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Study of ulcer healing property of
traditional deglycyrrhizinated
licorice compared with flavonoid de
glycyrrhizinated licorice
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ULCER :
A break in skin or mucous membrane with loss of surface tissue,disintegration and necrosis of epithelialtissue
Causes :Due to the imbalance of aggressive and defensive factors.
Aggressive factors:HCl, Pepsin, Bile and H.pyloi
Defensive factors :Gastric mucosa, bi carbonate secretion, prostaglandins,
nitricoxide, innate resistant of mucosal cells
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ETIOLOGY:
H.Pylori
Acid and Pepsin
NSAIDs
Smoking
caffeine
alcohol
Stress
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Clinical Features :
Abdominal pain, classically epigastric with severity relating to
mealtimes (duodenal ulcers are classically relieved by food,
while gastric ulcers are exacerbated by it);
Bloating and abdominal fullness
Water brash (bitter regurgitation)
Nausea, and sometimes vomiting
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Loss of appetite and weight loss;
Hematemesis (vomiting of blood);
Melena (tarry, foul-smelling feces due to oxidized iron
from hemoglobin);
Rarely, an ulcer can lead to a gastric or duodenal
perforation. This is extremely painful and requires
immediate surgery.
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DIAGNOSIS :
Biopsy during EGD;
Breath testing (does not require EGD);
Direct culture from an EGD biopsy specimen;
Direct detection of urease activity in a biopsy specimen;
Measurement of antibody levels in blood (does not require
EGD). It is still somewhat controversial whether a positive
antibody without EGD is enough to warrant eradication
therapy.
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TREATMENT :
The drug treatment of peptic ulcer is targeted at either
counteracting aggressive factors or stimulating the mucosal defenses.
The ideal aims of treatment are to relieve pain, heal the ulcer and
delay the ulcer recurrence.
Reduction of gastric acid secretion:
H2anti histamines
Proton pump inhibitors
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Anti cholinergics
Prostaglandin analogues
Neutralization of gastric acid secretion:
Systemic
Non systemic
Ulcer protective
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DRUGS :
H2-blocker
Ranitidine
Famotidine
Cimetidine
Nizatidine
Mechanism of action
Histamine H2-receptor antagonists inhibit secretion of acid by
the parietal cells in the stomach lining by blocking H2 receptors.
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Proton Pump Inhibitors :
Omeprazole
Lansoprazole
Esomeprazole
Rabeprazole
Pantoprazole.
Mechanism of action :
These proton pumps move hydrogen ions across cellmembranes into the stomach cavity, thereby lowering pH in the
stomach.
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PLANT REVIEW 17
Kingdom : Plantae
Division : Angiospermae
Class : Dicotyledoneae
Order : Fabales
Family : Fabaceae,Leguminosae
Genus : Glycyrrhiza
Species : glabra
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CHEMICALCONSTITUENTS:
1. GlycosideSaponin group : Glycyrrhizin 50 time sweeter
than sugar .
2. FlavonoidsLiquiritin & Isoliquiritin
3. Proteins .
4. Sugars (glucose ,sucrose ).
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USES:
1. Antihistaminic
2. Expectorant
3. Flavoringagentfor Aloe , Quinine , NH4CL , Chocolates
and others
4. Anti-inflammatory activity
5. Demulcent
6. Soft drink
7. Anti tumor activity:prosate cancer
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Anti ulcer.
Licorice helps heal ulcers by inactivating 15-
hydroxyprostaglandin dehydrogenase in the stomach lining.As with cortisol in the kidney, licorice locally extends thelife of prostaglandins that protect the stomach wall.
