Investigation of Anti-ulcer Activity of Aqueous Extract of Fagonia cretica
In Indomethican Induced Ulcers in Rats
A thesis submitted in the partial fulfilment for the degree of
Masters of Philosophy in Pharmacy
(Pharmacology)
Maseela Azam
Pharm. D (R.Ph.)
Session (2015-2017)
Punjab University College of Pharmacy,
University of the Punjab,
Lahore, Pakistan
Approval Certificate
The thesis entitled, “Investigation of anti-ulcer activity of aqueous extract of Fagonia cretica in
Indomethican induced ulcers in rats” is prepared by Maseela Azam under my supervision in the
partial fulfilment for the degree of Masters of Philosophy in Pharmacy (Pharmacology) is,
hereby, approved for submission.
Supervisor
Prof. Dr. Syed Nawazish-i-Husain
____
B.Pharmacy, University of the Punjab, Lahore, Pakistan
M.Phil, (Pharmacology), University of the Punjab, Lahore, Pakistan
Ph.D, (Pharmacology), University of Strathclyde, Glasgow, UK
Post-Doctoral Research positions. Glasgow, UK
1
________________
Roll Number
03MCOL-E15A
Research Supervisor &
______________________________
Internal Examiner
Prof. Dr. Syed Nawazish-i-Husain
Professor (Pharmacology)
Punjab University College of Pharmacy,
University of the Punjab, Lahore, Pakistan
Principal
______________________________
Prof. Dr. Nadeem Irfan Bukhari
Punjab University College of Pharmacy
University of the Punjab, Lahore, Pakistan
External Examiner
______________________________
Research Investigator
______________________________
Maseela Azam
Viva Held On
______________________________
2
Acknowledgements
In the name of ALLAH Almighty, the most merciful and beneficent, all the praises and thanks be
to Allah, the Lord of the 'Alamin’ who gave me the strength, resources and the will to complete
my research work and thesis.
Firstly, I would like to express my sincere and profound gratitude to my supervisor Prof. Dr.
Syed Nawazish-i-Husain, for his continuous support, patience, motivation, enthusiasm, and
immense knowledge that enabled me to complete my thesis. His critique helped me in improving
my work and in modifying the thesis in its best possible form. I am forever thankful to him for
having paved the way for me to complete my thesis by helping me over whenever I faced any
difficulty.
I acknowledge Prof. Dr. Nadeem Irfan Bukhari, Principal, Punjab University College of
Pharmacy and Prof. Dr. Khalid Hussain, Dean, University College of Pharmacy, University of
the Punjab, Lahore, Pakistan for the administrative support. Furthermore, I am extremely
thankful to the technical staff members of the Pharmacology Department for providing me with
their continuous assistance during my lab work and for taking such good care of the research
animals.
Lastly, I would want to appreciate and acknowledge my family and my husband for having
shown continuous support, love, encouragement and assistance whenever needed, without which
the completion of my thesis would have never been possible.
Maseela Azam
3
List of Abbreviations
F.C.
Fagonia Cretica
UI
Ulcer Index
Aq.
Aqueous
B.W.
Body weight
BSA
Bovine Serum Albumin
Ach
Acetylcholine
Conc.
Concentration
DTNB
Dithio-nitro-bis-benzoic acid
GERD
Gastroesophageal reflux disease
GSH
Glutathione
H .Pylori
Helicobactor pylori
HCl
Hydrochloric Acid
Kg
Kilogram
L
Liter
M
Molar
mg
Milligram
ml
Milliliter
N
Normal
NSAIDs
Non-steroid anti-inflammatory drugs
μM
Micromolar
μg
Microgram
μl
Microliter
S.E.M.
Standard error of the mean
4
Table of Contents
1. Introduction …………………………………………………………………… 11
1.1. Anatomy and Physiology Of Stomach …………………………………………….12
1.2. Pathophysiology of Gastric Ulcer………………………………………………….12
1.3. Treatments of Gastric Ulcer………………………………………………………...13
1.4. Models for Anti-ulcer Study………………………………………………………13
1.5. Fagonia Cretica Plant………………………………………………………………14
1.6. Constituents in Fagonia Cretica………………………………………………….....14
1.7. Medicinal Uses of Fagonia Cretica………………………………………………....15
1.8. Aqueous Extract of Fagonia Cretica………………………………………………..15
2. Materials and Methods…………………………………………………….
16
2.1. Collection And Identification Of Plant………………………………………….
17
2.2. Extraction Of Plant Material……………………………………………………
17
2.3. Chemicals and Drugs………………………………………………………….
18
2.4. Animal Model And Study Groups………………………………………….
18
2.5. Induction of Gastric Ulcers by Indomethacin………………………………...
19
2.6. Evaluation of Anti-ulcer Activity…………………………………………….
19
2.6.1. Macroscopic Examination of Stomach……………………………………. 20
2.6.2. Histopathology of Examination ………………………………………...
20
2.6.3. Ulcer Index……………………………………………………………….
20
2.6.4. Percentage Inhibition…………………………………………………….
21
2.6.5. Volume And pH Of Gastric Secretions……………………………………. 21
2.6.6. Total acidity of Gastric Juice………………………………………………. 22
2.6.7. Mucus Estimation…………………………………………………………
22
2.6.8. Estimation of Total Protein Content……………………………………….
23
2.6.9. GSH Estimation………………………...……………………………..
24
2.7 Statistical Analysis…………………………………………………
24
5
3. Results……………………………………………………………………………25
3.1. Macroscopic Examination of Stomach……………………………………………..26
3.2. Histopathology Evaluation………………………………………………………….31
3.3. Ulcer Index………………………………………………………………………….36
3.4. Percentage Inhibition………………………………………………………….
41
3.5. Volume of Gastric Juice………………………………………………………….
45
3.6. pH Of Gastric Juice……………………………………………………………
49
3.7. Total Acidity Of Gastric Contents………………………………………………
53
3.8. Mucus Estimation……………………….………………………………………… 57
3.9. Total Protein Content Estimation………………………………………………… 62
3.10. GSH Estimation……………………………………………………………………67
4. Discussion……………………………………………………………………….73
5. Conclusion………………………………………………………………………78
6. References………………………………………………………………………79
6
List of Figures
Figure 1. Macroscopic view of normal rat’s stomach.
Figure 2. Macroscopic view of rat’s stomach given Indomethacin 25mg/kg.
Figure. 3. Macroscopic view of rat’s stomach given Ranitidine.
Figure. 4. Macroscopic view of rat’s stomach given Omeprazole.
Figure. 5. Macroscopic view of rat’s stomach given Sucralfate.
Figure. 6. Macroscopic view of rat’s stomach treated with Aq. Extract of Fagonia cretica 200mg/kg.
Figure. 7. Macroscopic view of rat’s stomach treated with 300 mg/kg Fagonia cretica.
Figure.8. Macroscopic view of rat’s stomach treated with 400 mg/kg Fagonia cretica.
Figure. 9. Microscopic view of cross-section of a healthy rat’s stomach.
Figure. 10. Microscopic view of cross-section of stomach of a rat’s given Indomethacin
Figure. 11. Microscopic view of cross-section of stomach of a rat’s given Ranitidine.
Figure. 12. Microscopic view of cross-section of stomach of a rat’s given Omeprazole
Figure. 13. Microscopic view of cross-section of stomach of a rat’s given Sucralfate
Figure. 14. Microscopic view of cross-section of stomach of a rat’s given 200 mg/kg Fagonia cretica.
Figure. 15. Microscopic view of cross-section of stomach of a rat’s given 300 mg/kg Fagonia cretica.
Figure. 16. Microscopic view of cross-section of stomach of a rat’s given 400 mg/kg Fagonia cretica.
Figure 17. Effect of Fagonia cretica (F.C.) on the ulcer index (mm2) against Indomethacin
induced gastric ulcer in rats.
Figure. 18. Effect of standard drugs Ranitidine, Omeprazole and Sucralfate on Ulcer Index
(mm2) in Indomethacin induced gastric ulcers in rats.
