Study of ulcer healing property of

traditional deglycyrrhizinated

licorice compared with flavonoid de

glycyrrhizinated licorice

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

ETIOLOGY:

H.Pylori
Acid and Pepsin
NSAIDs Smoking caffeine alcohol

Stress

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

 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.

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.

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

 Anti cholinergics Prostaglandin analogues

Neutralization of gastric acid secretion:

Systemic Non systemic Ulcer protective

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.

Proton Pump Inhibitors :

Omeprazole Lansoprazole

Esomeprazole
Rabeprazole Pantoprazole.

Mechanism of action : These proton pumps move hydrogen ions across cell

membranes into the stomach cavity, thereby lowering pH in the

stomach.

PLANT REVIEW 17
Kingdom Division Class Order : Plantae : Angiospermae : Dicotyledoneae : Fabales

Family
Genus Species

: Fabaceae,Leguminosae
: Glycyrrhiza : glabra

CHEMICAL CONSTITUENTS:
1.

Glycoside Saponin group : Glycyrrhizin 50 time sweeter

than sugar .
2. 3. 4.

Flavonoids Liquiritin & Isoliquiritin Proteins . Sugars (glucose ,sucrose ).

USES:
1. 2.

Antihistaminic Expectorant

3.
4.

Flavoring agent for Aloe , Quinine , NH4CL , Chocolates and others

Anti-inflammatory activity

5.
6. 7.

Demulcent
Soft drink Anti tumor activity: prosate cancer

Anti ulcer. Licorice helps heal ulcers by inactivating 15hydroxyprostaglandin dehydrogenase in the stomach lining. As with cortisol in the kidney, licorice locally extends the life 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

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

Animal Models :
Surgical: Pylorus Ligation (PL); Physical: Cold Stress ulcer (CSU) Chemical: Indomethacin Induced ulcer (IND)

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

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.

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.

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.

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 in

acid-pepsin accumulation due to pyloric obstruction and

subsequent mucosal digestion

Anti-ulcer agents like omeprazole inhibit the acid secretion and prevent the ulcers.

Drugs used:
Omeprazole

Chemicals used:
Sodium hydroxide was 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.

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.

 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

Scoring:
0 - no ulcer 1 - petechial hemorrhages 2 ulcer<2mm 3 - ulcer >2<4mm 4- ulcers>4mm

Ulcer index(UI)=UN+US+UP*10-1

GROUP i ii Iii Iv V Vi Vii Viii ix

TREATMENT Vehicle control(10ml/kgb.w) Pylorus ligation control (4 h) Standard control (Omeprazole 10 mg/kg) Traditional DGL (37.5 mg/kg b.w.) Traditional DGL (75 mg/kg b.w.) Traditional DGL (150 mg/kg b.w.) Flavonoid-rich DGL (37.5 mg/kg b.w.) Flavonoid-rich DGL (75 mg/kg b.w.) Flavonoid-rich DGL (150 mg/kg b.w.)

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

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.

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.

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.

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.

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

RESULTS
Pylorus ligation induced ulcer DGL at the dose level of 75 mg/kg and 150 mg/kg. Total acidity was inhibited by Flavanoid rich DGL at all the dose levels. Total acidity was reduced by 45.30% and 30.66% by flavonoid-rich DGL and traditional DGL respectively at their highest dose. Flavanoid rich DGL at the dose level of 75 mg/kg and 150 mg/kg also inhibited ulcerogenic activity of pylorus ligation significantly. 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%.

Photograph 5.1: PYLORUS LIGATION INDUCED ULCER

NORMAL CONTROLPYLORUS LIGATION CONTROL 4h OMEPRAZOLE CONTROL 10mg/kg

TRADITIONAL DGL 150mg/kg

FLAVONOID-RICH DGL 150mg/kg

GRP S

TREATMENT

MEAN SEM MEAN SEM MEAN pH G.CONT TOTAL ENT ACIDITY

SEM MEAN ULCER INDEX

SEM

%INHIBITI ON OF ULCER INDEX

-

I Vehicle control (10 ml/kg)

3.16

II

Pylorus ligation control (4 h)

Omeprazole (10 mg/kg)

2.14

0.05

6.33

0.3

120.66

5.55

30

4.32

III

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) VI Traditional DGL (150 mg/kg)

2.33

0.12

4.58

0.5

103

9.36

7.66

1.3

74.46

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) VIII Flavonoid DGL (75 mg/kg) IX Flavonoid DGL (150 mg/kg)

2.45

0.2

4.25

0.4

87.33*

1.97

8.5

1.97

71.66

2.52

0.17

3.83*

0.3

72*

7.42

5.66*

0.8

81.66

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 using albino Wistar rats
7
#

Volume of gastric content (ml)

5 * 4 *

0 Treatment groups
I Vehicle control (10 ml/kg) VI Traditional DGL (150 mg/kg) II Pylorus ligation control (4 h) VII Flavonoid DGL (37.5 mg/kg) III Omeprazole (10 mg/kg) VIII Flavonoid DGL (75 mg/kg) IV Traditional DGL (37.5 mg/kg) IX Flavonoid DGL (150 mg/kg) V Traditional DGL (75 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
8
*

5
pH values

0 Treatment groups
I Vehicle control (10 ml/kg) VI Traditional DGL (150 mg/kg) II Pylorus ligation control (4 h) VII Flavonoid DGL (37.5 mg/kg) III Omeprazole (10 mg/kg) VIII Flavonoid DGL (75 mg/kg) IV Traditional DGL (37.5 mg/kg) IX Flavonoid DGL (150 mg/kg) V Traditional DGL (75 mg/kg)

*p < 0.05 Treated groups Vs Pylorus ligation control

Graph 5.3: Total acidity of gastric juice of treated groups in pylorus ligation induced ulcer model using albino Wistar rats
130 120 110 100 *
#

