Abstract
The aqueous extract of Rotula aquatica Lour (Boraginaceae) roots was investigated for its anti-inflammatory potential in acute and chronic inflammatory conditions in rats. The aqueous extract of the plant at doses 50, 100, and 200 mg kg−1, p.o., were screened against carrageenan-induced rat paw edema, cotton pellet-induced granuloma as well as crystal-induced inflammation in rats. The IL-6 levels in the exudates formed due to crystal-induced inflammation were also determined. The extract exhibited a statistically significant (p < 0.05) inhibition of rat paw edema as compared to the control group. With respect to crystal-induced inflammation, the extract demonstrated a statistically significant (p < 0.05) reduction in the neutrophil and monocyte count in the inflammatory exudates compared to the control group. The extract at a dose of 200 mg kg−1 also effectively inhibited IL-6 levels. The extract exhibited a statistically significant (p < 0.05) reduction in the weight of cotton pellet-induced granuloma at the doses of 100 and 200 mg kg−1 employed in the study compared to the control group. Supportive studies included determination of in vitro antioxidant potential of the extract by DPPH free radical scavenging method. The results revealed that the extract possesses appreciable anti-oxidant activity (IC50 = 11.07 μg mL−1). Collectively, the results indicated the extract of R. aquatica to have a potentially beneficial effect in relieving inflammation and providing a platform for the development of plant drugs for crystal-induced arthropathy.
Introduction
Rotula aquatica (R. aquatica) Lour (Boraginaceae) is a branched shrub, growing in the sandy and rocky beds of streams throughout India (CitationYoganarasimhan, 2000). Traditionally the roots of the plant are used as a diuretic and laxative. They are used in the treatment of piles, kidney stones and venereal diseases (CitationYoganarasimhan, 2000). In India, especially in the states of Kerala and Karnataka, R. aquatica is one of the most extensively used medicinal plants in the Ayurvedic system to dissolve urinary calculi and kidney stones (CitationSivarajan & Balachandran, 1994). R. aquatica has also demonstrated antimitotic (CitationPatil et al., 2004) and urolithitic (CitationChristina et al., 2002) properties.
The anti-inflammatory potential of R. aquatica has not yet been evaluated scientifically. Therefore, the present study investigated the anti-inflammatory activity in acute and chronic inflammatory conditions in rats.
Materials and methods
Plant material
R. aquatica roots were supplied by Zandu Pharma-ceutical Ltd., Mumbai, India and authenticated by Dr. V. Naik, Botanist, at Nicholas Piramal Research Centre, Mumbai, India, where a voucher specimen (no. 731) was deposited.
Preparation of the aqueous extract of R. aquatica roots
The roots were shade-dried and pulverized in a hammer mill. The root powder was passed through a 40-mesh sieve and then used for the extraction procedure. The powdered dried roots (50 g) were subjected to continuous Soxhlet extraction using 300 mL of distilled water for 48 h. The aqueous extract was evaporated in vacuo and the extractive yield was 9% w/w. The phytochemical screening (CitationKokate, 1994; CitationTrease & Evans, 1997) of the extracts revealed the presence of tannins, saponins, and reducing sugars.
The extracts were prepared as a suspension using 0.5% sodium carboxymethylcellulose solution (vehicle) for the efficacy studies.
Animals
Wistar rats of either sex, weighing 180-200 g (six animals per group), were used. They were kept in standard environmental conditions and fed with standard pellet diet and water ad libitum. All the experimental procedures and protocols used in this study were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of C.U.Shah College of Pharmacy (Mumbai, India).
Chemicals and materials
Carrageenan and DPPH (1,1-diphenyl-2-picrylhydrazyl) were purchased from Sigma Chemical Company (St. Louis, MO). Interleukin-6 (IL-6) ELISA kits were procured from Bender Med Systems (Vienna, Austria).
Acute toxicity studies
Acute toxicity studies were conducted on Wistar rats as per the Organization for Economic Co-operation and Development (OECD) 423 guidelines (CitationEcobichon, 1997). The aqueous extract of Rotula aquatica roots at doses of 5, 50, 300, and 2000 mg kg−1 body weight were administered to four groups of rats (n = 6) after overnight fasting.
The animals were observed twice on the day of the dosing and once daily thereafter for 14 days. Animals were observed daily for mortality and for gross changes in activity and behavioral pattern. They were also observed for the presence of tremors, convulsions, salivation, diarrhea, and lethargy.
Anti-inflammatory activity studies
Effect of R. aquatica on carrageenan-induced paw edema in rats
Acute inflammation was induced by injecting 0.1 mL of 1% w/v carrageenan into the sub-plantar region of the right hind paw of the rats. The rats were divided into five groups (n = 6). The aqueous extract of R. aquatica (50, 100, and 200 mg kg−1) were administered orally 1 h before the carrageenan injection (CitationWinter et al., 1962). The control animals received an equal volume (10 mL kg−1, p.o.) of vehicle, while ibuprofen (25 mg kg−1, p.o.) was used as the standard drug. The paw edema was measured with a micrometer (Michito, Japan) before and 3 h after the carrageenan injection.