Figure. 19. Effect of Aq. Extract of Fagonia cretica (F.C.) on ulcer index in Indomethacin
induced gastric ulcers in rats.
Figure 20. Effect of Fagonia cretica (F.C.) on the percentage protection against Indomethacin
induced gastric ulcer in rats.
Figure 21. Effect of Ranitidine, Omeprazole and Sucralfate on the percentage protection against
Indomethacin induced gastric ulcer in rats.
7
Figure. 22. Effect of aq. extract Fagonia cretica (F.C.) on the percentage protection against
Indomethacin induced gastric ulcer in rats.
Figure 23. Effect of Fagonia cretica (F.C.) on the volume of gastric juice against Indomethacin
induced gastric ulcer in rats
Figure. 24. Effect of standard drugs Ranitidine, Omeprazole and Sucralfate on Vol. of gastric
juice (ml) in Indomethacin induced gastric ulcers in rats.
Figure. 25. Effect of aq. extract Fagonia cretica (F.C.) on volume of gastric juice against
Indomethacin induced gastric ulcer in rats.
Figure. 26. Effect of aq. extract Fagonia cretica (F.C.) on pH of gastric juice against
Indomethacin induced gastric ulcer in rats
Figure. 27. Effect of standard drugs Ranitidine, Omeprazole and Sucralfate on pH of gastric juice
(ml) in Indomethacin induced gastric ulcers in rats
Figure. 28. Effect of aq. extract Fagonia cretica (F.C.) on pH of gastric juice against
Indomethacin induced gastric ulcer in rats.
Figure. 29. Effect of aq. extract Fagonia cretica (F.C.) on total acidity of gastric content against
Indomethacin induced gastric ulcer in rats.
Figure. 30. Effect of Ranitidine, Omeprazole, Sucralfate on total acidity of gastric content
against Indomethacin induced gastric ulcer in rats.
Figure. 31. Effect of aq. extract Fagonia cretica (F.C.) on total acidity of gastric content against
Indomethacin induced gastric ulcer in rats.
Figure. 32. The standard curve of Alcian Blue for the Estimation of Mucus
Figure. 33. Effect of aq. extract Fagonia cretica (F.C.) on adherence of mucus to gastric wall
against Indomethacin induced gastric ulcer in rats.
Figure. 34. Effect of standard drugs Ranitidine, Omeprazole, Sucralfate on adherence of mucus
to gastric wall against Indomethacin induced gastric ulcer in rats.
Figure. 35. Effect of aq. extract Fagonia cretica (F.C.) on adherence of mucus to gastric wall in
Indomethacin induced gastric ulcer in rats.
Figure. 36. BSA Standard curve for the Total Protein Concentration Estimation.
Figure. 37. Effect of aq. extract Fagonia cretica (F.C.) on total protein content in Indomethacin
induced gastric ulcer in rats.
8
Figure. 38. Effect of standard drugs Ranitidine, Omeprazole, Sucralfate on total protein content
in Indomethacin induced gastric ulcer in rats.
Figure. 39. Effect of aq. extract Fagonia cretica (F.C.) on total protein content in Indomethacin
induced gastric ulcer in rats.
Figure. 40. GSH Standard curve for the estimation of glutathione in all animal groups
Figure. 41. Effect of aq. extract Fagonia cretica (F.C.) on the concentrartion of GSH in
Indomethacin induced gastric ulcer in rats.
Figure. 42. Effect of Ranitidine, Omeprazole and Sucralfate on the concentrartion of GSH in
Indomethacin induced gastric ulcer in rats.
Figure. 43. Effect of aq. extract Fagonia cretica (F.C.) on the concentrartion of GSH in
Indomethacin induced gastric ulcer in rats
9
Abstract
Fagonia cretica is a plant from Zygophyllaceae family and is extensively found in dry regions of
Pakistan. It is commonly called as Suchi booti or Dhamasa in local areas. It has long been used
for various ailments in folk medication. The plant has multiple medical uses. In order to
investigate the presence of anti-ulcer activity in Fagonia cretica, a study was performed where
aqueous extract of the plant was tested against indomethacin induced ulcers in rats. Indomethacin
was administered to the rats by oral gavage according to 25 mg per kg of the body weight to
induce gastric damage. An increasing dose of aqueous extract of Fagonia cretica i.e., 200 mg/kg
B.W., 300 mg/kg B.W. and 400 mg/kg B.W. was used for the said study. Various parameters like
ulcer index, percentage inhibition, analysis of gastric content, macroscopic and microscopic
study, mucus estimation, total protein content estimation and glutathione estimation were
evaluated. Ulcer index was significantly reduced with the increasing dose of the extract,
percentage protection was increased which were initial indications that the extract possesses
ulcer curing properties. Further tests revealed that with the treatment of aq. extract of Fagonia
cretica against indomethacin induced ulcers the protein content in the gastric tissue, mucus
adherence to the gastric wall and concentration of GSH in the gastric tissue increased
significantly. The research proved that aqueous extract of Fagonia cretica possesses ulcer healing
and suggested further investigation to find the particular pharmacological moiety for this effect.
10
Chapter 1
Introduction
11
1. Introduction
1.1. Anatomy and Physiology of Stomach:
Stomach is a visceral organ and it is a part of the digestive system. The shape of the stomach is like a J. The
stomach is located in the upper part of the abdomen (www.cancer.ca, 2019). Upper part of the stomach is
joined with esophagus through the cardiac sphincter and is called cardiac region, bottom part joins with the
small intestine via pyloric sphincter and is called pyloric region. The body of the stomach is divided into
two parts i.e., Fundus and Corpus, where corpus makes the most part of the stomach by size
(www.laproscopic.md, 2019). The size of the stomach differs from person to person. The Stomach has the
capacity of 1000 to 1500 ml of fluids, therefore making it the most dilated part of the digestive tube. The
stomach is used for mixing and breaking down food. Digestion of the food takes place by secretion of HCl
and pepsin. The corpus and fundus in the stomach contain the acid secreting glands. The stomach is lined
with gastric mucosa which protects the stomach from its own acidic secretions. This mucosal barrier is
made up of surface epithelium. Histamine, Ach and Gastrin stimulate the secretion of acid by the gastric
mucosa (DI Soybel, 2005)
The stomach conditions which are very common and can be painful and problematic include Dyspepsia,
GERD (Gastro Esophageal Reflex Disease), Gastritis, Gastric ulcers, Gastroparesis (delayed gastric
emptying).
1.2. Pathophysiology of gastric ulcers:
One among the most common diseases of the alimentary canal is Ulcer. The painful blisters or lesions on
mucosal lining of esophagus, stomach or intestine are called Peptic Ulcers. Ulcers in the gastrointestinal
tract are of two types depending on their origin. Those in the stomach are called Gastric Ulcers and the
sores in initial segment of small intestine are called Duodenal Ulcers. Ranging from mild to severe, ulcers
12
effect a large number of populations including both men and women of different age groups. H. pylori and
NSAIDs are ascribed to the high occurrence rate of gastric ulcers. Peptic ulcers mainly are caused by the
imbalance between hostile factors like acid, pepsin, oxidative stress and protective factors such as mucus,
prostaglandins, antioxidants and bicarbonates. Researches have shown that there are various medicines
which cause lesions in the gastric mucosa which may lead to bleeding ulcers. Most common drugs in this
category are the NSAIDs e.g. Aspirin, Indomethacin, etc. (Sajjadi, 2017).
NSAIDs cause a high incidence of gastric and duodenal ulcers, which is considered to be due to inhibition
of production of prostaglandins and loss of their protective effects. Gastric ulcer frequently happens with
alteration in pepsin and acid activity, suggesting the involvement of mucosal defense impairment. Gastric
ulcers can be divided into three categories, ulcers caused by H. Pylori infection, ulcers caused by NSAIDs
and Ulcers due to hyper-secretion of acid and pepsin called Zollinger-Ellison syndrome (Mertz. 1991).
