Research & Reviews: A Journal of Pharmacology Volume 3, Issue 1, ISSN: 2230-9861 __________________________________________________________________________________________ Anti-nociceptive Potential of Scurrula parasitica: An Unexploited Parasitic Plant Nilesh Mahajan1*, Parag Joshi2, Manish Kondawar3, K. L. Senthil Kumar1, V. Vaidhyalingam4 1 Padmavathi College of Pharmacy, Periyanahalli, Dharmapuri, Tamil Nadu, India 2 Jubilant Drug Discovery Centre, Bangalore, Karnataka, India 3 Appasaheb Birnale College of Pharmacy, Sangali, Maharashtra, India 4 K. K. College of Pharmacy, Gerugambakkam, Chennai, Tamil Nadu Abstract Scurrula parasitica (Loranthaceae) is an herbaceous growing shrub parasitic on Dendrophthoe falcata (Loranthaceae) which is itself parasitic on Mangifera indica (Anacardiaceae). Scurrula parasitica is distributed throughout the western ghat region of Maharashtra, some parts of Uttar Karnataka and Tamil Nadu. Genus Scurrula comprising of around 91 species, however, only S. ferruginea, S. atropurpurea, S. fusca have been investigated systematically. No such pharmacological and phytochemical data are available on the plant Scurrula parasitica. So, the main intention of the current study is to investigate the unexploited angiospermic parasite plant, Scurrula parasitica for its ethno medical claims as well as pharmacological activities based on chemotaxonomic tracing, in experimental animals. The dried methanol extract (SPM) of the whole plant of Scurrula parasitica L was studied for its analgesic activity using animal models such as- Aceticacid-induced writhing test (chemical stimulation), Heat conduction method (thermal stimulation) in comparison with Diclofenac sodium as reference standards. The SPM was given in doses of 100, 200 and 400 mg/kg body weight in which dose 400 mg/kg given significant results in comparison with reference standard Diclofenac sodium. The study may conclude that some chemical entities transferred from host Dendrophthoe falcata to the parasite Scurrula parasitica are accountable for its anti-nociceptive potential. Keywords: Scurrula parasitica, anti-nociceptive, writhing, heat-conduction *Author for correspondence E-mail: nileshpharmacology@gmail.com INTRODUCTION Drug discovery is a starving process, which requires constant feeding with new chemical compounds from a suitable source. Several methods like, synthetic chemistry, combinatorial chemistry, molecular modeling and isolation from plants and other natural sources [1] have been adopted to get compounds for drug discovery. In recent years, there has been growing interest in alternative therapies and the therapeutic use of natural products, especially those derived from plants. It is well known that plants produce a wide range of structural type of molecules and they have a wide spectrum of pharmacological activities. Plants have been a source of therapeutic agents for more than 5000 years [2]. Plant parasite has originated multiple times during angiosperm evolution, and consequently, parasitic genera vary considerably in their habits and hosts ranges [3]. The aerial stem-parasites commonly known as mistletoes belong to several families, of which the major ones are Loranthaceae and Viscaceae which are considered to be Santalales. Loranthaceae consists of 75 genera and about 900 species, widely distributed from the tropics to temperate regions, particularly in the South. The genus Scurrula is assigned to family Loranthaceae [4]. Scurrula has about 91 species. Most of them in South East Asia, Malaysia and China whereas small number in some regions of India and Australia. The genus has spread into all sorts of wooded RRJoP (2013) 4-8 © STM Journals 2013. All Rights Reserved Page 4 Anti-nociceptive Potential of Scurrula parasitica Mahajan et al. __________________________________________________________________________________________ habitat and some species have become very specialized parasites of particular host, sometimes occurring only on other mistletoes, principally Loranthaceae. Scurrula parasitica (Loranthaceae) is an herbaceous growing shrub parasitic on Dendrophthoe falcata (Loranthaceae) which is itself parasitic on Mangifera indica (Anacardiaceae) [5, 6] Scurrula parasitica is distributed throughout the western ghat region of Maharashtra, some parts of Uttar Karnataka and TamilNadu [7]. Scurrula parasitica is only grown on Dendrophthoe falcata. Genus Scurrula comprising around 91 species, however, only S. ferruginea, S. atropurpurea, S. fusca has been investigated systematically. This plant reported to have many pharmacological activities such as anticancer, immunomodulatory, antidiabetic, antioxidant, antihepatotoxic and cytotoxic activity [8, 9]. Phytochemicals from different classes, responsible for various biological activities, like catechin, rutin, icariside, aviculin, flavonoids, oleanolic acid, lupeol etc. have been recovered from S. parasitica [10]; whereas phytochemicals like quercetin, 4-Oacetylquercitrin possesses cytotoxic activity. In case of S. ferruginea, S. atropurpurea and, S. fusca, except reports on ethno medicinal