Exploring the ameliorative potential of Punica granatum in dextran sulfate sodium induced ulcerative...

PUNICA GRANATUM IN ULCERATIVE COLITIS 1565

Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 23: 1565–1574 (2009)DOI: 10.1002/ptr

Copyright © 2009 John Wiley & Sons, Ltd.

PHYTOTHERAPY RESEARCHPhytother. Res. 23: 1565–1574 (2009)Published online 15 April 2009 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/ptr.2822

Exploring the Ameliorative Potential of Punicagranatum in Dextran Sulfate Sodium InducedUlcerative Colitis in Mice

Kavinder Singh, Amteshwar Singh Jaggi and Nirmal Singh*Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala 147002, India

The present study was designed to investigate the ameliorative potential of Punica granatum in dextran sulfatesodium (DSS) induced ulcerative colitis. DSS (2%) was administered orally in drinking water for 7 days toinduce ulcerative colitis. The extent and severity of ulceration was analysed macroscopically, histopathologicallyand using a disease activity index. Myeloperoxidase (MPO), a specific marker of inflammation; histamine, amarker of mast cell degranulation; superoxide anion generation and, lipid peroxides were analysed. Adminis-tration of DSS resulted in a significant development of ulceration in the colon along with a rise in histamine,MPO activity and oxidative stress. Treatment with Punica granatum extract and its ellagic acid rich fraction(100 mg/kg and 200 mg/kg p.o.) significantly attenuated DSS-induced colonic inflammation along with attenuationof histamine, MPO and oxidative stress. The antiulcerative effect of Punica granatum extract and its ellagicacid rich fraction were comparable to sulphasalazine (100 mg/kg, p.o.) and sodium cromoglycate (40 mg/kgi.p). It is concluded that Punica granatum has a potential for ameliorating DSS-induced colitis and its ellagicacid rich fraction may be responsible for this effect. Further, the antiulcerative effects may be attributed tomast cell stabilizing, antiinflammatory and antioxidant actions. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: Punica granatum; dextran sulfate sodium; ulcerative colitis; myeloperoxidase; mast cells.

Received 19 January 2009Revised 17 February 2009

Accepted 19 February 2009

* Correspondence to: Dr Nirmal Singh, Department of PharmaceuticalSciences and Drug Research, Punjabi University, Patiala 147002, Punjab,India.E-mail: [email protected]

INTRODUCTION

Ulcerative colitis is a worldwide chronic, idiopathic,inflammatory bowel disease of the rectal and colonicmucosa (Chung et al., 2007). The etiopathogenesishas not been fully elucidated, but is currently presumedto result from a complex interplay among genetic, envi-ronmental, microbial and immune factors (Baumgartand Carding, 2007). The current status of drug therapyfor ulcerative colitis is not very satisfactory. Though,presently available drugs such as 5-aminosalicylates, cor-ticosteroids and immunosuppressants have been foundbeneficial in controlling the disease, these are associatedwith unpredictable and serious side effects, particularlywith long term use. Consequently, there has been anurgent need for alternative medicine with high efficacyand fewer side effects.

Punica granatum L. (Punicaceae), commonly calledpomegranate, is a large deciduous shrub/small tree andits different parts are used in traditional medicine inEurope, India, China, the Philippine Islands and SouthAfrica. The flowers of this plant have been recom-mended in Unani literature for the treatment of diabetesand associated complications (Jurjani, 1878; Majoosi,1889). Experimental reports have indicated its protectiveeffects against liver fibrosis, prostate cancer, hypoxic-ischemic injury, ultraviolet-induced pigmentation, aller-

gic reactions (Kasai et al., 2006; Adhami and Mukhtar,2007; Toklu et al., 2007). Further, its antibacterial,anticonvulsant, antiinflammatory, antifungal, immuno-modulatory, cardioprotective, antimutagenic, anti-spasmodic and antidiabetic activities have also beendocumented (Ross et al., 2001; Aviram and Dornfeld,2003; Huang et al., 2005). Recently, the reduction ofgingivitis risk by mouth rinsing with Punica granatumhas also been documented (Disilvestro et al., 2009).

