Research Article [Kore et al.

, 2(7): June, 2011]

ISSN: 0976-7126

INTERNATIONAL JOURNAL OF PHARMACY & LIFE SCIENCES

Evaluation of antiulcer activity of protocatechuic acid ethyl
ester in rats
K. J. Kore*, P. P.Bramhakule, R.M. Rachhadiya and R.V.Shete
Department of Pharmacology, Rajgad Dnyanpeeth’s College of Pharmacy,
Bhor, Pune, (M.H.) - India
Abstract
To study the antiulcer activity of Protocatechuic acid ethyl ester using different models of gastric ulceration in rats.
Antiulcer activity of Protocatechuic acid ethyl ester was studied in rats in which gastric ulcers were induced by oral
administration of ethanol or aspirin or by pyloric ligation. Protocatechuic acid ethyl ester was administered in the
dose of 30 mg/kg and 60 mg/kg i.p. 30 min prior to ulcer induction. The antiulcer activity was assessed by
determining and comparing the ulcer index in the test drug group with that of the vehicle control group. Gastric total
acid output and pepsin activity were estimated in the pylorus ligated rats. Ranitidine and Sucralfate were used as a
reference drug. The ulcer index in the Protocatechuic acid ethyl ester treated animals was found to be significantly
less in all the models compared to vehicle control animals. This antiulcer property was more prominent in animals in
whom ulcers were induced by ethanol, aspirin and pyloric ligation. Ranitidine (30 mg/kg) produced a significant
gastric ulcer protection when compared with the control group. The anti-ulcer activity of Protocatechuic acid ethyl
ester was however, less than that of ranitidine. Our results suggest that Protocatechuic acid ethyl ester possesses
significant antiulcer property which could be either due to cytoprotective action of the drug or by strengthening of
gastric mucosa and thus enhancing mucosal defence.
Key-Words: Cyto protection, Mucosal defence, Ulcer Protection, Protocatechuic acid ethyl ester

Introduction
Peptic ulcer disease broadly refers to a group of The present study nominated modulatory role of
disorders characterized by the presence of ulcers in any protocatechuic acid ethyl ester in gastric ulcer
portions of g.i. tract, exposed to acid in sufficient treatment. Protocatechuic acid ethyl ester is white or
duration and concentration although these ulceration pale brownish yellow, crystalline powder; odourless or
most commonly occurs in the small intestine (duodenal has faint phenol-like odour Chemical name of
ulcer) or stomach (gastric ulcer). The most common Protocatechuic acid ethyl ester is Ethyl 3,4-
forms of peptic ulcers are gastric ulcer, duodenal ulcer, dihydroxybenzoate. Its molecular formula is C9H10O4
esophageal ulcer, meckel’s diverticulum ulcer. The and molecular weight is 182.2. Antioxidant1,
majority of gastric ulcer can be attributed to either H. Cardioprotective2, Iron deficiency3, Collagen
4
pylori or NSAIDs induced mucosal damage. Gastric production inhibitor , Hypoxia tolerance5,
ulcer that occurs in the pyloric area or those in the body Neuroprotective6, Antimicrobial & Antioxidant7,
associated with a duodenal ulcer. Gastric acid output Myoprotective activity8 of protocatechuic acid ethyl
tends to be normal or decrease in gastric acid animals. ester have already been reported. All the new alkyl
When gastric acid develops in the presence of minimal protocatechuate antioxidants studied possessed better
acid level, impairment of mucosal defense factor may radical-scavenging capacity than the natural
be present .Pathogenesis of gastric ulcer usually normal antioxidant protocatechuic acid. The antioxidant
to low acid levels, hyper acidity if present is due to activity indicates that protocatechuic acid ethyl ester
high serum gastrin, damage to mucus barrier. may posess antiulcer activity, so we designed the
protocol to evaluate the antiulcer activity
* Corresponding Author: protocatechuic acid ethyl ester.
E-mail: parnavi.b@gmail.com
Mob.: +91-9766000351