9.Skin problems:
emollient,eczema and psoriasis anti allergic
10.Harmonal Action:
pseudo aldosterone activity
Cortisone action estrogenic activity
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2. OBJECTIVE :
a. Procurement of Flavonoid-rich DGL & Traditional DGL
b. Standardization of anti-ulcer models.
c. Evaluation of anti-ulcer activity of Flavonoid-rich DGL and
Traditional DGL.
d. Comparative evaluation of anti ulcer potential of
Flavonoidrich DGL vs. Traditional DGL
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Animal Models :
Surgical: Pylorus Ligation (PL);
Physical: Cold Stress ulcer (CSU)
Chemical: Indomethacin Induced ulcer (IND)
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SCREENING PROCEDURES:
Pylorus ligation induced ulcer
NSAIDs induced ulcer (Indomethacin, Aspirin, Ibuprofen)
Cold stress induced ulcer
Alcohol induced ulcer
Sub acute gastric ulcer in rats
Gastric ischemia reperfusion injury in rats
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METHODOLOGY
ANIMALS
Species: Albino rats.
Strain: Wistar.
Sex: Either sex.
Source: Bred and reared at Natural Remedies Pvt. Ltd.
Body weight range: 180 - 200 g Identification: By cage card and corresponding picric acid colour
body markings.
Number of animals per dose group: 3 per sex.
Acclimation: One week in experimental room.
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Dosing of tests and standards
Dose formulation :
The test substance will be formulated as solution/suspension.
Formulations as above will be freshly prepared before dosing.
Administration of test substance
The test substance will be administered by oral gavage to each
rat as a single dose, using an intubation needle fitted onto a
syringe of appropriate size. The dose administered to
individual rat will be calculated according to its body weight
recorded on the day of test substance administration.
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Statistics
Values will be expressed as mean SEM. The data will be
analyzed by one way ANOVA. The statistical significance will
be set at p 0.05 followed by Dunnets T3.
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PYLORUS LIGATION INDUCED ULCER MODEL
Principle of the test
Pyloric ligation (PL) induced ulcers were caused due to
Imbalance between offensive and defensive mucosal factors.
PL-induced gastric ulcers occur because of an increase inacid-pepsin accumulation due to pyloric obstruction and
subsequent mucosal digestion
Anti-ulcer agents like omeprazole inhibit the acid
secretion and prevent the ulcers.
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Drugs used:
Omeprazole
Chemicals used:
Sodium hydroxidewas used at the conc. of 0.01M for
titration of gastric juices.
Phenolphthalein as indicator in acid base titration.
Observations: Ulcer index, pH of gastric content, total acidity and gastric
content.
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Pyrocedure:
Female, wister rats of 150-170gm, starved 48hr.
Avoid coprophagy.
six animals per dose and as control.
Midline abdominal incision, pylorus is ligated.
Closed by sutures , test compound given orally route.
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Placed 19hr in plastic cylinder d-45mm,closed ends
Animals are sacrificed in co2 anesthesia
Abdomen is opened and ligature is placed around
esophagus
Stomach is removed and contents are drained to centrifuge
tube
Stomach is opened along the greater curvature pined to
cork plate
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Scoring:
0 - no ulcer
1 - petechial hemorrhages
2ulcer24mm
Ulcer index(UI)=UN+US+UP*10-1
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GROUP TREATMENT
i Vehicle control(10ml/kgb.w)
ii Pylorus ligation control (4 h)
Iii Standard control (Omeprazole 10 mg/kg)
Iv Traditional DGL (37.5 mg/kg b.w.)
V Traditional DGL (75 mg/kg b.w.)
Vi Traditional DGL (150 mg/kg b.w.)
Vii Flavonoid-rich DGL (37.5 mg/kg b.w.)
Viii Flavonoid-rich DGL (75 mg/kg b.w.)
ix Flavonoid-rich DGL (150 mg/kg b.w.)
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COLD STRESS INDUCED ULCER MODEL
Principle of the test: Cold stress ulcer (CSU) is a well-accepted model for the
induction of gastric ulcer in which peripheral sympathetic
activation plays an important role in induction of ulcers.
