Gastroprotective Effect of Senecio Candicans DC on Experimental Ulcer Models

5/26/2018 Gastroprotective Effect of Senecio Candicans DC on Experimental Ulcer Model...

http:///reader/full/gastroprotective-effect-of-senecio-candicans-dc-on-experimen

Journal of Ethnopharmacology 140 (2012) 145150

Contents lists available at SciVerse ScienceDirect

Journal ofEthnopharmacology

j ournal homepage: www.elsevier .com/ locate / je thpharm

Gastroprotective effect ofSenecio candicans DC on experimental ulcer models

Lakshmanan Hariprasath, RamanJegadeesh, Nanjian Raaman

Centre forAdvanced Studies in Botany, University of Madras, GuindyCampus, Chennai 600025, India

a r t i c l e i n f o

Article history:

Received 2 June 2011

Received in revised form

29 September 2011

Accepted 2 January 2012

Available online 10 January 2012

Keywords:

Senecio candicans

Antiulcerogenic

Acute toxicity

Aqueous extract

a b s t r a c t

Ethnopharmacological relevance: Senecio candicans DC (Asteraceae) is used as a remedy for gastric ulce

and stomach pain in the Nilgiris district, Tamil Nadu for which no scientific evidence exists.

Aim ofthe study: The present study was performed to evaluate the gastroprotective effects and acute ora

toxicity ofaqueous leafextract ofSenecio candicans (AESC) in experimental models.Materials andmethods: The antiulcerogenic activity ofAESC was performed in two different ulcer model

viz., pylorus-ligated model and ethanol-induced model using Wistar albino rats. Acute toxicity study wa

also performed to get information on the admissible dose for treatment ofulcer. Preliminary phytochem

ical screening of AESC was performed to find the active principles present, which are thus responsibl

for the antiulcerogenic activity. DPPH assay was performed to confirm the antioxidant activity ofAESC

Results: The acute toxicity study did not show any mortality up to 2500 mg/kg b.w. of AESC. Both th

ulcer models showed gastroprotective effect comparable to that ofthe standard Omeprazole. The result

of antioxidant enzymes, histopathology sections, ATPase and mucus content ofgastric secretion showed

that several mechanisms are involved in the gastroprotective effect. The preliminary phytochemica

screening revealed the presence ofalkaloids, flavonoids and steroids in AESC. The DPPH assay confirme

the antioxidant activity ofAESC.

Conclusion:The traditional consumption ofAESC for the treatment ofgastric ulcer is thus true, the antiox

idant constituents present in the extract plays a major role in the gastroprotective activity, but sinc

Senecio species are known for the presence ofpyrrolizidine alkaloids, a detailed study in future is require

to describe the safe dose for a prolonged period.

2012 Elsevier Ireland Ltd. All rights reserved

1. Introduction

Peptic ulcer disease (PUD) encompassing gastric and duodenal

ulcer is the most prevalent gastrointestinal disorder (Valle, 2005).

Duodenal,gastric ulcersand gastric cancerare common and serious

diseases allover theworld(Calam andBaron, 2001). These patholo-

gies areamong themostimportantcausesof morbidityin theworld

population. Peptic ulcers are focal lesions of gastric or duodenal

mucosa, occurringat siteswhere the mucosal epitheliumis exposed

toacidand pepsinandare characterized bygnawingor burning sen-

sationintheabdomen(Dhuley andNaik, 1998). Traditionally peptic

ulcers have been described as an imbalance between the luminal

acid peptic attacks versus the mucosal defence (Mutra et al., 1996).

This life time prevalence of PUD is about 10% (Brunton, 1996). An

estimated 15,000 deaths occur each year as a consequence of PUD

(Valle, 2005). Despite the availability of many orthodox medica-

tions for PUD, the morbidity and mortality toll is still very high.

Corresponding author at: Centre for Advanced Studies in Botany, Life Sciences

Building (2nd floor), University of Madras, Guindy Campus, Chennai 600 025.

Tel.: +91 44 43833036.

E-mail address: [email protected](L. Hariprasath).

In the United States, about 6500 deaths occur each year on ulcer

related complications (Sonnenberg, 1994). Many pharmaceutica

products have been employed for the treatment of gastroduodena

and peptic ulcer, but these products are still too expensive for th

less wealthy population (Toma et al., 2004). Thus,in the foreseeabl

future, therapy for gastric and duodenal ulcers will be one of th

major research goals.