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Research Article [Kore et al., 2(7): June, 2011]
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Material and Methods Aspirin-induced gastric mucosal damage10,13
Experimental animals Albino rats (Wistar) of 200-250 g were selected, fasted
The study was conducted on Albino rats (Wistar) of for 36 h. Rats of either sex were randomly divided in to
200-250 g and maintained under standard conditions 5 groups of 6 animals in each group.Group-I : Normal
(room temperature 24- 27oC and humidity 60-65%) control (Normal saline), Group-II : Ulcerated control
with 12 h light and dark cycle. The food in the form of (30 mg/kg, p.o.), Group-III: Ranitidine (50
dry pellets (Amrut Lab., Pune) and water were mg/kg,i.p.), Group-IV :Protocatechuic acid ethyl ester
available ad libitum. The animal experiments were (30 mg/kg, i.p.), Group-V : Protocatechuic acid ethyl
approved by the ethics committee of the institute. ester (60 mg/kg, i.p.).
Chemicals and drugs After 30 min., aspirin suspended in 1% CMC in water
Protocatechuic acid ethyl ester, ethanol (Yash (20 mg/ml) at a dose of 30 mg/kg was administered
Chem.,Pune),aspirin (Research lab, Mumbai), orally to all the animals and 4 h later, the animals were
ranitidine (Cipla, Mumbai) sucralfate (Dr. Reddys lab, sacrificed. The stomach was removed and opens along
Mumbai), carboxy methyl cellulose (CMC), the greater curvature. The number of ulcer spots in the
trichloroacetic acid (Research lab, Mumbai), phenol glandular portion of the stomach were counted in both
reagent (Research lab, Pune), bovine albumin serum control and drug treated animals and the ulcer index
(Research lab. Fine chem. industries, Mumbai), std. was calculated.
phenol solution (Research lab, Pune), conc. HCl Gastric cytoprotection methods (Ethanol induced
(Research lab, Fine chem industries, Mumbai), NaOH ulcers)14-15
(Research lab Fine chem industries, Mumbai) were Albino rats (Wistar) of 200-250 g are maintained
used in the study. under standard conditions (room temperature 24- 27oC
Experiment design and humidity 60-65%) with 12 h light and dark cycle.
Acute Toxicity Studies (OECD 423)9 The food in the form of dry pellets (Amrut Lab., Pune)
The acute toxicity study was done as per the OECD and water were available ad libitum. Rats of either sex,
guidelines (423). The compounds were administered were randomly divided in to 5 groups of 6 animals in
i.p. in different doses, where 24 h toxicity was recorded each group.Group-I: Normal control (Normal saline),
to identify the toxic dose. No mortality and no signs of Group-II: Ulcerated control (1 ml ethanol, p.o.),
toxicity were found at the dose of 300 mg/kg body Group-III: Sucralfate (400 mg/kg,p.o.), Group-IV:
weight of protocatechuic acid ethyl ester. Therefore, it Protocatechuic acid ethyl ester (30 mg/kg, i.p.),
might be considered that protocatechuic acid ethyl ester Group-V: Protocatechuic acid ethyl ester (60 mg/kg,
have an LD50 value above 300 mg/kg. Two doses 30 i.p.).
mg/kg and 60 mg/kg were selected for present study. Thirty minutes after the test or reference drug or the
Pyloric ligation method10-12 control vehicle treatment, 1 ml of ethanol was orally
In this method, albino rats were fasted in individual administered to each rat. After 1 h the rats were
cages for 24 h. Care was being taken to avoid euthanized with excess of anesthetic ether and stomach
coprophagy. Rats of either sex, were randomly divided was cut open along the greater curvature, cleared of
in to 5 groups of 6 animals in each group. Group-I: residual matter with saline and the inner surface was
Normal control (Normal saline), Group-II : Ulcerated examined for ulceration. Ulcer index and % ulcer
control(1% CMC, 5 ml/kg, p.o.)., Group-III: protection were calculated by using the methods
Ranitidine(30 mg/kg,i.p.)., Group-IV: Protocatechuic described earlier.
acid ethyl ester (30 mg/kg, i.p.)., Group-V: Determination of various parameters
Protocatechuic acid ethyl ester (60 mg/kg, i.p.). Determination of ulcer index (UI)16-17
Protocatechuic acid ethyl ester or reference drug or The ulcerative index was calculated by severity of
control vehicle was administered 30 min prior to gastric mucosal lesions and graded as follows;
pyloric ligation. Under light ether anesthesia, the Erosions Score
abdomen was opened and the pylorus was ligated. The 1 mm or less 1
abdomen was then sutured. At the end of 4 h after 1-2 mm 2
ligation, the animals were sacrificed with excess of More than 2 mm 3
anesthetic ether, and the stomach was dissected out. Then the UI was calculated by using the formula:
Gastric juice was collected and its volume was UI = 1 (no. of lesions of grade 1) + 2 (no. of lesions of
measured. The glandular portion was then exposed and grade 2) + 3 (no. of lesions of grade 3). Then the
examined for ulceration. Ulcer index was determined. overall score was divided by a factor 10, which was