Mast cells are multi-effector cells playing an importantrole in inflammation by releasing histamine, serotonin,tryptase, chymase, elastase, chemotactic factors, plate-let activating factor, prostaglandins, leukotrienes andcytokines (Mekori and Metcalfe, 2000; Prussin andMetcalfe, 2006; Bischoff, 2007). It has been reportedthat mast cell degranulation with a consequent releaseof various inflammatory mediators may be responsiblefor the induction of inflammatory reactions in ulcera-tive colitis and hence, mast cells play a key role inits pathogenesis (Xu et al., 2002; Videla et al., 2007).Further, treatment with mast cell stabilizer has beendocumented to attenuate the severity of ulcerative colitispatients (Marshall and Irvine, 1998).

Traditionally, Punica granatum is used extensively forthe treatment of diarrhea, dysentery, oxyuriasis, colic,ulcer, urinary tract infections, syphilis and as an anthel-mintic and vermifuge (Duke and Ayensu, 1985; Naoviet al., 1991; Kirthikar and Basu, 2000). Punica granatumis rich in phenolic compounds, and is an importantsource of hydrolysable tannins, ellagitannins, or ellagicacid (Yoshimura et al., 2005; Kasai et al., 2006; Aviramet al., 2008). Tannin rich fractions, ellagic acid rich andellagitannins of Punica granatum have been reported


1566 K. SINGH ET AL.

to exert diverse actions such as antioxidant, antimicro-bial, anticancer and immunoprotection (Yoshimuraet al., 2005; Adams et al., 2006; Kasai et al., 2006;Reddy et al., 2007). Further, it has been reported thatmicrospheres of ellagic acid produce a beneficial effectin ulcerative colitis (Ogawa et al., 2002).

Therefore, the present study was designed to showameliorative potential of Punica granatum flowerextract and its ellagic acid rich fraction in dextran sulfatesodium induced ulcerative colitis in mice.

MATERIALS AND METHODS

Chemicals. Dextran sulfate sodium salt (molecular weight:36 000–50 000 Daltons) from MP Biomedicals, France;sodium cromoglycate from Pharma Science Inc., Montreal,Canada; nitroblue tetrazolium (NBT), o-Phthalaldehyde(OPT) from Loba Chem, Mumbai, India and o-dianisidine hydrochloride from S.D. Fine-Chemicals Ltd,Mumbai, India were employed in the present study. Allother chemicals were obtained from S.D. Fine-ChemicalsLtd, Mumbai, India and were of analytical grade.

Plant material. Punica granatum flowers were collectedlocally and a sample has been kept as a voucher speci-men (PuP-030/2007-2008) at Punjabi University, Patiala.

Preparation of the Punica granatum flower hydroalcoholextract. The flowers of Punica granatum were driedin shade and ground to make a coarse powder. Thepowder was then extracted thrice with methanol–watermixture (3:1) by stirring at room temperature for 1 heach time. The extract was filtered and the solvent wascompletely removed at 50 °C under reduced pressure.The yield of the extract was 15% (w/w) in terms ofdried starting material. The extract was standardizedusing HPTLC finger-printing taking chloroform andmethanol (80:20) as the mobile phase and usinganisaldehyde as the spraying agent. The bands weredetected at 254 nm, 366 nm and under white light.

Preparation of ellagic acid rich fraction of Punicagranatum flower. The ellagic acid rich fraction of Punicagranatum was prepared as described earlier (Yoshimuraet al., 2005). The dried powder of flowers of Punicagranatum was extracted thrice with 50% aqueous ethylalcohol at 60 °C–70 °C for 2 h each time. The alcoholsolvent was removed under reduced pressure. Theresulting aqueous solution was acidified with hydro-chloric acid and then refluxed at 70 °C for 6 h. Upondilution with water, ellagic acid was precipitated. Theprecipitate was collected by filtration and dried in avacuum oven.

Experimental animals. Swiss albino mice, weighing 25–30 g maintained on a standard laboratory diet (KisanFeeds Ltd, Mumbai, India) and having free access totap water, were employed in the present study. Theywere kept in the departmental animal house and wereexposed to normal light/dark cycle. The experimentalprotocol was duly approved by the Institutional AnimalEthical Committee (IAEC) and the care of animalswas carried out according to the guidelines of the Com-mittee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA), Ministry of Environ-ment and Forest, Government of India (Reg. No. 107/1999/ CPCSEA).

