Orient Pharm Exp Med DOI 10.1007/s13596-013-0143-1
RESEARCH ARTICLE
Evaluation of lyophilized extract of leaves of Tridax procumbens Linn. in rodent models of inflammatory and neuropathic pain Sudarshan Sawant & Virendra Chine & Annasaheb Kalange & Parag Joshi & Vaibhavkumar Gawali & Shuvranshu Narayan Praharaj & Chandraprabhu Jangme & Masood Siddiqui
Received: 8 July 2013 / Accepted: 5 December 2013 # Institute of Korean Medicine, Kyung Hee University 2014
Abstract The aim of present study was to evaluate effect of lyophilized extract of Tridax procumbens Linn. in rodent models of inflammatory and neuropathic pain. The antiallodynic and antihyperalgesic activity of Tridax procumbens was assessed in Chronic Constriction Injury of Sciatic Nerve in rats (CCI) and Scald pain model in rats (burning pain) respectively at 100,200 and 400 mg/kg, p.o. In CCI model, the extract shows the dose dependent antiallodynic activity 60 min. post dose. In Scald pain model, extract shows the significant antihyperalgesic activity at 200 and 400 mg/kg at 60 and 90 min. post dose. The lyophilized extract of Tridax procumbens was found to be effective in neuropathic and inflammatory pain, suggesting its possible action via peripheral mechanisms. The activity may be attributed to the presence of flavonoids such as Quercetin. Keywords Tridax procumbens . Quercetin . Chronic constriction injury . Scald pain . Neuropathic pain . Inflammatory pain . Lyophilization
Introduction TP (Tridax procumbens Linn.) (N.O. Compositae) is commonly known as Tikki Kasa in Hindi, (Yadava and Kumar 1998) a small herb having short, hairy blade like leaves. It is a S. Sawant (*) : V. Chine : A. Kalange : P. Joshi : V. Gawali : S. N. Praharaj : M. Siddiqui Jubilant Biosys Ltd, #96, Industrial suburb, 2nd stage, Yeshwanthpur, Bangalore 560022, Karnataka, India e-mail:
[email protected] C. Jangme Maharashtra College of Pharmacy, Nilanga, Maharashtra, India
very common weed found in open places and it has yellow corolla. It is widely distributed throughout Indo-Pak region and commonly known as ‘Ghamra’, ‘coat button’ in English. Traditionally, it has great value for the treatment of bronchial catarrh, dysentery, malaria, stomachache, diarrhea, high blood pressure, to check hemorrhage from cuts, bruises, wounds and to prevent falling of hair. It possesses antiseptic, insecticidal, parasiticidal and hepatoprotective properties (Salahdeen et al. 2004; Vilwanathan et al. 2005; Edeoga et al. 2005). The constituent of TP comprise large amount of fumaric acid, βsitosterol, lipids, unsaturated fatty acids, tannins and polysaccharides, also the TP is reported to have flavonoids like quercetin (Ikewuchi 2012), procumbenetin (Ali et al. 2001). In India TP also dispensed as ‘Bhringraj’ as ayurvedic medicine for liver disorders and also for traditional wound healing activities. TP is very effective in free radical scavenging, anti inflammatory activity, wound healing activity, anti-diabetic activity and blood pressure lowering effect, immunomodulatory activity, anti-bacterial activity, anti-hepatotoxic and antioxidant activity, topical analgesic activity (Pathak et al. 1991; Nia et al. 2003; Bhat et al. 2007; Diwan et al. 1989; Salahdeen et al. 2004; Oladunmoye 2006). Quercetin is reported to have activity in formalin induced nociception, diabetic neuropathic pain (Narenjkar et al. 2011) and in alcohol induced neuropathy (Raygude et al. 2012). Although several synthetic drugs are available for the treatment of pain, but still drug discovery approach continues with the finding of the appropriate drugs. Opioids, adjuvant analgesic and NSAIDs are the major drugs used for the treatment of neuropathic pain and inflammatory pain but drug dependency and side effect of these drugs limits their use (Gupta and Kulhara 2007). Since ancient time, people are using the medicinal plant as an arsenal to maintain their health. So there has been a growing interest on the medicinal plants and their constituents in the drug
S. Sawant et al.
discovery. Thus, the aim of the present study was to explore analgesic activity of TP in CCI induced neuropathic pain and scald inflammatory pain.
Materials and methods Chemicals Diclofenac Sodium (Sigma, Bangalore, India), Gabapentin (Fluorochem, Derbyshire, United Kingdom), Isoflorane (Abbott, Mumbai, India) Quercetin (Sigma, Bangalore, India).
1)°C, relative humidity (65±10) % and 12 h light, 12 h dark cycle. The animals were fed with standard pellet diet (Tetragon chemie Pvt. Ltd., Bangalore) and drinking water ad libitum. The animals were allowed to acclimatize to experimental conditions by housing them for 8–10 days prior to the experiments. Institutional animal ethics committee approval The experimental design and research protocol were prepared as per guidelines and approved from the institutional animal ethics committee. Analgesic activity
Plant collection and authentication
Effect of TP on CCI induced neuropathic pain
The leaves of TP were collected from Arsikere, DistHasan, Karnataka, and the authentification was done by Prof. P. Jayraman. PhD, Director of National Institute of Herbal Science. Plant Anatomy Research Center, West Tambram, Chennai—45.
