Download Title of the Topic ANTI-INFLAMMATORY AND ANTI

ANTI-INFLAMMATORY AND ANTI-ULCER ACTIVITY OF FRUIT
OF PUNICA GRANATUM LINN.
M. PHARM DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH
SCIENCES, KARNATAKA, BANGALORE
BY
ARJUN REDDY
B.Pharm.
UNDER THE GUIDANCE OF
Mr.TUKARAM TADMALLE
M.Pharm., (Ph.D).
P. G. DEPARTMENT OF PHARMACOLOGY
RRK’S COLLEGE OF PHARMACY,
BIDAR-585402
2010-11
Rajiv Gandhi University of Health Sciences,
Karnataka, Bangalore.
Annexure – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
01
Name and Address of the
Candidate
ARJUN REDDY
S/o HANMANTH REDDY
H.NO: 2-6-55, RAMNAGAR,
SANGAREDDY, MEDAK (DIST),
502001, ANDHRA PRADESH.
R.R.K’S COLLEGE OF PHARMACY,
NAUBAD,BIDAR
KARNATAKA
02
Name of the Institution
03
Course of the Study Branch
M.Pharm (Pharmacology)
04
Date of Admission to course
22-11-2010
05
Title of the Topic
Brief resume of the intended work
6.1. Need for the Study
06
Enclosure – I
6.2. Review of the Literature
Enclosure – II
6.3. Objective of the Study
Enclosure – III
Materials and Methods
Enclosure – IV
7.1. Source of data
07
ANTI-INFLAMMATORY AND
ANTI-ULCER ACTIVITY OF FRUIT
OF PUNICA GRANATUM LINN.
7.2. Methods of collection of data
Enclosure – V
7.3. Does the study require any
Investigations on animals?
Enclosure – VI
If yes give details
7.4. Has ethical clearance been
obtained form your institution
in case of 7.3.
08
List of References
Yes, Registration No: 361/01C/CPCSEA
(Copy enclosed)
Enclosure – VII
09
Signature of the Candidate
(ARJUN REDDY)
10
Remarks of the Guide
The present research work is original and not
published in any of the journals with best of
my knowledge upon extensive literature
review. This work will be carried out in the
Pharmacology laboratory by the above said
student under my supervision.
Name and Designation of
(in Block Letters)
11.1. Guide
Mr. TUKARAM TADMALLE
M. Pharm., (Ph. D)
Asst. Professor
P.G. Department of Pharmacology,
RRK’S College of Pharmacy,
Naubad, Bidar
11.2.Signature
-----------------------------
11.
11.3.Co-Guide (if any)
11.4.Signature
11.5. Head of the Department
Mr. Rajshekar Bhande
Professor & H.O.D.
P.G. Department of Pharmacology,
RRK’S College Of Pharmacy,
Naubad, Bidar, Karnataka.
11.6.Signature
Remarks of the Principal
12
The present study is permitted to perform in
the Pharmacology laboratory of our
institution and the study protocol has been
approved by IAEC.
12.1. Signature
(Dr. K. Sreenivasa Rao)
M. Pharm., Ph.D.
Principal
RRK’S College of Pharmacy,
Naubad, Bidar
ENCLOSURE: I
06. Brief resume of the intended work
6.1. Need for the study
Cyclooxygenase (Cox) catalyzes the metabolism of arachidonic acid (AA) to intermediate
prostaglandins (PG) H2, Which is then converted to the various PGs and thromboxanes (TX) by cell specific
enzymes. There are two distinct isoforms of Cox, namely, COX-1 and COX-2. COX-1 is expressed in most
tissues but expression of COX-2 is low under basal conditions. Increased expression of COX-2 is found in
certain pathophysiological conditions such as inflammation, tissue damage and malignant transformation. From
these findings it was proposed that COX-1 is responsible for the maintenance of homeostasis reactions. In
particular, the formation of mucosa protective PGs in the gastrointestinal tract (GIT) was exclusively to the
COX-1 isoform. Induction of COX-2 results in increased production of mediators that are involved in
inflammation.
Standard nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the activity of both COX-1 and COX2. NSAIDs have potent analgesic, antipyretic and anti-inflammatory potency but in some cases cause GIT side
effects. Long term NSAID use is associated with gastric erosions and 2-5 fold increase in risk and 30% risk of
ulcer perforation, upper GIT bleeding and death. NSAIDs also damage the small and large bowel.
