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Journal of Ethnopharmacology 132 (2010) 456460

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Anti-inammatory and analgesic properties of Drynaria quercifolia (L.) J. Smith


G.I. Anuja, P.G. Latha , S.R. Suja, S. Shyamal, V.J. Shine, S. Sini, S. Pradeep, P. Shikha, S. Rajasekharan
Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram 695 562, Kerala, India

a r t i c l e

i n f o

a b s t r a c t
Ethnopharmacological relevance: Drynaria quercifolia (L.) J. Smith (Polypodiaceae), has been widely used by ethnic groups of India to treat inammation, rheumatism, headache, bone fracture, jaundice, etc. Aim of the study: To evaluate the anti-inammatory and analgesic properties of the ethanolic extract of rhizome of Drynaria quercifolia (DQ) and its phytochemical prole. Materials and methods: DQ was used to evaluate the anti-inammatory and analgesic effects using carrageenan-induced paw oedema/cotton pellet-induced granuloma in Wistar rats and acetic acidinduced writhing/formalin-induced paw licking test in Swiss albino mice respectively. Results: Oral administration of DQ produced signicant inhibition of carrageenan-induced paw oedema and granuloma formation in rats, almost comparable to that caused by indomethacin. DQ signicantly attenuated acute and delayed phases of formalin-induced pain and acetic acid-induced writhing episodes in mice. The analgesia was comparable to that produced by sodium salicylate and aspirin respectively. Phytochemical analysis gave positive tests for catechin, coumarins, avonoids, phenolics, saponin, steroids, tannins, and triterpenes. The total phenolics in DQ was 244 mg/g and naringin content was 0.048%. Conclusion: The results suggest the presence of potent anti-inammatory and analgesic principles in DQ that justies its use for alleviating painful inammatory conditions. 2010 Published by Elsevier Ireland Ltd.

Article history: Received 1 October 2008 Received in revised form 9 August 2010 Accepted 16 August 2010 Available online 21 August 2010 Keywords: Analgesic Anti-inammatory Drynaria quercifolia Phytochemicals

1. Introduction Drynaria quercifolia (L.) J. Smith (Polypodiaceae) is an epiphytic medicinal pteridophyte, distributed widely in the evergreen forests of the Western Ghats of Kerala, locally called Marappannakizhangu or Attukalkizhangu. The rhizome is reported to be used by tribal communities of Tamil Nadu and Kerala to cure various diseases like phthisis, dyspepsia, cough (Caius, 1986). The leaves are used for poulticing swellings. The plant is used by the Nicobarese to treat body ache, headache and with other drugs in rheumatic pain (Dagar and Dagar, 1987). The whole plant of Drynaria quercifolia is anthelmintic, pectoral, expectorant and tonic, and is used to treat chest and skin diseases and loss of appetite (Koushik and Dhiman, 1995). According to Nadkarni (1976) and Dixit and Vohra (1984), the rhizome is used against typhoid fever. It is also used to treat jaundice and as a poultice and antifertility agent (Rajendran and Rajan, 1996) and antipyretic agent (Sajeev and Sasidharan, 1997; Khan et al., 2007). The tribals of Kolli hills of Tamil Nadu use the rhizome of the plant as an anti-inammatory agent (Irudayaraj and Senthamarai, 2004). The objective of the present study was to screen for anti-inammatory

and analgesic effects of Drynaria quercifolia rhizome ethanolic extract.

2. Materials and methods 2.1. Chemicals Acetyl salicylic acid, -amyrin, -sitosterol, carrageenan, indomethacin, and naringin were obtained from Sigma Chemicals Co., USA. Phosphotungstic acid and sodium salicylate were purchased from HiMedia, India and thiosol sodium from Neon Laboratory, India. Assay kits for serum alanine transferase (ALT), aspartate amino transferase (AST), and serum acid phosphatase were procured from Crest Biosystems, India. All the other chemicals were analytical and HPLC grade.