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Research Article [Kore et al., 2(7): June, 2011]
ISSN: 0976-7126
designed as ulcer index. % gastro protection was Results and Conclusion
calculated according to Effect of Protocatechuic acid ethyl ester in pylorus
% gastro protection = (UIC-UIT)/UIC*100 ligated rats
Where, UIC-ulcer index of control, UIT-ulcer index of Pylorus ligation in ulcerated control group had
test. produced ulcer in all animals and the mean ulcer index
Collection of gastric juice was 4.20±0.19 indicating the ulcerogenic effect.
Gastric juice was collected from pylorus ligated rats. Another ulcerogenic effect as compared to normal
The gastric juice collected was centrifuged at 60 rpm control was measured as follows; mean gastric content
for 10 min. and the volume of gastric juice was volume as 6.18±0.17, pH as 1.67±0.05, free acidity as
measured. The gastric juice was used for biochemical 36.23 ± 1.60, total acidity as 75.23 ± 1.50, pepsin
estimation. content as 5.16 ± 0.59, indicating the ulcer production
Determination of free acidity and total acidity11 in animals. Pylorus ligation also produced ulcers in all
Gastric juice (1 ml) was taken in to a 100 ml conical the protocatechuic acid ethyl ester pretreated animals.
flask, to this 2-3 drops of Topfer’s reagent was added However, the ulcer index showed significant dose
and titrated with 0.01 N NaOH until all traces of red dependent reduction in the animal pretreated with
colour disappears and the colour of the solution turns protocatechuic acid ethyl ester 30 mg/kg (UI; 3.02 ±
yellowish orange (end point). The volume of alkali 0.10) and 60 mg/kg (UI; 2.15 ± 0.08).It indicated
added was noted. This volume corresponds to free 28.09% gastroprotection at 30 mg/kg and 48.80%
acidity. Then 2-3 drops of phenolphthalein solution gastroprotection at 60 mg/kg as compared with
was added and titration was continued until a definite ulcerated control. The results indicate that the higher
red tinge reappears. The volume of alkali added was dose of protocatechuic acid ethyl ester i.e. 60 mg/kg
noted which corresponds to total acidity. Acidity was was effective in protecting ulcers in pylorus ligated
calculated by using the formula; rats. Pylorus ligation had produce ulcers in all animals
Acidity (mEq/litre) = Volume of NaOH* Normality of pretreated with Ranitidine 30 mg/kg. However, ulcer
NaOH * 100/0.1 index (1.58 ± 0.09) showed significant reduction as
Estimation of pepsin12,18-19 compared with ulcerated control and showed 62.38 %
For estimation pepsin, placed 4 test tubes (1) and (2) gastroprotection.
containing 5 ml of 1% bovine albumin in 0.01 M Effect of Protocatechuic acid ethyl ester in Aspirin
HCL, (3) and (4) containing 10 ml of 0.35 M induced gastric ulcer
trichloroacetic acid. The gastric juice was mixed with Aspirin induced ulcerated control group had produced
an equal volume of 0.01 M HCL, warmed to 370 c. 1 ulcer in all animals and the mean ulcer index was 2.15
ml of this mixture was added to each of test tubes of ± 0.11 indicating the ulcerogenic effect. Pylorus
(1) and (4). Incubated for 15 min. At the end mixed ligation also produced ulcers in all the protocatechuic
content of tube (1) with (3). Allow to stand for about 4 acid ethyl ester pretreated animals. However, the ulcer
min. (1)+(3) gives test and (2)+ (4) gives blank.The index showed significant dose dependent reduction in
mixture was filtered. To 2 ml of the filtrate, 10 ml of the animal pretreated with protocatechuic acid ethyl
NaOH was added. Then 1 ml of phenol reagent was ester 30 mg/kg (UI; 1.15 ± 0.05) and 60 mg/kg (UI;
added and mixed by gentle rotation. After 30 min.,the 0.75 ± 0.12).It indicated 46.51% gastro protection at 30
absorbance was measured at 680 nm. The difference mg/kg and 65.11% gastro protection at 60 mg/kg as
between test and blank gives the measures of peptic compared with ulcerated control. The results indicate
activity. As standard, mixed 2 ml of freshly prepared that the higher dose of protocatechuic acid ethyl ester
phenol solution containing 50 mcg/ml with 10 ml i.e. 60 mg/kg was effective in protecting ulcers in
NaOH and 1 ml of phenol reagent was addd and was pylorus ligated rats. Pylorus ligation had produce
measured at 680 nm after 5 to 10 min. ulcers in all animals pretreated with Ranitidine 30
Statistical analysis mg/kg. However; ulcer index (0.25 ± 0.02) showed
The Statistical analysis was performed by using One significant reduction as compared with ulcerated
Way ANOVA followed by Dunnet’s comparision test control and showed 88.37 % gastro protection.
and student t-test (unpaired).The values are expressed Effect of Protocatechuic acid ethyl ester in ethanol
as mean ± SEM and the P<0.05 was taken as induced gastric ulcer
significant. Ethanol induced ulcerated control group had produced
ulcer in all animals and the mean ulcer index was 4.42
± 0.05 indicating the ulcerogenic effect. Pylorus
ligation also produced ulcers in all the protocatechuic

