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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.2012.01.0025/26/2018 Gastroprotective Effect of Senecio Candicans DC on Experimental Ulcer Model...
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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
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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
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148 L. Hariprasath et al. / Journal of Ethnopharmacology 140 (2012) 145150
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
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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
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