EVALUATION OF ANTI-INFLAMMATORY AND ANTI-ULCER PROPERTIES OF ROSA CENTIFOLIA Linn. FLOWERS IN EXPERIMENTAL RATS.
M.Pharm. Dissertation Protocol
Submitted to the
RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA.
By
SHAH CHANDRAGOPAL SURESHCHANDRA.
B. Pharm.
Under the Guidance Of
Mr. SHIV KUMAR
Assistant Professor,
Department of Pharmacology,
NETPharmacyCollege, Raichur
DEPARTMENT OF PHARMACOLOGY
N.E.T.PHARMACYCOLLEGE,
RAICHUR – 584103
2009-2010.
RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA.
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1. / Name of the candidateAnd Address:
/ SHAH CHANDRAGOPAL SURESHCHANDRA,S/O. Shah Sureshchandra S.
Nr. Modh Ni Wadi,
Talav Road,
Godhra-389001
Panchmahal(PMS)
Gujarat.
2. / Name of the Institution: / N.E.T.PharmacyCollege,
Raichur- 584103
3. / Course of study and Subject: / Master of Pharmacy in Pharmacology.
4. / Date of admission to the Course: / 10-11-2009
5. / Title of the topic:
Evaluation of Anti-inflammatory and Anti-ulcer properties of Rosa centifolia Linn. flowers in experimental rats
6 / Brief resume of the intended work:
6.1:Need for the study: ENCLOSURE -I
6.2:Review of the literature: ENCLOSURE -II
6.3:Main objective of the study: ENCLOSURE –III
7 / Material and methods:
7.1: Source of the data: ENCLOSURE -IV
7.2: Methods of the collection of the data: ENCLOSURE - V
7.3: Does the study require any investigations or interventions to be conducted on
Patients other human or animals? If so, please describe briefly.
YES (Wistar Rats and Mice)
7.4: Has ethical clearance been obtained from your institute in case of 7.3?
YES: IAEC NO. : 576/2002/bc/IAEC/CPCSEA
8 / List of References: ENCLOSURE-VI
9. / Signature of the candidate: / (SHAH CHANDRAGOPAL S.)
10. / Remarks of the guide: / The proposed research work can be carried out in our institute.
11. / Name and designation of
11.1 Guide:
11.2Signature
11.3 Co-guide (if any):
11.4 Signature
11.5 Head of the department:
11.6Signature / Mr. SHIV KUMAR
Assistant Professor,
Department of Pharmacology,
NETPharmacyCollege, Raichur
------
------
Dr. BHEEMACHARI
Professor & HOD
Department of Pharmacology,
N.E.T.PharmacyCollege,
Raichur.
12. / 12.1 Remarks of the Chairman
and Principal
12.2 Signature / The work proposed in this synopsis can be carried out at our institute
Dr. H. DODDAYYA
PRINCIPAL,
N.E.TPHARMACYCOLLEGE,
RAICHUR-584103.
ENCLOSURE –I
6. BRIEF RESUME OF THE INTENDED WORK:
6.1: Need for the study:
Inflammation (Latin, inflammare, to set on fire) is the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. The causes of inflammation are burns, chemical irritants, frost bite, toxins, infection by pathogens and hypersensitivity reactions.1
For the management as well as treatment of various inflammatory disorders, more frequently Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), Ibuprofen, Diclofenac are used. Akin to any other NSAIDs, these drugs act by inhibiting the synthesis of prostaglandins.2
Long-term use of NSAIDs can cause gastric erosions, which can become stomach ulcers and in extreme cases can cause severe haemorrhage resulting in death. The risk of death as a result of use of NSAIDs is 1 in 12,000 for young adults aged 16–45. The risk increases almost twenty-fold for those over 75. Over use of acetaminophen (paracetamol) causes liver damage and is the most common cause of liver failure in the United States, according to a 2009 report from the federal Food and Drug Administration.3
A peptic ulcer, also known as ulcus pepticum, PUD or peptic ulcerdisease, is an ulcer (defined as mucosal erosions equal to or greater than 0.5cm) of an area of the gastrointestinal tract that is usually acidic and thus extremely painful. As many as 80% of ulcers are associated with helicobacter pylori, a spiral-shaped bacterium that lives in the acidic environment of the stomach. Contrary to general belief, more peptic ulcers arise in the duodenum (first part of the small intestine, just after the stomach) rather than in the stomach. About 4% of stomach ulcers are caused by a malignant tumor. The causes of ulcers are stress, smoking, diet, helicobacter pylori bacterium, excess secretion of HCl, genetic predisposition, use of anti-inflammatory medication such as aspirin, indomethacin.4
In Western countries the prevalence of Helicobacter pylori infections roughly matches age (i.e., 20% at age 20, 30% at age 30, 80% at age 80 etc). Prevalence is higher in third world countries. Transmission is by food, contaminated groundwater, and through human saliva (such as from kissing or sharing food utensils.)4
Though there are available different anti-ulcer agents like proton pump inhibitors, gastro protective agents, antacids, H2 receptor antagonists etc, they have their own serious side effects on chronic use; hence it may lead to discontinuation of treatment and reoccurrence of ulcer.5
The upper gastrointestinal toxicity is one of the most common side effects associated with the use of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs). Many attempts to prepare potent NSAIDs free from gastrotoxicity have failed. The treatment with these drugs is known to produce gastric mucosal injury comprising a spectrum of lesions, which are known as “NSAID gastropathy". Recent studies have suggested that the use as well as the prevalence of NSAID induced lesions in stomach is increasing at a faster rate. The incidence of NSAID gastropathy is especially higher in older patients. It is generally accepted that aspirin and indomethacin are most likely representatives of the NSAID group to cause upper gastrointestinal ulceration and bleeding . It has been reported that besides gastric lesions, indomethacin also produce ileum and jejunum ulcers and this drug is almost equally toxic to the stomach whether given parenterally or orally.6
Hence, there is an increasing demand for the alternative therapies for anti-inflammatory drugs with gastro protective effects, particularly herbal therapies that are believed to be potent, safe and economical.
