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Journal of Ethnopharmacology 133 (2011) 303–307

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

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valuation of the disease modifying activity of Colchicum luteum Baker inxperimental arthritis

inod Nair, Surender Singh ∗, Y.K. Guptaepartment of Pharmacology, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi 110029, India

r t i c l e i n f o

rticle history:eceived 26 April 2010eceived in revised form 1 July 2010ccepted 8 September 2010vailable online 20 October 2010

eywords:djuvant arthritisnti-inflammatoryolchicum luteumisease modifyingroinflammatory cytokines

a b s t r a c t

Ethnopharmacological relevance: Colchicum luteum (CL) has been traditionally used in the Unani systemof medicine as a chief ingredient of many polyherbal formulations for the treatment of joint pain andrheumatoid arthritis (RA).Aim of the study: To evaluate the antiarthritic activity of CL hydroalcoholic extract (CLHE) in formaldehydeand complete Freund’s adjuvant (CFA) induced arthritis.Materials and methods: Arthritis was induced by administration of either formaldehyde (2% v/v) or CFAinto the subplantar surface of the hind paw of the animal. Joint swelling was measured on days 8, 9 and10 in formaldehyde induced arthritis and days 3, 7, 14 and 21 in CFA induced arthritis. In order to evaluatethe effect of CLHE on disease progression, serum TNF-� level and synovial expression of proinflammatorymediators (TNF-R1, IL-6 and IL-1�) was determined in CFA induced arthritis.Results: CLHE produced a significant and dose dependent inhibition of joint swelling during the entire

duration of the study in both, formaldehyde and CFA induced arthritis. Serum TNF-� level was alsoreduced significantly in a dose dependent manner in all the CLHE treated groups. The expression ofproinflammatory mediators (TNF-R1, IL-6 and IL-1�) was also found to be less in the CLHE treated groupas compared to control.Conclusion: We believe that the antiarthritic activity of CLHE was due to its modulatory effect on theexpression of proinflammatory cytokine in the synovium. Our results contribute towards validation of

n the

the traditional use of CL i

. Introduction

Rheumatoid arthritis (RA) is a progressive, disabling, chronicultisystem disease that is characterized by pain, swelling and

tiffness of the synovial joints. The exact aetiology of this debili-ating disease is not known, but it is believed to be the result ofn autoimmune response of the body which can be triggered by aariety of external and genetic factors (Lipsky, 2005).

Conventional drugs used in the treatment of RA are NSAIDs,lucocorticoids, IL-1 receptor antagonist, and anti-TNF-� drugs. Ofhese, the most effective in limiting the progression of the diseasere glucocorticoids and cytokine antagonists. However, their use islso associated with a plethora of side effects including immuno-uppression and emergence of opportunistic infections which can

dd to morbidity and mortality. Owing to the chronic nature ofhe disease and side effects associated with long term use of thesemmunomodulatory agents, patients with RA often seek alternative

ethods for symptomatic relief and are amongst the highest users

∗ Corresponding author. Tel.: +91 011 26546460.E-mail address: surenderaiims@gmail.com (S. Singh).

378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2010.09.027

treatment of RA and other inflammatory joint disorders.© 2010 Elsevier Ireland Ltd. All rights reserved.

of complementary and alternative Medicine (Cronan et al., 1989;Eisenberg et al., 1998).

In traditional systems of medicine, many formulations are usedfor the treatment of joint pain and RA. Most of these formulationscontain Colchicum luteum (Liliaceae) as the main ingredient (Said,1997). Colchicum luteum Baker (CL) is an annual herb that is widelydistributed in the Western Himalayas (China, India, Pakistan andAfghanistan) at an altitude of 600–2700 m. Its corms and seeds areused by practitioners of traditional medicine for the treatment ofgout, rheumatism and diseases of liver and spleen (Singh and Aswal,1994; Kapur and Singh, 1996). Despite its wide spread use in tradi-tional medicine for treatment of gout and rheumatism, only a fewstudies have evaluated the efficacy of this medicinal plant in exper-imental and clinical settings. The methanolic extract of this planthas been shown to possess potent lipoxygenase inhibitory activityin in-vitro experiments (Ahmad et al., 2006). A few clinical studiesin alternative medicine have reported symptomatic improvement

in patients with RA after the use of polyherbal formulations con-taining CL extract (Joshi et al., 1973; Javed et al., 2005). However,there is a dearth of scientific evidence regarding the mechanism ofantiarthritic activity of this medicinal plant. Therefore, the presentstudy was carried out with an aim to scientifically evaluate the tra-

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itional use of CL and to elucidate the underlying mechanism forts antiarthritic activity.