The selective COX-2 inhibitors have potent anti-infammatory and analgesic activities, but induce
considerably less gatro intestinal injury than standard NSAIDs in experimental animals and man.
The comparison of selectivity of various NSAIDs for the COX isoenzymes with the incidence of
gastro intestinal side effects in human in vivo experiments suggested that, the ulcerogenicity of the drugs
correlates with the selectivity for COX-1 but not COX-2. Both in experimental animals and humans COX-2
inhibitors did not cause measurable inhibition of gastric PG formation. This is in contrast to standard NSAIDs
that potently suppress gastric PGs. Taken together, there findings led to the concept that NSAID induced
gastrointestinal side effects are due to blockade of COX-1 and inhibition of COX-2 does not contribute to gastro
mucosal damage1.
On the contrary, 5 Lipoxygenase (LO) inhibitors – REV 5901 and l651, 392 and leukotriene (LT)antagonists FPL 55, 712, L7-171 883 significantly inhibit aspirin (ASP) and indomethacin-induced ulceration in
rats. Studies with MK886 (LT antagonist) revealed that the compound significantly decreased mucosal LTB4
generation following administration of ASP and indomethacin2.
Punica granatum L. fruit, popularly known as Anar, Pomegranate, Dadima, in India is commonly
used in treatment of various diseases like dysentery, chronic diarrhoea fevers, leucorrhea, lowering body heat,
gastric and asthamic fever, inflammation of urinary tract, hemorrhages, indigestion, inflammation of the colon
and the problems with digestive tract.
The seeds of the plant have been reported to posses antioxidant activity3. The peel of Punica granatum
L. plant have been reported for its antibacterial activity4.
How ever, there is no scientific data available on fruits for anti-inflammatory and anti-ulcer activity.
Hence, the present investigation has been undertaken to study the anti-inflammatory and anti-ulcer activity of
Punica Granatum L. (Fruit) in experimental animals.
ENCLOSURE: II
6.2. REVIEW OF LITERATURE5:
The plant Punica granatum Linn. Belonging to family Punicaceae. It is commonly called as Anar in
Hindi, Anarmitha in Urdu, Dalimba in Marathi, Dalim in Assamese and Bengali, Dalimbo in Oriya, Dadam in
Gujarati, and Dadimba in Telugu, Dalimba in Kannada and Marathi, Matalam in Malayalam, Madulam in Tamil.
Description6:
The plant Punica granatum Linn. (Family-Punicaceae) commonly known as Pomegranate, Anar,
Matalam, is a shrub, usually with multiple stems. The pomegranate is native to Asia, from the Middle East, to the
Himalayas, Where it grows in sandy or rocky scrublands. It has been widely cultivated throughout India and
drier parts of Southeast Asia, Malaya, the East Indies and tropical Africa. It is cultivated for its fruit and showy
flowers in much of the Mediterranean region and tropical America. The Pomegranate has escaped cultivation and
become established in parts of southern Europe and the American South West. ,
An attractive shrub or small tree, to 20 or 30 ft (6 or 10 m) high, the pomegranate is much-branched,
more or less spiny, and extremely long-lived, some specimens at Versailles known to have survived two
centuries. It has a strong tendency to sucker from the base. The leaves are evergreen or deciduous, opposite or in
whorls of 5 or 6, short-stemmed, oblong-lanceolate, 3/8 to 4 in (1-10 cm) long, leathery. Showy flowers are
home on the branch tips singly or as many as 5 in a cluster. They are 1 1/4 in (3 cm) wide and characterized by
the thick, tubular, red calyx having 5 to 8 fleshy, pointed sepals forming a vase from which emerge the 3 to 7
crinkled, red, white or variegated petals enclosing the numerous stamens.
Nearly round, but crowned at the base by the prominent calyx, the fruit, 2 1/2 to 5 in (6.25-12.5 cm) wide, has a
tough, leathery skin or rind, basically yellow more or less overlaid with light or deep pink or rich red. The
interior is separated by membranous walls and white spongy tissue (rag) into compartments packed with
transparent sacs filled with tart, flavorful, fleshy, juicy, red, pink or whitish pulp (technically the aril). In each
sac, there is one white or red, angular, soft or hard seed. The seeds represent about 52% of the weight of the
whole fruit.
Medicinal uses of different parts of Punica granatum Linn.5
Leaves
: Dysentry, Conjunctivitis
Fruits
: Strengthening and giving tone to the stomach, lowering
The body heat, gastric and asthamatic fever, Inflammation of
The urinary tract, hemorrhages
Peel
: Chronic diarrhea,
Bark
: For fevers, Leukorrhea, Malaria
Root
: Most effectively for Tape worm.