Medicinal plants are reservoirs for drugs and lead compound

for many therapeutic agents. The treatment of peptic ulcers wit

plant products used in folk medicine and the protection of induced

gastric ulcer in laboratory animals using medicinal plants was we

documented (Disi et al., 1998). There are avalanche of scientifi

support on the efficacy of medicinal plants in the management o

ulcers of different aetiologies (Austin and Jegadeesan, 2000; Akah

et al., 2001; Nwafor and Akah, 2003; Nwafor and Okoye, 2005).

Senecio candicans DC (Asteraceae) is a sub-shrubby climber

endemic to the Western Ghats, India. Water boiled with leaves i

used to treat gastric ulcer and stomach pain in various parts of Nil

giris district, Tamil Nadu, India. No evidence or detailed scientifi

investigations have been carried out to define the antiulcerogeni

activities ofSenecio candicans. Thus, the present work sets out t

study the antiulcerogenic activity of aqueous leaf extract ofSeneci

candicans (AESC). The effect produced by theextract was compare

0378-8741/$ see front matter 2012 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.jep.2012.01.002
http://localhost/var/www/apps/conversion/tmp/scratch_8/dx.doi.org/10.1016/j.jep.2012.01.002http://localhost/var/www/apps/conversion/tmp/scratch_8/dx.doi.org/10.1016/j.jep.2012.01.002http://www.sciencedirect.com/science/journal/03788741http://www.elsevier.com/locate/jethpharmmailto:[email protected]://localhost/var/www/apps/conversion/tmp/scratch_8/dx.doi.org/10.1016/j.jep.2012.01.002http://localhost/var/www/apps/conversion/tmp/scratch_8/dx.doi.org/10.1016/j.jep.2012.01.002mailto:[email protected]://www.elsevier.com/locate/jethpharmhttp://www.sciencedirect.com/science/journal/03788741http://localhost/var/www/apps/conversion/tmp/scratch_8/dx.doi.org/10.1016/j.jep.2012.01.002



146 L. Hariprasath et al. / Journal of Ethnopharmacology 140 (2012) 145150

with the standard antiulcerogenic drug Omeprazole. We investi-

gated the acute toxicity of AESC and also performed a preliminary

phytochemical screening to identify the active principles responsi-

ble for the gastroprotective effect.

2. Materials and methods

2.1. Chemicals

Assay kits for the activity of catalase (CAT), superoxide

dismutase (SOD) and lipid peroxidation (LPO), 1,1-diphenyl-

2-picrylhydrazyl (DPPH), quercetin were obtained from

SigmaAldrich (Mumbai, India). All other chemicals used for

the studies were of analytical grade.

2.2. Preparation of crude aqueous leaf extract

Fresh leaves were collected from Kundah and Oddayaratty hills

of the Nilgiris, India during the month of July and were authen-

ticated by the Botanical Survey of India, Coimbatore, India. A

herbarium has been submitted to the Centre for Herbal Sciences,

University of Madras with accession number CHS-SC-203. The

cleaned leaves were shade dried at room temperature and pow-

dered. Thedried powder of leaf (500g) was boiled in 2 L of distilled

water for 1h and filtered. This was repeated for 23 times with

the same powder. The filtrate was lyophilized and finally 68g of

aqueous extract was obtained.

2.3. Experimental animals

Healthy male and female Wistar albino rats weighing between

180 g and 200 g were used in this study. The female animals

used were nulliparous and non-pregnant. The animals were fed

pellet feed (purchased from Tamil Nadu Veterinary and Animal

Sciences University, Chennai, India). Food and water were pro-

vided ad libitum during acclimation and throughout the study. The

animals were maintained at 22C (3 C) and relative humidity

around 5060%. Animals were housed in polypropylene cages overhusk beddings and 12h light and 12h dark cycle was maintained

throughout the experimental period. All the animal experiments

were performed after getting necessary approval from the Insti-

tutional Animal Ethical Committee (IAEC No. 03/013/08, approval

date 28.04.2008) of University of Madras, governed by the guide-

lines of Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA), Govt. of India. All efforts were

made to minimize both the number of animals used and unwanted

stress or discomfort to the animals throughout experimental pro-

cedures.