Induction of ulcerative colitis. Dextran sulfate sodium(DSS) was employed for the induction of ulcerative colitisas described previously (Okayasu et al., 1990). The drink-ing water for mice was replaced by 2% DSS solutionand mice received DSS orally for 7 days. On day 8, theanimals were killed and colonic portions were taken formacroscopic, histopathological and biochemical analyses.

Macroscopic analysis. The colonic portion was tran-sected and placed on filter papers for determinationof colon length. The colon was then cut open longitudi-nally to expose the entire surface. Thereafter, mucosalinjury was assessed macroscopically using the gradingscale of Millar et al. (1996). No macroscopic changes(score 0); mucosal erythema only (score 1); mild mucosaledema, slight bleeding or small erosions (score 2);moderate edema, bleeding ulcers or erosions (score 3);severe ulceration, erosions, edema and tissue necrosis(score 4).

Disease activity index. DSS-induced disease severityof ulcerative colitis was analysed in terms of diseaseactivity index (DAI), which in turn was calculatedon the basis of a reduction of body weight, stool con-sistency and bleeding on different days (Okayasu et al.,1990; Murthy et al., 1993).

Histopathological analysis. Colonic specimens werefixed in 10% formalin followed by wax embedding,sectioning (4 mm) and staining with hematoxylin andeosin for histopathogical evaluation of colonic damage.The extent of colonic damage was scored as describedby Wallace et al. (1989): normal histological appear-ance (score 0); histological damage limited to surfaceepithelium (score 1); focal ulceration and cell disruptionlimited to mucosa (score 2); focal transmural inflamma-tion and ulceration (score 3); extensive transmuralinflammation bordered by areas of normal mucosa(score 4); extensive transmural inflammation involvingentire section from epithelium to serosa (score 5).

Determination of total protein. Total protein contentwas determined by the method of Lowry et al. (1951)using bovine serum albumin (BSA) as a standard.

Determination of lipid peroxidation. Estimation oflipid peroxidation was performed by measuring thelevels of thiobarbituric acid reactive substances (TBARS),an index of lipid peroxidation in colon, performedaccording to the method of Ohkawa et al. (1979). Theconcentration of TBARS in the colonic homogenatewas expressed in terms of nmol/mg protein.

Determination of superoxide anion generation. Thecolon tissue superoxide anion generation was estimatedusing the method of Wang et al. (1998) and the resultswere expressed as pmol of reduced NBT/min/mg oftissue.

Disease activity index =

Weight loss + Stoolconsistency + bleeding

3



Determination of myeloperoxidase (MPO) activity. Asmall portion of colonic tissue was taken and rinsedwith ice-cold saline, blotted dry, weighed, minced andhomogenized in 10 volumes of ice-cold potassium phos-phate buffer (pH 7.4). The homogenate was centrifugedat 3500 rpm for 30 min at 4 °C. The supernatant wasdiscarded and 10 mL of ice-cold 50 mM potassiumphosphate buffer (pH 6.0) containing 0.5% hexadecyl-trimethylammonium bromide (HTAB) and l0 mM EDTAwas added to the pellet. It was then subjected to onecycle of freezing and thawing and a brief period (l5 s)of sonication. After sonication, the solution was againcentrifuged at 15 000 rpm for 20 min at 4 °C and myelo-peroxidase activity in the supernatant was measuredspectrophotometrically at 460 nm (Bradley et al., 1982;Krawisz et al., 1984).

Determination of tissue histamine content. The histaminecontent in tissue was estimated spectrofluorometricallyas described by Shore et al. (1959). Colonic tissue washomogenized in 9 volumes of 0.4 N perchloric acidand was then centrifuged at 5000 rpm. A 4 mL aliquotof the supernatant fluid was transferred to a flaskcontaining 0.5 mL of 5 N sodium hydroxide solution(NaOH), 1.5 g of solid NaCl and 10 mL of n-butanol.The flask was shaken for 5 min to extract the histamineinto the butanol, and after centrifugation at 4000 rpm,the aqueous phase was removed by aspiration. Theorganic phase was then shaken for about 1 min with 5 mLof salt-saturated 0.1 N NaOH, and then centrifuged.8 mL aliquot of the butanol was transferred to a flaskcontaining 3.5 mL of 0.1 N HCl and 15 mL of n-heptane.After shaking for about 1 min, the mixture was centri-fuged at 4000 rpm and the histamine in the aqueousphase was assayed spectrofluorometrically by reactingwith o-phthalaldehyde (OPT) reagent.

