Av

SMa

b

a

ARR1AA

KAAAAT

1

tto(egaoa

nti

(

0d

Journal of Ethnopharmacology 138 (2011) 741– 747

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

journa l h o me page: www.elsev ier .com/ locate / je thpharm

nti-inflammatory and anti-nociceptive activities of ethanolic extract and itsarious fractions from Adiantum capillus veneris Linn.

aqlain Haidera, Syed Nazreena, Mohammad Mahboob Alama, Amit Guptab, Hinna Hamida,ohammad Sarwar Alama,∗

Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi 110 062, IndiaIndian Institute of Chemical Technology, Hyderabad 500007, India

r t i c l e i n f o

rticle history:eceived 18 June 2011eceived in revised form9 September 2011ccepted 10 October 2011vailable online 15 October 2011

eywords:diantum capillus veneris Linn.diantaceaenti-inflammatorynti-nociceptive activity

a b s t r a c t

Aim of the study: To investigate the anti-inflammatory and anti-nociceptive activities of the crude ethano-lic extract of Adiantum capillus veneris Linn. (Adiantaceae) and its various fractions.Materials and methods: The ethanolic extract and its fractions were given at a dose of 200 mg/kg po and300 mg/kg po for testing their anti-inflammatory activity by carrageenan induced hind paw edema. Theanalgesic activity of the ethanolic extract and its fractions has been carried out by tail-flick method andwrithing test at a dosage of 300 mg/kg po. Gastric ulceration studies have been further carried out tostudy the antiulcer effect of the ethanolic extract and its various fractions at dose of 900 mg/kg bodyweight.Results: Amongst the tested fractions, the ethyl acetate fraction exhibited better inhibition (67.27%) at300 mg/kg po dosage when compared to the standard drug Indomethacin (63.63%) after 3 h in the car-rageenan induced hind paw edema. The anti-inflammatory activity of the ethanolic extract and its various

umor necrosis factor-� fractions appear to be related to the inhibition of NO release, and the decreasing TNF-� level. The ethano-lic extract and all its fractions especially the ethyl acetate (p < 0.01) showed significant analgesic activitywith insignificant ulceration as compared to the standard drug, i.e. ibuprofen. The histopathological studyof ethanolic extract and its fractions reveals that none of them cause ulcer.Conclusion: The present study indicates that Adiantum capillus veneris Linn. has significant anti-inflammatory and analgesic effect.

. Introduction

Adiantum is a large genus of ferns which are widely distributedhroughout the world. Ethno medicinally, the genus is impor-ant and popularly known as “Hansraj” in the Ayurvedic Systemf Medicine. About nine species of Adiantum are found in IndiaMohini et al., 1990). A. capillus veneris Linn. has been shown toxhibit antimicrobial activity against different bacterial and fun-al strains (Singh et al., 2007). This genus has been used as tonic

nd diuretic; in treatment of cold, fever, cough and bronchial dis-rders, as stimulant, emollient, purgative, demulcent, general tonicnd hair tonic, in addition to skin diseases, tumors of spleen, liver

Abbreviations: NO, nitric oxide; LPS, lipopolysaccharide; NED, N (1-aphthyl ethylene diamine dihydrochloride); PBS, phosphate buffer saline; TMB,ri-methyl benzidine; TNF-�, tumor necrosis factor-�; NSAIDS, non-steroidal anti-nflammatory drugs; NOS, NO synthase.∗ Corresponding author. Tel.: +91 11 26059688x5555; fax: +91 11 26059663.

E-mail addresses: msalam@jamiahamdard.ac.in, msalam747@yahoo.co.inM.S. Alam).

378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2011.10.012

© 2011 Elsevier Ireland Ltd. All rights reserved.

and other viscera (Singh et al., 2008) and in treatment of jaundiceand hepatitis (Abbasi et al., 2009). A. capillus veneris Linn. is tra-ditionally used in the Unani system of medicine for the treatmentof inflammatory diseases (Kabeeruddin, 1937; Farah et al., 2005).Chemical analysis of A. capillus veneris Linn. reveals an array of com-pounds including triterpenes, flavonoids, phenylpropanoids andcarotenoids (Berti et al., 1969; Takahisa et al., 1999). Khare (1996)and Vasudeva (1999) have described the use of Adiantum species forthe treatment of various diseases. On the basis of the common usesof this plant in traditional folk medicine, the present investigationwas carried out to evaluate the anti-inflammatory and antinoci-ceptive potential of Adiantum capillus veneris Linn. in experimentalanimal models.