2.4. Acute toxicity studies

Animals were divided into six groups, each group containing sixanimals. Group 1 animals served as control which received normal

saline. Group 2Group 6 animals received AESC in single doses of

500, 1000, 1500, 2000 and 2500 mg/kg b.w., respectively. The AESC

was suspended in normal saline (0.9% NaCl solution) and admin-

istered by gavage (P.O.). The animals were observed continuously

for 1 h after treatment, then intermittently for 4 h, and thereafter

for over a period of 14 days after administration (Silva et al., 1997)

for behavioural changes, signs of toxicity and/or death.

2.5. Gastroprotective activity

2.5.1. Pylorus-ligated ulcer model

The antiulcerogenic activity of AESC was performed in pylorus-

ligated ulcer model as described by Oliveira et al., 2004. Animals

were divided into 4 groups each of six rats. Group 1 animals

received normal saline and served as control. Group 2 animals

received Omeprazole (40mg/kg) orally and served as the refer-

ence drug for comparison. Groups 3 and 4 animals received 250

and 500mg/kg b.w. of AESC, respectively.

Rats in all the groups were fasted in individual cages for 24h

before the administration of AESC. All the above treatments were

made 1 h prior to pyloric ligation. Pyloric ligation was performed

in these animals under mild ether anaesthesia. The animals weredeprived of food and water during post-operative period. The

pyloric portion of the stomach was identified, slightly lifted out

and ligated, avoiding traction to the pylorus or damage to the

blood supply. The stomach was then replaced carefully and the

abdominal wall closed by interrupted sutures. Four hours after

ligation, all the animals were sacrificed. The stomach was cut

along the greater curvature and separated from the surrounding

tissues and thus brought out as a whole along with its con-

tents. The content (gastric juice) was subjected to centrifugation

at 3000rpm for 10min and the clear supernatant was then ana-

lysed for its volume, pH, the total acidity, free acidity, ulcer index

and percent inhibition. The status of lipid peroxidation (LPO),

catalase (CAT) and superoxide dismutase (SOD) were estimated

by its appropriate kits (SigmaAldrich, Mumbai, India). A por-

tion of the stomach was subjected to histopathology. The pH

was estimated using pH strips with pH ranges of 2.04.5 and

5.08.5.

2.5.2. Ethanol-induced ulcer model

The antiulcerogenic activity of AESC was performed in ethanol-

induced ulcer model as described by Hollander et al. (1985). In

this study, rats were divided into 4 groups. Group 1 animals

received normal saline and served as control. Group 2 animals

received Omeprazole (40mg/kg) orally and served as the refer-

ence drug for comparison. Groups 3 and 4 animals received 250

and 500mg/kg b.w. of AESC, respectively.

Gastric lesions were induced by oral administration of 1 mL of

absolute ethanol per rat. Test substances were given 30min beforethe ulcerative agent and after 1 h animals were sacrificed by cervi-

cal dislocation and stomach was incised alongthe greater curvature

and examined for ulcers in the glandular region. Erosions formed

on the glandular portions of the stomach were counted and each

given a severity rating on a 15 scale based on the diameter of

the ulcer. The status of antioxidant enzymes SOD, CAT and LPO

were determined. Stomach of all treated and control rats were

subjected to visual macroscopic examination and ulcer score was

calculated.

2.5.3. Determination of free and total acidity in gastric juice

The free and total acidity of gastric juice collectedfrom pylorus-

ligated rats was determined by volumetric analysis as detailed by

Hawk (1947). One mLof gastric juice was pipetted into a 100 mLconical flask and 23 drops of Topfers reagent (HiMedia Pvt., Ltd.,

Mumbai) was added and titrated with 0.01N NaOH (which was

previously standardized with 0.01N oxalic acid) until all traces of

red colour disappears and turned to yellowish orange. The volume

of alkali was noted. This volume corresponds to free acidity. Then

23drops of phenolphthalein solution wasaddedand titration was

continued until a definite red tinge reappears. Again, the total vol-

ume of alkali added was noted. This volume corresponds to total

acidity. The free and total acidity of gastric juice was calculated by

using the following formula:

Acidity =volume of NaOH normality of NaOH

0.1

100meq/L per 100 g



L. Hariprasath et al. / Journal of Ethnopharmacology 140 (2012) 145150 14

2.5.4. Determination of ulcer index (UI) and percentage inhibition

Ulcer index and percentage inhibition was calculated in both

pylorus-ligated and ethanol-induced rats. For the determination of

ulcer index, the stomach was cut open along the greater curvature

and the inner surface was examined for ulceration with the help

of a simple dissecting microscope. Usually, circular lesions were

observed but, sometimes, linear lesions were also seen. The ulcer

index was calculated according to the scoring method ofTan et al.