Experimental design. Nine groups, each having six mice,were employed in the present study.

Group I (normal control): mice were kept for 7 dayswithout any treatment and on day 8, the mice werekilled and macroscopic, histopathological and biochemi-cal analyses were performed in colonic tissue.Group II (DSS colitis control): dextran sulfate sodium(2%) was administered to mice in drinking water for7 days. On day 8, the mice were killed and macro-scopic, histopathological and biochemical analyses wereperformed.Group III (vehicle treated): 0.5% carboxymethyl cellu-lose (CMC) was administered once daily, starting 2 daysbefore the induction of colitis and continued for 7consecutive days. On 8 day, the mice were killed andmacroscopic, histopathological and biochemical analy-ses were performed.Group IV (Punica granatum treated, 100 mg/kg):hydroalcohol extract of Punica granatum (100 mg/kgp.o.) was suspended in 0.5% CMC and was adminis-tered once daily, starting 2 days before the induction ofcolitis and continued for 7 consecutive days. On day 8,the mice were killed and macroscopic, histopathologicaland biochemical analyses were performed.Group V (Punica granatum treated, 200 mg/kg):hydroalcohol extract of Punica granatum (200 mg/kgp.o.) was suspended in 0.5% CMC and administeredonce daily, starting 2 days before the induction of colitis

and continued for 7 consecutive days. On day 8, themice were killed and macroscopic, histopathological andbiochemical analyses were performed.Group VI (ellagic acid rich fraction of Punica granatumtreated, 100 mg/kg): ellagic acid rich fraction of Punicagranatum (100 mg/kg p.o.) was suspended in 0.5% CMCwater and administered once daily starting 2 daysbefore the induction of colitis by and continued for7 consecutive days. On day 8, the, mice were killedand macroscopic, histopathological and biochemicalanalyses were performed.Group VII (ellagic acid rich fraction of Punica granatumtreated, 200 mg/kg): ellagic acid rich extract of Punicagranatum (200 mg/kg p.o.) was suspended in 0.5% CMCand administered once daily, starting 2 days beforethe induction of colitis and continued for 7 consecutivedays. On day 8, the mice were killed and macrosco-pic, histopathological and biochemical analyses wereperformed.Group VIII (sodium cromoglycate treated, 40 mg/kg):sodium cromoglycate (40 mg/kg i.p.) was dissolved indistilled water and administered once daily, starting2 days before the induction of colitis and continuedfor 7 consecutive days. On day 8, the mice were killedand macroscopic, histopathological and biochemicalanalyses were performed.Group IX (sulfasalazine treated group, 100 mg/kg):sulfasalazine (100 mg/kg p.o.) was dissolved in distilledwater and administered once daily, starting 2 daysbefore the induction of colitis continued for 7 consecu-tive days. On day 8, the mice were killed and macro-scopic, histopathological and biochemical analyses wereperformed.

Statistical analysis. The results were expressed as mean± standard error of means (SEM). The data obtainedfrom various groups were statistically analysed usingone-way analysis of variance (ANOVA) followed byTukey’s multiple range test. Disease activity index wasanalysed by two-way analysis of variance (ANOVA)followed by Bonferonni’s post test. A value of p < 0.05was considered to be statistically significant.

RESULTS

Effect of Punica granatum on dextran sulfate sodiuminduced macroscopic changes in colon

Administration of dextran sulfate sodium (DSS) resultedin severe macroscopic changes in the colon in terms

Table 1. Criteria for scoring disease activity index

Disease activity index

Weight Stool Occult blood/Score loss (%) consistency gross bleeding

0 None Normal Normal1 1–52 6–10 Loose stool Guiac (+)3 11–154 >15 Diarrhea Gross bleeding

Normal stools, well formed pellets; loose, pasty stools that do notstick to the anus; diarrhea, liquid stools that stick to the anus.



