|Year : 2011 | Volume
| Issue : 2 | Page : 146-151
Tephrosia purpureaLinn leaves attenuate pain and inflammation in experimental animals
Vishal Gulecha1, Thangavel Sivakumar2, Aman Upaganlawar3, Rakesh Khandare3, Chandrashekhar Upasani3
1 Department of Pharmacology, SNJB'S SSDJ College of Pharmacy, Neminagar, Chandwad, Nashik; Department of Pharmacology, Nandha College of Pharmacy, Erode, Tamil Nadu, India
2 Department of Pharmacology, Nandha College of Pharmacy, Erode, Tamil Nadu, India
3 Department of Pharmacology, SNJB'S SSDJ College of Pharmacy, Neminagar, Chandwad, Nashik, India
|Date of Submission||18-Dec-2010|
|Date of Acceptance||05-Feb-2011|
|Date of Web Publication||23-Aug-2011|
Department of Pharmacology, SNJB'S SSDJ College of Pharmacy Neminagar, Chandwad, Nashik
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim : Present study evaluates the analgesic and anti-inflammatory activities of different fractions of Tephrosia purpurea using various experimental models. Materials and Methods: The analgesic activity of T. purpurea carried out using acetic acid-induced writhing in mice and tail flick test in rats. The anti-inflammatory activity was evaluated using carrageenan-induced rat paw edema and cotton pellet-granuloma formation in rats. The effects of the administration of reference standard (Ibuprofen and hydrocortisone) were also evaluated. Results: Five different fractions (TPI, TPII, TPIII, TPIV, and TPV) of T. purpurea at the dose level of 20 and 40 mg/kg, p.o. were tested. The fraction TPI (40 mg/kg, p.o.) and TPIII (40 mg/kg, p.o.) were found to be more effective in preventing carrageenan induced rat paw edema, cotton pellet granuloma formation and acetic acid-induced writhing; however, TPI (20 mg/kg, p.o.) and TPIII (20 mg/kg, p.o.) were found to be more effective in increasing latency period in tail flick method. Conclusions: Out of five different fractions of T. purpurea tested, TPI and TPIII possess potent analgesic and anti-inflammatory activities against different models of inflammation and pain.
Keywords: Acetic acid, carrageenan, granuloma formation, Tephrosia purpurea,tail flick
|How to cite this article:|
Gulecha V, Sivakumar T, Upaganlawar A, Khandare R, Upasani C. Tephrosia purpureaLinn leaves attenuate pain and inflammation in experimental animals. Int J Nutr Pharmacol Neurol Dis 2011;1:146-51
|How to cite this URL:|
Gulecha V, Sivakumar T, Upaganlawar A, Khandare R, Upasani C. Tephrosia purpureaLinn leaves attenuate pain and inflammation in experimental animals. Int J Nutr Pharmacol Neurol Dis [serial online] 2011 [cited 2020 Jan 21];1:146-51. Available from: http://www.ijnpnd.com/text.asp?2011/1/2/146/84205
| Introduction|| |
Chronic inflammatory diseases remain one of the world's major health problems. , Inflammation is the response of living tissues to injury. It involves a complex array of enzyme activation, mediator release, extravasations of fluid, cell migration, tissue breakdown and repair. , A large number of herbal drugs are presumed to have excellent medicinal value and are used for treating different types of inflammatory, arthritic, and pain conditions with considerable success. Scientific studies are, therefore, required to judge their efficacy and some of the medicinal properties popularly claimed, as well as other limitations to widen the scope of these drugs.
Tephrosia purpurea (TP) L. (Fabaceae), commonly known as "Sharapunkha" in Sanskrit, is a copiously branched, suberect, herbaceous perennial plant, which occurs through out the Indian.  Whole plant has been used to cure tumors, ulcers, leprosy, allergic, and inflammatory conditions such as rheumatism, asthma, and bronchitis.  The aqueous extract of Tephrosia purpurea seeds has shown significant in vivo hypoglycemic activity in diabetic rabbits  and the ethanolic extracts possess potential antibacterial activity. The flavanoids isolated from the plant has been reported to have antimicrobial activity.  It has also been reported to acquire hepatoprotective, mast cell stabilizing and erythrocyte membrane integrity enhancing effect in various animal models. , Phytochemical investigations on T. purpurea have revealed the presence of various phytoactive constituents such as glycosides, rotenoids, isoflavones, flavanones, chalcones, flavanols, flavones and sterols. 