2.2. Plant material The rhizomes of Drynaria quercifolia were collected from the forest within the Institute, and authenticated by Dr Mathew Dan, plant taxonomist of the institute. A voucher specimen has been deposited at the Herbarium of the Institute (TBGT 57025 dtd 05/05/06).

Corresponding author. Tel.: +91 472 2869226; fax: +91 472 2869646. E-mail addresses: lathagopalakrishnan@yahoo.com, plathagopalakrishnan@gmail.com (P.G. Latha). 0378-8741/$ see front matter 2010 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2010.08.038

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2.3. Preparation of the plant extract The rhizomes of Drynaria quercifolia were washed thoroughly with tap water, shade-dried and powdered. The powder (100 g) was extracted with ethanol (1000 ml) for 24 h, at room temperature with constant stirring. The extract was ltered and the ltrate was concentrated at 30 C under reduced pressure in a rotary evaporator. The yield (w/w) of the crude extract was found to be 4%. The crude ethanolic extract (DQ) was suspended in 10% Tween-80 to required concentrations and used for the experiments. 2.4. Experimental animals Wistar rats (150250 g) and Swiss albino mice (2735 g) were housed in poly acrylic cages (two animals per cage) and maintained under standard laboratory conditions (temperature 2428 C, relative humidity 6070% and 12 h light/dark cycles). They were fed commercial rat feed (Lipton India Ltd, Mumbai) and boiled water, ad libitum. All experiments involving animals were done according to NIH guidelines, after getting the approval of the Institutes Animal Ethics Committee. 2.5. Anti-inammatory activity 2.5.1. Carrageenan-induced paw oedema in rats Anti-inammatory activity of DQ was assessed by the carrageenan-induced paw oedema method (Winter et al., 1962). Rats were divided into 5 groups of 6 animals each. Group 1 animals (carrageenan control, CC) received p.o., 10% Tween-80, 30 min prior to carrageenan injection, group 2, the standard group was given p.o., an aqueous solution of indomethacin (IM-10 mg/kg). Groups 3, 4 and 5 were given 125, 250, 500 mg/kg of DQ p.o., respectively, 30 min prior to carrageenan injection. The paw volume was measured plethysmographically, just before and 3 h after carrageenan injection. The percentage inhibition of oedema was calculated for each group with respect to the vehicle treated control group. 2.5.2. Cotton pellet granuloma formation in rats The effect of DQ on granuloma formation in rats was studied as described by Schiatti et al. (1986). Sterile cotton pellets (100 1 mg) were inserted in the inter-scapular region, on either side of a midline incision (1 cm) made on the dorsum of rats and incision closed by interrupted sutures. The extract DQ (125, 250, 500 mg/kg) was administered p.o., daily for 7 consecutive days starting from the day of pellet implantation. IM (10 mg/kg p.o.) was given to the standard group while the control group received p.o., an equal quantity of distilled water containing 10% Tween-80. The animals were sacriced on the 8th day, the cotton pellets removed, cleaned of extraneous tissue, weighed and dried in a hot air oven at 80 C for 24 h. Blood was collected for the estimation of different serum biochemical parameters like serum glutamate pyruvate transaminase (SGPT) and glutamate oxaloacetate transaminase (SGOT) as described by Reitman and Frankel (1957) and acid phosphatase and serum orosomucoid levels (Varley et al., 1980). 2.6. Analgesic activity 2.6.1. Acetic acid-induced writhing Analgesia of DQ was assessed by the writhing test in mice using the method of Koster et al. (1959). Mice were divided into 6 groups (6/group). All the groups received i.p., 0.5% aqueous solution of acetic acid. Group 1, the acetic acid control (AC) group received a single dose of 10% Tween-80 (0.5 ml) p.o., 20 min prior to the administration of acetic acid. Group 2, the standard control group received p.o., acetyl salicylic acid (AS100 mg/kg). Groups 3, 4 and 5 received p.o., 100, 200 and 300 mg/kg

of DQ respectively, 20 min prior to the administration of acetic acid. The number of writhes per animal was recorded during the 20 min period, beginning 5 min after the injection of acetic acid.