. Material and methods

.1. Animals

The experimental protocol was approved by the Institutionalnimal Ethics Committee, All India Institute of Medical Sciences,ew Delhi, India. Adult male Wistar albino rats (150–180 g) fromur institutional breeding stock were used in the study. Animalsere housed at 25 ± 2 ◦C inside clean polypropylene cages in groups

f three. They had access to food and water ad libitum through-ut the duration of the study. All experiments were carried out inccordance with ‘Guidelines for care and use of animals in scientificesearch (Indian National Science Academy 1998, Revised 2000)’.

.2. Test drug

Dried corms were obtained and authenticated by Prof. Moham-ad Ali, pharmacognosist and phytochemist at Jamia Hamdardniversity, New Delhi, India, as Colchicum luteum Baker (Liliaceae)nd a voucher specimen (PRL/JH/08/02) was submitted at theerbarium for future reference. The corms were coarsely groundnd extracted by cold maceration with 50% methanol for 72 h. Thelurry was filtered through cotton wool to separate the suspendedarticles. The solvent was then evaporated under reduced pres-ure till a resinous extract was obtained. The extract was goldenrown in colour and the total quantitative yield was 12.50% (w/w).olchicum luteum hydroalcoholic extract (CLHE) was further sub-

ected to pharmacognostical standardization for detection of sec-ndary plant metabolites (Dey and Harborne, 1987) and was foundo contain alkaloids, tannins, and sugars. The total alkaloid contentf CL was determined by gravimetry and was found to be 0.90%w/w). Quantitative estimation of colchicine was done by HPTLCensitometry using a precoated silica gel 60 F254 TLC plate (Merck)f 0.2 mm thickness in a chloroform:methanol (9:1) solvent systematurated with strong ammonia solution. The plates were dried in aot air oven, scanned at 243 nm and a chromatogram was recorded.he total colchicine content in CL was found to be 0.19% (w/w).

.3. Formaldehyde induced arthritis

Animals were divided into five groups (n = 6) and the base-ine ankle joint diameter was measured by using a micrometercrew gauge. All the drug suspensions were freshly prepared in% gum acacia (vehicle) at the time of administration. Group Ieceived the vehicle (2 ml/kg body weight) and served as the con-rol, group II received 3 mg/kg body weight indomethacin (Rasoolnd Varalakshmi, 2006) and groups III, IV and V received CLHE inoses of 17 mg, 34 mg and 68 mg/kg body weight respectively forperiod of 10 days. Dose selection for the test drug was done on

he basis of a pilot study carried out in our laboratory. Thirty min-tes after administration of the vehicle/drug, arthritis was inducedy subplantar administration of 0.1 ml formaldehyde (2% v/v) intohe left hind paw of all the animals using a 26 gauge needle onays 1 and 3 (Brownlee, 1950; Gujral et al., 1959). Increase in jointiameter was measured on days 8, 9 and 10.

.4. Complete Freund’s adjuvant (CFA) induced arthritis

Animals were divided into five groups with respective treat-ents as mentioned under Section 2.3 and the baseline joint

iameter was recorded with a micrometer screw gauge. Thirtyinutes after oral administration of vehicle/drug, 0.1 ml of CFA

Difco: 0.05% (w/v) Mycobacterium butyricum in mineral oil) was

acology 133 (2011) 303–307

injected into the subplantar surface of the left hind paw by usinga 26 gauge needle (Newbould, 1963). This was designated as day0. Vehicle/drug treatment was continued for 20 more days. Jointsize measurements were carried out on days 3, 7, 14 and 21. Onday 21, the animals were sacrificed and terminal blood collectionwas done. The serum was separated and TNF-� levels were esti-mated by capture ELISA (U-CyTech Biosciences, The Netherlands).The ankle joints were collected and stored at −80 ◦C for immuno-histochemistry studies.