2. Materials and methods

2.1. Plant preparation and ethanolic extraction

The fronds of Adiantum capillus veneris Linn. were collected froma nursery in Ashok Vihar area of New Delhi in the month of April.

7 ophar

TCw(pefaa(

2

CwwtTIC

2

lpCm

2

aTbCte1cmioHtw

2

0tItowoip

2

pa

42 S. Haider et al. / Journal of Ethn

he plant was authenticated by Dr. H.B. Singh, taxonomist, NISCAIR,SIR, New Delhi (Voucher Number 2009-10/1199/03). The plantas shade dried and powdered. It was then extracted with ethanol

95% v/v) in a Soxhlet apparatus and dried under vacuum at a tem-erature of 45 ◦C in a rotary evaporator. The yield of the ethanolicxtract was (11.43% w/w). The extract was then fractionated by dif-erent solvents of increasing polarity using petroleum ether, ethylcetate and methanol. The yields of petroleum ether, ethyl acetatend methanolic fractions were 35% (w/w), 31.25% (w/w) and 33.75%w/w) respectively.

.2. Animals

Albino Wistar rats of either sex (150–200 g) were obtained fromentral Animal House, Hamdard University, New Delhi. The animalsere kept in cages at the room temperature and fed with food andater ad libitum. Fourteen hours before the start of the experiment

he animals were sent to lab and fed only with water ad libitum.he experiments were performed in accordance with the rules ofnstitutional Animals Ethics Committee (registration number 173-PCSEA).

.3. Drugs

Indomethacin, Ibuprofen, Carrageenan, Carboxymethylcellu-ose, N (1-naphthyl ethylene diamine dihydrochloride), glutamine,enicillin, streptomycin were purchased from Sigma–Aldrichhemicals Pvt. Limited, Bangalore, India. ELISA kits for the deter-ination of TNF-� were provided by e-Bioscience (San Diego, CA).

.4. Toxicity study

The selected albino rats were used to determine the dose. Thenimals were fasted overnight before the start of the experiment.he animals were divided in to six groups of six in each. The kar-ers method (Kale and Kale, 1994) was used to determine the dose.arboxymethylcellulose (1% w/v) was used as vehicle to suspendhe extracts and administered orally. The other group received thextract in one of the following doses – 50, 100, 200, 300, 500 and000 mg/kg. Immediately after dosing, the animals were observedontinuously for first four hours for behavioral changes and forortality at the end of 24 h, 48 h and 72 h respectively. The tox-

city study showed that the ethanolic extract at a minimum dosef 300 mg/kg onwards shows the reaction in experimental animals.owever, no mortality was reported even after 72 h. This indicates

hat the ethanolic extract is safe up to a single dose of 3 g/kg bodyeight.

.5. Anti-inflammatory activity

The rat paw edema was induced by subcutaneous injection of.1 mL of 1% freshly prepared saline solution of carrageenan intohe right hind paw of rats (Winter et al., 1962). The standard drugndomethacin (20 mg/kg po) was given orally as a positive con-rol. The control group was administered orally with 0.9% of 0.1 mLf saline solution only. The test groups were administered orallyith the ethanolic extract and its various fractions at the dosages

f 200 and 300 mg/kg body weight, one hour before the admin-stration of carrageenan. The paw volumes were measured usinglethysmometer (Ferreira, 1979) at interval of 3 h and 5 h.

.6. Isolation of peritoneal macrophages

Balb/C mice were injected i.p. with 10 mL (ice cold) of incom-lete medium RPMI containing antibiotic and antimycotic mixtures well as heparin (5 U/mL) or ice cold PBS. The abdomen was

macology 138 (2011) 741– 747

gently massaged and peritoneal cells were lavaged out in tubes.The peritoneal cells were washed thrice with incomplete RPMI bycentrifugation at 1000 rpm, 10 min in cold and finally suspendedat 2 × 106 cells/mL in complete RPMI containing 10% FCS (heatinactivated). 100 �L cell suspension/well were dispensed in trip-licates using a 96 well flat-bottomed culture plate. Macrophageswere allowed to adhere to the bottom of the wells at 37 ◦C for24 h in CO2 (5%) incubator. Non-adherent cells were removed andmacrophages were stimulated by LPS (10 �g/mL) in 100 (l volumefor 24 h incubation at 37 ◦C in CO2 incubator.