Effect of R. aquatica on cotton pellet-induced granuloma in rats
Adsorbent cotton wool was cut into pieces weighing 20 mg and made into a pellet. The pellets were then sterilized in a hot air oven at 120°C for 2 h. Two pellets were implanted bilaterally in the axilla regions of the rat under light ether anesthesia and sterile technique (CitationSwingle & Shideman, 1972). The animals were divided into five groups (n = 6). Aqueous extract (50, 100, and 200 mg kg−1), prednisolone (5 mg kg−1) and vehicle (10 mL kg−1) were administered orally daily for 7 days. On day 8 after implantation, rats were anesthetized by ketamine hydrochloride (50 mg kg−1, intraperitoneally) and the implanted pellets were dissected and dried overnight at 60°C. The dried pellets were weighed and percentage inhibition of granuloma was determined.
Effect of R. aquatica on crystal-induced inflammation in rats
Rat air pouches were formed subcutaneously by injecting 20 mL of sterile air into the back of the animals, after anesthetizing the animals with ketamine hydrochloride (50 mg kg−1 i.p.). Three days later, 10 mL of filtered air was reinjected to keep the pouch inflated. On the sixth day, inflammation was induced in the air pouches by injecting monosodium urate monohydrate crystals (15 mg mL−1) in sterile saline (CitationLiote et al., 1996). The animals were divided into five groups (n = 6). The aqueous extract of R. aquatica (50, 100, and 200 mg kg−1), colchicine (0.1 mg kg−1) and vehicle (10 mL kg−1) were administered orally 1 h prior to introduction of crystals into the pouches. After 24 h of crystal administration the exudates were subjected to total white blood cell count and differential white blood cell count.
Effect of R. aquatica on IL-6 levels of the aspirated exudates
The aspirated exudates collected from the above groups were subjected to cytokine IL-6 assay using enzyme- linked immunosorbent assay (ELISA) kits according to the recommended procedure (Bender Med Systems, Vienna, Austria). The IL-6 levels were measured by pipetting 50 μL of sample and 100 μL of standard diluent buffer into the wells of a microtiter plate coated with an antibody specific to rat IL-6. After adding biotin- conjugate to all the wells, the plate was incubated at 18-25°C for 2 h. Streptavidin peroxidase HRP was then added and incubated for 30 min to bind to the biotinylated antibody. After two more washings with assay buffer to remove unbound enzymes, color was developed by adding stabilized chromogen tetramethylbenzidine, and a stop solution. Finally, the optical density was calculated with an Elisa microplate reader at 450 nm and the IL-6 was quantified by comparing the sample to the standard curve generated from the kit. The results were expressed as cytokine concentrations (pg/mL).
Effect of R. aquatica on DPPH radical scavenging activity
DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging activity was measured by the spectrophotometric method (CitationSoni et al., 2003). To a methanol solution of DPPH (200 μM), test extracts of R. aquatica dissolved in distilled water were added at different concentrations (4-25 μg/mL). After 30 min, the decrease in absorbance of test mixtures (due to quenching of DPPH free radicals) was read at 517 nm and the percentage inhibition was calculated. Ascorbic acid was used as a reference free radical scavenger.
Statistical analysis
The results were expressed as mean ± SEM. The data were analyzed for statistical significance by one way analysis of variance (ANOVA) followed by Dunnett’s t-test for the comparison with the control group. Bonferroni t-test was performed for the pairwise comparison between the extract and reference standard. The difference was considered to be significant at 5% level (p < 0.05).
Results
Acute toxicity studies
Acute toxicity studies revealed that R. aquatica extract was safe at all doses when administered orally to rats, up to a dose of 2000 mg kg−1. No mortality was observed during the 14 days of the observation period.
Effect of R. aquatica on carrageenan-induced paw edema in rats
The aqueous extract of R. aquatica (50, 100, and 200 mg kg−1, p.o.) produced a significant (p < 0.05) inhibition of carrageenan-induced paw edema as compared to the control animals (). The aqueous extract at doses 50, 100, and 200 mg kg−1 demonstrated a statistically significant (p < 0.05) inhibition of edema to the extent of 26.82, 28.56, and 34.64%, respectively as against ibuprofen (25 mg kg−1, p.o.) that showed a significant (p < 0.05) inhibition of edema to the extent of 61.78%.
Table 1. Effect of aqueous extract of R. aquatica roots on carrageenan-induced paw edema in rats.
Effect of R. aquatica on cotton pellet-induced granuloma in rats
The aqueous extract (100 and 200 mg kg−1, p.o.) administered daily for 7 days significantly inhibited the cotton pellet-induced granuloma as compared to control animals. The extract at the dose of 50, 100, and 200 mg kg−1 showed significant (p < 0.05) inhibition of granuloma to the extent of 17, 23, and 26%, respectively, as against the reference standard prednisolone (5 mg kg−1, p.o.) that showed significant (p < 0.05) inhibition of granuloma to the extent of 55% ().
Table 2. Effect of aqueous extract of R. aquatica roots on cotton pellet induced granuloma in rats.