(1996).

Score 0 no ulcer;

Score 1 vessel dilation and pointed ulcers;

Score 2.5 small ulcers 5mm long.

UI for each animal was calculated as mean ulcer score. The per-

centage of inhibition was calculated by the following formula:

UI control UI treated

UI control 100

2.5.5. Histopathological studies

The freshly excised stomachs were washed with saline and pre-served in 10% formaldehyde solution for histopathological studies.

The sections of stomachswere stained withhaematoxylinand eosin

and permanent mounts of the tissues were prepared (Bancroft

and Cook, 1984) to investigate the histopathological changes. The

microscopic slides were photographed.

2.5.6. Estimation of H+K+ATPase activity

The H+K+ATPase activity was assayed in ethanol-induced ulcer

animals (Nagayaet al., 1987). HundredL of thetissue homogenate

was taken in centrifuge tube and incubated in the assay medium

(70mM Tris buffer, 5mM MgCl2, 10 m M KCl in a total volume of

1mL; pH 6.8) for 1h. The reaction was initiated by adding 2mM

ATP, incubated at 37 C for 20min and the reaction was stopped by

10% TCA. Theprecipitates formedon addition of TCAin both thetestand tissue control tubes were removed by centrifugation and their

supernatants were transferred to fresh tubes. The reagent blank

contained 1.8mLof TrisHCl buffer. The standard tubes containing

Pi taken at a concentration range of 210g/mL were placed in

dist. water and were made up to 1.8 mLwith TrisHCl buffer. To all

the above tubes, 0.5 mLof ammonium molybdate and 0.2mLof 1-

amino-2-naphthol-4-sulphonic acid was added and left for 20min

forthe blue colourto develop, which wasreadat 620nm against the

reagent blank using a spectrophotometer. Results are expressed as

mmol of Pi liberated/min/mg protein.

2.5.7. Determination of mucus in gastric content

The assay was performed in ethanol-induced ulcer animals

according to the methodology described by Sun et al. (1991)with some modifications. The gastric contents of the stomach was

immersed in 10mL 0.02% Alcian blue in 0.16M sucrose/0.05M

sodium acetate, pH 5.8, and incubated for 24h at 20C. The Alcian

blue binding extract was centrifuged at 3000rpm for 10min. The

absorbance of the supernatant was measured at 615nm using a

spectrophotometer. The free mucus in the gastric content was cal-

culated from the amount of Alcian blue binding (mg/wet tissue

(g)).

2.6. Preliminary phytochemical screening

The crude AESC was subjected to qualitative chemical screen-

ing for the identification of major chemical constituents such as

alkaloids (Dragendorffs test: Waldi, 1965), phenolic constituents

(Ferric chloride test: Mace, 1963), flavonoids and phytosterol

(Asongalem et al., 2004).

2.7. In vitro antioxidant assay

The scavenging activity of DPPH radicals by AESC wasmeasured

according to the method reported by Yen and Hsieh (1998). Assay

were performed in 1.5 mLreaction mixtures containing 1.0mLo

0.1mM DPPH ethanol solution and 1.0 mLof different concentrations of crude extracts and fractions ofSenecio candicans (500, 250

125, 63, 32, 16 and 8g/mL) or 0.5 mLethanol (as control). Afte

30min of incubation at room temperature under dark condition

the absorbance of the reaction mixtures were measured at 517nm

Quercetin was used as the standard antioxidant. The inhibitor

effect of DPPH was calculated according to the following formula:

Inhibition (%) =

absorbancecontrol absorbancesample

absorbancecontrol

100

Axy scatter graph was plotted to determine the inhibitory con

centration (IC50) according to the regression equation.