2.6.1. The estimation of release of nitric oxide (NO) ex vivoNitrite estimation was done by Griess reagent for assaying the

macrophage function in the supernatant of unstimulated and stim-ulated macrophages (Anamika et al., 2007). Griess reagent wasprepared by mixing N (1-naphthyl ethylene diamine dihydrochlo-ride) (NED) (0.1%) in distilled water and sulfanilamide (1%) in 5%phosphoric acid in equal volume (prepared fresh). 50 �L of testsupernatant from macrophages was added to 96 well microtitreplate in triplicate with complete medium as blank. 50 �L of sul-fanilamide solution was added and incubated for 5 min and then50 �L of NED was added to reaction mixture in each well. Platewas kept for 5 min at room temperature for colour developmentand stabilization. Absorbance at 550 nm was measured using anELISA reader. A standard curve was generated using sodium nitriteto quantitate the test results. Results are expressed in (M.

2.7. Analysis of cytokines in spleen cell culture supernatant(TNF-˛) ex vivo

Spleens from Balb/C mice were removed aseptically and sin-gle splenocyte suspension in RPMI 1640 containing 10% FBS, 1 MHEPES, 2 mM glutamine, 100 U/mL penicillin and 100 mg/mL strep-tomycin was obtained by passage through a stainless mesh. Redblood cells were lysed with lysis buffer for 5 min. After washingwith PBS, cells were cultured in 96-well plates at 2 × 106 cells/mLand cytokines were then measured from the supernatants by ELISA.The assay was performed according to the manufacturer’s instruc-tion with multipoint analysis (Malik et al., 2007).

Briefly, 100 �L of diluted capture antibody was added to eachwell in a 96 well plate and was allowed to adhere overnight for 4 ◦C.Plates were washed and then blocked with 1× PBS supplementedwith 10% FBS for 1 h at room temperature. After washing, serialdilutions of the standard and samples were prepared in the platesand were then incubated for 2 h at room temperature. Then, plateswere washed and working detector solution (including detectorantibody and avidin–horse radish peroxidase reagent) was addedinto each well. Plates were then sealed and incubated for 1 h at roomtemperature. After washing, 100 �L of tri-methyl benzidine (TMB)substrate was added into each well. Stop solution (2 N H2SO4)was finally added after incubation in the dark for 30 min at roomtemperature. The absorbance was read at 450 nm. The result wasanalyzed using softmax program and values determined againstthe standard provided by the manufacturer.

2.8. Antinociceptive activity

2.8.1. Tail immersion methodIn the present study analgesia was assessed by employing tail

immersion method (Distasi et al., 1988). Prior to the experiment theanimals were screened for the sensitivity test by immersing the tailof the rats gently in hot water maintained at 55 ◦C (Agrahari et al.,

2010). The animals flicking their tail from hot water in 5 s wereselected for the study in order to avoid any thermal injury to thetail. The selected rats were then divided into six groups of six ratseach. The control group was administered orally with 0.9% of 0.1 mL

ophar

ooabwc

2

KiiToupta

2

idup9ta

2

teR

3

3

aiiihHodawI

3

fmiecac(e

S. Haider et al. / Journal of Ethn

f saline solution only. The standard drug, i.e. ibuprofen was givenrally at a dose of 20 mg/kg body weight. The ethanolic extractnd its various fractions were given orally at a dose of 300 mg/kgody weight. After administration of the drugs, the reaction timeas measured at 30, 60 and 120 min. The basal reaction time was

alculated as the reaction time prior to the drug administration.

.8.2. Writhing testThe writhing test in mice was carried out using the method of

oster et al. (1959). The writhes were induced by intraperitonealnjection of 0.6% acetic acid (v/v) (80 mg/kg). The standard drug,.e. ibuprofen was given orally at a dose of 20 mg/kg body weight.he ethanolic extract and its various fractions were administeredrally to groups of five animals each, 30 min before chemical stim-lus. The numbers of muscular contractions were counted over aeriod of 20 min after acetic acid injection. The data represents theotal number of writhes observed during 20 min and is expresseds writhing numbers.

.9. Ulcerogenic activity

The ethanolic extract and its various fractions having anti-nflammatory & analgesic activities comparable with the standardrugs were further tested for their anti-ulcerogenic activity. Thelcerogenic activity was done at three times higher dose in com-arison to the dose used for anti-inflammatory activity, i.e. dose of00 mg/kg body weight and 60 mg/kg body weight of tested frac-ions and standard respectively were used. Each group had threenimals which were later sacrificed (Fig. 1 ).