1.3. Treatments for gastric ulcers:
Various treatment options are available these days for the treatment of gastric ulcers. Antacids are used
when ulcers are caused due to increased acid secretion. Prostaglandin analogues, Proton pump inhibitors,
histamine receptor blockers and cytoprotectants are used widely for the treatment and prevention of ulcers.
1.4. Models for anti-ulcer study:
In order to evaluate the protective and healing effects of drugs on ulcers, various experimental models are
used. These methods include induction of ulcer by using NSAIDs. Induction of ulcers by ligation method,
13
ulcer induction by using acetic acid and induction of ulcer by using ethanol, ulcers by the influence of H.
Pylori infection (Okabe, 2005). (Susumu Okabe, 2005).
1.5. Fagonia Cretica Plant:
Plants are being used for centuries for curing diseases and also heal wounds. One of the many plants being
used is Fagonia Cretica. Fagonia cretica is a specie of plant from Zygophyllaceae family. This plant is
known to be used for cancer and breast cancer treatment in particular (Ahmed et al, 2013). It is also said to
be used in the various treatments for digestive problems and also blood and vascular diseases. The petals
of Fagonia Cretica are purplish pink in color. It is mostly found in the dry regions of the world. It is known
to be widely collected in India and Pakistan. The plant is widely found on the rocky soils in cities of Punjab,
KPK. In local language the plant is called ‘Dhamasa’ and ‘Suchi Booti’ The plants which are in the same
family as the Fagonia, are known to provide possible treatments for many kinds of health issues (Ahmed et
al. 2013).
1.6. Constituents in Fagonia Cretica:
Plants have been used in healing and curing many human diseases because of that they are known
as medicinal plants. Since they are medicinal plants, they have phytochemical constituents in them.
Phytochemicals are found naturally in the plants, whether they are either leaves, fruit or roots.
They have defensive mechanism, which can be used in protecting from different types of diseases
(Nostro A, 2000). The phytochemical analysis of Fagonia cretica has shown the presence of high
levels of terpenoids and flavonoids. These constituents have anti-malarial, cholesterol inhibiting,
analgesic and anti-inflammatory properties (Wadood, 2013).
14
1.7. Medicinal uses of Fagonia Cretica:
Since Fagonia Cretica has anti-bacterial and anti-inflammatory properties, it is being used in the Indo-Pak
region for treatments for fever, digestion disorders, liver ailments, asthma, and bronchitis (Marwat et
al,2008). It is also used to cure and heal snake bites. Fagonia cretica is known to have anti-diabetic and
blood thinning properties. Fagonia cretica is known to cure cancer by causing DNA damage and apoptosis
in cancerous cells.
1.8. Aqueous extract of Fagonia Cretica:
The aqueous extract of Fagonia Cretica is widely used in treatments for cancer. One of the treatments for
breast cancer contains extracts of Fagonia Cretica and it is a herbal tea based product. The aqueous extract
is effectual therapy to be used in the early stages of cancer. The extract is also used for the treatment of
inflammation of the stomach, which is known to be the cause of gastritis (Shah et al, 2011). The aqueous
extract of Fagonia Cretica is known to help decrease the size of the cancer tumor and it also helps in
improving the life of the patients who have breast cancer. The extract does not cause cancer patients to have
effects like vomiting, diarrhea or the spot baldness. These are some of the common side effects, which are
experienced when cancer patients are going through the standard chemo treatment.
15
Chapter 2
Materials and Methods
16
2. Materials and Methods:
2.1. Collection and Identification of Plant:
Fagonia cretica plant was collected from the city of Rabwah in Sargodha District. It was
identified at the Botany Department of Government College University Lahore, a voucher
number (GC. Herb. Bot. 3545) was allotted to the specimen and it was saved in the herbarium.
2.2. Extraction of Plant Material:
Fagonia cretica plant was altogether washed with tap water to free it from dirt and other
undesirable materials amassed on it from its natural habitat. It was then kept in shade at the
Pharmacology laboratory in College of Pharmacy, Punjab University for about 10 days till it was
completely dried. The dried plant was then converted into a fine powder and the aqueous
extraction was carried out by maceration process. 450g of powdered F. cretica was immersed in
2250 ml of distilled water in conical flasks. The mouths of flasks were covered with aluminum
foil and were kept for five days while stirring it daily. The material was then filtered using a
muslin cloth followed by filtration with Whatman filter paper. The solvent from the concentrate
was then removed using a rotary vacuum evaporator over a water bath of 60°C temperature
(Nagappan, 2012). A brownish concentrated extract weighing 43 gm was obtained and stored in
refrigerator for further experimental use.
17
2.3. Chemicals and Drug:
Sucralfate ( Zhei Jing Pharma, China ), Ranitidine ( Saraca Lab, India), Omeprazole (SigmaAldrich, Germany), Albumin Bovine Serum ( Bioshop Canada), Alcian blue 8GX ( UNIChem Chemical Reagents, China), Diethyl ether (Labscan Asia Co. Ltd, Thailand),
Glutathione (Merck Laboratories Pvt Ltd, Germany), DTNB ( Chem
Impex Intl., USA),
Formalin ( Merck,, KGaA-64721 Darmstadt, Germany), Folin and Ciocalteu’s phenol reagent
(Central Drug House Ltd. New Delhi, India), Sodium bicarbonate, Sodium hydroxide,
Magnesium chloride ( BDH Chemicals Ltd, Poole, England), Potassium Sodium Tartarate (
Sigma, Germany), Sodium chloride (Sigma-Aldrich, Germany), Tris hydrochloride (
Bioworld Bioplus fine chemicals, India), Sucrose (Riedel-de Haen, GmbH).
2.4. Animal Model and Study Groups:
48 Albino Wistar rats weighing between 175 and 225g of either sex were purchased from the
Animal House of College of Pharmacy, University of the Punjab, Lahore. The animals were
housed in stainless steel cages, kept under optimum environmental conditions of temperature
(23°C ± 3°C), humidity (40-60 %) and 12hr light/dark cycle. The animals were provided with
distilled water and fodder. Experimental group was fasted for 24hr before the study given
sufficient supply of water. The experimentation was carried out according to the rules and
regulations set by the Ethical Committee of the College of Pharmacy, University of the Punjab.
18
The animals were randomly divided into 8 groups, each containing 6 rats. Group 1 as normal
study group having healthy rats and were only given water and fodder. Group 2 as Ulcer control
where rats were given Indomethacin 25mg/kg. Group 3 where animals are given 25mg/kg
Ranitidine before the induction of ulcer. Group 4 was given 25mg/kg Omeprazole. Group 5
animals were given Sucralfate 100mg/kg. Group 6,7 and 8 were treatment groups and were given
Aq. Extract of Fagonia cretica 200mg/kg B.W., 300mg/kg B.W. and 400mg/kg B.W.
respectively.
2.5. Induction of Gastric Ulcers by Indomethacin:
The animals were kept on fasting for 24hrs with access to water only prior to the
experimentation. Animals were given standard drugs and extract doses according to their
relevant groups for seven consecutive days before inducing ulcers ( Paseban, Niazmand 2014).
Standard groups received Ranitindine 25mg/k, Omeprazole 25mg/kg and Sucralfate 100mg/kg
orally. Treatment groups were administered 200mg/kg, 300mg/kg and 400 mg/kg extract of F.
cretica. All treatments were done p.o an hour before the induction of ulcer by Indomethacin
25mg/kg. 6 h after the administration of Indomethacin, the animals were anesthetized and
dissected to ligate and excise the stomach to evaluate the ulcers and perform further tests.
2.6. Evaluation of Anti-Ulcer Activity:
The protective effect of aq. Extract of Fagonia cretica against indomethacin induced gastric
damage was evaluated by the following parameters,
19
2.6.1. Macroscopic Examination of Stomach:
The stomach after dissecting the animal was ligated with the help of surgical sutures at both
cardiac and pyloric ends. The stomach was excised, gastric contents emptied, and stomach
was opened along the greater curvature. Washed the stomach thoroughly with normal saline,
placed it over a plain surface and fixed with a few pins. The stomach was then observed to
identify ulcers which were visible in the form of mucosal erosion, spot ulcer, lesions and
hemorrhagic streaks.