2.8. Statistical analysis

The data were subjected to One-way Analysis of Varianc

(ANOVA) and Tukeys Multiple Comparison Test was done to eval

uate the significance of difference of means of various treatmen

groups, using SPSS statistical software package (Version: 10). Th

values are presented as meanSD and *P




Table 1

Effectof aqueous leaf extract ofSenecio candicans on various gastric parameters of pylorus-ligated and ethanol-induced rats.

Treatment Group 1 (control) Group 2 (Omeprazole 40 mg/kg) Group 3 (AESC 250 mg/kg) Group 4 (AESC 500 mg/kg)

Pylorus ligated ulcer model

Gastric volume (mL) 8.250.26 6.560.22 a*** 7.820.13 a**; b** 7.270.37 a***; bns

pH 2.290.2 4.750.17 a*** 4.090.33 a***; bns 4.480.32 a***; bns

Free acidity (mEq L1 100g1) 56.671.15 27.931.47 a*** 42.672.31 a***; b 38.533.56 a***; b

Total acidity (mEqL1 100g1 ) 109.333.06 75.331.15 a*** 99.336.11 a**; b*** 82.533.23 a***; bns

Ulcer index 31.752.3 2.172.17 a*** 16.582.29 a***; b*** 8.750.94 a***; b***

Percent ulcer inhibition (%) 93.18 47.26 72.39Ethanol-induced ulcer model

Ulcer index 32.422.82 2.420.80 a*** 10.752.72 a***; b*** 4.330.52 a***; bns

Ulcer inhibition (%) 92.59 66.73 83.52

Values presented are meanS.D. of sixnumbersof animals in each group (n= 6). Multiple comparisons between treatmentgroups wereperformed by Tukeys test. a: Group

1 compared with Groups24; b: Group 2 compared with Groups3 and4. ns: not significant. *P



L. Hariprasath et al. / Journal of Ethnopharmacology 140 (2012) 145150 14

Fig.2. Status of antioxidant enzymesin gastric juice of ethanol-induced ratstreated

with aqueous leaf extract ofSenecio candicans. (A) Superoxide dismutase; (B) lipid

peroxidation and (C) catalase. Values presented are meanS.D. of six numbers of

animals in each group. Multiple comparisons were performed by Tukeys test. a:

Group 1 compared with Groups 24; b: Group 2 compared with Groups 3 and 4.

*P




References

Akah, P.A., Nwafor, S.V., Okoli, C.O., Orji, U.I., 2001. Evaluation of anti-ulcer proper-ties ofPseudocedrela kotschyi stems bark extract. Discovery and Innovation 13,132135.

Asongal em, E.A., Foyet, H.S., Ekobo, S., Dimo, T., Kamtchouing, P., 2004.Anti-inflammatory, lack of central analgesia and antipyretic properties ofAcanthus montanus (Ness) T. Anderson. Journal of Ethnopharmacology 95,6368.

Austin, A., Jegadeesan, M., 2000.Gastric and duodenalanti-ulcer and cytoprotectiveeffects ofCissus quadrangularis L. variant in rats. Nigerian Journal of NaturalProducts and Medicine 6, 1014.

Bancroft, J.D., Cook, B.C., 1984. Manual of Histological Techniques. Churchill Living-stone, Edinburgh, NY.

Brunton,L.L.,1996.Agentsfor controlof gastric acidityand treatment of pepticulcer.In: Hardman,J.G., Limbird, L.E., Molinoff, P.B., Ruddon, R.W., Gilman, A.G.(Eds.),ThePharmacological Basis of Therapeutics. , 9thed. McGraw-Hill, NewYork, pp.901915.

Calam, J., Baron, J.H.,2001. ABCof the upper gastrointestinal tract: pathophysiologyof duodenal and gastric ulcer and gastric cancer. British Medical Journal 323,980982.

Conforti, F., Loizzo, M.R., Statti, G.A., Houghton, P.J., Menichini, F., 2006. Biologicalproperties of different extracts of two Senecio species. International Journal ofFood Science and Nutrition 57, 18.

Dhuley, J.N., Naik, S.R., 1998. Protection by Rhinax in various models of ulcerationin rats. Journal of Ethnopharmacology 63, 219225.