2.5. Immunohistochemistry

The ankle joints were decalcified with 10%EDTA (pH 7.4) andfrozen sections (6 �m) were made. After acetone fixation, the sec-tions were processed for immunohistochemical analysis of TNF-R1,IL-1� and IL-6 expression by using anti-rat antibodies (Santa CruzBiotech Inc.) and an avidin–biotin based detection kit (Vector Labs,USA). Expression of the cytokines/cytokine receptor was visualizedby colour development using DAB (diaminobenzidine) substrate kit(Vector Labs, USA) and counterstaining with haematoxylin.

2.6. Toxicity studies of the plant extract

Evaluation of acute oral toxicity of CLHE was carried out accord-ing to the OECD guidelines for testing of chemicals – 425 (OECD,2001). A limit test (2000 mg/kg body weight) was performed usingfive male Wistar rats (150–180 g) from our breeding stock. All theanimals were observed for behavioral changes and mortality till 14days after administration of the dose.

Evaluation of 28 day oral toxicity of CLHE was carried out accord-ing to the OECD guidelines for testing of chemicals – 407 (OECD,2008). Sixteen male Wistar rats (150–180 g) from our breedingstock were divided into two groups (n = 8). Group I received thevehicle (2 ml/kg body weight, 1% gum acacia) and served as nor-mal control and group II received CLHE in a dose of 340 mg/kgbody weight (five times the maximum dose tested in antiarthriticstudies). Drug/vehicle was administered daily for a duration of 28days.

2.7. Statistical analysis

All data is represented as Mean ± SE (6 animals per group). Com-parison between groups was done by One-way ANOVA followed byDunnett’s multiple comparison test. P < 0.05 was considered signif-icant.

3. Results

3.1. Effect of CLHE on joint swelling in formaldehyde inducedarthritis

An increase in joint diameter was seen in all the animalsthroughout the observation period (Table 1). Although all drugtreated groups showed a decrease in joint swelling as comparedto control, the difference was significant only in Group V (CLHE68 mg/kg) on all the observational days. Groups II (Indoemthacin)and IV (CLHE 34 mg/kg) showed significant reduction in jointswelling only on Days 9 and 10. Even though CLHE at a dose of17 mg/kg (Group III) produced a reduction in joint swelling, it wasnot statistically significant. Maximum inhibition of joint swellingwas produced by CLHE (68 mg/kg) on all the observational days.

3.2. Effect of CLHE on joint swelling in CFA induced arthritis

As in the formaldehyde induced arthritis model, an increase injoint diameter was seen in all the animals throughout the observa-

V. Nair et al. / Journal of Ethnopharmacology 133 (2011) 303–307 305

Table 1Antiarthritic activity of CLHE in formaldehyde induced arthritis.

Group Increase in joint diameter (mm) [% inhibition of joint swelling]

Day 8 Day 9 Day 10

I – Control (2 ml/kg vehicle) 1.27 ± 0.16 1.59 ± 0.13 1.45 ± 0.17II –Indomethacin (3 mg/kg) 0.96 ± 0.14 [24.40] 1.07 ± 0.07b [32.70] 0.98 ± 0.02a [32.41]III – CLHE (17 mg/kg) 1.07 ± 0.11 [15.74] 1.29 ± 0.09 [18.86] 1.12 ± 0.08 [22.75]IV – CLHE (34 mg/kg) 0.99 ± 0.09 [22.04] 1.14 ± 0.13a [28.30] 0.96 ± 0.07a [33.79]V – CLHE (68 mg/kg) 0.70 ± 0.18a [44.88] 1.06 ± 0.05b [33.33] 0.89 ± 0.13b [38.62]

All values are mean ± SE. Statistical analysis by one-way ANOVA followed by Dunnett’s Multiple Comparison.a P < 0.05.b P < 0.01.

Table 2Antiarthritic activity of CLHE in CFA induced arthritis.