.10. Statistical analysis

Data was analyzed by one way ANOVA followed by Dunnett’s ‘t’est (n = 6), *p < 0.05, **p < 0.01 significant from control; ***p < 0.001xtremely significant from control; ns, not significant (Sokal andohlf, 1995). It is shown in Tables 1 and 2.

. Results

.1. Anti-inflammatory activity

The results of the anti-inflammatory activity of ethanolic extractnd its various fractions of Adiantum capillus veneris Linn. are shownn Table 1. The anti-edema activity is expressed as “mean increasen paw volume ± SEM” in terms of mL and percentage inhibitionn paw volume by different doses of the extract. All the extractsave shown optimal anti-edema activity at 300 mg/kg dosages.owever they exhibited moderate anti-edema activity at dosagef 200 mg/kg. Amongst these fractions ethyl acetate fraction pro-uced significant inhibition of paw edema at 300 mg/kg dosages its p value is extremely significant (p < 0.001) at the end of 3 hith an inhibition of 67.27% as compared to the standard drug

ndomethacin (63.63%).

.2. Ex vivo TNF- ̨ and NO activity

We also examined the effect of ethanolic extract and its variousractions on the level of TNF-�, in LPS-treated mouse peritoneal

acrophages. The results are shown in Table 2. The reductionn the amount of TNF-� in the serum of the mice treated withthyl acetate fraction at a dosage of 100 �g/mL is highly signifi-ant (p < 0.001). The rest of the fractions also exhibited inhibitory

ctivity in LPS-induced TNF-� production. Inhibition was signifi-ant with methanol fraction at doses of 50 �g/mL and 100 �g/mLp < 0.01). The petroleum ether fraction at a dose of 100 �g/mL alsoxhibited significant inhibition of TNF-� (p < 0.01). Macrophages

macology 138 (2011) 741– 747 743

released low levels of NO, although, upon stimulation with LPS, theproduction of NO increased markedly (Fig. 2). Indomethacin is usedas standard.

3.3. Antinociceptive activity

The results of antinociceptive activity by the tail immersionmethod are shown in Table 3. The analgesic activity is expressedas “mean increase in latency after drug administration ± SEM” rel-ative to controls. Since all the fractions show optimal anti-edemaactivity at 300 mg/kg dosage, the same dosage is used for anal-gesic studies. The results of analgesic studies revealed that the ethylacetate fraction (3.48 ± 0.27 min, p < 0.01) showed significant anal-gesic activity after 120 min. The petroleum ether fraction exhibitedmoderate analgesic (3.19 ± 0.26 min, p < 0.05) effect along withmethanolic fraction (3.23 ± 0.35 min, p < 0.05) and ethanolic extract(3.22 ± 0.41 min, p < 0.05) respectively, after 120 min. The resultsof the writhing test are shown in Table 4. The ethyl acetate frac-tion showed significant protection (47.8%, p < 0.001) as comparedto ibuprofen (47.6%, p < 0.001).

3.4. Ulcerogenic activity

Very low power (4×) photomicrograph of stomach wall fromibuprofen group animals showed ulceration (six ulcers) of themucosa involving the upper half of the mucosa. High power (40×)photomicrograph of the same section showed the inflammatorycells in the sub mucosa. High power photomicrograph (40×) ofstomach wall from same group of animals showed intact surfacelining epithelium and superficial glands in the stomach mucosa.Low power photomicrograph (10×) of stomach wall from ethylacetate group animals showed very superficial ulceration of themucosal layer of the stomach. High power photomicrograph (40×)of stomach wall from ethyl acetate group animals showed theloss of epithelial cells from the superficial layer of the stomachmucosa. The ethanolic extract and its fractions did not cause anymucosal or epithelial gastric damage. The active ethyl acetate frac-tion induced only surface epithelial damage but that too is less thanthat induced by the standard drug, i.e. ibuprofen. When comparedwith ibuprofen, these fractions did not cause any gastric ulcerationand disruption of gastric epithelial cells at the above mentionedoral dose. Hence gastric tolerance towards the ethanolic extractand its fractions was better than that of ibuprofen indicating thatcarboxylic group present in ibuprofen is responsible for ulceration.