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acid ethyl ester pretreated animals. However, the ulcer act on tissue to disrupt the hydrophobic protective
index showed significant dose dependent reduction in lining of the mucus gel layer20-21.
the animal pretreated with protocatechuic acid ethyl It is evident from the present result that protocatechuic
ester 30 mg/kg (UI; 2.28 ± 0.17) and 60 mg/kg (UI; acid ethyl ester has potent ulcer protective activity at a
2.10 ± 0.09).It indicated 44.41% gastro protection at 30 dose 30 mg/kg and 60 mg/kg, but at the dose 60 mg/kg
mg/kg and 52.48% gastro protection at 60 mg/kg as was more potent. The ulcer index of protocatechuic
compared with ulcerated control. The results indicate acid ethyl ester treated rat was comparable to those of
that the higher dose of protocatechuic acid ethyl ester ulcerated control rats. There was significant decrease in
i.e. 60 mg/kg was effective in protecting ulcers in ulcer index in protocatechuic acid ethyl ester treated
pylorus ligated rats. Pylorus ligation had produce rats and ranitidine treated rats.
ulcers in all animals pretreated with Sucralfate 400 Ethanol produce necrotic lesions in the gastric mucosa
mg/kg. However, ulcer index (1.05 ± 0.19) showed by its direct toxic effect reducing the secretion of
significant reduction as compared with ulcerated bicarbonate and production of mucus, increase vascular
control and showed 76.24 % gastro protection. permeability and decreases non-protein sulf-hydryl
The finding of the present study demonstrated that groups (NP-SH) of gastric mucosa. Also, increase
protocatechuic acid ethyl ester possess antiulcer xanthine oxidase activity and malonyl-dialdehyde
activity against the ulceration caused by pylorus level. The ethanol also depresses tissue level of DNA,
ligation , aspirin and ethanol. In pylorus ligated rats, RNA and proteins, leading to flow stasis and injured
gastric acid is associated with sever ulceration of the area.
rat gastric mucosa .The activation of vagus –vagal- In the present study, protocatechuic acid ethyl ester has
reflux by stimulation of pressure receptors in the antral potent ulcer protective activity at a dose 30 mg/kg and
gastric mucosa is believed to increase gastric acid 60 mg/kg, but at the dose 60 mg/kg was more potent.
secretion. The digestive site of accumulated gastric The ulcer index of protocatechuic acid ethyl ester
juice and interference of gastric blood circulation treated rat was comparable to those of ulcerated control
responsible for ulceration. It is evident from the present rats. There was significant decrease in ulcer index in
result that protocatechuic acid ethyl ester has potent protocatechuic acid ethyl ester treated rats and
ulcer protective activity at a dose 30 mg/kg and 60 ranitidine treated rats.
mg/kg, but at the dose 60 mg/kg was more potent. The In conclusion, it appears that protocatechuic acid ethyl
ulcer index of protocatechuic acid ethyl ester treated rat ester possess anti-ulcerogenic property against gastric
was comparable to those of ulcerated control rats. mucosal damage induced by pylorus ligation, aspirin
There was significant decrease in ulcer index in and ethanol, through inhibition of gastric acid, pepsin,