Rosa centifolia Linn. [Family: Rosaceae] is one such plant that is commonly found through out India. It is extensively used as traditional medicine in Uttar Pradesh and Bihar. A decoction of flowers of rose is prescribed for inflammation of the mouth and pharynx, and ulcers of the intestine. Powder of rose buttons and seeds is used as astringent in haemorrhage and diarrheoa.7 The roots of the plant are used for astringent and useful in intestinal ulcers, rickets, haemorrahages, and diarrhoea.The leaves are useful in treating wounds, ophthalmia, hepatopathy and haemorrhoids.8 However, the anti-inflammatory and anti-ulcer property of the Rosa centifolia flowers is not scientifically documented. In this context, an attempt has been proposed to evaluate the anti-inflammatory and anti-ulcer properties of Rosa Centifolia Linn. flowersin animal models of inflammation and ulcers.
ENCLOSURE –II
6.2: Review of Literature
Rosa centifolia Linn. [Family: Rosaceae] is one of the plant species that is extensively used in traditional medicine in Uttar Pradesh and Bihar. It is found in common throughout India.7
Prickles unequal, large hooked; bristles numerous. Leaflets usually 5, pubescent on both sides or only beneath. Rhachis not prickly. Flowers usually pink, very double, on long and slender pedicels, nodding, fragment; petals inflexed; sepals persistent.9
There are two kinds: Red and white –The white flower is acrid with flavour; cooling laxative, aphrodiasic; cures “tridosha”, biliousness, leprosy,”kapha”.The red flower cures diseases of blood and scorpion-sting according to ayurveda.9
The flowers and leaves contain 1.3 and 8.5 % of saponin respectively. Petals are reported to contain methionine sulphoxide. Cabbage rose yields a volatile oil (0.2%) consisting mainly of citronellol, geraniol, nerol, phenylethanol, linalool and citral. It contains 15% tannins (oligomericproanthocyanidins).7
Actions of flowers of rose are- a decoction is prescribed for inflammation of the mouth and pharynx, and ulcers of the intestine. Powder of rose buttons and seeds—astringent in haemorrhage and diarrheoa.7
The other parts of the plant such as roots are used for astringent and vulnerary disease and useful in intestinal ulcers, rickets, haemorrahages, and diarrhoea. The leaves are useful in treating wounds, ophthalmia, hepatopathy and haemorrhoids.8
Several rose products are used to make creams, lotions, and other cosmetic uses. It is also used in potpourri as a pleasant cent. It is mixed with vegetable gylcerine for moisturizing use. It is also used in toilet preparations, lozenges and toothpaste. Rose water is used in desserts, pastries and cakes. Gulkand made from petals possesses mild laxative properties and is useful in sore throat and enlarged tonsils.8
ENCLOSURE -III
6.3 Main objective of study:
The central objective of the study is to evaluate the anti-inflammatory and anti-ulcer properties of the flower extract of Rosa centifolia Linn. in validated animal models of inflammation and ulcers.
The whole study is divided into two phases for the sake of convenience
Phase I:-
Preparation of various solvent extracts of flowers of Rosa centifolia Linn.using Soxhlet apparatus.
To carry out preliminary phytochemical investigations of the extracts obtained.
Determination of LD 50 and selection of experimental dose for the study.
Phase II:-
1. To evaluate anti-inflammatory activity of the extracts in various experimental
animals models like
Carrageenan induced paw oedema in rats.
Adjuvant induced arthritis like inflammation in rats.