Table 2. Effect of hydroalcohol extract of Punica granatum and its ellagic acid rich fraction, sodiumcromoglycate and sulphasalazine on macroscopic and histopathological score of dextran sodium sulfateinduced colitis in mice

Dose Macroscopic HistopathologicalGroup Treatment (mg/kg p.o.) score score

1 Normal control – 0 ± 0 0 ± 02 DSS colitis control – 3.2 ± 0.5a 3.8 ± 0.4a

3 Punica granatum 100 1.5 ± 0.4b 2.2 ± 0.4b



(ellagic acid rich fraction)6 Punica granatum 200 0.5 ± 0.3b 1.2 ± 0.3b

(ellagic acid rich fraction)7 Sodium cromoglycate 40 (i.p.) 1.5 ± 0.4 1.5 ± 0.3b

8 Sulphasalazine 100 0.5 ± 0.3b 1.3 ± 0.4b

Values are expressed as mean ± SEM of six animals per group.a p < 0.05 in comparison with normal control group.b p < 0.05 in comparison with DSS colitis control group.

of mucosal erythema, edema, bleeding, erosions, ulcera-tion and tissue necrosis compared with normal mice(Table 2). Further, DSS administration also markedlyreduced the colonic length in the disease control groupcompared with the normal control group (8.1 ± 0.2 cmvs 10.6 ± 0.4 cm) (Table 3). However, treatment withthe hydroalcohol extract of Punica granatum (100 mg/kg and 200 mg/kg p.o.) and the ellagic acid rich fractionof Punica granatum (100 mg/kg and 200 mg/kg p.o.)significantly attenuated DSS-induced macroscopical changes.Treatment with sodium cromoglycate, a mast cell stabi-lizer, (40 mg/kg i.p.) and sulphasalazine, a standard forulcerative colitis, (100 mg/kg p.o.) also reduced the DSS-induced severity in gross lesion score.

Further, these interventions also attenuated theeffect of DSS on shortening of colonic length, thoughthe significant effects could not be obtained (Table 3).

Effect of Punica granatum on DSS-induced diseaseseverity

Administration of DSS significantly increased diseaseseverity in terms of the disease activity index (DSI),

Table 3. Effect of hydroalcohol extract of Punica granatum andits ellagic acid rich fraction, sodium cromoglycate and sulphasalaz-ine on colon length in dextran sodium sulfate induced colitis inmice

Dose Colon lengthGroup Treatment (mg/kg p.o.) (cm)

1 Normal control – 10.6 ± 0.42 DSS colitis control – 8.1 ± 0.23 Punica granatum 100 9.6 ± 0.64 Punica granatum 200 10.1 ± 0.55 Punica granatum 100 9.2 ± 0.3

(ellagic acid rich extract)6 Punica granatum 200 9.6 ± 0.4

(ellagic acid rich extract)7 Sodium cromoglycate 40 (i.p.) 9.4 ± 0.48 Sulphasalazine 100 9.2 ± 0.4

Values are expressed as mean ± SEM of six animals per group.

with severity at its peak on day 5. However, treatmentwith the hydroalcohol extract of Punica granatumand its ellagic acid rich fraction significantly attenuatedDSS-induced disease severity. Treatment with sodiumcromoglycate and sulphasalazine also attenuated DSS-induced increase in disease activity index (Fig. 1).

Effect of Punica granatum on DSS-inducedhistopathological changes in colon

Administration of DSS resulted in significant histopatho-logical changes in the colon as in terms of markeddestruction of surface epithelium, focal ulceration,cell disruption, transmural inflammation involving theentire section from epithelium to serosa. However, treat-ment with the hydroalcohol extract of Punica granatumand its ellagic acid rich fraction, sodium cromoglycateand sulphasalazine attenuated DSS-induced histopatho-logical changes in colon (Table 2, Figs 2 and 3).

Effect of Punica granatum on myeloperoxidase(MPO) activity

Administration of DSS significantly increased MPO acti-vity in colonic tissue compared with the normal controlgroup. However, treatment with Punica granatum andits ellagic acid rich fraction, sodium cromoglycate andsulphasalazine significantly attenuated the DSS-inducedrise in colonic MPO activity in colonic tissue (Fig. 4).

