant effect on mechanical hyperalgesic test (Fig. 3).However, statistically significant attenuation was recorded only with medium and high dose of Butea monosperma.Further, vehicle, Butea monosperma per se and pregabalin did not show any significant effect on mechanical hyperalgesic test (Fig. 4).
EFFECT OF BUTEA MONOSPErMA ON TAIL HEAT HYPERALGESIC TEST Chronic constriction injury of sciatic nerve resulted in significant development of noxious tactile mechanical hyperalgesia, indicated by decrease in tail withdrawal threshold, after the 3 rd day of surgery as compared to sham control.Administration of ethanolic extract of Butea monosperma (BM 200, 300, and 400 mg/kg, p.o.) attenuated CCI induced decrease in the nociceptive threshold for mechanical hyperalgesia in a dose dependent manner.Treatment of pregabalin also produced similar effects.However, statistically significant attenuation was recorded only with medium and high dose of Butea monosperma.Further, vehicle, Butea monosperma per se and pregabalin did not show any significant effect on tail heat hyperalgesic test (Fig. 5).Sciatic nerve ligation resulted in significant rise in TBARS, total calcium levels and decreased in the levels of reduced glutathione, after the 21 st day of surgery as compared to sham control.Administration of the ethanolic extract of Butea monosperma (BM 200, 300, and 400 mg/kg, p.o.) attenuated CCI induced rise in sciatic nerve tissue thiobarbituric reactive substances (TBARS), total calcium and decrease in reduced glutathione levels in dose dependent manner.Treatment of pregabalin also produced similar effects.Further, vehicle, Butea monosperma per se and pregabalin did not show any significant effect on biochemical levels (Table I).
EFFECT OF BUTEA MONOSPErMA ON HISTOPATHOLOGICAL CHANGES Chronic constriction injury of sciatic nerve resulted in significant histopathological changes assessed in transverses sections of the sciatic nerve.In transverse section, nerve derangement, axonal swelling, increase in number of Schwann & satellite cells were also noted.Administration of the ethanolic extract of Butea monosperma (200, 300, and 400 mg/ kg, p.o.) significantly attenuated CCI induced axonal degeneration and histopathological alterations (Fig. 6).