protocatechuic acid ethyl ester treated rats and histamine and free radical and stimulation of mucus
ranitidine treated rats. secretion.
There was significant decrease in the volume of gastric Acknowledgement
content, free acidity, total acidity, peptic activity and We are very thankful to Dr. R.V.Shete, Principal of
increase in pH in protocatechuic acid ethyl ester treated R.D.’s college of Pharmacy, Bhor, Pune and also staff
rats in dose dependent manner as compared with members for providing facilities.
ulcerated control rats. The activity of protocatechuic
acid ethyl ester was less than ranitidine ( 30 mg/kg ). References
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Table 1: Effect of protocatechuic acid ethyl ester on volume and pH of gastric content in pylorus
ligated rats
S/No. Treatment (mg/kg) Volume of gastric juice (ml) pH of gastric juice
1. Normal control 1.06±0.07 3.77±0.03
2. Ulcerated control 6.18±0.17 1.67±0.05
3. Ranitidine (30) 3.00±0.12** 3.50±0.06**
4. Protocatechuic acid ethyl ester 4.10±0.09* 2.38±0.07*
(30 mg/kg)
5. Protocatechuic acid ethyl ester 3.22±0.08** 3.34±0.10**
(60 mg/kg)
Values are expressed as mean ± S.E.M., n=6, Ranitidine 30 mg/kg, Protocatechuic acid ethyl ester 30,60
mg/kg, *p<0.05, **p<0.01, as compared with ulcerated control using one way ANNOVA followed by
Dunnet test.
Table 2: Effect of protocatechuic acid ethyl ester on free acidity and total acidity in pylorus ligated
rats
S/ No. Treatment (mg/kg) Free acidity (meq/L/100gm) Total
acidity(meq/L/100gm)
1. Normal control 9.54 ± 0.40 24.6 ± 0.65
2. Ulcerated control 36.23 ± 1.60 75.23 ± 1.50
3. Ranitidine (30 mg/kg) 14.30 ± 0.36** 31.5 ± 0.66**
4. Protocatechuic acid ethyl ester 33.88 ± 0.55 70.05 ± 0.50
(30 mg/kg)
5. Protocatechuic acid ethyl ester 22.1 ± 0.95* 45.7 ± 1.21**
(60 mg/kg)
Values are expressed as mean ± S.E.M., n=6, Ranitidine 30 mg/kg, Protocatechuic acid ethyl ester 30,60
mg/kg, *p<0.05, **p<0.01, as compared with ulcerated control using one way ANNOVA followed by
Dunnet test.
Table 3: Effect of protocatechuic acid ethyl ester on pepsin content in pylorus ligated rats
S/ No. Treatment (mg/kg) Pepsin content (mcg/ml)
1. Normal control 0.77 ± 0.03
2. Ulcerated control 5.16 ± 0.59
3. Ranitidine (30 mg/kg) 2.29 ± 0.63**
4. Protocatechuic acid ethyl ester (30 4.27 ± 0.09*
mg/kg)
5. Protocatechuic acid ethyl ester (60 2.98 ± 0.12**
mg/kg)
Values are expressed as mean ± S.E.M., n=6, Ranitidine 30 mg/kg, Protocatechuic acid ethyl ester 30,60
mg/kg, *p<0.05, **p<0.01, as compared with ulcerated control using one way ANNOVA followed by
Dunnet test.
Table 4: Effect of protocatechuic acid ethyl ester on ulcer index and % gastro protection in pylorus
ligated rats
S/ No. Treatment (mg/kg) Ulcer index (Mean± SEM) % Gastro protection
1. Normal control - 100
2. Ulcerated control 4.2 ± 0.19 -
3. Ranitidine (30 mg/kg) 1.58 ± 0.09 62.38**
4. Protocatechuic acid ethyl ester 3.02 ± 0.10* 28.09*
(30 mg/kg)
5. Protocatechuic acid ethyl ester 2.15 ± 0.08** 48.80**
(60 mg/kg)
Values are expressed as mean ± S.E.M., n=6, Ranitidine 30 mg/kg, Protocatechuic acid ethyl ester 30,60
mg/kg, *p<0.05, **p<0.01, as compared with ulcerated control using one way ANNOVA followed by
Dunnet test.