2.6.2. Formalin-induced paw licking in mice The method of Hunskaar and Hole (1987) was used for the study. Mice were divided into 6 groups of 6 animals each. Group 1, the control group received Tween-80, p.o., group 2, the standard group received sodium salicylate (SS-100 mg/kg). Groups 3, 4, and 5 received 100, 200, and 300 mg/DQ extract p.o., 30 min prior to formalin injection. 20 l of 1% formalin was injected into the dorsal surface of the left hind paw. The time spent licking the injected paw was recorded. Animals were observed for the 5 min post formalin (acute phase) or for 1 min starting at 20th min post formalin (delayed phase).

2.7. Phytochemical analysis of DQ DQ was subjected to preliminary phytochemical analysis and TLC studies, following the methodology of Harborne (1984).

2.7.1. Estimation of total phenolics in DQ The total phenolic content in DQ was determined by FolinCiocalteus method (Singleton and Rossi, 1965). A standard curve was prepared using different concentrations of gallic acid (10, 25, 50, 100, 110, 125, 150, and 200 mg/ml) in ethanol. To the above standard solutions of gallic acid (1 ml each), 5 ml FolinCiocalteus reagent (1:10 dilution in distilled water) was added and after 8 min, 4 ml of 7.5% sodium carbonate was added. These solutions were incubated at room temperature for 2 h and their absorbance at 765 nm was measured spectrophotometrically (UV-1650 PC, Shimadzu, Japan). For the test solution, 1 ml of DQ (1 mg in 10 ml ethanol) was added to 5 ml of FolinCiocalteus reagent. 4 ml of 7.5% sodium carbonate was added after 8 min and this was incubated at room temperature for 2 h and its absorbance was measured at 765 nm. The gallic acid equivalent (GAE) was plotted and the total phenolic content of DQ was calculated in terms of gallic acid equivalent.

2.7.2. High performance liquid chromatography (HPLC) analysis of DQ The HPLC analysis of DQ was carried out at the Department of Biotechnology of the Institute, following the methodology of Okpuzor et al. (2009) on HPLC system (Gilson, France) driven by a UNIPOINT system software and the chromatographic separations were performed using a C18 Kromacil Column (250 4.6 mm), with a ow rate of 1 ml/m and a sample size of 15 l. The mobile phase used was methanol, acetonitrile, and ammonium acetate buffer (5:11:4) and isocratic elution was performed. The sample was monitored with UV detection at 260 nm at the ow rate of 1 ml min1 at ambient temperature. Retention time for naringin was 2.1 min.

2.8. Behavioural and toxic effects Six groups of 10 mice each were administered p.o., 500, 1000, 2000, 3000, 4000, and 5000 mg/kg respectively of DQ, maintaining appropriate controls. The animals were observed continuously for 1 h for any gross behavioural changes, symptoms of toxicity and mortality if any and intermittently for the next 6 h and then again, 24 h after dosing with DQ.

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Fig. 1. Effect of varying doses of Drynaria quercifolia rhizome ethanolic extract (DQ)/IM (10 mg/kg) on carrageenan-induced paw oedema in rats (values are the mean S.D., n = 6. ANOVA **P 0.01 vs carrageenan control).

Fig. 2. Effect of different doses of Drynaria quercifolia rhizome ethanolic extract (DQ)/AS (100 mg/kg) on acetic acid-induced writhing response in mice (values are the mean S.D., n = 6. ANOVA **P 0.01 vs acetic acid control).

2.9. Statistical analysis Statistical comparison between control and treated groups was made using ANOVA, followed by multiple comparisons (Armitage and Berry, 1985).

3. Results 3.1. Anti-inammatory activity 3.1.1. Carrageenan-induced paw oedema The group treated with indomethacin showed maximum inhibition of oedema formation (88.24%). DQ at all the doses (125, 250 and 250 mg/kg) studied signicantly inhibited the carrageenaninduced paw oedema in rats in a dose dependent manner, i.e. 80.00%, 83.53%, and 85.88% respectively. The indomethacin group and DQ at 500 mg/kg showed almost equal amount of inhibition of oedema (Fig. 1).
Fig. 3. Effect of different doses of Drynaria quercifolia rhizome ethanolic extract (DQ)/SS (100 mg/kg) on the formalin test (acute and delayed phases) (values are the mean S.D., n = 6. ANOVA **P 0.01 vs vehicle control).