DISCUSSION
In the present study, Butea monosperma attenuated chronic constriction injury of sciatic nerve induced Traditional medicines prepared from Emblica officinalis, Piper longum and Butea monosperma has been documented as a nervine tonic and used for a life span of 100 years with full vigour, cognitive functions and to preserve youth (Adams et al. 2007, Manyam 1999).Pippali rasayana, an ayurvedic herbal medicine, prepared from Piper longum and Butea monosperma has been proven for its traditional claim like immunomodulatory property (Agarwal et al. 1994(Agarwal et al. , 1997)).Methanolic extract of the flowers of this plant has been reported for in vitro anti-oxidant activity (Lavhale and Mishra 2007, Kostoff et al.An Acad Bras Cienc (2012) 84 (4) 1101 ROLE OF Butea monosperma IN NEUROPATHIC PAIN 2008).Further, leaves of this plant have also been used as a nerve tonic to alleviate disorders related to nerves and as analgesic in muscular pain, joint pain and severe headache.Ethanolic extract of leaves of Butea monosperma have shown to possess the anti-oxidant and anti-diabetic activity (Sharrna and Garg 2009).
Free radical mediated oxidative damage has played major role in the pathogenesis of neurodegenerative disease (i.e., amyotrophic lateral sclerosis [ALS], Alzheimer's disease and Parkinson's disease) (Honda et al. 2004).Reactive oxygen and reactive nitrogen species have been documented to contribute the pathophysiological changes in diabetes and its complications (Gao et al. 2007), toxin, Freund's adjuvant induced inflammation, CCI and vincristine mediated neuropathy (Otto et al. 2003).The present study resulted in TBARS levels rise (an index of lipid peroxidation) & total calcium and fall in the reduced glutathione (GSH, an endogenous anti-oxidant level).Thus supporting the contention that free radicals may contribute in pathogenesis of neuropathy.Moreover, the administration of ethanolic extract of Butea monosperma attenuated the CCI induced alterations of peripheral and central behavioural changes associated with oxidative stress marker changes in rats (Bandyopadhyay et al. 1999).Butea monosperma has been documented to decrease free radical generation via enhancement of anti-oxidant mechanisms (Sai Krishna et al. 2010).Free radicals have been reported to increase intracellular Ca 2+ concentration along with activation of NMDA receptor, which has been considered having consanguineous relation with pain modulation (Stanciu et al. 2000).CCI induced increase in calcium levels in sciatic nerve has been documented in the present study as well as in earlier reports (Muthuraman et al. 2008b, Muthuraman andSingh 2011).Increase in calcium ions has been noted to induce electrical hyper-excitability, deplete ATP and activate calpains (Xie and Barrett 1991).Calcium induced activation of calpain is also associated with generation of reactive oxygen species form mitochondria (Carriedo et al. 2000).
Calcium-induced activation of calpains has been shown as responsible for the axonal degeneration by alteration of stability of axonal cytoskeleton protein (Glass et al. 2002, Muthuraman andSood 2010).
Administration of Butea monosperma attenuated CCI induced rise in calcium ion, and oxidative stress markers which it may play a critical role in its anti-nociceptive effects in the development of painful neuropathy.The noted decrease in calcium levels with Butea monosperma may be attributed to its anti-oxidant effects as free radicals are well reported to increase calcium ions (Glass et al. 2002).However, the possibility of direct action of Butea monosperma on decrease in calcium levels may not be ruled out.Moreover, increase in calcium ions is also associated with increase in oxidative stress (Carriedo et al. 2000).So, the noted antioxidant effects of Butea monosperma may also be ascribed secondary to decrease in calcium ion levels.However, number of experimental reports indicating anti-oxidant effects of Butea monosperma in various studies (Lavhale and Mishra 2007, Sharrna and Garg 2009, Sai Krishna et al. 2010).Similar results were obtained in the pregabalin treated animals.Pregabalin is a potential voltage dependent calcium channel (α2-δ subunit) antagonist (Kumar et al. 2010).It has also been reported to possess the potential role in the management of painful neuropathy in human (Kumar et al. 2010) and in experimental animal (Bender et al. 2010, Park et al. 2010).Ameliorative effect of Butea monosperma against chronic constriction injury of sciatic nerve induced neuropathic pain may be due to its potential of anti-oxidative, neuroprotective and inactivation of calcium channel opening.Nevertheless, further studies are needed to substantiate these findings.
Butea monosperma were collected from Madurai and authenticated by Dr. D. Stephen, Asst Prof., Department of Botany, American College, Madurai.Plant sample have been kept in the Department of Pharmacognosy, (Voucher specimen n#: BM. 001/2007-2008), College of Pharmacy, Madurai Medical College, Madurai.

Fig. 1 -Fig. 2 -
Fig. 1 -Effect of Butea monosperma on paw heat hyperalgesia.Digits in parenthesis indicate dose in mg/kg.Data were expressed as mean ± SEM, n=6 rats per group.a p<0.05 vs sham control group.b p<0.05 vs CCI control group.c p<0.05 vs pregabalin treated group.

Fig. 3 -
Fig. 3 -Effect of Butea monosperma on paw mechanical hyperalgesia Digits in parenthesis indicate dose in mg/kg.Data were expressed as mean ± SEM, n=6 rats per group.a p<0.05 vs sham control group.b p<0.05 vs CCI control group.c p<0.05 vs pregabalin treated group.

Fig. 4 -
Fig. 4 -Effect of Butea monosperma on paw mechanical allodynia Digits in parenthesis indicate dose in mg/kg.Data were expressed as mean ± SEM, n=6 rats per group.a p<0.05 vs sham control group.b p<0.05 vs CCI control group.c p<0.05 vs pregabalin treated group.

Fig. 5 -
Fig. 5 -Effect of Butea monosperma on tail heat hyperealgesia Digits in parenthesis indicate dose in mg/kg.Data were expressed as mean ± SEM, n=6 rats per group.a p<0.05 vs sham control group.b p<0.05 vs CCI control group.c p<0.05 vs pregabalin treated group.
Data were expressed as mean ± SEM for 6 rats per group.
b Digits in parenthesis indicate dose in mg/kg.a p<0.05 vs sham control group.b p<0.05 vs CCI control group.c p<0.05 vs pregabalin treated group.