Effect of Punica granatum on histamine content incolonic tissue

Administration of DSS resulted in significant elevationof histamine in colonic tissue compared with the normalcontrol group. However, treatment with Punica granatumand its ellagic acid rich fraction significantly attenuatedthe DSS-induced rise in colonic histamine level. Sulphas-alazine and sodium cromoglycate also attenuated theDSS-induced rise in histamine level in colonic tissue(Fig. 5).



Figure 1. Effects of Punica granatum (PG) hydroalcohol extract (100 and 200 mg/kg p.o.), Punica granatum ellagic acid rich extract(100 and 200 mg/kg p.o.), sodium cromoglycate (40 mg/kg i.p.) and sulfasalazine (100 mg/kg p.o.) on severity of disease measured interms of disease activity index (DAI) in dextran sulfate sodium (DSS) induced colitis in mice. Results are expressed as mean ± SEMof six observations. a p < 0.05 compared with the normal control group; b p < 0.05 compared with the DSS colitis control group.

Figure 2. Effect of Punica granatum and mast cell stabilizer on dextran sodium sulfate induced histopathological changes in colon:In normal control group (A), the hematoxylin and eosin stained section of mice colon showed intact epithelial surface; ¥100. In DSScolitis control group (B), the colonic mucosa showed massive necrotic destruction of epithelium, submucosal edema and inflamma-tory cellular infiltration; ¥100. Treatment with Punica granatum hydroalcohol (C: 100 mg/kg p.o. and D: 200 mg/kg p.o.) showed aprotective effect against DSS-induced damage.

Effect of Punica granatum on oxidative stress markers

Administration of DSS resulted in a significant increasein the levels of thio-barbituric acid reactive substance(TBARS) and superoxide anions, when compared withthe normal control group. Administration of Punica

granatum and its ellagic acid rich fraction attenuatedthe DSS-induced increase in TBARS and superoxideanion generation in a significant manner. Treatmentwith sodium cromoglycate and sulphasalazine alsoattenuated the DSS-induced rise in oxidative stressin colonic tissue (Figs 6 and 7).



Figure 3. Treatment with Punica granatum ellagic acid rich extracts (E: 100 mg/kg p.o. and F: 200 mg/kg p.o.), showed a protectiveeffect against DSS-induced damage. Almost complete protection was observed with the highest dose of Punica granatum ellagic acidextract (200 mg/kg p.o.), sodium cromoglycate (G: 40 mg/kg i.p.) and sulphasalazine treated group (H: 100 mg/kg p.o.); ¥100.

Figure 4. Effects of Punica granatum (PG) hydroalcohol extract (100 and 200 mg/kg p.o.), Punica granatum ellagic acid rich extract(100 and 200 mg/kg p.o.), sodium cromoglycate (40 mg/kg i.p.) and sulfasalazine (100 mg/kg p.o.) on colonic myeloperoxidase (MPO)levels in dextran sulfate sodium (DSS) induced colitis in mice. Results are expressed as mean ± SEM of six observations. a p < 0.05compared with the normal control group; b p < 0.05 compared with the DSS colitis control group.



Figure 5. Effects of Punica granatum (PG) hydroalcohol extract (100 and 200 mg/kg p.o.), Punica granatum ellagic acid rich extract(100 and 200 mg/kg p.o.), sodium cromoglycate (40 mg/kg i.p.) and sulfasalazine (100 mg/kg p.o.) on colonic histamine contents indextran sulfate sodium (DSS) induced colitis in mice. Results are expressed as mean ± SEM of six observations. a p < 0.05 comparedwith normal control group; b p < 0.05 compared with DSS colitis control group.

Table 4. HPTLC fingerprinting profile of Punica granatumextract taking chloroform and methanol (80:20) as mobile phaseand using anisaldehyde as spraying agent

S. no of UV UVresolving bands 254 nm 366 nm White light

1 0.13 – 0.122 0.61 – 0.223 – – 0.344 – – 0.425 – – 0.636 – – 0.727 – – 0.79

DISCUSSION

Dextran sulfate sodium (DSS) induced ulcerative colitisin mice is a simple, reproducible and a well establishedmodel, which shares many characteristic features withhuman colitis (Cheon et al., 2006; Hirata et al., 2007).Experimentally, colitis is characterized macroscopicallyby mucosal erythema, bleeding, erosion, ulceration andtissue necrosis; while histopathologically the diseaseis associated with mucosal and epithelial damage, celldisruption, transmural ulceration and inflammation(Wallace et al., 1989; Millar et al., 1996). In the presentstudy, administration of DSS resulted in the development