2.6.2 Histopathology Examination:
Small sections of stomach were cut and kept in 10% buffered formalin solution. Using rotary
microtome thin slices of 5m thickness from the stomach were obtained and stained with
eosin and hematoxylin and the slides were analyzed by a pathologist for necrosis,
inflammation and erosion (Bancroft and Layton, 2013)
2.6.3. Ulcer Index:
After spreading and fixing the ulcerous stomach on a planar surface, the total area and
ulcerated areas were drawn on a cellophane sheet. This cellophane sheet was placed over a
20
millimeter paper and the sum of total area and ulcerous area was taken in mm2 (Dengiz, 2007
). The ulcer index was calculated by the formula,
Ulcer Index (UI) = Ulcerated Area (mm²) x 100
Total Area (mm²)
2.6.4. Percentage Inhibition:
The protective effect of the drugs ranitidine, omeprazole, sucralfate and F. cretica extract
against indomethacin induced ulcer was determined using the following formula,
% Inhibition = Ulcer Index (C) - Ulcer Index (T) x 100
Ulcer Index (c)
2.6.5. Volume and pH of Gastric Secretions:
The gastric content was emptied after cutting the stomach out. The fluid’s volume and pH
were measured after being centrifuged using a centrifuge machine (B. Hernle GmbH Co.,
Germany) for ten minutes at 3000 rpm. Volume of the fluid was measured with a graduated
cylinder and pH of gastric juice was determined using a pH meter (InoLab pH 720,
Germany).
21
2.6.6. Total Acidity of Gastric Juice:
The method of Shay (Shay, 1945) was used with slight modifications to perform the assay.
The collected gastric juice after centrifugation was mixed with 1 ml of distilled water. Two
drops of phenolphthalein as an indicator were added to the samples and their titration against
0.01N NaOH solution was carried out. When a persistent pink colour appeared, the volume
of 0.01N NaOH was noted from the burette. Total acidity was calculated by the following
formula and the results were expressed in mEq/L.
Total Acidity = Volume of NaOH x N x 100
0.1
2.6.7. Mucus Estimation:
Corne method (Corne et al., 1974) was used to estimate the amount of mucus on gastric wall.
Mucus content was determined from the Alcian blue standard curve. The glandular part of
the stomach after being weighed was immersed for 2 hrs. in 10ml of 0.1% w/v alcian blue in
0.16 mol/l sucrose solution. The tissue was rinsed twice by putting it in 0.25 mol/l sucrose
solution for 15min both times to remove excess dye. The dye attached to gastric mucus was
extracted by immersing the tissue in 0.5 mol/l magnesium chloride solution. For a period of 2
hrs, it was vigorously shaken for a minute after an interval of 30 minutes. After this, the blue
extract was shaken with diethyl ether. The emulsion was then centrifuged at 3600 rpm for 10
minutes and a spectrophotometer (Shimadzu Corp., Japan) was used to measure the optical
density of the aqueous phase at 600nm. The results are expressed as absorbance per gram of
the wet tissue.
22
2.6.8. Estimation of Total Protein Content:
Lowry method (Lowry et al., 1951) was used to determine the total protein content of the
gastric tissue. Protein content was calculated using the Bovine Serum Albumin standard
curve. BSA stock solution and two reagents were needed for this test which were prepared as
follows
Reagent 1: 1ml of 0.5% CuSO4.5H2O in solution, 1ml of 1% NaK Tartarate solution, 48ml of
2% Na2CO3 solution in 0.1N NaOH.
Reagent 2: One part of F.C Reagent in one part of water.
BSA Stock Solution: 1mg/1ml
For the preparation of tissue homogenate, tissue samples were weighed and diluted with tris
HCl buffer in a ratio of 1:3. The tissues were then finely chopped in the petri dishes with the
help of scissors and scalpel and homogenized using a homogenizer (WiseStar, Daithan
Scientific Co., Ltd., Korea). The tissue homogenates were centrifuged in a Refrigerator
Centrifuge machine at 4C temperature for half an hour at 12000 rpm. Supernatant was
separated for tests to quantify for the total protein content.
25, 50, 75, 100, 125, 150 g/ml of BSA stock solution was taken in 6 separate test tubes and
volume was made upto 1ml by adding 0.02 M solution of tris HCl buffer. A test tube used as
blank contained 1ml of 0.02 M tris HCl buffer of 7.6 pH. Incubated the six test tubes for 10
minutes after adding 4.5ml of reagent 1. Afterwards added 0.5ml of reagent 2 and incubated
for another 30 min.
23
2.6.9. GSH Estimation:
Ellman and Sedlak and Lindsay’s method (Ellman 1959, Sedlak & Lindsay 1968) was used
to perform the glutathione essay. 2.5ml solution of 0.02M EDTA was taken in a test tube and
mixed well with 2ml of 10% homogenate solution. 2 ml from this mixture was taken, 4 ml
distilled water and 1ml of 50% Trichloroacetic acid solution were further added to this. After
centrifuging it for 10 minutes at 3000 rpm, supernatant was withdrawn. 0.4M Tris-HCl buffer
at pH 8.9 and 0.1 ml of DTNB (5,5-dithiobis 2-nitrobenzoic acid) were added to 2ml of the
supernatant and shaken vigorously. After this the absorbance was measured at 412nm using a
spectrophotometer against blank sample. The amount of GSH was calculated using the GSH
standard curve and results were expressed as mol/mg.
2.7. Statistical Analysis:
The results of all observations are expressed as mean of readings of 6 animals  SEM. Statistical
comparison between the mean values was carried out by One-Way ANOVA followed by
Tukey’s test for multiple comparisons. Statistical significance was taken as P<0.05.
24
Chapter 3
Results
25
3. Results:
3.1. Macroscopic Examination of Stomach:
The stomach of rats from each group were observed with the help of a magnifying glass to observe the
changes in the mucosa.
Group 1 included healthy rats which were only given distilled water, no damage was found in this group,
Figure 1 shows a picture of stomach taken with a high-resolution camera (Nikon D3200, Japan). Figure 2
is from group 2 where rats were given 25 mg/kg Indomethacin for the induction of ulcer. Severe damage
to the gastric mucosa in this group was found and ulcers were present in the form of hemorrhage, spot
ulcers and redness.
Figures 3, 4 and 5 show effects of ranitidine, omeprazole and sucralfate respectively. Ulcers were reduced
and there was lesser mucosal damage as compared to the ulcer control group.
An increasing dose of 200 mg/kg, 300 mg/kg and 400 mg/kg were given to the rest of the groups and a
dose dependent reduction in gastric damage was observed. Figure 6 represents the stomach of a rat from
group 5, who were administered 200 mg/kg of aq. Extract of Fagonia cretica. Fig. 7 and 8 are from the
rats who were treated with 300 mg/kg and 400 mg/kg of Fagonia cretica.
26
(Fig.1)
(Fig.2)
Figure 1. Macroscopic view of normal rat’s stomach.
Figure 2. Macroscopic view of rat’s stomach given Indomethacin 25mg/kg.
27
(Fig. 3)
(Fig. 4)
Figure. 3. Macroscopic view of rat’s stomach given Ranitidine.
Figure. 4. Macroscopic view of rat’s stomach given Omeprazole.
28
(Fig. 5)
(Fig. 6)
Figure. 5. Macroscopic view of rat’s stomach given Sucralfate.
Figure. 6. Macroscopic view of rat’s stomach treated with Aq. Extract of Fagonia cretica 200mg/kg.
29
(Fig. 7)
(Fig. 8)
Figure. 7. Macroscopic view of rat’s stomach treated with 300 mg/kg Fagonia cretica.
Figure.8. Macroscopic view of rat’s stomach treated with 400 mg/kg Fagonia cretica.