No, scientific reports is observed till date regarding the effects of T. purpurea fractions on analgesic and anti-inflammatory activity. So, an attempt was made to screen the different fraction of TP for their analgesic and anti-inflammatory activities using different models in rats and mice.
| Materials and Methods|| |
The leaves of T. purpurea were collected from local region of Nashik, India in the month of July 2008. The plant material was identified and authenticated by Prof. P.G. Diwakar, Botanical survey of India, Pune and the Voucher No. BSI/WC/Tech/08/340.
Preparation of T. purpurea fractions
The extraction was carried out using pet ether initially, then it was allowed to evaporate slowly in shallow dish and resinous mass was discarded and yield obtained was 14.8 g (2.96%). The remaining material was further fractionated into acetone soluble and acetone insoluble portions. These two portions were dissolved in benzene and subjected to column chromatographic separation as detailed below:
Chromatographic separation of acetone soluble and insoluble part
For column chromatography Silica gel c was first activated at 150 °C for 3 h in an oven. After cooling, slurry was prepared in benzene, it was poured in glass column and set aside for 2 h. The residue of pet ether extract was dissolved in minimum volume of benzene, and it was mixed thoroughly with silica gel. It was air dried and charged into the column. The elution was carried out first with benzene (TPI) and successively eluted with ethyl acetate (TPII). The solvents were changed when portion of eluent showed absence of residue. The separation of acetone insoluble part was carried out as described above with benzene (TPIII) followed by ethyl acetate.
Preparation of fractions of alcoholic extract
The dried alcoholic extract was separated into water soluble and water insoluble portion (TPIV) using minimum quantity of distilled water. Only about 10% extract was soluble in water, which was vigorously shaken repeatedly with small volumes of ethyl acetate (TPV) till the ethyl acetate layer becomes colorless. On the preliminary phytochemical screening,  theses fractions gave positive tests for, flavonoids, glycosides, steroids, tannins, and saponins.
Albino rats of Wistar strain (150--200 g) and Swiss albino mice (25--30 g) of either sex were used in the entire study and were procured from Yash farm, Pvt.Ltd., Pune. They were housed in standard polypropylene cages and kept under controlled room temperature (24 ± 2 °C; relative humidity 60%--70%) in a 12 h light-dark cycle. The animals were fed with standard laboratory diet and water ad libitum. Food was withdrawn 12 h before and during the experimental hours. The experimental protocol was approved by Institutional Animal Ethical Committee.
Studies on anti-inflammatory activity
Rat paw edema induced by Carrageenan
The anti-inflammatory activity of these fractions (TPI, TPII, TPIII, TPIV, and TPV) was assessed using carrageenan induced rat paw edema. The fractions were suspended in 0.5% (w/v) gum acacia and were administered in doses of 20 and 40 mg/kg, p.o., respectively, 1 h prior to carrageenan injection (0.1 ml of 1% solution). The paw volume was measured at an interval of 0, 1, 2, and 3 h using Plethysmometer (UGO Basil, Italy, Model no. 7130). 
Granuloma formation induced by cotton pellet in rats
Male rats of 125--150 g weight were divided into seven groups containing five animals in each group. The cotton pellet weighing 50 ± 1 mg was sterilized in an autoclave (Lab Hosp., Mumbai, India) handled with sterile instrument. The pellet was inserted in each animal on the back. Control group received vehicle. Group II, III, IV, and V were treated with TPI (20 and 40 mg/kg, p.o.) and TPIII (20 and 40 mg/kg, p.o.) whereas group VI and VII were treated with reference standard, i.e.., hydrocortisone (30 mg/kg, p.o.) and ibuprofen (40 mg/kg, p.o.,) for consecutive 6 days. , The animals were sacrificed on seventh day and cotton pellet along with granuloma mass were collected, it was weighted and dried at 60 °C. Results of the assay were calculated as %inhibition of dry weight of granuloma formation by using the formula: 100(A -- B)/A, where A = gain in dry weight of control pellet (mg), B = gain in dry weight of drug treated (mg).