Stress has been reported to have an important role in
etiopathology of gastro-duodenal ulceration, increase in gastric
motility, vagal over activity, mast cell degranulation; decreased
gastric mucosal blood flow and decreased prostaglandin
synthesis
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Significantly, when source of stress is cold as in CSU,
incidence of ulcers is mainly due to increased acid secretion
and generation of free radicals etc
Drugs used:
Omeprazole
Observations
Ulcer index, pH of gastric content.
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STRESS ULCER BY COLD WATER IMMERSION
PROCEDURE:
Groups of 6 wistard rats(150-200).
Oral test drug, placed in restraint cages, 22degc/1hr
Inject i.v via tail vein with 30 mg/kg evans blue.
After 10 min stomach is removed by co2 anaesthesia Formol saline is injected, storage overnight
Then next day stomach are opend with greatercurvature, washed.
lesions(lengths) are measuerd.
EVALUATION: inhibition of the lesion porduction isexpressed as
percentage value.
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INDOMETHACIN INDUCED ULCER MODEL
Principle of the test
Anti-inflammatory drugs like Indomethacin when administered
produce visible gastric ulcers in animals.
Indomethacin is a potent inhibitor of prostaglandin biosynthesis
and prostaglandins are known to play an important role in
maintaining mucosal integrity.
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Drugs used:
Omeprazole
Chemicals used:
Indomethacin was used at the dose levels of 40 mg/kg rat body
weight.
Sodium Carbonate
Observations
Ulcer index, pH of gastric content.
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Indomethacin induced ulcer:
6 wister rats(150-200g)
Test-20mg/kg indomethacin,
After 10 min oraly, test drug in 0.1%tween 80
6hr later , sacrifice in co2,stomaach is removed
Stomach Inject formal saline, kept for over night
Stomach is opened, examined under microscope
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RESULTS
Pylorus ligation induced ulcer
DGL at the dose level of 75 mg/kg and 150 mg/kg. Totalacidity was inhibited by Flavanoid rich DGL at all the doselevels.
Total acidity was reduced by 45.30% and 30.66% byflavonoid-rich DGL and traditional DGL respectively at their
highest dose.
Flavanoid rich DGL at the dose level of 75 mg/kg and 150mg/kg also inhibited ulcerogenic activity of pylorus ligationsignificantly.
Flavonoid-rich DGL at a dose of 150 mg/kg b.w inhibitedulcer index by 88.33% compared to traditional DGL, which atthe same dose level inhibited ulcer index by 77.8%.
Ph t h PYLORUS LIGATION INDUCED ULCER
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Photograph 5.1: PYLORUS LIGATION INDUCED ULCER
NORMAL CONTROL PYLORUS LIGATION CONTROL 4hOMEPRAZOLECONTROL 10mg/kg
TRADITIONAL DGL 150mg/kgFLAVONOID-RICH DGL 150mg/kg
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GRP
S
TREATMENT MEAN
pH
SEM
MEAN
G.CONT
ENT
SEM
MEAN
TOTAL
ACIDITY
SEM
MEAN
ULCER
INDEX
SEM
%INHIBITI
ON OF
ULCER
INDEX
IVehicle control (10 ml/kg)
2 0 0 0 0 0 3.16 0 -
II Pylorus ligation control (4
h)
2.14 0.05 6.33 0.3 120.66 5.55 30 4.32 -
III Omeprazole (10 mg/kg) 6.89* 0.13 2.41* 0.23 15.33* 0.91 3.33* 1.38 88.9