Adult male SD rats were used in this study. All surgical procedures were carried out under sterile conditions. CCI was performed according to the method described by Bennett and Xie. Briefly, rats were anaesthetized with isoflurane (2.5 % in Oxygen). The left common sciatic nerve was exposed at the middle of the thigh by blunt dissection through biceps femoris. Proximal to the sciatic trifurcation, about 7 mm of nerve was freed of adhering tissue and 4 loose ligatures (5-0, Ethicon, Johnson & Johnson) were tied loosely around sciatic nerve with about 1 mm spacing. The ligatures were loosely tied in order to minimize nerve constriction. After surgery, the skin was closed by double knots with the use of surgical thread (3-0, Ethicon, Johnson & Johnson) (Bennett and Xie 1988).
Leaves extraction Extraction procedure of TP was done according to previously reported literature (Diwan et al. 1989; Kokate et al. 2004). Briefly, fresh leaves of the plant were collected, cleaned and wiped dry. The 700 g leaves were ground in a mixer without adding water or any other vehicle. From this 600 ml leaves juice was obtained after straining extract from muslin cloth. Then 300 ml filtrate was centrifuged for 15 min. at 1,000 rpm (Eppendorff Centrifuge). From this 225 ml supernatant was obtained. This supernatant solution was frozen with dry ice and acetone for 15–20 min. Then the frozen compound kept in freeze dryer for Lyophilization at −47 °C and vacuum for 24 h. After completely drying near about 5 g water soluble powder of leaves extract was obtained. Animals Adult male Sprague Dawley rats weighing 150–220 g were obtained from a registered local breeder. Animal house condition The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care. The animals were housed under good hygienic conditions in the Animal house of Jubilant Biosys Ltd, Bangalore. India. The animals were housed under standard conditions of temperature (24±
Behavioral tests Measurement of mechanical allodynia Mechanical allodynia was measured at day 14 post surgery by using previously described Up-down method von Frey filaments (Bioseb, France) were used to assess mechanical allodynia. The animals were placed in a plexiglass cage (16 × 24 × 14 cm) with a grid bottom and adapted for at least 10 min. Mechanical stimuli were generated by touching the plantar region of the left hind paw of the rat with a continuous increasing/ decreasing pressure changes by the pattern of (xoxoxo). For the paw withdrawal threshold the mean of two independent measurements was calculated. Filaments used in experiment were 3.61, 3.84, 4.08, 4.31, 4.56, 4.74, 4.93 and 5.18 g. The values of the paw withdrawal thresholds were manually recorded and noted in result sheet (Bennett and Xie 1988).
Evaluation of lyophilized extract of leaves of Tridax procumbens
The basal value was recorded and selected animals were divided into five groups (n =6). Group I and II were administered with water (10 mL/kg, PO) and Gabapentin dissolved in water (150 mg/kg, PO) respectively, while groups III, IV and V were treated with
extract of TP dissolved in water (100, 200 and 400 mg/kg, PO). Post 60 min. of dosing animals were taken for observation. The observer was blinded to pharmacological treatments. The % MPE was calculated by using following formula,
% MPE ¼ ððLog PWT−Avg: Log PWT of vehicleÞ=ðLogð15Þ−Avg: Log PWT of vehicleÞÞ*100
To assess the effect of TP on Scald pain induced inflammatory pain In this test, under the light Isoflorane anesthesia the right hind paw was scalded by immersing in the hot water maintained at 57 °C for 6 s, after 2 h the same scalded paw was immersed in the warm water maintained at 40 °C, then immediately rats were returned to their home cage. Animals were selected on the basis of baseline latency divided into five groups (n =6). Group I and II were administered with water (10 mL/kg, PO) and Diclofenac sodium dissolved in water (15 mg/kg, PO) respectively, while groups III, IV and V were treated with extract of TP dissolved in (100, 200 and 400 mg/kg, PO). The rats were placed on the hot plate (Ugo-Basile) that was thermostatically maintained at 55±0.1 °C. A Plexiglas cylinder was used to confine the animal in the hot plate. The reaction time for each animal (Either hind paw licking or jumping) was considered as pain response. The latency to reaction was measured. When none of the animal response occurred within 30 s exposure, the test was terminated in order to avoid further tissue damage (Hamura et al. 2004). The % MPE was calculated by using following formula, % MPE ¼ ðPost dose latency − Basal latencyÞ =ðCut off time − Basal latencyÞ*100
Camag glass twin—through chamber previously saturated with mobile phase vapour for 20 min. After development, the plates were dried with a hair dryer and then scanned at 380 nm with a Camag TLC scanner using the Deuterium lamp (Rakesh et al. 2009). Statistical analysis In CCI study, One-way ANOVA was used for comparison test of significant differences among groups followed by Dunnett post test. A level of significance (p