Disi, A.M., Tamimi, S.O., Abuereish, G.M., 1998. Effects ofAnchusa strigosa rootaque-ous extract on gastric ethanol-induced ulcer in laboratory animals. Journal ofEthnopharmacology 60, 189198.

Hawk, S., 1947. Hawks Physiological Chemistry. McGraw-Hill Book Company, NewYork, 147.Hollander, D., Taranawski, A., Krause, W.J., Gergely, H., 1985. Protective effect of

sucralfate against alcohol-induced gastric mucosal injury in the rat. Gastroen-terology 88, 366374.

Mace, M.E., 1963. Histochemical localization of phenols in healthy and diseasedtomato roots. Phytopathology 16, 915925.

Mutra, S.K., Gopumadhavan, S., Hemavathi, T.S., Muralidhar, T.S., Venkataranganna,M.V., 1996.Protective effect ofUL-409,a herbalformulationagainst physicaland

chemical factor induced gastric and duodenal ulcers in experimental animals.Journal of Ethnopharmacology 52, 165169.

Nagaya, H., Satoh, H., Maki, Y., 1987. Actions of antisecretory agents on protontransports in hog gastric microsomes. Biochemical Pharmacology 36, 513519.

Nwafor, S.V., Okoye, C.F., 2005. Antiulcer properties of ethanol root extract ofCis-sampelosmucronata. Pharmaceutical Biology 43, 396403.

Nw afo r, S. V. , Akah, P .A., 20 03. E ffe ct o f methan ol leaf extract of Cissampelosmucronata A. rich against indomethacin induced ulcer in rats. Indian JournalofMedical Research 41, 181183.

Oliveira, F.A., Vieira@@Jr., G.M., Chaves, M.H., Almeida, F.R.C., Florencio, M.G.,Lima@@Jr., R.C.P., Silva, R.M., Santos, F.A., Rao, V.S.N., 2004. Gastroprotective

and anti-inflammatory effects of resin from Protium heptaphyllum in mice andrats. Pharmacological Research 49, 105111.

Silva, O.,Daurk, A., Pimentel,M., Viegas,S., Barroso,H., Machado,J., Pires, I., Carbrita,J., Gomes, E., 1997. Antimicrobial activity ofTerminilia macroptera root. JournalofEthnopharmacology 57, 203207.

Sonnenberg, A., 1994. Peptic ulcer. In: Digestive Diseases in the United States: Epi-demiology and Impact, No. 94-1447. US Department of Health and HumanServices, Public Health Services, National Institute of Health, pp. 359408.

Sun,X.B., Matsumoto, T., Yamada, H., 1991. Effects of a polysaccharidefractionfromthe roots ofBupleurum falcatum L. on experimental gastric ulcer models in ratsand mice. Journal of Pharmacy and Pharmacology 43, 699704.

Tan, P.V., Nditafon, G.N., Yewah, M.P., Dimo, T., Ayafor, J.F., 1996. Eremomastaxspeciosa: effects of leafaqueousextracton ulcer formation and gastric secretionin rats. Journal of Ethnopharmacology 54, 139142.

Toma, W., Trigo, J.R., Bensuaski de Paula, A.C., Souza Brito, A.R.M., 2004. Modulationofgastrin and epidermal growth factor by pyrrolizidine alkaloidsobtained fromSenecio brasiliensis in acute and chronic induced gastric ulcers. CanadianJournalofPhysiology and Pharmacology 82, 319325.

Valle, D.L., 2005. Peptic ulcerdiseases andrelateddisorders. In:Braunwald, E.,Fauci,A.S., Kasper, D.L., Hauser, S.L., Longo, D.L., Jameson, J.L. (Eds.), Harrisons Princi-ples of Internal Medicine. , 16th ed.McGraw-Hill, New York, pp.17461762.

Waldi, D., 1965. Spray reagents for thin-layer chromatography. In: Egon Stahl (Ed.),Thin-Layer Chromatography A Laboratory Handbook. Academic Press Inc.,Publishers, New York, U.S.A., p. 491.

Yen, G., Hsieh, C., 1998. Antioxidant activity of extracts from Duzhong (Eucom-mia ulmoides) towards various lipid peroxidation models in vitro. Journal ofAgricultural Food Chemistry 46, 39523957.

Documents

Gastroprotective Effect of Senecio Candicans DC on Experimental Ulcer Models