IV Traditional DGL (37.5
mg/kg)
2.2 0.06 5.41 0.35 113.33 2.99 18.66 1.54 37.80
V
Traditional DGL (75 mg/kg)
2.33 0.12 4.58 0.5 103 9.36 7.66 1.3 74.46
VI Traditional DGL (150
mg/kg)
2.39 0.07 4.83 0.33 83.66 8.98 6.66 1.52 77.80
VII
Flavonoid DGL (37.5 mg/kg)
2.45 0.2 4.25 0.4 87.33* 1.97 8.5 1.97 71.66
VIII
Flavonoid DGL (75 mg/kg)
2.52 0.17 3.83* 0.3 72* 7.42 5.66* 0.8 81.66
IX
Flavonoid DGL (150 mg/kg)
2.72 0.21 3.33* 0.35 66* 7.16 3.5* 1.5 88.33
Graph 5.1: Volume of gastric content of treated groups in pylorus ligation induced ulcer model
i lbi Wi t t
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using albino Wistar rats
0
1
2
3
4
5
6
7
Treatment groups
Volu
meofgastriccontent(ml)
I Vehicle control (10 ml/kg) II Pylorus ligation control (4 h) III Omeprazole (10 mg/kg) IV Traditional DGL (37.5 mg/kg) V Traditional DGL (75 mg/kg)
VI Traditional DGL (150 mg/kg) VII Flavonoid DGL (37.5 mg/kg) VIII Flavonoid DGL (75 mg/kg) IX Flavonoid DGL (150 mg/kg)
# p < 0.05 Pylorus ligation control Vs Vehicle control*p < 0.05 Treated groups Vs Pylorus ligation control
#
*
*
*
Graph 5.2: pH values of treated groups in pylorus ligation induced ulcer model using albino Wistar rats
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0
1
2
3
4
5
6
7
8
Treatment groups
pHvalues
I Vehicle control (10 ml/kg) II Pylorus ligation control (4 h) III Omeprazole (10 mg/kg) IV Traditional DGL (37.5 mg/kg) V Traditional DGL (75 mg/kg)
VI Traditional DGL (150 mg/kg) VII Flavonoid DGL (37.5 mg/kg) VIII Flavonoid DGL (75 mg/kg) IX Flavonoid DGL (150 mg/kg)
*p < 0.05 Treated groups Vs Pylorus ligation control
*
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Graph 5.3: Total acidity of gastric juice of treated groups in pylorus ligation induced ulcer model
using albino Wistar rats
0
10
20
30
40
50
60
70
80
90
100
110
120
130
Treatment groups
Totalacidity(mEq/L/100g)
I Vehicle control (10 ml/kg) II Pylorus l igation control (4 h) III Omeprazole (10 mg/kg) IV Tradit ional DGL (37.5 mg/kg) V Tradit ional DGL (75 mg/kg)
VI Tradit ional DGL (150 mg/kg) VII Flavonoid DGL (37.5 mg/kg) VIII Flavonoid DGL (75 mg/kg) IX Flavonoid DGL (150 mg/kg)
# p < 0.05 Pylorus ligation control Vs Vehicle control*p < 0.05 Treated groups Vs Pylorus ligation control
#
*
*
*
*
Graph 5.4: Ulcer index of treated groups in pylorus ligation induced ulcer model using albino Wistar rats
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0
5
10
15
20
25
30
35
Treatment groups
U
lcerindex
(scores)
I Vehicle control (10 ml/kg) II Pylorus ligation control (4 h) III Omeprazole (10 mg/kg) IV Traditional DGL (37.5 mg/kg) V Traditional DGL (75 mg/kg)
VI Traditional DGL (150 mg/kg) VII Flavonoid DGL (37.5 mg/kg) VIII Flavonoid DGL (75 mg/kg) IX Flavonoid DGL (150 mg/kg)
# p < 0.05 Pylorus ligation control Vs Vehicle control*p < 0.05 Treated groups Vs Pylorus ligation control
#
*
*
*
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Cold stress induced ulcer:
Flavanoid rich DGL at all dose levels inhibited ulcerogenicactivity of cold stress significantly.
Flavonoid-rich DGL at a dose of 150 mg/kg b.w inhibited
ulcer index by 92.71% compared to traditional DGL, which at
the same dose level inhibited ulcer index by 86.51%.