2. To evaluate anti-ulcer activity of the extracts in various experimental animal
models like
- Pylorus ligation model
- Indomethacin induced gastric lesions.
- Ethanol induced mucosal damage in rats.
- Cold restraint stress induced ulcers.
ENCLOSURE – IV
7. Materials and Methods:
7.1: Source of the data:
Whole work is planned to generate data from laboratory based studies as described in objectives of the study and experimental studies in journals and text books available with college and other institutions, National/International journals (IJP, IJPP, Ethanopharmacol, Indian J Exp Biol.) and through e-publishing and Helinet consortium of RGUHS, Bangalore.
ENCLOSURE – V
7.2: Methods of collection of the data (including sampling procedure if any):
Data will be generated from experimental animal studies and results are to be subjected for statistical analysis by ANOVA followed by Dunnett’s ‘t’ test and p values less than 0.05 will be considered as statistically significant.
PHASE-1:
- Preparation of various solvent extracts of Rosa Centifolia Linn flowers: -
Flowers of Rosa centifolia Linn. will be charged in a thimble of soxhlet apparatus and extracted with water, ethanol and petroleum ether. Each time before extracting with the next solvent, powder will be dried in air-oven below 500C. Each extract will be concentrated by distilling off the solvent and then evaporating to dryness on the water bath.
- Preliminary phytochemical Screening:10
The preliminary phytochemical investigations will be carried out with the flowers extract of Rosa Centifolia Linn. forqualitative identification of phytoconstituents.
3. Experimental animals:
The healthy albino wistar rats and mice of either sex will be obtained from the central animal house of N.E.T Pharmacy college, Raichur and kept in 12:12 hr. light and dark cycle, and will be used throughout the experimental study. Before initiation of experiment, animals will be acclimatized to laboratory conditions for one week. During this period, laboratory pellet chow and water ad libitum will be provided to animals under hygienic conditions.
4. Determination of LD50 of flower extracts of Rosa centifolia Linn: 11
The acute oral toxicity of Rosa centifolia Linn. extractswill be determined by using albino mice of either sex (20-25 g), maintained under standard conditions. The animals will be fasted for 3 h prior to the experiment. Animals will be administered with single dose of flower extract ofRosa Centifolia Linn. and observed for its mortality up to 48 h study period (short term toxicity). Based on the short-term toxicity profile, the next dose will be determined as per OECD guidelines No 425. From the LD50 dose, the experimental dose will be calculated.
PHASE-II
7.2.1: DETERMINATION OF ANTI-INFLAMMATORY ACTIVITY
1) CARRAGEENAN INDUCED PAW OEDEMA IN RATS.12,13
Male albino rats weighing between 120-150 g will be divided into 6 groups of 6 animals each
Group 1: Normal control (water) p.o.
Group 2: Positive control (Carrageenan)
Group 3: Carrageenan + Standard (Indomethacin 10mg/kg) p.o.
Group 4: Carrageenen + Aqueous extract of Rosa centifolia Linn. p.o.
Group 5: Carrageenan + Ethanol extract of Rosa centifolia Linn. p.o.
Group 6: Carrageenan + Petroleum ether extract of Rosa centifolia Linn. p.o.
Acute inflammation will be produced by injecting 0.1 ml of 1% carrageenan into subplantar region of rats hind paw. The test extracts, standard drug and control vehicle will be administered 60 min before carrageenan injection. The paw volume will be measured at 0, 0.5, 1, 2, 3, 4 and 5 h interval using plethysmograph apparatus.
2) ADJUVANT INDUCED ARTHRITIS LIKE INFLAMATION IN RATS.14
Male albino rats weighing between 150-200 g will be divided into 6 groups of 6 animals each.
Group 1: Negative control (water) p.o.
Group 2: Positive control (Freunds complete adjuvant)
Group 3: Freunds complete adjuvant + Standard (Indomethacin 10mg/kg) p.o.
Group 4: Freunds complete adjuvant + Aqueous extract of Rosa centifolia Linn. p.o.
Group 5: Freunds complete adjuvant + Ethanol extract of Rosa centifolia Linn. p.o.
Group 6: Freunds complete adjuvant + Petroleum ether extract of Rosa centifolia Linn. p.o.
Rats will be injected with Freunds complete adjuvant containing 10 mg of heat killed mycobacterium tuberculosis in 1 ml paraffin oil (0.1 ml) into the left paw intradermally.14 Group of animals will be treated with extracts of Rosa centifolia Linn, distilled water and standard drug respectively everyday for 14 days from day of injection of Freunds complete adjuvant and the following parameters will be recorded
1. Body Weight:15
Body weight for each group of rats will be recorded for alternative days during the period of arthritis. The difference between mean body weights in each group will be calculated to determine the change in body weight between the first day and fourteenth day.