Effect of R. aquatica on crystal-induced inflammation in rats
The aqueous extract of R. aquatica (200 mg kg−1, p.o.) exhibited a statistically significant (p < 0.05) reduction in total white blood cell count (WBC) to the extent of 13.33% as compared to the control animals (). The extract at a dose of 200 mg kg−1 also showed a statistically significant reduction (p < 0.05) in total neutrophil and monocyte cell count to the extent of 20 and 62.11%, respectively, as compared to control group. Colchicine (0.1 mg kg−1, p.o.) demonstrated significant (p < 0.05) reduction in the total white blood cell count, neutrophils, and monocyte count to the extent of 29.96, 19.52, and 56.28%, respectively.
Table 3. Effect of aqueous extract of R. aquatica roots on crystal induced inflammation in rats at 24 h.
Effect of R. aquatica on IL-6 levels of the aspirated exudates
The aqueous extract of the plant at 100 and 200 mg kg−1 showed a statistically significant (p < 0.05) decrease in IL-6 levels of the exudates to the extent of 40 and 60%, respectively. However, colchicine demonstrated a significant (p < 0.05) reduction in IL-6 levels to the extent of 80% ().
Table 4. Effect of aqueous extract of R. aquatica roots on IL-6 levels in the rat pouch exudates at 24 h.
Effect of R. aquatica on DPPH radical scavenging activity
The aqueous extract of R. aquatica showed an appreciable free radical scavenging effect in the DPPH assay with an IC50 of 11.07 μg/mL. Ascorbic acid, which is a known antioxidant, demonstrated an IC50 of 1.07 μg/mL.
Discussion
In the present study, the anti-inflammatory activity of the aqueous extract of R. aquatica has been presented. The extract was found to significantly inhibit carrageenan- induced paw edema in rats, a test which has significant predictive value for anti-inflammatory agents acting by inhibiting the mediators of acute inflammation. Carrageenan-induced inflammation is useful in detecting orally active anti-inflammatory agents. Edema formation due to carrageenan in the rat paw is a biphasic event. The initial phase is attributed to the release of histamine and serotonin. The second phase of edema is due to the release of prostaglandins, proteases, and lysosomes (CitationOlajide et al., 2000).
In our experimental investigation we have tried to establish the efficacy of the aqueous extract of R. aquatica in cotton pellet-induced granuloma as well as in crystal-induced inflammatory conditions. The inflammatory granuloma is a typical feature of established chronic inflammatory conditions (CitationIsmail et al., 1997). The dry weight of the pellets was found to correlate well with the amount of granulomatous tissue (CitationSwingle & Shideman, 1972). The efficacy of the aqueous extract R. aquatica in our study may be due to the reduction in the number of fibroblasts and synthesis of collagen and mucopolysaccharide, which are natural proliferative events of granulation tissue formation (CitationDunne, 1990; CitationIonac et al., 1996; CitationSethuraman et al., 1984).
Depositions of monosodium urate crystals play a vital role in the pathogenesis of acute and chronic articular syndromes, which cause inflammatory responses (CitationBryan & Liote, 2005). In crystal-induced inflammation, the extract demonstrated marked reduction in total white blood cell count. This is attributed to the decrease in total neutrophil and monocyte count indicative of its anti-inflammatory activity.
Pro-inflammatory cytokines, such as IL-1, IL-6, IL-8 and TNF-α, are induced on interaction of monosodium urate crystals with monocytes. These proinflammatory cytokines have been measured in the pouch exudates of rats induced by injection of the monosodium urate crystals. IL-6 is a pluripotent mediator of crystal- induced inflammation (CitationBryan & Liote, 2005; CitationNishimoto & Kishimoto, 2004). The overproduction of IL-6 is a key feature observed in the pathology of crystal-induced inflammation and leads to massive infiltration of neutrophils and monocytes within the articulation. This leads to further enhancement of the inflammatory response (CitationBryan & Liote, 2005). Thus, IL-6 is a putative marker in the crystal-induced inflammation model and the effect of extract on the IL-6 levels in the exudates was determined. The extract of R. aquatica exhibited a reduction in IL-6 in the pouch exudates of the rats, demonstrating its activity in crystal-induced inflammation.
The role of oxidants in inflammatory reactions has been amply demonstrated. In inflammation, infiltration of leukocytes and other phagocytic cells readily defend the host from further injury. They do this by releasing powerful oxidant mixtures of NO, O2− and H2O2 (CitationChoi & Hwang, 2003). Antioxidants are known to inhibit the actions of some oxidants generated in inflammation (CitationOmisore et al., 2004). In the present study, the extract demonstrated a significant free radical scavenging effect as was evidenced by DPPH scavenging assay. The phytochemical analysis of R. aquatica revealed the plant to be rich in polyphenols (tannins), which could be the plausible explanation offered for its antioxidant activity (CitationFenglin et al., 2004).
The results of the study demonstrate the anti-inflammatory potential as well as anti-oxidant activity of the aqueous extract of R. aquatica and provide a rational and scientific basis for the selection of the plant in the management of crystal-induced arthropathy.
Acknowledgement
The authors are thankful to the Department of Biotechnology (DBT), New Delhi, India for providing financial assistance.
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