COLD STRESS ULCER (CSU)
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NORMAL CONTROL COLD STRESS CONTROL 4h OMEPRAZOlCONTRol10mg/kg
TRADITIONAL DGL 150mg/kg FLAVONOID-RICH DGL 150mg/kg
Graph 5.7: pH values of treated groups in cold stress induced ulcer model using albino Wistar rats
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0
1
2
3
4
5
6
7
Treatment groups
pHvalues
I Vehicle control (10 ml/kg) II Cold stress control (4 h) III Omeprazole (10 mg/kg)
IV Traditional DGL (37.5 mg/kg) V Traditional DGL (75 mg/kg) VI Traditional DGL (150 mg/kg)
VII Flavonoid DGL (37.5 mg/kg) VIII Flavonoid DGL (75 mg/kg) IX Flavonoid DGL (150 mg/kg)
*
*p < 0.05 Treated groups Vs Cold stress control
Graph 5.8: Ulcer index of treated groups in cold stress induced ulcer model using albino Wistar rats
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0
5
10
15
20
25
30
35
Treatment groups
U
lcerindex(scores)
I Vehicle control (10 ml/kg) II Cold stress control (4 h) III Omeprazole (10 mg/kg)
IV Traditional DGL (37.5 mg/kg) V Traditional DGL (75 mg/kg) VI Traditional DGL (150 mg/kg)
VII Flavonoid DGL (37.5 mg/kg) VIII Flavonoid DGL (75 mg/kg) IX Flavonoid DGL (150 mg/kg)
*
# p < 0.05 Cold stress control Vs Vehicle control*p < 0.05 Treated groups Vs Cold stress control
#
*
*
*
*
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Indomethacin induced ulcer:
Traditional DGL at the dose level of 37.5 mg/kg and 75 mg/kgsignificantly inhibited ulcer induction. Flavanoid rich DGL at
the dose level of 75 mg/kg and 150 mg/kg inhibited
ulcerogenic activity of indomethacin significantly.
Flavonoid-rich DGL at a dose of 150 mg/kg b.w inhibited
ulcer index by 65.51% compared to traditional DGL, which at
the same dose level inhibited ulcer index by 44.05%.
Photograph 5.2: INDOMETHACIN INDUCED ULCER
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VEHICLE CONTROL INDOMETHACIN CONTROL 40mg/kg OMEPRAZOLE CONTROL 10mg/kg
TRADITIONAL DGL 150mg/kg FLAVONOID-RICH DGL 150mg/kg
TABLE 5.2: Anti-ulcer activity of DGL in Indomethacin induced ulcer model using albino Wistar rats.
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GRPS TREATMENT MEAN
pH
SEM
MEAN
ULCER
INDEX
SEM
% INHIBITION
OF ULCER INDEX
IVehicle control (10 ml/kg) 2 0 0.83 0.4
-
IIIndomethacin control (40
mg/kg)2
0 71.5 3.87-
IIIOmeprazole (10 mg/kg)
6.5*0.22 2.83* 1.45
96.04
IVTraditional DGL (37.5 mg/kg)
20 27.83* 8.02
61.07
V Traditional DGL (75 mg/kg) 2.33 0.33 27.66* 5.54 61.31
VITraditional DGL (150 mg/kg)
2.660.42 40 6.06
44.05
VIIFlavonoid DGL (37.5 mg/kg)
2.660.42 54.5 4.02
23.77
VIIIFlavonoid DGL (75 mg/kg)
3.330.42 32.16* 6.23
55.02
IX
Flavonoid DGL (150 mg/kg)
3.33
0.42 24.66* 2.13
65.51
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Graph 5.5: pH values of treated groups in indomethacin induced ulcer model using albino Wistar rats
0
1
2
3
4
5
6
7
Treatment group
pHvalues
I Vehicle control (10 ml/kg) II Indomethacin control (40 mg/kg) III Omeprazole (10 mg/kg)
IV Traditional DGL (37.5 mg/kg) V Traditional DGL (75 mg/kg) VI Traditional DGL (150 mg/kg)
VII Flavonoid DGL (37.5 mg/kg) VIII Flavonoid DGL (75 mg/kg) IX Flavonoid DGL (150 mg/kg)
*p < 0.05 Treated groups Vs Indomethacin control
*
Graph 5.6: Ulcer index of treated groups in indomethacin induced ulcer model using albino Wistar rats