2. Body Temperature:15
Body temperature, as an index of inflammation will be monitored for rats from day 0 to day 14 using rectal thermometer on every alternative days and compared with that of day 0.
- Arthritis Index and hind paw volume:15
The paw volume will be measured on alternative days from day 0 to day 14 using plethysmograph apparatus.
Arthritis Index will be calculated by the following equation:
AI = (Hind paw volume on day x)-(Hind paw volume on day 0)
(Hind paw volume on day 0)
- Ankle diameter:15
Changes in the ankle diameter of both injected and non injected paws from day 0 to day 14 on alternative days will be assessed using a vernier scale.
- Serum bio-chemical examination:16
On 14th day, Rats will be anaesthetized with anaesthetic ether and blood samples will be collected from the retro-orbital route without any anti-coagulant. After one hour serum will be separated by centrifugation and maintained at –4oC until further use. The serum will be subjected for determination of Blood Urea Nitrogen (BUN), Total protein, Albumin, Alanine amino transferases (ALT), Aspartate amino transferases (AST) and Alkaline Phosphatase (ALP) by using semi auto-analyser.
- Haematological examination:17
On 14th day, Rats will be anaesthetized with anaesthetic ether and blood samples will be collected by retro-orbital route using EDTA as anticoagulant and subjected to estimation of RBC, WBC (Total and Differential count) and mean hemoglobin concentration.
7.2.2: DETERMINATION OF ANTI-ULCER ACTIVITY
1. PYLORIC LIGATION MODEL.18
Albino rats of either sex weighing between 150 to 200 g were divided into five different groups as shown below. Each group will be consisting of 6 animals.
Group 1: Pyloric ligation control (10 ml/kg of 2% w/v of gum acacia/tween 20) p.o.
Group 2: Standard drug (Lansoprazole 8mg/kg) p.o.
Group 3: Aqueous extract of Rosa centifolia Linn. p.o.
Group 4: Ethanol extract of Rosa centifolia Linn. p.o.
Group 5: Petroleum ether extract of Rosa centifolia Linn. p.o.
In this method, the rats will be fasted in individual cages for 24 h. Care will be taken to avoid Coprophagy. The vehicle, standard drug (Lansoprazole) and different extracts of Rosa centifolia Linn. will be administered by oral route. After 30 minutes, pyloric ligation will be carried out under light ether anesthesia, (the abdomen will be cut opened and the pylorus will be ligated by black thread) the abdomen will be then sutured. After 4h of pyloric ligation, the animals will be sacrificed with excess of anesthetic ether, and the stomach will be dissected out to determine ulcer index.18 Gastric juice will be collected and its volume, pH, free and total acidity will be measured.
A) Evaluation of ulcer index: 19
The glandular portion of the stomach will be opened along the greater curvature, and the severity of haemorrhagic erosions in the acid secreting mucosa will be assessed on a scale of 0 to 3.
Mean ulcer score for each animal is expressed as Ulcer Index.
Ulcer Index = UN + US + UP +100 X 10-1
Where, UN = Average number of ulcer per animal.
US = Severity of ulcer
UP = Percentage of animals with ulcers
The percentage protection will be calculated using the formula –
Where Ut = Ulcer index of treated group
Uc = Ulcer index of the control group
Ulcer scores19
Sr. No. / Stomach colours / Ulcer score1 / Normal colour / 0
2 / Red colour / 0.5
3 / Red spots / 1
4 / Hemorrhagic streaks / 1.5
5 / 3 - 5 ulcers / 2
6 / 5 ulcers / 3
Determination of free acidity and total acidity: 19
1ml of gastric juice will be pipetted out into a 100ml conical flask and 2-3 drops of Topfer’s reagent will be added, titrated with 0.01 N sodium hydroxide until all traces of red color disappeared and the color of the solution turns to yellowish orange. The volume corresponds to free acidity. Then 2-3 drops of phenolpthalein solution will be added and titration will be continued until a definite red tinge reappears. Again the total volume of alkali added will be noted. This volume corresponds to total acidity.
Acidity will be calculated by using the formula:
Acidity = Volume of NaOH x Normality of NaOH ×100 mEq/L/100 g
0.1
2. Indomethacin induced gastric ulcers.20,21
Albino rats of either sex weighing between 150-200 g will be divided into 6 groups of six rats each.
Group 1: Normal control (10 ml/kg of 2% w/v of gum acacia/tween 20) p.o.
Group 2: Positive control (Indomethacin 30 mg/kg in 1 % acacia suspension) p.o.
Group 3: Indomethacin + Standard drug (Lansoprazole 8mg/kg) p.o.
Group 4: Indomethacin + Aqueous extract of Rosa centifolia Linn. p.o.
Group 5: Indomethacin + Ethanol extract of Rosa centifolia Linn. p.o.