4. Discussion

Exploring the healing power of plants is an ancient concept andfor centuries people have been trying to reduce the side effects anddevelop effective anti-inflammatory agents using different plantextracts and formulations (Cowan, 1999). People are getting moreinclined towards natural sources of drugs because the non-steroidalanti-inflammatory drugs (NSAIDS) present in the market induceseveral side effects like gastric ulcer (Alsarra et al., 2010) and hep-atotoxicity (Tan et al., 2007).

Carrageenan-induced hind paw edema is the standard experi-mental model of acute inflammation. Carrageenan is the phlogisticagent of choice for testing anti-inflammatory drugs as it is notknown to be antigenic and is devoid of apparent systemic effects(Vadivelan et al., 2007). Moreover, the experimental model exhibitsa high degree of reproducibility (Winter et al., 1962). Carrageenaninduced edema is a biphasic response. The first phase is mediated

through the release of histamine, serotonin and kinins whereasthe second phase is related to the release of prostaglandin andslow reacting substances which peak at 3 h (Vinegar et al., 1969).In the present study, ethyl acetate fraction has shown better

744 S. Haider et al. / Journal of Ethnopharmacology 138 (2011) 741– 747

Fig. 1. Histopathology of rat stomach in Albino Wistar rats. Low power (10×) and high power photomicrographs (40×) of stomach wall of the animal groups administeredwith ethanolic extract, petroleum ether, ethyl acetate, methanol fractions, standard drug ibuprofen and control respectively.

S. Haider et al. / Journal of Ethnopharmacology 138 (2011) 741– 747 745

Fig. 1. (Continued).

Table 1Anti-inflammatory activity of Adiantum capillus veneris extract and its various fractions.

Group Dose (mg/kg po) Change in paw volume (ml) Mean ± SEM % Inhibition

3 h 5 h 3 h 5 h

Control 2 mL/kg 0.550 ± 0.105 0.550 ± 0.105 – –Indomethacin 20 0.200 ± 0.036** 0.216 ± 0.030** 63.63 57.00Ethanolic extract 200 0.283 ± 0.040* 0.300 ± 0.036* 48.54 43.39

300 0.266 ± 0.049* 0.216 ± 0.030** 51.50 47.90Petroleum ether fraction 200 0.300 ± 0.057* 0.28 ± 0.060* 45.45 47.16

300 0.233 ± 0.033** 0.216 ± 0.030** 58.80 47.90Ethyl acetate fraction 200 0.216 ± 0.047** 0.213 ± 0.048** 60.72 59.81

300 0.183 ± 0.030*** 0.183 ± 0.0307** 67.27 51.92Methanol fraction 200 0.283 ± 0.047* 0.258 ± 0.045** 48.54 51.32

300 0.250 ± 0.042** 0.233 ± 0.049* 54.55 44.70

Data is analyzed by one way ANOVA followed by Dunnett’s ‘t’ test and expressed as mean ± SEM from six observations.* p < 0.05.

** p < 0.01.*** p < 0.001.

Table 2Th1 (TNF �) cytokine level in the serum of Balb/C mice administered with ethanolic extract of Adiantum capillus veneris and its various fractions.

S. no Sample Conc (�g/mL) Conc. of TNF-� (pg/mL)

1 Control 87.2 ± 2.462 LPS 1 457.4 ± 3.44*

3 Indomethacin 2 307 ± 6.23**

4 P1 10 602.4 ± 5.785 P2 50 452.4 ± 6.21*

6 P3 100 316.6 ± 7.21**

7 E1 10 587.4 ± 5.448 E2 50 486.2 ± 4.68*

9 E3 100 364 .4 ± 5.56**

10 M1 10 512.2 ± 5.7811 M2 50 384.8 ± 7.23**

12 M3 100 290.2 ± 3.44**

13 Et1 10 470.6 ± 4.56*

14 Et2 50 310.4 ± 5.78**

15 Et3 100 230.6 ± 4.66***

P, petrol fraction; E, ethanolic extract; M, methanolic extract, Et, ethyl acetate extract. The values are presented as mean ± S.E. (n = 10). Value for the concentration is Th1(TNF alpha) cytokine expressed in pg/mL. The results are presented as Mean ± S.E.

* p < 0.05 compared to control.** p < 0.01 compared to control.

*** p < 0.001 compared to control.

Table 3Analgesic effect of Adiantum capillus veneris by tail flick method.