30
3.2. Histopathology Evaluation:
Histological examination was carried out on the gastric tissue of rats to assess the damage on microscopic
level. Figure 9 from the healthy rat shows completely normal morphology while figure 10 has necrosis,
inflammation, hemorrhage and degeneration caused by Indomethacin.
In figure 11, there is a cross-section of gastric tissue from the group to which Ranitidine was
administered. There was slightest damage in the tissues of animals who were pre-treated with standard
drugs, their morphological features were close to normal group. Figure 12 contains the microscopic view
of a rat’s stomach from the group treated with omeprazole and figure 13 shows the slide from Sucralfate
treated group. Figure 14 shows reduced degeneration, necrosis and edema in the gastric wall where 200
mg/kg extract of Fagonia cretica was given. Figure 15 has the slide from group 7 which was administered
with 300 mg/kg F.C. extract and figure 16 contains the microscopic picture where the animal was treated
with 400 mg/kg aq. extract of Fagonia cretica.
31
(Fig. 9)
(Fig. 10)
Figure. 9. Microscopic view of cross-section of a healthy rat’s stomach.
Figure. 10. Microscopic view of cross-section of stomach of a rat’s given Indomethacin.
32
(Fig. 11)
(Fig. 12)
Figure. 11. Microscopic view of cross-section of stomach of a rat’s given Ranitidine.
Figure. 12. Microscopic view of cross-section of stomach of a rat’s given Omeprazole.
33
(Fig. 13)
(Fig. 14)
Figure. 13. Microscopic view of cross-section of stomach of a rat’s given Sucralfate
Figure. 14. Microscopic view of cross-section of stomach of a rat’s given 200 mg/kg Fagonia cretica.
34
(Fig. 15)
(Fig. 16)
Figure. 15. Microscopic view of cross-section of stomach of a rat’s given 300 mg/kg Fagonia cretica.
Figure. 16. Microscopic view of cross-section of stomach of a rat’s given 400 mg/kg Fagonia cretica.
35
3.3. Ulcer Index:
Ulcer index in all the animals was calculated by measuring the ulcerated area against total area of the
stomach in mm2. The results in figure 17 show mean  SEM of the ulcer index calculated for all the study
groups. The ulcer index of ulcer control group was ( 11.80.79 ). Ulcer index in the groups treated with
standard drugs and extract treated group was reduced significantly ( p<0.0001 ) as compared to the
control group. Ulcer index of Ranitidine treated group came out to be ( 1.810.37 ) , for omeprazole it
was ( 0.470.08 ) and for the group given sucralfate was ( 1.180.17 ). The results from omeprazole were
comparable to healthy group. For the groups treated with extract doses, the ulcer index was decreased
with increasing dose. The group which received 200 mg/kg B.W dose of aq. extract of Fagonia cretica
had ulcer index ( 6.240.47 ), for the animals who received 300 mg/kg B.W dose UI ( 4.940.39 ) and
maximum reduction in UI ( 4.260.34 ) was found to be in the group given 400 mg/kg extract of F.C.
Figure 18 compares ulcer control group with standard drugs and figure 19 reveals a comparison between
ulcer control and doses of aq. extract of Fagonia cretica.
36
Ulcer Index (mm2)
15
10
***
***
***
5
***
***
***
0
al
m
r
l
e
e
te
kg
kg
kg
ro
in
ol
a
/
/
/
t
f
z
d
g
g
g
l
a
ra
on
iti
p
C
No
cr
0m
0m
0m
n
e
r
0
0
0
a
u
S
2
3
4
R
ce
Om
C.
C.
C.
.
.
.
Ul
F
F
F
Figure 17. Effect of Fagonia cretica (F.C.) on the ulcer index (mm2) against Indomethacin induced
gastric ulcer in rats.
Each bar shows the mean ± S.E.M. of ulcer index of 6 animals where *** P<0.0001. The data was found
to be statistically significant when compared to ulcer control group by using one-way ANOVA followed
by Tukey’s test.
37
38
Ulcer Index (mm2)
15
10
5
***
***
***
te
al
fa
Su
cr
az
ol
e
ep
r
ni
tid
in
e
Ra
Om
Ul
ce
rC
on
t
ro
l
0
Figure. 18. Effect of standard drugs Ranitidine, Omeprazole and Sucralfate on Ulcer Index (mm2)
in Indomethacin induced gastric ulcers in rats.
Each bar represents the ulcer index of 6 rats as mean ± S.E.M. In this graph the ulcer control group is
compared with the groups given standard drugs. The data was found to be statistically significant where
*** p<0.0001 by using one-way ANOVA followed by Tukey’s test.
39
Ulcer Index (mm2)
15
10
***
***
***
5
kg
F.
C.
40
0m
g/
kg
F.
C.
30
0m
g/
kg
g/
0m
20
C.
F.
Ul
ce
rC
on
t
ro
l
0
Figure. 19. Effect of Aq. Extract of Fagonia cretica (F.C.) on ulcer index in Indomethacin induced
gastric ulcers in rats.
Each bar represents the ulcer index of 6 rats as mean ± S.E.M. In this graph the ulcer control group is
compared with the groups treated with aq. Extract of Fagonia cretica. The data was found to be
statistically significant where *** p<0.0001, using one-way ANOVA followed by Tukey’s test.
40
3.4. Percentage Inhibition:
The percentage protection against indomethacin induced ulcers by various drugs was calculated by the
ulcer index of control group and treatment groups. Figure 20 represents % protection for each group. The
inhibition by omeprazole was highest among all treatment groups. The percentage inhibition by ranitidine
was ( 84.7%  2.4 ), for omeprazole it was estimated to be ( 95.98%  0.50 ) and for the group treated
with sucralfate the % inhibition was 89.9%  1.36 ). The percentage inhibition for the treatment groups
was found to be directly proportional to the dose of extract. Maximum inhibition ( 63.9%  0.89 ) took
place at 400 mg/kg dose of aq. extract of Fagonia cretica. The result for animals given 300 mg/kg dose
was ( 57.74%  1.62 ) and for 200 mg/kg the percentage inhibition was calculated to be ( 47.46%  3.77
). The results revealed that aq. extract of F.C. was successful in preventing the gastric damage caused by
indomethacin.
41
Percentage Inhibition (%)
100
80
60
40
20
0
al
rm
l
e
e
te
kg
kg
kg
ro
in
ol
a
/
/
/
t
f
z
d
g
g
g
l
ra
on
iti
ra
p
C
No
c
0m
0m
0m
n
e
r
0
0
0
a
u
S
2
3
4
R
ce
Om
C.
C.
C.
.
.
.
Ul
F
F
F
Figure 20. Effect of Fagonia cretica (F.C.) on the percentage protection against Indomethacin
induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the percentage inhibition of ulcers by treatment doses and standard
drugs.
42
Percentage Inhibition (%)
100
80
60
40
20
te
al
fa
cr
ep
r
Om
Su
az
ol
e
e
in
iti
d
Ra
n
No
r
m
al
0
Figure 21. Effect of Ranitidine, Omeprazole and Sucralfate on the percentage protection against
Indomethacin induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the percentage inhibition of ulcers by standard drugs.
43
100
% inhibition
80
60
40
20
g/
kg
F.
C.
40
0m
g/
kg
F.
C.
30
0m
g/
kg
20
0m
F.
C.
No
r
m
al
0
Figure. 22. Effect of aq. extract Fagonia cretica (F.C.) on the percentage protection against
Indomethacin induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the percentage inhibition of ulcers by treatment doses.