Group Increase in joint diameter (mm) [% inhibition of joint swelling] TNF-alpha levels(pg/ml of serum)

Day 3 Day 7 Day 14 Day 21

I – Control (2 ml/kg vehicle) 3.10 ± 0.12 2.15 ± 0.15 2.06 ± 0.21 2.15 ± 0.17 60.25 ± 1.31II – Indomethacin (3 mg/kg) 1.38 ± 0.09b [55.48] 0.97 ± 0.08b [54.88] 0.71 ± 0.03b [65.53] 0.59 ± 0.02b [72.55] 166.83 ± 4.28b

III – CLHE (17 mg/kg) 2.50 ± 0.12b [19.64] 1.65 ± 0.07b [23.25] 1.21 ± 0.07b [41.26] 1.41 ± 0.10b [34.41] 47.00 ± 1.15b

IV – CLHE (34 mg/kg) 2.40 ± 0.07b [22.58] 1.50 ± 0.05b [30.23] 0.90 ± 0.04b [56.31] 0.69 ± 0.05b [67.90] 32.33 ± 3.28b

V – CLHE (68 mg/kg) 2.37 ± 0.05b [23.54] 1.32 ± 0.03b [38.60] 0.70 ± 0.04b [66.01] 0.68 ± 0.02b [68.37] 29.66 ± 2.33b

All values are mean ± SE. Statistical analysis by one-way ANOVA followed by Dunnett’s Multiple Comparison.b P < 0.01.

Fig. 1. Effect of 21 day CLHE treatment on cytokine/cytokine receptor expression in synovial joint of rats in CFA induced arthritis – Sections are 6 �m thick and photomicro-graphs are taken at 40×. A, control animal; B, indomethaicn treated animal; C, CLHE (68 mg/kg) treated animal; 1, IL-1� expression in the synovial joint; 2, IL-6 expressionin the synovial joint; 3, TNF-R1 expression in the synovial joint; bold arrows, DAB staining of the synovial membrane depicting presence of respective cytokines.

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ion period. Maximum joint swelling was observed on Day 3 afterhich there was a gradual reduction, except in Group I (control)

nd III (CLHE17 mg/kg) where there was a slight increase in jointiameter after Day 14 (Table 2). There was a significant reduction

n joint swelling in all drug treated groups as compared to control.LHE produced a significant and dose dependent reduction of jointwelling throughout the study. However, on Day 21 the percent-ge inhibition of joint swelling seen in the two higher dose CLHEreated groups was similar (67.90 and 68.37 respectively). Maxi-

um inhibition of joint swelling was produced by indomethacin72.55) on Day 21.

.3. Effect of CLHE on serum TNF-˛ levels in CFA induced arthritis

All the drug/vehicle treated animals showed an increase inerum TNF-� level (Table 2). CLHE treatment produced a signif-cant and dose dependent reduction in the serum TNF-� levelss compared to control. However, indomethacin treated animalshowed a significant and >2.5 fold increase in serum TNF-� levelss compared to control. TNF-� level in normal rats was not in theetectable range with the ELISA kit that was used in the study.

.4. Immunohistochemistry of the synovial joint

Immunohistochemical staining demonstrated the localizationf large amounts of proinflammatory cytokines (IL-1�, IL-6) andNF-R1 in the synovial membrane of the control animals (Fig. 1).reduction in the expression of these cytokines/cytokine receptoras observed in the CLHE (68 mg/kg) treated group as compared

o control. The expression of these mediators in the indomethacinreated group was intermediate to that of control and CLHE68 mg/kg) treated groups.

.5. Toxicity profile of the test plant

Administration of CLHE in a dose of 2000 mg/kg body weight didot produce any behavioural abnormalities in any of the animalsested. However, there was one mortality out of five animals thatere tested. The mortality was observed 48 h after drug adminis-

ration. As more than 50% of the tested animals survived, the oralD50 of CLHE in rats was found to be >2000 mg/kg body weight.

Chronic administration of CLHE in a dose of 340 mg/kg bodyeight for 28 days did not produce any significant physiological

hanges in the tested animals as compared to normal control (dataot shown). There was a marginal increase in body weight, bleedingime, RBC count and SGOT (serum glutamate oxaloacetate transam-nase) levels as compared to normal control, but this increase wasot statistically significant. WBC count, %Hb, SGPT (serum gluta-ate pyruvate transaminase) levels and percentage organ weight

f liver showed a marginal decrease from normal control, but thisifference was also not statistically significant. All other parametersemained unaltered.