Studies on analgesic activity
Tail flick latency period in rats
Male rats of 125--150 g weight were divided into six groups containing five animals in each group. A tail flick response was evoked by placing each rat tail over the wire heated electrically, using Analgesiometer (Space Scientific, Nashik, India). The intensity of heat was adjusted so that baseline tail flick latency averaged 3--4 s in all animals. Cut-off time was 15 s in order to avoid injury to tail. The fractions TPI, TPIII, and reference standard ibuprofen were administered orally in their respective doses 1 h prior to the test. 
Acetic acid-induced writhing in mice
The writhing syndrome was elicited by intraperitoneal injection of acetic acid (0.1 ml of 0.6% solution) and numbers of writhes displayed from 5 to 20 min were recorded.  The fractions TPI, TPIII, and reference standard ibuprofen were administered orally in their respective doses 30 min prior to the test.
| Statistical Analysis|| |
Results of all the above estimations have been indicated in terms of mean ± SEM. Difference between the groups was statistically determined by analysis of variance (ANOVA) with Dunnett's test multiple comparisons test using GraphPad InStat version 5.00, GraphPad Software, CA, USA. The level of significance was set at P < 0.05.
| Results|| |
Effect of T. purpurea fractions on rat paw edema induced by carrageenan
Different fractions (TPI, TPII, TPIII, TPIV, and TPV) of T. purpurea were tested against carrageenan induced rat paw edema [Table 1]. The fractions TPI and TPIII of T. purpurea significantly (P < 0.01, P < 0.05) reduced the rat paw edema induced by carrageenan. TPI and TPIII (40 mg/kg, p.o.) reduced inflammation by 63.75% and 50%, respectively. However, other fractions (TPII, TPIV, TPV) showed mild inhibition to the paw edema induced by carrageenan during the three time points from 1 to 3 h.
|Table 1: Effect of T. purpurea fractions on rat paw edema induced by carrageenan|
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Effect of T. purpurea fractions on cotton pellet granuloma formation in rats
The effect of T. purpurea fractions on cotton pellet granuloma formation is shown in [Table 2]. Treatment with TPI and TPIII (40 mg/kg, p.o.) to rats showed a significant (P < 0.01) inhibition in the weight of cotton pellet in rats and the percentage inhibition was found to be 24.84 and 21.25, respectively. Treatment with the reference standard, i.e., hydrocortisone (30 mg/kg, p.o.) and ibuprofen (40 mg/kg, p.o.) also showed significant inhibition in cotton pellets granuloma formation as compared to control group.
|Table 2: Effect of T. purpurea fractions in granuloma formation induced by cotton pellet in rats|
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Effect of T. purpurea fractions on tail flick latency period
Treatment of TPI and TPIII (20 mg/kg, p.o.) significantly inhibited nociception in rats by 17.60% and 22.02%, respectively. Whereas, TP I and TPIII (40 mg/kg, p.o.) significantly inhibited pain perception by 16.58% and 12.53%, respectively. Ibuprofen treatment (40 mg/kg, p.o.) significantly inhibited pain perception by 27.92%.[Table 3]
Effect of T. purpurea fractions in acetic acid induced writhing in mice
[Figure 1] shows the effect of different fractions of T. purpurea against acid induced writhing in mice. It was observed that mice treated with TPI 20 (39.86%) and TPIII 20 (34.88%) shows significant (P < 0.01) protection compared to control group, however TPI 40 (49.16%) and TPIII 40 (48.50%) was found to be more significant (P < 0.01) in protecting acetic acid induced writhing compared to control group. Ibuprofen showed 56.14% protection against acetic acid induced writhing in mice.