44
3.5. Volume of Gastric Juice:
The volume of the gastric fluid was measured for each animal is all 8 groups and results were represented
as mean  S.E.M. A comparison of all groups is shown in figure. 23. The volume (ml) increased upto (
4.3  0.17 ) in ulcer control group while the measurement for normal group was ( 1.53  0.13 ). In figure
24 ranitidine ( 2.2  0.17 ), omeprazole ( 1.81  0.13 ), sucralfate ( 2.0  0.24 ) are compared with ulcer
control group, the volume of gastric juice decreased significantly ( p<0.0001 ) as compared to control
group. Figure 25 shows a comparison of ulcer control group with treatment groups, the volume for 200
mg/kg Fagonia cretica ( 3.7  0.14 ) decreased at a significance level ( p<0.05 ). The volumes for 300
mg/kg dose ( 3.1  0.17 ) and 400 mg/kg (2.8  0.2 ) were reduced significantly ( p< 0.0001 ) as
compared to ulcer control group.
45
Volume of Gastric Juice (ml)
5
*
4
***
***
3
***
***
***
2
1
kg
40
0m
g/
kg
g/
C.
F.
F.
C.
30
0m
g/
kg
te
F.
C.
20
0m
al
fa
cr
Su
ep
r
Om
Ra
ni
tid
in
az
ol
e
e
ro
l
rC
on
t
Ul
ce
No
r
m
al
0
Figure 23. Effect of Fagonia cretica (F.C.) on the volume of gastric juice against Indomethacin
induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the volume of gastric juice calculated from 6 rats in the group,
where *** p<0.0001. The results revealed decrease in volume as compared to the ulcer control group. The
data was found to be statistically significant when compared to ulcer control group by using one-way
ANOVA followed by Tukey’s test.
46
Volume of Gastric Juice (ml)
5
4
3
***
***
***
2
1
fa
te
cr
Su
zo
ep
ra
Om
al
le
e
in
ni
tid
Ra
Ul
ce
rC
on
t
ro
l
0
Figure. 24. Effect of standard drugs Ranitidine, Omeprazole and Sucralfate on Vol. of gastric juice
(ml) in Indomethacin induced gastric ulcers in rats.
Each bar represents mean  S.E.M. of the volume of gastric juice calculated from 6 rats in the group,
where *** p<0.0001. There was reduction in the gastric fluid when compared with ulcer control group.
The data was found to be statistically significant when compared to ulcer control group by using one-way
ANOVA followed by Tukey’s test.
47
Volume of Gastric Juice (ml)
5
*
4
***
***
3
2
1
g/
kg
F.
C
.4
00
m
g/
kg
F.
C
.3
00
m
g/
kg
00
m
.2
F.
C
Ul
ce
rC
on
tro
l
0
Figure. 25. Effect of aq. extract Fagonia cretica (F.C.) on volume of gastric juice against
Indomethacin induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the volume of gastric juice calculated from 6 rats in the group,
where *** p<0.0001 and * p<0.05. The data was found to be statistically significant when compared to
ulcer control group by using one-way ANOVA followed by Tukey’s test.
48
3.6. pH of Gastric Juice:
The pH of gastric fluid was measured using a pH meter and mean  S.E.M was calculated for rats ( n=6 )
for each group. Figure 26 compares the mean pH of all the groups. The pH in control group was very low
( 0.98  0.1 ) than the normal group ( 2.63  0.12 ). For the groups treated with ranitidine ( 4.51  0.2 ),
omeprazole ( 5.03  0.12 ) and sucralfate ( 4.2  0.16 ) the pH was significantly ( p<0.0001 ) increased as
compared to ulcer control. The comparison is shown in figure 27. In figure 28, there is a comparison
between ulcer control and treatment groups for the doses of Fagonia cretica. There was an increase for
extract dose of 200 mg/kg ( 1.55  0.17 ) at significance level of p<0.05. A significant increase (
p<0.0001 ) was observed in groups treated with 300 mg/kg extract dose ( 2.04  0.14 ) and 400 mg/kg
dose ( 2.62  0.16 ) as compared to ulcer control group.
49
6
***
***
pH of Gastric Juice
***
4
***
***
2
*
g/
kg
40
0m
g/
kg
C.
F.
F.
C.
30
0m
g/
kg
te
F.
C.
20
0m
al
fa
cr
Su
ep
r
Om
Ra
ni
tid
in
az
ol
e
e
ro
l
rC
on
t
Ul
ce
No
r
m
al
0
Figure. 26. Effect of aq. extract Fagonia cretica (F.C.) on pH of gastric juice against Indomethacin
induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the volume of gastric juice calculated from 6 rats in the group,
where *** p<0.0001 and * p<0.05. The pH in ulcer control group was lower than the normal group. In
groups treated with standard drugs, the pH was increased tremendously. The data was found to be
statistically significant when compared to ulcer control group by using one-way ANOVA followed by
Tukey’s test.
50
6
***
pH of Gastric Juice
***
***
4
2
te
cr
al
fa
Su
az
ol
e
Om
ep
r
in
ni
tid
Ra
Ul
ce
rC
on
t
ro
l
e
0
Figure. 27. Effect of standard drugs Ranitidine, Omeprazole and Sucralfate on pH of gastric juice
(ml) in Indomethacin induced gastric ulcers in rats.
Each bar represents mean  S.E.M. of the volume of gastric juice calculated from 6 rats in the group,
where *** p<0.0001. The pH in ulcer control group was lower than the normal group. In groups treated
with standard drugs, the pH was increased. The data was found to be statistically significant when
compared to ulcer control group by using one-way ANOVA followed by Tukey’s test.
51
3
***
pH of Gastric Juice
***
2
*
1
g/
kg
F.
C.
40
0m
g/
kg
F.
C.
30
0m
g/
kg
20
0m
C.
F.
Ul
ce
rC
on
t
ro
l
0
Figure. 28. Effect of aq. extract Fagonia cretica (F.C.) on pH of gastric juice against Indomethacin
induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the volume of gastric juice calculated from 6 rats in the group,
where *** p<0.0001, * p<0.05. The data was found to be statistically significant when compared to ulcer
control group by using one-way ANOVA followed by Tukey’s test.
52
3.7. Total Acidity of Gastric Contents:
The gastric content from the animals was assayed to calculate the total acidity and the results were noted
as mean  S.E.M. Figure 29 shows a comparison of all the groups and the results reveal that total acidity
of the gastric content was reduced with increasing dose from ( 71  1.91 ) for 200 mg/kg dose of fagonia
cretica treated group to ( 40.2  1.57 ) for group treated with 400 mg/kg. This revealed a significant (
p<0.0001 ) decrease in comparison with ulcer control group ( 83.2  1.77 ). The comparison between
control and treatment doses is shown in figure 31. For the groups treated with standard drugs the acidity
was massively reduced and the results were significant as compared to control group. Figure 30 reveals a
comparison among ranitidine treated group ( 25.2  1.34 ), omeprazole treated group ( 21.3  1.1 ),
sucralfate treated group ( 27.2  1.1 ) and ulcer control group. The total acidity was significantly (
p<0.0001 ) decreased.
53
100
Total Acidity (mEq/l)
80
***
***
60
***
40
***
***
***
20
kg
40
0m
g/
kg
g/
F.
C.
0m
30
C.
F.
F.
C.
20
0m
g/
kg
te
al
fa
Su
cr
ep
ra
zo
le
in
e
Om
Ra
ni
tid
ro
l
rC
on
t
Ul
ce
No
r
m
al
0
Figure. 29. Effect of aq. extract Fagonia cretica (F.C.) on total acidity of gastric content against
Indomethacin induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the total acidity of the gastric gastric content calculated from 6 rats
in the group, where *** p<0.0001. The data was found to be statistically significant when compared to
ulcer control group by using one-way ANOVA followed by Tukey’s test.
54
100
Total Acidity (mEq/l)
80
60
40
***
***
***
20
te
al
fa
cr
Su
az
ol
e
ep
r
Om
ni
tid
Ra
Ul
ce
rC
on
t
ro
l
in
e
0
Figure. 30. Effect of Ranitidine, Omeprazole, Sucralfate on total acidity of gastric content against
Indomethacin induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the total acidity of the gastric gastric content calculated from 6 rats
in the group, where *** p<0.0001. The data was found to be statistically significant when compared to
ulcer control group by using one-way ANOVA followed by Tukey’s test.