Group Dose/route Basal reaction time (min) Reaction time (min)

30 60 120

Control 2 mL/kg po 2.11 ± 0.27 2.65 ± 0.25 1.75 ± 0.04 1.77 ± 0.07Ibuprofen 20 mg/kg po 2.39 ± 0.15 2.62 ± 0.38 3.88 ± 0.16** 3.69 ± 0.22**

Ethanolic extract 300 mg/kg po 2.46 ± 0.22 2.86 ± 0.53 3.05 ± 0.42 3.22 ± 0.41*

Ethyl acetate fraction 300 mg/kg po 2.61 ± 0.19 2.29 ± 0.24 3.58 ± 0.38* 3.48 ± 0.27**

Methanol fraction 300 mg/kg po 2.02 ± 0.21 2.83 ± 0.45 2.78 ± 0.58 3.23 ± 0.35*

Petroleum ether fraction 300 mg/kg po 2.45 ± 0.15 2.75 ± 0.36 3.64 ± 0.39* 3.19 ± 0.26*

Data is analyzed by one way ANOVA followed by Dunnett’s ‘t’ test and expressed as mean ± SEM from six observations.* p < 0.05.

** p < 0.01.

746 S. Haider et al. / Journal of Ethnopharmacology 138 (2011) 741– 747

Fig. 2. Effect of ethanolic extract of Adiantum capillus veneris and its various fractions on NO production. S1, petroleum ether fraction; S2, ethanol extract; S3, methanolfraction; S4, ethyl acetate fraction; standard, Indomethacin.

Table 4Analgesic effect of Adiantum capillus veneris by acetic acid induced writhing method.

Group Dose Number of writhes in 10 min % Protection

Control 2 mL/kg po 99.4 ± 2.7 –Ibuprofen 20 mg/kg po 52.0 ± 8.7*** 47.6Ethanolic extract 300 mg/kg po 72.0 ± 2.8** 27.5Petroleum ether fraction 300 mg/kg po 77.0 ± 2.47* 22.5Ethyl acetate fraction 300 mg/kg po 51.8 ± 2.63*** 47.8Methanol fraction 300 mg/kg po 71.2 ± 2.49** 28.3

Data is analyzed by one way ANOVA followed by Dunnett’s ‘t’ test and expressed as mean ± SEM from five observations.

iIrcee

iaMra�pesdda

fsmewt

AuTaf

* p < 0.05.** p < 0.01.

*** p < 0.001.

nhibition of inflammation when compared to the standard drugndomethacin where as the other fractions have shown compa-able activity. On preliminary phytochemical screening Adiantumapillus veneris Linn. was found to contain triterpenes (Jankowskit al., 2004) which were probably responsible for cyclooxygenasenzyme inhibiting activity.

Macrophages are the first line of defense against microbialnvaders and malignancies by nature of their phagocytic, cytotoxicnd intracellular killing capacities (Adams and Hamilton, 1984).acrophage activation by lipopolysaccharide (LPS) results in the

elease of several inflammatory mediators such as nitric oxide (NO)nd the proinflammatory cytokines, tumor necrosis factor-� (TNF-) (Schimmer and Parker, 2001). NO is a highly reactive moleculeroduced from guanidine nitrogen of NO synthase (NOS). How-ver, overproduction of NO can be harmful and may result in septichock, neurologic disorders, rheumatoid arthritis, and autoimmuneiseases (O’Shea et al., 2002). Therefore, inhibition of NO pro-uction is an important therapeutic target in the development ofnti-inflammatory agents.

In this study, we demonstrated that the ethanolic extract and itsractions suppressed NO production and tumor necrosis factor-�ecretion at higher doses, both of which are crucial in the inflam-atory and healing mechanism (Shen et al., 1994). As seen in this

xperiment, the ability of these fractions to suppress inflammationhen it is applied after the onset of inflammation is likely to be due

o the genuine anti-inflammatory activity.The anti-nociceptive activity of the crude ethanolic extract of

diantum capillus veneris Linn. and its various fractions was eval-

ated using both chemical and thermal methods of nociception.hese methods are used to detect the central and peripheral mech-nisms of analgesia. Acetic acid induced writhing test is usedor detecting both central and peripheral analgesia, whereas tail

flick test is sensitive to centrally acting analgesia. Intraperitonealadministration of acetic acid releases prostaglandins and sympa-thomimetic system mediators like PGE2 and PGF2� and their levelsare increased in the peritoneal fluid of the acetic acid induced mice(Deraedt et al., 1980). Thermal induced nociception indicates nar-cotic involvement (Besra et al., 1996). Thermal nociceptive tests aremore sensitive to opioid � receptors and non-thermal tests are toopioid � receptors (Abbott and Young, 1988; Furst et al., 1988).