|Figure 1: Effect of T. purpurea fractions in acetic acid induced writhing in mice. Data were analyzed using ANOVA and expressed as Mean ± SEM (N = 5) followed by Dunnett's test and differences between means were regarded signifi cant at * (P < 0.05), ** (P < 0.01)|
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| Discussion|| |
T. purpurea is a traditional herbal medicine, which has been used for the treatment of various kinds of inflammatory diseases including tumors, ulcers, leprosy, rheumatism, and bronchitis.  Analgesic and anti-inflammatory effects of flavonoids, steroids and tannins have been reported  hence the analgesic and anti-inflammatory effects produced by these fractions may be attributed individually or collectively to the flavonoids and steroids. However, its pharmacological actions and mechanisms have not been precisely documented in spite of its increasing usage recently. Present work reported the potential effects of the T. purpurea fractions, as an anti-inflammatory and analgesic agent using both in vivo and in vitro models.
Carrageenan-induced paw edema and cotton pellet granuloma formation in rats reflect the edematous stages during acute and chronic inflammation. , In the present study, five different fractions of T. purpurea were tested. Carrageenan induced rat paw edema has been a popular inflammatory model to investigate nonsteroidal anti-inflammatory effect of compounds  Serotonin, histamine, bradykinin, and prostaglandin have been identified as a mediators for carrageenan induced rat paw edema. 
Among these fractions only TPI and TPIII were found to posses a prominent anti-inflammatory activity, where as other fractions were gentle inhibition to the paw edema induced by carrageenan during the three time points from 1 to 3 h. In cotton pellet granuloma model, the fraction TPI and TPIII showed significant inhibition. The effectiveness of these fractions at 1 and 3 h in carrageenan induced paw edema indicates their antagonist effect at Serotonin, histamine, bradykinin and prostaglandin. Because the release of serotonin and histamine occurs 1 h after carrageenan whereas bradykinin and prostaglandin are released 2 and 3 h, respectively, after carrageenan injection.  The cotton pellet granuloma is a model of chronic inflammation, and dry weight has been shown to correlate with the amount of granulomatous tissue formed.  In the present study animals treated with TPI and TP III showed significant inhibition of granuloma formation. Both hydrocortisone and ibuprofen were found to be more effective in preventing granuloma formation compared to TPI and TPIII respectively.
Since inflammation is also associated with pain, majority of anti-inflammatory drug posses analgesic activity. The peripheral analgesic effect of drugs may be mediated through inhibition of cyclo-oxygenases and/or lipoxygenases (and other inflammatory mediators), while the central analgesic action may be mediated through inhibition of central pain receptors. This hypothesis is in line with previous reports e who have postulated that acetic acid-induced writhing and tail flick methods are useful techniques for evaluation of peripherally and centrally acting analgesic drugs, respectively. Present study also showed the effects of T. purpurea fractions on acetic acid-induce writhing and tail flick latency test. Treatment of TPI and TPIII (20 mg/kg, p.o.) significantly inhibited nociception. Whereas TP I and TPIII (40 mg/kg, p.o.) inhibited pain perception respectively in tail flick latency test and acetic acid-induce writhing. These results indicated TPI and TPIII fractions might produce the analgesic effect peripherally as well as centrally.
Flavonoids isolated from some medicinal plants have been proven to posses antinociceptive and/ or anti-inflammatory effects.  It has been shown by Meli et al., Dicarlo et al. that flavonoids also inhibit gastric motility in a dose - dependent, manner. It is therefore possible that the inhibitory effects on anti-nociceptive and anti-inflammatory effects observed in these fractions may be attributed in part to its flavonoid content. Flavonoids also inhibit the phosphodiesterases involved in cell activation.  Much of this effect is upon the biosynthesis of protein cytokines that mediates adhesion of circulating leukocytes to sites of injury. Flavonoids inhibit biosynthesis of prostaglandins, which are involved in various immunologic responses and are the end products of the cyclooxygenase and lipoxygenase parthways.  Protein Kinases are another class of regulatory enzymes affected by flavonoids. Inhibition of these enzymes provides the mechanism by which flavonoids inhibit inflammatory processes. ,
| Conclusions|| |
From the present study, it is concluded that T. purpurea fractions are capable of inhibiting inflammatory reactions as well as pain. The results provided experimental evidence for its traditional use in treating various diseases associated with inflammation and pain.
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[Table 1], [Table 2], [Table 3]
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