55
100
Total Acidity (mEq/l)
80
***
***
60
***
40
20
kg
F.
C.
40
0m
g/
kg
F.
C.
30
0m
g/
kg
g/
0m
20
C.
F.
Ul
ce
rC
on
t
ro
l
0
Figure. 31. Effect of aq. extract Fagonia cretica (F.C.) on total acidity of gastric content against
Indomethacin induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the total acidity of the gastric gastric content calculated from 6 rats
in the group, where *** p<0.0001. The data was found to be statistically significant when compared to
ulcer control group by using one-way ANOVA followed by Tukey’s test.
56
3.8. Mucus Estimation:
The mucus content on the gastric wall was calculated from the Alcian Blue standard curve. The standard
curve for alcian blue is shown in figure 32. The results for the amount of mucus on gastric wall were
expressed as g/g of the gastric tissue. Figure 33 shows the mean  S.E.M. of the results obtained for
mucus adherence to gastric wall for all the study groups. The results showed that there was a significant (
p<0.0001 ) increase in the mucus concentration with increasing dose of aq. extract of Fagonia cretica. The
concentration of mucus was higher in ranitidine ( 85  2.5 ), omeprazole ( 781.84 ) and sucralfate (
902.6 ) as that of ulcer control group ( 280.82 ), revealed in figure 34. The results for ranitidine and
omeprazole were comparable with normal group ( 80  2.5 ), while the results for sucralfate results were
even higher than the normal group. Figure 35 contains the results for 200 mg/kg aq. extract of Fagonia
cretica ( 451.15 ), 300 mg/kg BW Fagonia cretica ( 551.19 ), 400 mg/kg BW Fagonia cretica ( 681.45
) compared to ulcer control group.
57
Figure. 32. The standard curve of Alcian Blue for the Estimation of Mucus.
58
100
***
Mucus Estimation (g/g)
***
***
80
***
***
60
***
40
20
g/
kg
40
0m
g/
kg
C.
F.
F.
C.
30
0m
g/
kg
te
F.
C.
20
0m
al
fa
cr
Su
ep
r
Om
Ra
ni
tid
in
az
ol
e
e
ro
l
rC
on
t
Ul
ce
No
r
m
al
0
Figure. 33. Effect of aq. extract Fagonia cretica (F.C.) on adherence of mucus to gastric wall against
Indomethacin induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the concentration of mucus on gastric wall calculated from 6 rats in
each group, where *** p<0.0001. The data was found to be statistically significant when compared to
ulcer control group by using one-way ANOVA followed by Tukey’s
test.
59
100
***
***
Mucus Estimation (g/g)
***
80
60
40
20
te
al
fa
cr
ep
r
Su
az
ol
e
in
ni
tid
Ra
Om
Ul
ce
rC
on
t
ro
l
e
0
Figure. 34. Effect of standard drugs Ranitidine, Omeprazole, Sucralfate on adherence of mucus to
gastric wall against Indomethacin induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the concentration of mucus on gastric wall calculated from 6 rats in
each group, where *** p<0.0001. The data was found to be statistically significant when compared to
ulcer control group by using one-way ANOVA followed by Tukey’s test.
60
80
Mucus Estimation (g/g)
***
***
60
***
40
20
kg
F.
C.
40
0m
g/
kg
F.
C.
30
0m
g/
kg
g/
20
0m
C.
F.
Ul
ce
rC
on
t
ro
l
0
Figure. 35. Effect of aq. extract Fagonia cretica (F.C.) on adherence of mucus to gastric wall in
Indomethacin induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the concentration of mucus on gastric wall calculated from 6 rats in
each group, where *** p<0.0001. The data was found to be statistically significant when compared to
ulcer control group by using one-way ANOVA followed by Tukey’s test.
61
3.9. Total Protein Content Estimation:
Total protein content was estimated with the help of bovine serum albumin standard curve, which is
shown in figure 36. The results for protein content were expressed as mean  SEM for each group where
n=6 in g/mg of the gastric tissue. Figure 37 shows the results of each group in the form of a bar graph
where they are statistically analyzed using one-way ANOVA. The figure reveals a significant increase in
all standard drug and treatment groups as that of ulcer control group. Protein content in ulcer control (
0.270.02 ) plummeted from normal group ( 0.840.023 ). In figure 38 there is a comparison between
ulcer control and ranitidine ( 0.680.022 ), omeprazole ( 0.700.027 ), sucralfate ( 0.660.026 ). This
comparison reveals a significant ( p<0.0001 ) increase from the ulcer control group. Figure 39 shows
significant increase in protein content of 200 mg/kg dose of Fagonia cretica ( 0.410.032 ), 300 mg/kg
dose of Fagonia cretica ( 0.50.032 ) and 400 mg/kg aq. extract of Fagonia cretica ( 0.540.018 ) in
comparison with control group. The difference between 300 mg/kg and 400 mg/kg dose of aq. extract of
Fagonia cretica was non-significant.
62
Figure. 36. BSA Standard curve for the Total Protein Concentration
Estimation.
63
Protein Estimation ( g/mg)
1.0
0.8
***
***
***
0.6
***
***
***
0.4
0.2
g/
kg
40
0m
g/
kg
C.
F.
F.
C.
30
0m
g/
kg
te
F.
C.
20
0m
al
fa
cr
Su
ep
r
Om
ni
tid
in
az
ol
e
e
ro
l
Ra
Ul
ce
rC
on
t
No
rm
al
0.0
Figure. 37. Effect of aq. extract Fagonia cretica (F.C.) on total protein content in Indomethacin
induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the total protein concentration calculated from 6 rats in each group,
where *** p<0.0001. The data was found to be statistically significant when compared to ulcer control
group by using one-way ANOVA followed by Tukey’s test.
64
0.8
***
Protein Estimation ( g/mg)
***
***
0.6
0.4
0.2
te
al
fa
cr
ep
r
Su
az
ol
e
in
ni
tid
Ra
Om
Ul
ce
rC
on
t
ro
l
e
0.0
Figure. 38. Effect of standard drugs Ranitidine, Omeprazole, Sucralfate on total protein content in
Indomethacin induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the total protein concentration calculated from 6 rats in each group,
where *** p<0.0001. The data was found to be statistically significant when compared to ulcer control
group by using one-way ANOVA followed by Tukey’s test.
65
0.6
***
Protein Estimation ( g/mg)
***
***
0.4
0.2
g/
kg
F.
C.
40
0m
g/
kg
F.
C.
30
0m
g/
kg
20
0m
C.
F.
Ul
ce
rC
on
t
ro
l
0.0
Figure. 39. Effect of aq. extract Fagonia cretica (F.C.) on total protein content in Indomethacin
induced gastric ulcer in rats.
Each bar represents mean  S.E.M. of the total protein concentration calculated from 6 rats in each group,
where *** p<0.0001. The data was found to be statistically significant when compared to ulcer control
group by using one-way ANOVA followed by Tukey’s test.
66
3.10. GSH Estimation:
Total glutathione in the gastric tissues of rats’ stomach for each group was calculated using the GSH
standard curve, which is presented in figure 40. The results are expressed as mean of 6 animals from each
group in figure 41. There was a significant ( p<0.0001 ) increase in the GSH content for all the test and
standard groups against control group. The result for normal group was ( 0.0870.002 ) and there was a
decline in the group given indomethacin to induce ulcers ( 0.0390.003 ). An increase in the GSH conc.
was observed with an increasing dose of 200 mg/kg ( 0.0450.002 ), 300 mg/kg ( 0.0520.002 ), 400
mg/kg aq. extract of Fagonia cretica ( 0.0550.001 ). Figure 42 shows a significant ( p<0.0001 ) increase
in the amount of GSH in ranitidine ( 0.0690.003 ), omeprazole ( 0.0720.002 ), sucralfate ( 0.0660.003
) as compared to ulcer control group.
67
Figure. 40. GSH Standard curve for the estimation of glutathione in all animal
groups.