3.2. Analgesic activity 3.2.1. Acetic acid-induced writhing Intraperitoneal injection of acetic acid produced 54.0 2.0 writhes in the control group, 20 min after injection. The percent inhibition of writhing by groups pretreated with DQ (125, 250 and 250 mg/kg) and aspirin (100 mg/kg) was almost similar, i.e. 77.41%, 79.81%, 83.15%, and 88.33% respectively (Fig. 2). 3.2.2. Formalin test in mice SS did not show any signicant effect on the acute phase of the formalin test (40.48%), but DQ at 100, 200, and 300 mg/kg reduced the nociception in this phase in a dose dependent manner (52.06%, 56.29%, and 61.63%). In the delayed phase of formalin test, SS produced only 63.55% inhibition, while DQ reduced the nociception signicantly i.e. 77.48%, 90.60%, 93.87%, dose dependently. All the three doses of DQ were more potent than SS in this phase (Fig. 3).

3.1.2. Cotton pellet granuloma DQ produced a signicant dose dependent inhibition of granuloma formation. It was almost equally potent as indomethacin in inhibiting both exudative and proliferative phases of granuloma formation. At 500 mg/kg, DQ produced 55.56% inhibition in the exudative phase and 62.83% in the proliferative phase (Table 1). A signicant increase in SGPT, SGOT, acid phosphatase and orosomucoid levels was observed in the pellet implanted rats in comparison with normal control group. There was a dose dependent decrease in these parameters in the DQ/IM administered rats (Table 1).

Table 1 Effect of Drynaria quercifolia ethanolic extract (DQ)/indomethacin (IM) on granuloma tissue and serum parameters of cotton pellet-induced granuloma in rats. Treatment Dose (mg/kg) Granuloma tissue Wet wt (mg) Normal control Pellet control IM 10 DQ 125 DQ 250 DQ 500 990 414 519 510 440 5.0 4.5** (58.18) 7.6** (47.58) 5.3** (48.49) 2.3** (55.56) Dry wt (mg) 565 195 290 240 210 6.8 3.1** (65.49) 7.2** (48.67) 10.1** (57.52) 4.3** (62.83) 36.1 98.6 86.0 89.0 62.5 50.4 7.6 6.5 9.4** 5.6** 7.2** 6.4** 63.0 128.3 56.7 57.2 51.4 40.8 7.4 9.6 10.1** 4.3** 8.7** 5.1** 7.0 13.1 6.8 10.6 8.6 9.3 0.3 0.4 0.2** 0.1** 0.1** 0.2** 0.3 1.63 0.84 0.98 0.58 0.76 0.03 0.01 0.02** 0.04** 0.01** 0.02** SGPT (IU/l) SGOT (IU/l) Acid phosphatase (IU/l) Serum orosomucoid level (g/l)

Figures in parenthesis indicate % of inhibition of granuloma formation (values are the mean S.D., n = 6. ANOVA **P 0.01 vs pellet control).