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Table 5: Effect of protocatechuic acid ethyl ester on ulcer index and % gastro protection in aspirin
induced gastric lesion in rats
S/ No. Treatment (mg/kg) Ulcer index (Mean± SEM) % Gastro protection
1. Normal control - 100
2. Ulcerated control 2.15 ± 0.11 -
3. Ranitidine (30) 0.25 ± 0.02** 88.37**
4. Protocatechuic acid ethyl ester 1.15 ± 0.05* 46.51*
(30 mg/kg)
5. Protocatechuic acid ethyl ester 0.75 ± 0.12** 65.11**
(60 mg/kg)
Values are expressed as mean ± S.E.M., n=6, Aspirin 30 mg/kg, Ranitidine 30 mg/kg, Protocatechuic acid
ethyl ester 30,60 mg/kg, *p<0.05, **p<0.01, as compared with ulcerated control using one way ANNOVA
followed by Dunnet test.
Table 6: Effect of protocatechuic acid ethyl ester on ulcer index and % gastro protection in ethanol
induced gastric ulcer in rats.

S/No. Treatment (mg/kg) Ulcer index (Mean± SEM) % Gastro protection

1. Normal control - 100
2. Ulcerated control 4.42 ± 0.05 -
3. Sucralfate (400 mg/kg) 1.05 ± 0.19 76.24
4. Protocatechuic acid ethyl ester 2.28 ± 0.17* 44.41*
(30 mg/kg)
5. Protocatechuic acid ethyl ester 2.10 ± 0.09** 52.48**
(60 mg/kg)
Values are expressed as mean ± S.E.M., n=6, Sucralfate 400 mg/kg, Protocatechuic acid ethyl ester 30,60
mg/kg, *p<0.05, **p<0.01, as compared with ulcerated control using one way ANNOVA followed by
Dunnet test.

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