#
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0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
Treatment group
Ulcerindex(scores)
I Vehicle control (10 ml/kg) II Indomethacin control (40 mg/kg) III Omeprazole (10 mg/kg)
IV Traditional DGL (37.5 mg/kg) V Traditional DGL (75 mg/kg) VI Traditional DGL (150 mg/kg)
VII Flavonoid DGL (37.5 mg/kg) VIII Flavonoid DGL (75 mg/kg) IX Flavonoid DGL (150 mg/kg)
# p < 0.05 Indomethacin control Vs Vehicle control
*p < 0.05 Treated groups Vs Indomethacin control
*
*
*
*
#
*
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Conclusion:
Hypersecretion of gastric acid is a pathological condition, which
occurs due to uncontrolled secretion of hydrochloric acid from the
parietal cells of the gastric mucosa through the proton pumping H+
K+ ATPase
Flavonoids are known to inhibit several enzymes e.g. alkaline
phosphatase, cAMP phosphodiesterase, lipases, hydrolases,
lysosomal H+ - ATPase and Na+ / K+ - ATPase.
Licorice extracts contained several potent antioxidant constituents asdetermined by their ability to inhibit -carotene consumption and
LDL oxidation.
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Pyloric ligation induced ulcers caused due to imbalance
between offensive and defensive mucosal factors
DGL with multiple mechanisms Total acidity was reduced by
45.30% and 30.66% by flavonoid-rich DGL and traditional
DGL respectively at their highest dose.
Flavonoid-rich DGL at a dose of 150 mg/kg b.w inhibited
ulcer index by 88.33% compared to traditional DGL, which at
the same dose level inhibited ulcer index by 77.8%.
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The indomethacin-induced ulcer model was employed because
the model shows drugs effect on cytoprotection and gastric
acid secretion.
Flavonoid-rich DGL inhibited ulcer index by 65.51% compared
to traditional DGL, which at the same dose level inhibited ulcerindex by 44.05%.
Stress can cause abnormalities in acid secretion, bile and
pancreatic juice reflux; these are factors that can lead to ulcer
formation
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Flavonoid-rich DGL at a dose of 150 mg/kg b.w inhibited ulcer
index by 92.71% compared to traditional DGL, which at the
same dose level inhibited ulcer index by 86.51%.
On the basis of the data presented here, it can be concluded that
the gastro protective effect elucidated by DGL could be mainly
due to the modulation of defensive factors through an
improvement of gastric cytoprotection and partly due to acid
inhibition and free radical scavenging properties.
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CONCLUSIONS
Flavonoid-rich DGL at a dose of 150 mg/kg b.w was found toinhibit ulcers in PL (88.33%), IND (65.51%), and CSU
(92.71%). While traditional DGL at the same dose level of 150 mg/kg b.w
reduced ulcers in PL (77.8%), IND (44.05%), and CSU(86.51%) induced ulcer models.
Total acidity was reduced by 45.30% and 30.66% by flavonoid-rich DGL and traditional DGL respectively in PL induced ulcermodel.
Conclusively, the ulcer protective effect of Flavonoid-rich DGLmay be due to its anti-oxidant27 along with cytoprotective25
mechanism. Therefore Flavonoid-rich DGL have more potentanti-ulcerogenic as well as ulcer-healing properties thantraditional DGL and could act as a potent therapeutic agentagainst peptic ulcer disease.
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