The subsequent histopathological studies reveals that theethanolic extract and all its fractions except ethyl acetate fractiondo not cause any ulceration. The extent of ulceration in the caseof ethyl acetate fraction is restricted to the surface epithelial layerand that too is less than the standard drug, i.e. ibuprofen (which isless ulcerogenic compared to Indomethacin).

5. Conclusion

It may be concluded that Adiantum capillus veneris Linn. showedanti-edema and antinociceptive effects, similar to those observedfor non-steroidal anti-inflammatory agents, such as Indomethacin.The anti-inflammatory activity of the ethanolic extract and itsfractions may be related to the inhibition in the functions ofmacrophages wherein the extract inhibits the release of inflam-matory mediators viz. NO and TNF-�. Histopathological studieshave shown that the ethanolic extract and its fractions are lessulcerogenic when compared to standard drug ibuprofen. Finally

we conclude that Adiantum capillus veneris Linn. may be a potentmedicinal plant for the treatment of anti-inflammatory diseaseslike hay fever, atherosclerosis and rheumatoid arthritis. The workis in progress to isolate and to characterize the active compounds

ophar

pa

A

vHt

R

A

A

A

A

A

A

B

B

C

D

D

F

F

F

S. Haider et al. / Journal of Ethn

resent in the ethanolic extract and the various fractions of Adi-ntum capillus veneris Linn.

cknowledgements

The authors are thankful to Jamia Hamdard University for pro-iding the required facilities. MA and SN are also thankful toamdard National Foundation for providing the financial assis-

ance.

eferences

bbasi, M., Khan, M.A., Ahmad, M., Zafar, M., Khan, H., Muhammad, N., Sultana,S., 2009. Medicinal plants used for the treatment of jaundice and hepatitisbased on socio economic documentation. African Journal of Biotechnology 8,1643–1650.

bbott, F., Young, S.N., 1988. Effect of 5-hydroxy tryptanin precursors on morphineanalgesia in the formalin test. Pharmacology Biochemistry and Behaviour 31,855–860.

dams, D.O., Hamilton, T.A., 1984. The biology of macrophage activation. AnnualReview of Immunology 2, 283–318.

grahari, A.K., Khaliquzzama, M., Panda, S.K., 2010. Evaluation of analgesic activity ofmethanolic extract of Trapa natans lvar. Bispinosa Roxb. roots. Journal of CurrentPharmaceutical Research 1, 8–11.

lsarra, A.I., Ahmed, O.M., Alanazi, K.F., ElTahir, H.E.K., Alsheikh, M.A., Neau, H.S.,2010. International Journal of Medical Sciences 7, 232–239.

namika, K., Amit, G., Pankaj, S., Surjeet, S., Fayaz, M., Jawant, S., Gupta, B.D., 2007.Immunomodulatory activity of biopolymeric fraction BOS 2000 from Boswelliaserrate. Phytotherapy Research 22, 340–348.

erti, G., Bottari, F., Marsili, A., 1969. Structure of triterpenoids epoxide from Adi-antum capillus veneris. Tetrahedron 25, 2939–2947.

esra, S.E., Sharma, R.M., Gomes, A., 1996. Anti-inflammatory effect of petroleumether extract of leaves of Litchi Chinensis Gaertn (Sapinadaceae). Journal ofEthnopharmacology 54, 1–6.

owan, M.M., 1999. Plants products antimicrobial agents. Clinical MicrobiologyReviews 14, 564–584.

eraedt, R., Joughney, S., Delevakee, F., Falhour, M., 1980. Release of prostaglandinE and F in an algogenic reaction and its inhibition. European Journal of Pharma-cology 51, 17–24.

istasi Costa, L.C., Mendacolli, M., Kirzawa, S.J.L., Games, M., Trolin, C.G., 1988.Screening in mice of some medicinal plants used for analgesic purposes in thestate of Sao Paulo. Journal of Ethnopharmacology 24, 205.

arah, A., Qudsia, N., Aslam, M., 2005. Classification of Unani Drugs, first ed. Fine

Offset Works, Delhi, p. 39.

erreira, S.H., 1979. A new method for measuring the variations of rat paw volume.Journal of Pharmaceutical and Pharmacology 31, 648.

urst, S., Gyires, K., Knoll, J., 1988. Analgesic profile of rimazolium as compared todifferent classes of painkillers. Drug Research 4, 552–557.

macology 138 (2011) 741– 747 747

Jankowski, C.K., Aumelas, A., Thuery, P., Reyes-Chilpa, R., Jimenez-Estrada, M., 2004.X-ray, 1H/13C 2D and 3D NMR studies of the structures of davallene (i) andadipedatol(ii) triterpenes isolated from American Adiantum capillus-veneris. Pol-ish Journal of chemistry 78, 389–408.