Figure 6. Effects of Punica granatum (PG) hydroalcohol extract (100 and 200 mg/kg p.o.), Punica granatum ellagic acid rich extract(100 and 200 mg/kg p.o.), sodium cromoglycate (40 mg/kg i.p.) and sulfasalazine (100 mg/kg p.o.) on colonic TBARS level in dextransulfate sodium (DSS) induced colitis in mice. Results are expressed as mean ± SEM of six observations. a p < 0.05 compared withnormal control group; b p < 0.05 compared with DSS colitis control group.



Figure 7. Effects of Punica granatum (PG) hydroalcohol extract (100 and 200 mg/kg p.o.), Punica granatum ellagic acid rich extract(100 and 200 mg/kg p.o.), sodium cromoglycate (40 mg/kg i.p.) and sulfasalazine (100 mg/kg p.o.) on superoxide anion generationmeasured in terms of nitroblue tetrazolium (NBT) reduction in colonic tissue in dextran sulfate sodium (DSS) induced colitis in mice.Results are expressed as mean ± SEM of six observations. a p < 0.05 compared with the normal control group; b p < 0.05 comparedwith DSS colitis control group.

of colitis as assessed macroscopically, histopathologicallyand in terms of disease activity index. However, treat-ment with hydroalcohol extract of flowers of Punicagranatum (100 and 200 mg/kg p.o.) attenuated the DSS-induced macroscopical and histopathological alterationsin colon, along with reduction in disease severity. Theobserved antiulcerative actions of Punica granatum werecomparable to that of sulphasalazine, a standard drugfor the treatment of colitis, indicating the potentialof Punica granatum in ameliorating the disease stateof ulcerative colitis. Traditionally, Punica granatum hasbeen used for the treatment of colitis (Duke and Ayensu,1985) and experimentally it was found effective incolon cancer (Adams et al., 2006), diarrhea and ulcers(Alkofahi and Atta, 1999). However, it is the first re-port indicating the ameliorative potential of Punicagranatum in DSS-induced ulcerative colitis in mice.

Punica granatum has been documented to containhydrolysable tannins, ellagic acid being the predominantone (Kasai et al., 2006; Wang et al., 2006; Yoshimuraet al., 2005). Earlier, it was documented that oraladministration of microspheres of ellagic acid providedprotection against DSS-induced ulcerative colitis inrats (Ogawa et al., 2002). Therefore, the ellagic acidrich fraction of Punica granatum extract was employedto explore the active constituent responsible for itsobserved protective activity. Treatment with the ellagicrich fraction of Punica granatum attenuated the diseasestate of ulcerative colitis in mice induced due to DSSadministration and its effects were comparable tothe hydroalcohol extract of Punica granatum. Based onthese results, it may be tentatively suggested that ellagicacid is the predominant constituent of Punica granatumresponsible for the antiulcerative activity in DSS-induced ulcerative colitis.

Myeloperoxidase (MPO) is an enzyme released fromneutrophils and has been used as an index of inflamma-tion and neutrophil influx (Boughton-Smith et al., 1988).

In the present investigation, administration of DSSwas associated with significant rise in the colonic MPOactivity indicating the acute inflammation, with intenseinfiltration of neutrophils in colonic region. However,treatment with Punica granatum hydroalcohol and itsellagic acid rich fraction significantly attenuated colonicMPO activity associated with ulceration. Inflammationhas been documented to play a key role in the patho-genesis of colitis in experimental as well as in clinicalsetup (Boismenu and Chen, 2000). Moreover, there havebeen a number of reports suggesting antiinflammatoryactivities of Punica granatum (Jung et al., 2006). There-fore, it may be possible to suggest that Punica granatummediated antiinflammatory actions are responsible itsantiulcerative effects in DSS-induced colitis.