68
GSH Estimation (mmol/mg)
0.10
0.08
***
***
***
0.06
***
***
**
0.04
0.02
kg
40
0m
g/
kg
g/
C.
F.
30
0m
g/
F.
C.
0m
F.
C.
20
cr
Su
kg
te
al
fa
az
ol
e
ep
r
Om
Ra
ni
tid
in
e
ro
l
rC
on
t
Ul
ce
No
r
m
al
0.00
Figure. 41. Effect of aq. extract Fagonia cretica (F.C.) on the concentrartion of GSH in
Indomethacin induced gastric ulcer in rats.
Each bar represents mean  S.E.M. for the estimation of glutathione calculated from 6 rats in each group,
where *** p<0.0001. The data was found to be statistically significant when compared to ulcer control
group by using one-way ANOVA followed by Tukey’s test.
69
0.08
***
***
GSH Estimation (mmol/mg)
***
0.06
0.04
0.02
te
al
fa
cr
Su
az
ol
e
ep
r
ni
tid
Ra
Om
Ul
ce
rC
on
t
ro
l
in
e
0.00
Figure. 42. Effect of Ranitidine, Omeprazole and Sucralfate on the concentrartion of GSH in
Indomethacin induced gastric ulcer in rats.
Each bar represents mean  S.E.M. for the estimation of glutathione calculated from 6 rats in each group,
where *** p<0.0001. The data was found to be statistically significant when compared to ulcer control
group by using one-way ANOVA followed by Tukey’s test.
70
***
0.06
GSH Estimation (mmol/mg)
***
***
0.04
0.02
kg
F.
C.
40
0m
g/
kg
F.
C.
30
0m
g/
kg
g/
0m
20
C.
F.
Ul
ce
rC
on
t
ro
l
0.00
Figure. 43. Effect of aq. extract Fagonia cretica (F.C.) on the concentrartion of GSH in
Indomethacin induced gastric ulcer in rats.
Each bar represents mean  S.E.M. for the estimation of glutathione calculated from 6 rats in each group,
where *** p<0.0001. The data was found to be statistically significant when compared to ulcer control
group by using one-way ANOVA followed by Tukey’s test.
71
Chapter 4
Discussion
72
4. Discussion:
Alternative Medicine is becoming famous and common these days. People want treatments
which have lesser adverse effects. Alternative medicine is a term used for treatments from
natural sources like plants. A huge variety of plants have been identified as medicinal plants and
their scope in future remains vast because there are millions of plants on earth. Phytochemical
and biochemical analysis are being performed on medicinal plants all around the world to
identify the constituents in different parts of plants responsible for relative medicinal use.
Various parts of plants and weeds are used as raw material for extraction of active ingredients,
which are then used to manufacture medicine. Medicinal plants may be used to support the ongoing drug therapy for serious diseases like cancer and as preventive medicine to avoid
appearance of a disease or adverse effect from synthetic treatments. Medicinal plants have an
important role in present day treatment of diseases, and they can help to synthesize more
effective drugs with fewer side effects (Hassan, 2012). A very common problem of the
alimentary canal these days affecting huge number of people of either sex and different age
groups is the Gastric Ulcer. Ulcers are perforations in the gastric mucosa. The mucosa normally
maintains a balance between damaging factors such as acid, pepsin and protective factors like
bicarbonates, mucus. During ulceration this balance is disrupted, and the mucosa is damaged
causing erosions. The factors which cause ulcers include H. Pylori infection, excessive secretion
73
of acid in the stomach, extensive use of NSAIDs including aspirin and indomethacin. The
mechanisms involved in NSAIDs induced ulcers are suggested to be over-production of TNF-α
(tumor necrosis factor) and inhibition of prostaglandins (Omran, Raheem, 2016). Ulcers cause
heartburn, pain, nausea and bleeding in severe cases.
In order to study the ulcers and anti-ulcer properties of drugs, rodents are used as study model.
In current study an investigation was made to evaluate the anti-ulcer property in aqueous extract
of Fagonia cretica in Indomethacin induced ulcers in rats. Fagonia cretica is a plant which
possesses multiple medicinal properties like analgesic, anti-diabetic, inhibition of cholesterol
synthesis, anti-bacterial, anti-malarial and anti-cancer, etc. Fagonia cretica tea is known to be
used for the treatment of breast cancer in Indo-Pak region. These properties in the plant are
because of the presence of glycosides, flavonoids, alkaloids and terpenoids (Wadood, 2013). The
rats were fasted for 24 hrs. and administered with 25 mg/kg body weight Indomethacin by oral
gavage. The animals were then euthanized after an hour and stomach was excised to check for
the ulcers. The group of animals given only indomethacin for ulcer induction were labeled as
ulcer control group. Three groups were takes as standard groups which were given Omeprazole,
Ranitidine and Sucralfate before the induction of ulcer. Similarly, three groups were taken as
treatment groups which were treated with 200 mg/kg aq. extract of Fagonia cretica, 300 mg
mg/kg and 400 mg/kg B.W. of aq. extract of Fagonia cretica. The results later revealed the
presence of ulcer curing property in Fagonia cretica. The number of lesions were reduced in the
74
animals treated with extract doses with maximum results at 400 mg/kg dose. The stomachs from
all the 48 animals were opened and evaluated for the degree of gastric damage comparing normal
group, ulcer control group, standard and treatment groups. Histological examination was also
carried out on all groups to check for necrosis, degeneration, edema and inflammation at
microscopic level. Ulcer index is calculated by measuring the total area of stomach and ulcerated
area and expressed as mm2. By taking difference between the ulcer index for control group and
treatment group, percentage inhibition was calculated for treatment and standard groups.
Percentage inhibition tells how much the drug was effective in preventing the gastric damage
from indomethacin. For 400 mg/kg aq. extract of Fagonia cretica the percentage inhibition was
63.9%.
Further tests included analysis of gastric fluid. Volume, pH and total acidity of gastric juice. In
ulcerated stomach, volume of fluid and total acidity increases, and pH decreases as compared to
normal stomach. For the animals treated with aqueous extract of fagonia cretica volume and
acidity of the gastric fluid decreased and pH increased, and the results were significantly close to
the normal group. Mucosal lining on the stomach wall in healthy stomach protects it against the
acid. In ulcerated stomach the amount of mucus adhered to the gastric wall decreases and thus
lesions and perforations are caused. When the test for mucus adherence was performed on
animals treated with Fagonia cretica, mucus adherence to gastric wall was found to have been
increased as compared to ulcer control group. The results were maximum for 400 mg/kg dose of
75
aqueous extract of Fagonia cretica. Similar were the results for total protein content were
estimated, the protein content was increased in the animals who were treated with Fagonia
cretica further confirming the anti-ulcer property of aqueous extract of the plant. Glutathione is
the biomarker for oxidative stress, it decreases in ulcer. Total glutathione content increased with
increasing the dose of fagonia cretica as treatment against ulcers. Maximum concentration was
found in the animals treated with 400 mg/kg extract dose. All these results proved that ulcer
curing properties are present in aqueous extract of Fagonia cretica and the study suggests further
research on the topic which can lead to a inventing a better and safe treatment for gastric ulcers.
76
Chapter 5
Conclusion
77
Conclusion:
The study was performed to investigate for the anti-ulcer activity present in the aqueous extract
of Fagonia cretica. The aqueous extract of Fagonia cretica protected the stomach against ulcers
induced by indomethacin by reducing the acid secretion, increasing the mucus content and
reducing ulcer index. The inhibition of ulcers was demonstrated by increase in protein content
and total glutathione and a reduction in acidity of gastric fluid. These initial findings prove the
presence of ulcer healing activity in Fagonia cretica. This property may mainly be due to the
presence of flavonoids and glycosides, these constituents possess strong anti-oxidant properties.
Further research is required to find the exact mechanism and to investigate for the particular
agent which contains this activity and also to develop a treatment protocol for curing ulcers using
Fagonia cretica.
78
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