Flowers
: Stopping nose bleeds, Gum bleeds, toning skin and
Treating hemorrohids.
Seeds
: Congestion of liver, Indigestion, Inflammation of colon and
Problems with Digestive tract.
PHYTOCHEMICAL CONSTITUENTS
POMEGRANATE FRUIT PARTS AND CONSTITUENTS7
PLANT
COMPONENT
Pomegranate juice
Pomegranate seed oil
Pomegranate pericarp
(peel, rind)
Pomegranate leaves
Pomegranate flower
Pomegranate roots and
bark
CONSTITUENTS
Anthocyanins, glucose, ascorbic acid, ellagic acid, Gallic acid,
caffeic acid, catechin, EGCG, quercetin, rutin, numerous,
minerals, particularly iron, amino acids.
95-percent punicic acid, other constituents, including ellagic
acid, other fatty acids, sterols.
Phenolic punicalagins; Gallic acid and other fatty acids;
catechin, EGCG; quercetin, rutin, and other flavonols;
flavones, flavonones; anthocyanidins.
Tannins (punicalin and punicafolin); and flavone glycosides,
including luteolin and apigenin
Gallic acid, ursolic acid; triterpenoids, including maslinic and
aciatic acid; other unidentified constituents.
Ellagitannins, including punicalin and punicalagin, numerous
piperidine alkaloids.
CHEMICAL CONSTITUENTS
The literature reveals that Punica Granatum L. has been posses anti-microbial activity8, Wound healing activity9,
Molluscicidal activity10. How ever the literature reveals that there is no scientific data on anti-inflammatory and
anti-ulcer activity.
Hence purpose of safe compound with lesser side-effects to treat gastro intestinal disorders by using
herbal plants.
Hence Punica granatum L. is used as herbal drug in ayurveda traditional medicinal system, Hence
present study is purpose to investigate the Punica granatum L. fruit for anti-inflammatory and anti-ulcer activity.
Review of literature till date regarding Punica granatum L. was carried out by referring Chemical
abstracts, Biological abstracts, Medicinal and Aromatic plant abstracts and other scientific journals.
In view of this the present study is taken up to investigate the anti-inflammatory and anti-ulcer
activity of punica granatum L., So this study is essential and Justifiable.
ENCLOSURE: III
OBJECTIVES OF THE STUDY:
Despite the number of pharmacological studies on Punica granatum L. carried out, search of published articles
reveals that there is a need to investigate its anti-inflammatory and anti-ulcer activity.
The present study is, therefore aimed to evaluate the preliminary anti-inflammatory and antiulcer
properties of Punica granatum L.
In view of this, the fruit of Punica granatum L. have been selected for the present study.
.
ENCLOSURE: IV
7. Materials and methods
7.1 Source of data
Whole work is intended to explore the results from the pharmacology laboratory of our institution. Albino
rats and mice will be used for the screening purpose.
The experiments, which involves the following steps
1. Collection of the fruit of Punica granatum Linn. after the authentication from the botanist / pharmacognocist.
2. Extraction of Fruit of Punica granatum using Methanol.
3. Detection of presence of phytochemicals in the crude extract.
4. Evaluation of the crude extract of Punica granatum Linn. for anti-inflammatory and anti-ulcer activity in rats.
ENCLOSURE: V
7.2 Method of collection of data
A) Chemical studies
For this study, Fruits of Punica Granatum L. will be collected from the surrounding gardens of the Bidar. Fresh
Fruits will be cleaned, shade dried at room temperature and coarse powdered. Then the powdered material will
be extracted with Methanol by Soxhlet’s extraction method. Thereafter, the extract will be concentrated using
rotary flash evaporator and percentage yield of the same will be recorded. The crude extract obtained will be
subjected to preliminary phytochemical screening following the standard procedures described in the literature.
B) Determination of acute toxicity11
Fixed dose method (OECD guide line No. 420) of CPCSEA will be followed to carry out LD 50 of the extracts.
For this purpose albino mice of either sex (20-25gm) will be used
2) Evaluation of the extract for anti-inflammatory and antiulcer activities by different experimentally induced
and ulcer models. The experimental models (screening models) will be used in the study are outlined as below.