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3.3. Behavioural and toxic effects In the acute toxicity study, no mortality occurred within 24 h with the six doses of DQ (500, 1000, 2000, 3000, 4000, and 5000 mg/kg) tested. The LD50 was therefore, greater than 5000 mg/kg p.o., in mice (data not shown). 3.4. Phytochemical screening of DQ Preliminary phytochemical analysis showed the presence of catechin, coumarins, avonoids, phenolics, saponin, steroids, triterpenes, and tannin in DQ. TLC studies of DQ revealed the presence of -amyrin, -sitosterol, and catechin. Total phenolic content of DQ was determined as 244 mg/g. The presence of the avanone glycoside, naringin in DQ was established by HPLC and quantied as 0.048% (data not shown). 4. Discussion Oral administration of DQ suppressed the oedematous response in a dose dependent manner, 3 h after carrageenan injection. The observed result could be compared to that caused by indomethacin. It is inferred that the inhibitory effect of DQ on carrageenan-induced inammation in rats may be due to inhibition of cyclooxygenase, leading to inhibition of prostaglandin synthesis, as reported elsewhere (Phadke, 1988). In experimental inammatory conditions, transaminase activities have been reported to be increased (De et al., 1994). In the present study, signicant increase was noted in the serum transaminase activities of cotton pellet implanted rats and the elevated levels were decreased signicantly by DQ, at all the dose levels studied. A marked increase was observed in the serum acid phosphatase level in pellet implanted rats, when compared to normal rats. The extract signicantly reduced the serum acid phosphatase activity at all the three doses studied. This indicates that DQ may have an enhancing effect on membrane stabilization or it may possibly be inhibiting the released factors. The effect of DQ on orosomucoid, one of the acute-phase proteins, observed in experimental inammation was used as a marker to assess the disease modifying anti-rheumatic drug (DMARD) potential of DQ. A marked increase in serum orosomucoid level was observed in cotton pellet implanted rats in comparison with normal rats. Treatment with DQ/IM led to a decrease in serum orosomucoid levels. These results indicate that DQ may have a DMARD effect. DQ was shown to possess anti-nociceptive activity to acetic acid at all the doses tested. Pretreatment with DQ reduced the number of acetic acid-induced writhes in mice signicantly. These writhes may be related to the increase in peritoneal uid level of PGE2 and PGF2 as postulated by Deraedt et al. (1980). The results of the present study indicate that the analgesic effect of DQ may possibly be by inhibiting synthesis or action of prostaglandin/cyclooxygenase/lipoxygenase. DQ was found to be effective in both the phases of formalin response, viz. acute and delayed (Hunskaar and Hole, 1987). Our results indicate the nociceptive effect of DQ in the rst phase of formalin test and propose that its activity partially results from the central action and the anti-inammatory effects of DQ extract proposes its anti-nociceptive effects in the second phase. Catechin, coumarins, avonoids, phenolics, saponin, steroids, and tannins were detected in DQ. The total phenolic content of DQ was very high (244 mg/g). TLC studies also helped to conrm the presence of -amyrin and -sitosterol, which is reported to reduce prostaglandin synthesis (Khan et al., 2007), which in turn helps to reduce inammation and pain. Khan et al. (2007) also reported that

Drynaria quercifolia rhizome signicantly reduced pyrexia in rabbit, induced by i.p. administration of boiled milk, due to the presence of -sitosterol in it. Flavonoids have been reported to have signicant antiinammatory activity in animal models (Narayana et al., 2001). We conrmed the presence of the avanone glycoside naringin, in DQ at a concentration of 0.048%, and this partially explains its anti-inammatory activity. Naringin is reported to be a strong antioxidant with anti-inammatory property (Pereira et al., 2007). However, detailed studies are warranted to decipher the exact nature and mechanism of action of the phytochemical compounds responsible for the anti-inammatory and analgesic effects of DQ. The acute toxicity study indicated that DQ is fairly nontoxic. This is not surprising, as Drynaria quercifolia is being used extensively by different ethnic groups of India to treat various ailments. Based on the present study, it can be concluded that Drynaria quercifolia rhizome ethanolic extract (DQ) has potent anti-inammatory and analgesic properties and thus support its claimed use by different ethnic groups of India for alleviating painful inammatory conditions and to treat rheumatic disorders. Acknowledgements Financial assistance from the Council of Scientic and Industrial Research (CSIR), New Delhi as Senior Research Fellowship to G. I. Anuja, is thankfully acknowledged. The authors also wish to thank Dr S. Ganeshan Director, TBGRI, for facilities, Mr. Gopan Raj S, Dr Satheesh Kumar K, and Mr. Binoy Jose, TBGRI for phytochemical assistance and Mr. S. Radhakrishna Pillai and Mr. K P Pradeep Kumar, TBGRI for technical assistance. References
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