claims, no other information is available. No such pharmacological and phytochemical data are available on the plant Scurrula parasitica. So, the main objective of the current study was to investigate the unexploited angiospermic parasite plant, Scurrula parasitica for its ethno medical claims as well as pharmacological activities based on chemotaxonomic tracing, in experimental animals. MATERIALS AND METHODS The plant material Scurrula parasitica (Loranthaceae) is hyperparasitic on Dendrophthoe falcata L. f. (Loranthaceae) which is itself parasitic on Mangifera indica L. (Anacardiaceae) was collected from Kherad – vita, Tal. Kadegaon, District Sangali, Western Ghat region of Maharashtra (16° 52'N Latitude and 74° 36' 0E Longitude and 1100 m Altitude) in November 2009. The plant specimen (Voucher no. 239) was authenticated by Head of the department, Botany, Padmabhushan Dr. Vasantraodada Patil Mahavidyalaya, Tasgaon, Dist. Sangali, India. Preparation of Extract The shade dried material was coarsely powdered (500 gm) and extracted with methanol (1.5 L) by cold maceration method. The extract was filtered, concentrated on rotary vacuum evaporator and further dried in vacuum dryer and weighed (10.17 %w/w). Dosages of different concentrations of extract (SPM) were dispensed from solution prepared from normal saline. The vehicle alone served as control. Animals Swiss albino mice (20–25 gm) were procured from National Toxicology Centre, Pune. The animals were housed under standard conditions of temperature (22 ± 1ºC), relative humidity (55 ± 10%), and 12 h light/dark cycles and fed with standard pellet diet (Amrut feeds, Pune, India) and water ad libitum. All the experiments performed are approved by the Institutional Animal Ethical Committee. Grouping of Animals For acetic-acid-induced writhing test and heat conduction method, the animals were divided into six groups each containing six animals. Group 1 served as untreated control and received normal saline (5 ml/kg, p.o.), group 2 served as positive control and received Diclofenac sodium (5 mg/kg, i.p.) and group 3, 4 and 5 were treated with 100, 200, and 400 mg/kg of SPM, respectively. Acute Toxicity Test The acute toxicity of Scurrula parasitica methanol extract was determined in swiss albino mice according to the method with slight modifications [11]. Mice fasted for 16 h were randomly divided into groups of six per group. Gradual doses of the extract (250, 500, 1000 and 2000 mg/kg p.o.) were separately administered to the mice and rats in each of the groups by means of bulb steel needle. All the animals were then allowed free access to food and water and observed over a period of 48 h for sign of acute toxicity. The number of deaths within this period was recorded. Anti-Nociceptive Activity Acetic-acid induced writhing test (Chemical Stimulation) The anti-nociceptive activity of the samples was evaluated using acetic acid induced RRJoP (2013) 4-8 © STM Journals 2013. All Rights Reserved Page 5 Research & Reviews: A Journal of Pharmacology Volume 3, Issue 1, ISSN: 2230-9861 __________________________________________________________________________________________ writhing method in mice [12]. In this method, acetic acid is administered intraperitoneally to the experimental animals to create pain sensation. As a positive control, any standard NSAIDs drug can be used. In our present study, Diclofenac sodium was used to serve the purpose. The SPM was administered orally in three different doses (100, 200 and 200 mg/kg body weight) to the swiss albino mice after an overnight fast. Test and vehicle samples were administered orally 30 minutes prior to intraperitoneal administration of 0.7% v/v acetic-acid solution (0.1ml/10gm) and Diclofenac sodium was administered 15 minutes prior to acetic acid injection. Then, the animals were placed on an observation table. Each mouse of all groups were observed individually for counting the number of writhing they made in 15 minutes commencing just 5 minutes after the intraperitoneal administration of acetic-acid solution. Full writhing was not always accomplished by the animal, because sometimes the animals started to give writhing but they did not complete it. This incomplete writhing was considered as half writhing. Accordingly, two half-writhing were considered as one full writhing. The number of writhes in each treated group was compared to that of a control group while Diclofenac sodium (5 mg/kg) was used as a reference standard. Results are given in Table 1 and Graph No.1. Table 1: Anti-Nociceptive Effect of Methanol Extract of Scurrula parasitica on Swiss Albino Mice Using Acetic-Acid Induced Writhing Test. Number of Writhes Percentage Treatment Dose (mg/kg) observed Inhibition Negative Control 5 ml/kg 38.55 ± 3.70 -(Normal Saline) Positive Control (Diclofenac sodium) SPM 5 mg/kg 14.35 ± 1.50*** 69.25 % 100 mg/kg 36.23 ± 1.20 6% 200 mg/kg 30.54 ± 2.50 21 % 400 mg/kg 18.48 ± 2.80*** 52 % Values are expressed as Mean ± SEM, n=6, and by One-way ANOVA followed by Dunnette’s test ***P<0.001, SPM (Scurrula parasitica methanol extract). Effect of S. parasitica