Mast cells are multi-effector cells playing an importantrole in inflammation by releasing histamine, serotonin,tryptase, chymase, elastase, carboxypeptidase A and B,cathepsin, b-galactosidase, b-glucuronidase, chemotacticfactors, platelet activating factor, prostaglandins, leuko-trienes and cytokines such as IL-1, 5, 6, TNF-a, TGF-b and other growth factors (Mekori and Metcalfe, 2000;Prussin and Metcalfe, 2006; Bischoff, 2007). Histamineis the predominant vasoactive amine localized in themast cells and its release has been used as a specificmarker of mast cell degranulation (Hogan and Schwartz,1997). In the present study, administration of DSSwas also associated with an increase in histamine con-tent in colonic tissue, indicating mast cell degranulation.It suggests that mast cell degranulation with the conse-quent release of various inflammatory mediators maybe responsible for the pathogenesis of DSS-inducedcolitis. Earlier, it was documented that mast cells playa key role in the pathogenesis of DSS-induced colitis(Videla et al., 2007). Further, treatment with sodiumcromoglycate, a mast cell stabilizer, also attenuated theseverity of colitis associated with DSS administration.The present study results are in consensus with earlier



reports documenting the effectiveness of mast cellstabilizers in ameliorating the disease status of colitis(Marshall and Irvine, 1998). Moreover, treatment withthe hydroalcohol extract of Punica granatum and itsellagic acid fraction also attenuated DSS-inducedincrease in the histamine level in a significant manner.These results suggest that Punica granatum may havestabilized the mast cells and prevented their degranula-tion, which may also be a possible mechanism for itsnoted antiulcerative effects. Based on these results, theprotective effects of Punica granatum in DSS-inducedulcerative colitis may be possibly attributed to its anti-inflammatory and mast cell stabilizing activities.

During inflammation reactions, polymorphonuclearcells elaborate reactive oxygen species (Hasegawaet al., 1997), which further contribute significantly intissue injury. It has been well reported that reactiveoxygen species are produced in excess in the inflamedmucosa and thus, play a crucial role in the pathobiologyof colitis (Grisham, 1994). Even sulphasalazine, amongthe currently available treatments, has been documentedto modify the disease state through an antioxidantmechanism (Simmonds et al., 1992). In the present studyalso, DSS-induced colitis was associated with a signifi-cant rise in TBARS levels and superoxide anion gen-eration, markers of oxidative stress and reactive oxygenspecies generation. Treatment with Punica granatumand its ellagic acid rich fraction markedly attenuatedthe DSS-induced rise in oxidative stress. Punica granatumis well documented to exhibit potent antioxidant actionsboth in vitro and in vivo systems (Chidambara Murthyet al., 2002). Therefore, it is suggested that antioxidantactions of Punica granatum also contribute in a signifi-cant manner to its protective effects in DSS-inducedcolitis.

Collectively, the ameliorative actions of Punica granatumin DSS-induced colitis may be attributed to mast cell

stabilizing, antiinflammatory and antioxidant activities.These contributing factors may be actually interlinkedas mast cell degranulation has been reported to triggerand amplify oxidative stress and inflammation (Swindleand Metcalfe, 2007). Accordingly, sodium cromoglycatemediated antioxidant and antiinflammatory actions maybe linked indirectly to mast cell stabilizing activity.Conversely, oxidative stress and inflammation has alsobeen documented to trigger mast cell degranulation(Suzuki et al., 2005). On this basis, the sulphasalazineinduced decrease in mast cell degranulation may possiblybe attributed indirectly to a decrease in inflammationand oxidative stress. However, further studies are requiredto identify the primary target site of Punica granatummediated antiulcerative actions in DSS-induced cascadeof reactions involving mast cell degranulation, inflamma-tion and oxidative stress.

CONCLUSION

Punica granatum has ameliorative potential in attenu-ating DSS-induced ulcerative colitis and its ellagic acidmay be the predominant constituent responsible forantiulcerative actions. Further, Punica granatum medi-ated mast cell stabilization along with antiinflammatoryactions and antioxidant effects may be responsible forits beneficial effect.

Acknowledgements

The authors are grateful to Dr Ramesh Kundal, Associate Professor,Government Medical College Patiala, for assisting in the histopatholo-gical analysis of colonic tissue. The authors are also grateful to DrAshok Kumar Tiwary, Head, Department of Pharmaceutical Sciencesand Drug Research, Punjabi University, Patiala, India for supportingthis study and providing technical facilities for the work.

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