Total acidity (mEq/L/100 g)

90 * 80 70 60 50 40 30 * 20 10 0 Treatment groups
I Vehicle control (10 ml/kg) VI Traditional DGL (150 mg/kg) II Pylorus ligation control (4 h) VII Flavonoid DGL (37.5 mg/kg) III Omeprazole (10 mg/kg) VIII Flavonoid DGL (75 mg/kg) IV Traditional DGL (37.5 mg/kg) IX Flavonoid DGL (150 mg/kg) V Traditional DGL (75 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

35

30

25
Ulcer index (scores)

20

15

10

* *
5

0 Treatment groups
I Vehicle control (10 ml/kg) VI Traditional DGL (150 mg/kg) II Pylorus ligation control (4 h) VII Flavonoid DGL (37.5 mg/kg) III Omeprazole (10 mg/kg) VIII Flavonoid DGL (75 mg/kg) IV Traditional DGL (37.5 mg/kg) IX Flavonoid DGL (150 mg/kg) V Traditional DGL (75 mg/kg)

# p < 0.05 Pylorus ligation control Vs Vehicle control

*p < 0.05 Treated groups Vs Pylorus ligation control

Cold stress induced ulcer:

Flavanoid rich DGL at all dose levels inhibited ulcerogenic

activity 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)

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
7

pH values

0 Treatment groups

I Vehicle control (10 ml/kg) IV Traditional DGL (37.5 mg/kg) VII Flavonoid DGL (37.5 mg/kg)
*p < 0.05 Treated groups Vs Cold stress control

II Cold stress control (4 h) V Traditional DGL (75 mg/kg) VIII Flavonoid DGL (75 mg/kg)

III Omeprazole (10 mg/kg) VI Traditional DGL (150 mg/kg) IX Flavonoid DGL (150 mg/kg)

Graph 5.8: Ulcer index of treated groups in cold stress induced ulcer model using albino Wistar rats
35

30

25
Ulcer index (scores)

20

15

*
10

* *

* *

0 Treatment groups

I Vehicle control (10 ml/kg) IV Traditional DGL (37.5 mg/kg) VII Flavonoid DGL (37.5 mg/kg) # p < 0.05 Cold stress control Vs Vehicle control
*p < 0.05 Treated groups Vs Cold stress control

II Cold stress control (4 h) V Traditional DGL (75 mg/kg) VIII Flavonoid DGL (75 mg/kg)

III Omeprazole (10 mg/kg) VI Traditional DGL (150 mg/kg) IX Flavonoid DGL (150 mg/kg)

Indomethacin induced ulcer:

Traditional DGL at the dose level of 37.5 mg/kg and 75 mg/kg

significantly 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

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.

GRPS

TREATMENT

MEAN pH

SEM

MEAN ULCER INDEX 0.83 71.5 2.83* 27.83* 27.66* 40 54.5 32.16*

SEM

% INHIBITION OF ULCER INDEX

I II III IV V VI VII VIII IX

Vehicle control (10 ml/kg) Indomethacin control (40 mg/kg) Omeprazole (10 mg/kg) Traditional DGL (37.5 mg/kg) Traditional DGL (75 mg/kg) Traditional DGL (150 mg/kg) Flavonoid DGL (37.5 mg/kg) Flavonoid DGL (75 mg/kg)

2 2 6.5* 2 2.33 2.66 2.66 3.33 3.33

0 0 0.22 0 0.33 0.42 0.42 0.42

0.4 3.87 1.45 8.02 5.54 6.06 4.02 6.23

96.04 61.07 61.31 44.05 23.77 55.02 65.51

Flavonoid DGL (150 mg/kg)

0.42

24.66*

2.13

Graph 5.5: pH values of treated groups in indomethacin induced ulcer model using albino Wistar rats
7

pH values

0 Treatment group

I IV

Vehicle control (10 ml/kg) Traditional DGL (37.5 mg/kg)

II V

Indomethacin control (40 mg/kg) Traditional DGL (75 mg/kg)

III IX

Omeprazole (10 mg/kg) Flavonoid DGL (150 mg/kg)

VI Traditional DGL (150 mg/kg)

VII Flavonoid DGL (37.5 mg/kg)

VIII Flavonoid DGL (75 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
#
75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0

Ulcer index (scores)

* * *

Treatment group

I IV

Vehicle control (10 ml/kg) Traditional DGL (37.5 mg/kg)

II V

Indomethacin control (40 mg/kg) Traditional DGL (75 mg/kg)

III IX

Omeprazole (10 mg/kg) Flavonoid DGL (150 mg/kg)

VI Traditional DGL (150 mg/kg)

VII Flavonoid DGL (37.5 mg/kg)

VIII Flavonoid DGL (75 mg/kg)

# p < 0.05 Indomethacin control Vs Vehicle control *p < 0.05 Treated groups Vs Indomethacin control

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 as

determined by their ability to inhibit -carotene consumption and

LDL oxidation.

 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%.

 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 ulcer

index by 44.05%.
Stress can cause abnormalities in acid secretion, bile and

pancreatic juice reflux; these are factors that can lead to ulcer formation

 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.

CONCLUSIONS Flavonoid-rich DGL at a dose of 150 mg/kg b.w was found to inhibit 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 flavonoidrich DGL and traditional DGL respectively in PL induced ulcer model. Conclusively, the ulcer protective effect of Flavonoid-rich DGL may be due to its anti-oxidant27 along with cytoprotective25 mechanism. Therefore Flavonoid-rich DGL have more potent anti-ulcerogenic as well as ulcer-healing properties than traditional DGL and could act as a potent therapeutic agent against peptic ulcer disease.