. Discussion and conclusion

The main phytochemical principles in CL are alkaloids, viz.olchicine, 2-desmethylcolchicine, �-lumicolchicine, N-desacetyl--formylcolchicine, and luteidine (Chommadov et al., 1970; Koulnd Thakur, 1977). Colchicine is considered to be the mostmportant anti-inflammatory compound obtained from CL and its

nti-inflammatory activity is attributed to the inhibition of micro-ubules in proinflammatory cells including macrophages (Rao etl., 1997). In the present study we have evaluated the antiarthriticctivity of CLHE by using two experimental models of arthritis, viz.ormaldehyde and CFA induced arthritis.

acology 133 (2011) 303–307

In formaldehyde induced arthritis, CLHE in the highest dosetested demonstrated good antiarthritic efficacy and was superior toindomethacin in reducing the joint swelling throughout the obser-vation period. To further confirm the antiarthritic activity of thetest drug, we evaluated its efficacy in reducing joint inflammationin CFA induced arthritis. This model is widely used for pharma-cological evaluation of anti-arthritic agents as it has been shownto share a number of clinical and immunological features withhuman arthritis, and therefore has a relatively high degree of valid-ity (Newbould, 1963). In our study, along with the measurementof joint swelling, we also evaluated three key cytokines primar-ily secreted by macrophages viz. TNF-�, IL-6 and IL-1� as theyhave been found to be expressed at significantly higher levels inchronic state of the disease (Ulfgren et al., 1995). Additionally, wealso checked the expression of TNF-R1 in the synovium as most ofthe pathophysiological effects of TNF-� are known to be mediatedthrough this receptor subtype (Feldmann and Maini, 2001; Shibataet al., 2009).

In our study, there was a consistent decrease in joint swellingwith higher two doses (34 mg and 68 mg/kg body weight) of CLHE.However, in the lowest dose (17 mg/kg body weight) treated groupthere was a slight increase in joint swelling from day 14 to 21. Thisincrease was also seen in the control group and could have been dueto the heightened immune response seen during the late phase ofCFA induced arthritis. The serum TNF-� level was also found tobe reduced in all the CLHE treated groups as compared to con-trol. However, the TNF-� level were higher in the indomethacintreated group. This could probably have been the result of the gas-trointestinal damage caused by long term use of indomethacin asreported in earlier studies (Saud et al., 2005). Immunohistochem-istry studies also correlated with the findings, as the expression ofTNF-R1, IL-1� and IL-6 was found to be reduced in the synovialmembrane of CLHE (68 mg/kg) and indomethacin treated groups.Even though indomethacin was more effective in reducing the jointswelling as compared to CLHE (68 mg/kg) treated group, the expres-sion of cytokines/cytokine receptor in the synovial membrane ofindomethacin treated group was intermediate to that of controland CLHE treated groups. This indicates that CLHE may be actingthrough a different mechanism as compared to indomethacin.

It is well established that only those agents which modulatethe immunological status have an effect on the progression of dis-ease activity in RA. Colchicine, the active ingredient of CL, has beenshown to inhibit proinflammatory cells including macrophages dueto its interaction with cellular tubulin protein (Rao et al., 1997).Immunohistochemistry studies clearly demonstrate that CLHE pro-duced its antiarthritic effect, at least in part, by modulation ofcytokine profile in the inflamed joint. However, due to the presenceof multiple alkaloid principles in CLHE, this immunomodulatoryeffect cannot be solely attributed to colchicine. Studies evaluat-ing individual fractions of CLHE in similar experimental models aretherefore required to identify all the immunomodulatory principlesthat could have contributed to this antiarthritic effect.

In our study CLHE was found to have an oral LD50 value above2000 mg/kg body weight. Chronic administration of CLHE also didnot produce any pathological changes in the tested animals, thusdemonstrating its safety on long term administration. Based on ourresults, we believe that further studies involving detailed cytokineprofiling and respective cellular characterization could conclu-sively establish CLHE as a potentially safer disease modifying agentin the treatment of RA.

Acknowledgement

The study was funded by an extra-mural research grant fromIndian Council of Medical Research, New Delhi, India. The funding

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