Kabeeruddin, M., 1937. Advia ki Do Taqseemain, first ed. Daftar-ul Maseeh, Hyder-abad, p. 13.

Kale, S.R., Kale, R.R., 1994. Practical Pharmacology & Toxicology, first ed. NiraliPrakashan, Pune, pp. 56–57.

Khare, P.B., 1996. Ferns and fern allies-their significance and fantasies. AppliedBotany NBRI Lucknow 16, 1.

Koster, R., Anderson, M., Beer, E.J., 1959. Acetic acid for analgesic screening. Feder-ation Proceeds 18, 412–416.

Malik, F., Jaswant, S., Anamika, K., Krishan, A.S., Naresh, K.S., Surjeet, S., Maharaj,K.K., Arun, K., Aruna, B., Ghulam, N.Q., 2007. A standardized root extract ofWithania somnifera and its major constituent withanolide-A elicit humoral andcell-mediated immune responses by up regulation of Th1-dominant polariza-tion in BALB/c mice. Life Sciences 80, 1525–1538.

Mohini, G., Anjana, B., Roy, S.K., 1990. Chemical constituents of a member of Adi-antum caudatum complex. Journal of Chemical Society 67, 86–89.

O’Shea, J.J., Ma, A., Lipsky, P., 2002. Cytokines and autoimmunity. Nature Reviews.Immunology 2, 37–45.

Schimmer, B.P., Parker, K.L., 2001. Adrenocorticotropic hormone; adrenocorticalsteroids and their synthetic analogs; inhibitors of the synthesis and actions ofadrenocortical hormones. In: Hardman, J.G., Limbird, L.E., Goodman Gilman, A.(Eds.), The Pharmacological Basis of Therapeutics. , tenth ed. McGraw-Hill, NewYork, pp. 1649–1677.

Shen, H.M., Kenned, J.L., Ou, D.W., 1994. Inhibition of cytokine release by cocaine.International Journal of Immunopharmacology 16, 295–300.

Singh, M., Singh, N., Khare, P.B., 2007. Antimicrobial activity of some important Adi-antum species used traditionally in indigenous systems of medicine. Journal ofEthnopharmacology 114, 327–329.

Singh, M., Singh, N., Khare, P.B., Rawat, A.K.S., 2008. Antimicrobial activity ofsome important Adiantum species used traditionally in indigenous systems ofmedicine. Journal of Ethnopharmacology 115, 327–329.

Sokal, R., Rohlf, F.J., 1995. Biometry, third ed. W.H. Freeman and Co., New York, p.887.

Takahisa, N., Yoko, A., Kazuo, M., Yuh, I., Hiroyuki, A., Kenji, S., 1999. Fern con-stituents: six new triterpenoid alcohols from Adiantum capillus veneris. Chemical& Pharmaceutical Bulletin 47, 543–547.

Tan, H.H., Ong, W.M.C., Lai, S.H., Chow, W.C., 2007. Nimesulide-induced hepatotox-icity and fatal hepatic failure. Singapore Medical Journal 48, 582–585.

Vadivelan, S., Sinha, B.N., Kalyan, S., Betanabhatla, Christina, A.J.M., Pillai, R.N., 2007.Anti-inflammatory activity of Spermacocearticularis on carrageenan inducedpaw edema in Wistar male rats. Pharmacology Online 3, 478–484.

Vasudeva, S.M., 1999. Economic importance of pteridophytes. Indian Fern Journal16, 130–152.

Vinegar, R., Schreiber, W., Hugo, R., 1969. Biphasic development of carrageenan

edema in rats. Journal of Pharmacology and Experimental Therapeutics 166,96–103.

Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenan induced edema in hind pawof rat as an assay for anti-inflammatory drugs. Proceedings of the Society forExperimental Biology and Medicine 111, 544–546.