on acetic acid induced writhing in mice number of Writhes 50 Negative Control 100 mg/kg 200 mg/kg 400 mg/kg Diclofenac 40 30 *** 20 *** 10 0 1 2 3 4 Animal Groups 5 Fig. 1: Effect of Methanol Extract of S. parasitica Using Acetic-acid Induced Writhing Test. Heat Conduction Method (Thermal Stimulation) The animals were divided into five groups of 6 animals each. Group 1 served as negative control. Group 2 served as positive control and were injected with Diclofenac sodium (5 mg/kg) intraperitoneally. Groups 3, 4 and 5 were treated with 100, 200, and 400 mg/kg body weight of SPM, respectively. After half, RRJoP (2013) 4-8 © STM Journals 2013. All Rights Reserved Page 6 Anti-nociceptive Potential of Scurrula parasitica Mahajan et al. __________________________________________________________________________________________ avoid damage to the tail for all groups. The time required for flicking of the tail, was recorded, to assess response to noxious stimulus [13]. Results are given in Table 2 and Graph No. 2. 1, 2 and 3 h, the tip of tail was dipped upto 5 cm into hot water maintained at 58°C. The response time was noted as the sudden withdrawal of the tail from the hot water. Cut off time of 10 seconds was maintained to Table 2: Anti-Nociceptive Effect of Methanol Extract of S. parasitica using Heat Conduction Method. Reaction time in sec. Dose Treatment (mg/kg) 0 Hour 1 Hour 2 Hour 3 Hour Negative Control (Normal Saline) 5 ml/kg 4.5±0.52 5.3±0.34 4.8±0.72 5.1±0.33 Positive Control (Diclofenac sodium) 5 mg/kg 4.8±0.60 8.2±0.40*** 6.2±0.26 5.3±0.34 100 mg/kg 4.7±0.28 5.4 ±0.38 5.1±0.28 4.5±0.25 200 mg/kg 4.8±0.36 6.2±0.52 5.8±0.27 5.1±0.32 400 mg/kg 4.8±0.38 7.3±0.42** 6.5±0.63* 5.8±0.47 SPM Values were expressed as Mean ± SEM, n=6, and by Two-way ANOVA followed by Bonferroni’s test ***P<0.001, * * P< 0.01, *P< 0.05 SPM (Scurrula parasitica methanol extract). tail withdrawal latancy (sec) Analgesic effect of methanol extract of S. parasitica using Heat conduction method 10 *** ** 8 Negative Control 100 mg/kg 200 mg/kg 400 mg/kg Diclofenac * 6 4 2 0 0 1 2 3 Animal Groups Fig. 2: Effect of Methanol Extract of S. parasitica Using Heat Conduction Method. STATISTICAL ANALYSIS All the values were statistically analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s test. Comparison between control and drug treated groups were considered to be significant. All values were expressed as mean ± SEM. RESULTS Oral administration of gradual doses (250, 500, 1000 and 2000 mg/kg p.o.) of the methanol extract of Scurrula parasitica to mice produce no significant changes in behavior. No mortality was however recorded for all the doses after 72 h of administration of the extract to the animal. Acetic-acid induced writhing in mice attributed visceral pain finds much attention of screening anti-nociceptive drugs [14]. The crude extract of the plant showed significant analgesic action compared to the reference drug Diclofenac sodium against acetic-acid induced pain (chemical stimulation) and heat conduction method (thermal stimulation) in mice at dose of 400 mg/kg body weight. An increase in reaction time and decrease in writhing are generally considered as important parameters of antinociceptive activity in heat conduction method and acetic-acid induced writhing test, respectively. Scurrula parasitica is feeding on its host; in the present study Scurrula parasitica is parasitic on Dendrophthoe falcata which is itself parasitic on Mangifera indica. So, it may conclude that chemical constituents of Dendrophthoe falcata are accountable for anti-nociceptive activity of RRJoP (2013) 4-8 © STM Journals 2013. All Rights Reserved Page 7 Research & Reviews: A Journal of Pharmacology Volume 3, Issue 1, ISSN: 2230-9861 __________________________________________________________________________________________ Scurrula parasitica due to their transfer from host to parasite [15]. CONCLUSION The methanol extract of Scurrula parasitica showed significant results for anti-nociceptive activity. As the hemi-parasite the plant get feeds on its host plant. Many of the primary as well as secondary metabolites may transfer from host to parasite. The anti-nociceptive effect of Scurrula parasitica may be due to the metabolites transfer from Dendrophthoe falcata. The present study may conclude that pharmacological activities of parasitic plants are depending on their hosts. If same parasite grown on different hosts then it may have different pharmacological actions or same pharmacological action with varying results. ACKNOWLEDGEMENT The authors would like to thank the Padmavathi College of pharmacy, Dharmapuri for providing the necessary facilities and support. The authors also wish to thank Appasaheb Birnale College of pharmacy, Sangali for encouraging and providing their scientific resources. REFERENCES 1. Balunas M. J et al. J. Life Sciences 2005; 78: 431–441p. 2. Goldman P et al. J. Annals of Internal Medicine 2001; 135: 594–600p. 3. Kuijt J et al. University of California Press, Berkeley 1969; 246p. 4. Elizabeth M. et al. 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