C) Anti-inflammatory activity12,13,14
a) Carrageenan induced Paw Oedema
b) Cotton-Pellet Granuloma
c) Formalin induced inflammation
D) Anti-ulcer activity15,16,17,18
a) Indomethacin induced gastric ulcer
b) Ethanol-induced ulcer
c) Pylorus ligation ulcer
F) Work plan details
Total duration for the completion of proposed research work may be ten months
1. Authentication and collection of plant material
- One month.
2. Duration of experimentation on animals including
preparation of crude extracts
- Five months.
3. Literature review
- Two months.
4. Dissertation writing and communication of research
- Two months.
papers.
ENCLOSURE-VI
7.3. Does the study require any investigation or interventions to be conducted on
patients or other humans and animals? if so please describe briefly.
The above study requires investigation on animals i.e albino mice for determination of anti-inflammatory
and anti-ulcer potential on albino rats and mice.
7.4. Has ethical clearance been obtained from your institution in case of 7.3
The present study protocol is approved from Institutional Animal Ethics
Committee (IAEC certificate is enclosed).
ENCLOSURE-VII
8. List of references:
01. Brigitta M, Peskar. Role of cyclooxygenase isoforms in gastric mucosal defence. Journal of physiology 2001;
95: 3-9.
02. Sen T, Abdul Salam CA, Siddhartha Pal, Suchandra Sen, Nag chaudhuri AK. Effect of dothiepin on gastric
ulceration mediated by lipid derived eicosanoids. Life Sciences 2000; 66(23): 325-30.
03. Yasoubi P, Barzegarm, Sahari MA, Azizi MH. Total phenolic contents and Antioxidant Activity of
Pomegranate (Punica granatum L.) Peel Extracts. J. Agric. Sci, Technol, 2007; 9: 35-42.
04. Pradeep BV, Manojbabu MK, Palaniswamy M. Antibacterial activity of Punica granatum L. against Gastro
Intestinal Tract Infection Causing Organisms. Ethanobaotanical Leaflets 2008; 12: 1085-89.
05. Dr. kurian JC. Plants that heal. Pune: Oriental Watchman publishing house: 1995; 231.
06. Julia F, Morton, Miami FL, Morton J. Fruits of warm climates. Pomegranate 1987; 352-355.
07. Julie Jurenka, MT (ASCP). Therapeutic Applications of Pomegranate (Punica granatum L.). Alternative
Medicine Review, 2008; 13: 2.
08. Ahmet D. Duman, Mustafa Ozgen, Kenan S. Dayisoyulu, Nurcan Erbil, Coskun Durgac. Antimicrobial
activity of Six Pomegranate (Punica granatum L.) varieties and Their Relation to Some of Their Pomological
and Phytonutrient Characteristics. Molecules 2009; 14: 1808-1817.
09. Murthy KN, Reddy VK, Veigas JM, Murthy UD. Study on Wound healing activity of Punica granatum Peel.
J Med Food, 2004; 7(2): 256-9.
10. Tripathi SM, Singh DK. Molluscicidal activity of Punica granatum bark and canna indica root. Brazilian
Journal of Medical and Biological research 2000; 33: 1351- 1355.
11.Mrs. Prema veeraraghavan. Expert consultant, CPCSEA, OECD guideline No.420.
12. Winter CA, Risley EA, Nuss GW. Carrageenin-induced Oedema in hind paw of the rat as an assay for
antiinflammatory drugs. Proc. Soc. Exp. Biol. Med 1962; 111: 544-47.
13. Shanahan RW. Local activity of anti-inflammatory and irritant agents on rat paw oedema induced by
Carrageenin. Arch. Int. Pharmacodyn 1968; 175:186-92.
14. Winter CA, Portal CC. Effect of alterations in side chain upon anti-inflammatory and liver glycogen
activities of hydrocortisone esters. J. Amer pharm. Assoc 1957; 46: 515-19.
15. Kulkarni SK. Handbook of experimental pharmacology. 3rd ed. New Delhi: Vallabha Prakashan; 1999:
128-31.
16. Somogi A, Kovacs K, Selye H. Jejunal ulcer produced by indomethacin, Journal of pharmacy and
Pharmacology 1969; 21: 122-23.
17. Robert A, Nezamis JE, Lancaster C, Haucher AJ. Cytoprotection by Prostaglandins in rats. Prevention of
gastric necrosis produced by alcohol, Hcl, NaoH, hypertonic Nacl and thermal injury. Gastroenterology
1979; 77: 433-43.
18. Shay M, Komanov SA, Flez D. A simple method for the uniform production of the gastric ulceration in the
rat. Gastroenterology 1945; 5: 43-1.