Rajiv Gandhi University of Health Sciences s145

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE.

ANNEXURE II

SYNOPSIS FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1 NAME OF THE CANDIDATE DR. RASHMI R RAO POSTGRADUATE STUDENT AND ADDRESS DEPT OF PHARMACOLOGY A.J.INSTITUTE OF MEDICAL SCIENCES MANGALORE- 575004

A.J.INSTITUTE OF MEDICAL SCIENCES 2 NAME OF THE INSTITUTION MANGALORE- 575004

3 COURSE OF STUDY AND MD COURSE IN PHARMACOLOGY

SUBJECT

4 DATE OF ADMISSION TO 10TH MAY 2010

COURSE

5 TITLE OF THE TOPIC “A COMPARATIVE STUDY OF ANALGESIC

ACTIVITY OF AQUEOUS EXTRACT OF

ANDROGRAPHIS PANICULATA AND

DICLOFENAC SODIUM IN MICE” 6 BRIEF RESUME OF INTENDED WORK: 6.1. NEED FOR THE STUDY: Pain is defined by the International association for the study of pain (IASP) as, “An unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage.”1 Pain is a disabling accompaniment of many medical conditions. Pain control is one of the most important therapeutic priorities. Analgesics are drugs that selectively relieve pain by acting on the central nervous system or peripheral pain mechanism without altering consciousness. Opiod and non opioid analgesics (NSAIDs) are the most commonly employed agents for symptomatic relief of pain. Diclofenac is a phenyl acetic acid derivative – a drug belonging to the Non steroidal anti- inflammatory group. It is relatively nonselective as a COX inhibitor. 2 It is the most commonly used NSAID. It is approved for long term treatment of Rheumatoid arthritis, Osteoarthritis and Ankylosing spondylytis. Diclofenac has analgesic, antipyretic and anti-inflammatory activities. Its potency against COX 2 is substantially greater than that of Indomethacin, Naproxen or several other NSAIDs.3 NSAIDs provide effective management of pain and inflammation, but are associated with the formation of peptic ulcers and an increased risk of peptic ulcer haemorrhage and perforation. Andrographis paniculata (AP) referred to as “Kalmegh” is a shrub that has been traditionally used over centuries for different medicinal purposes. A study done on the active principle of Andrographis paniculata -Andrographolide has shown to have analgesic property.4 Moreover, very scanty studies are reported worldwide on its analgesic activity. There is a need for better analgesic which could effectively relieve pain without causing much adverse effects. Apart from this, the plant is cost effective, easily available and has insignificant side effects. Hence, I have chosen to compare the analgesic activity of Andrographis paniculata with that of Diclofenac. 6.2. REVIEW OF LITERATURE:

Andrographis paniculata, also known commonly as “King of Bitters” is a member of the plant family Acanthaceae. It grows extensively in Southeast Asia , i.e. India, Srilanka, Pakistan, Java, Malaysia and Indonesia, it is cultivated extensively in India, China and Thailand.5 It is an annual herb extremely bitter in every part of the plant.5 Previous investigations on the chemical composition of Andrographis paniculata showed that it is a rich source of diterpenoids and 2’- oxygenated flavanoids. 5 The aerial parts of the plant (leaves and stem) are used to extract the active phytochemicals. The primary bioactive component of the plant Andrographis paniculata is Andrographolide. 5 It is a colourless crystalline bicyclic diterpenoid lactone. It is present in all the parts of the plant. 5 Andrographis paniculata has anti-inflammatory, antidiarrhoeal, antiviral, antimalarial, hepatoprotective, cardiovascular, anticancer, immunostimulant property. According to previous studies Andrographolide, the active constituent of Andrographis paniculata showed significant analgesic activity in acetic acid induced writhing in mice and Randall selitto’s test in rats. But, analgesic activity was weaker than aspirin.4 Another study done on ethanol extract of Andrographis paniculata showed that it has analgesic activity and potentiated the analgesic effect of Aspirin.6 Andrographolide is reported to inhibit NF-kB binding to DNA, thus reducing the expression of pro-inflammatory proteins such as cyclooxgenase-2 ( COX- 2).7 Diclofenac, other than Rheumatoid arthritis, Osteoarthritis and Ankylosing spondylitis is also useful for short term treatment of musculoskeletal pain, post operative pain and dysmenorrhoea. Usual daily dose is 100- 200 mg. A 0.1% of ophthalmic preparation is recommended for prevention of post operative ophthalmic inflammation. A topical gel containing 3% Diclofenac is effective for Solar keratosis.2 Diclofenac produces gastrointestinal side effects in 20% of patients. Moderate elevation of hepatic transaminase in plasma occurs in 5-15% of patients. Therefore transaminase should be measured during the first 8 weeks of therapy of diclofenac and the drug discontinued if abnormal values persist.4 Rarely CNS effects, rashes, allergic reactions, fluid retention, edema, impairment of renal function may be seen.3 There is no conclusive data regarding effects of Andrographis paniculata on pain upon chronic administration. Hence in the following study, the hot plate and acetic acid induced writhing tests will be employed to evaluate the analgesic activity of aqueous extract of Andrographis paniculata with that of Diclofenac sodium in mice.

6.3. AIMS & OBJECTIVES OF STUDY

1. To evaluate the acute and chronic effects of aqueous extract (Aq) of Andrographis paniculata

on thermal and chemical induced pain in mice.

2. To compare the analgesic effects of aqueous extract of Andrographis paniculata with that of

Diclofenac sodium in mice.

7 MATERIALS AND METHODS: 7.1. SOURCE OF DATA: Type of study: Animal study Site of study : A J Institute of Medical Science, Mangalore Plant material: Aqueous extract of Andrographis paniculata Preparation of extract: Aqueous extract of Andrographis paniculata will be obtained by maceration Standard Drug : Diclofenac sodium Instrument used: Eddy’s Hot Plate Drug used to induce pain & inflammation: Acetic Acid Animals: Inbred Albino ice (Swiss strain) weighing between 25-30gms of either sex will be used for the study. They will be housed in clean cages and maintained at room temperature between 27- 31° C with standard laboratory feed and water ad libitum. .

7.2. METHOD OF COLLECTION OF DATA:

Animals will be divided into 2 sets each consisting of 4 groups with 6 animals in each group. Group 1 will serve as control and receive normal saline. Group 2 will receive the drug

Diclofenac sodium(100mg).8 Group 3 will receive aqueous extract of Andrographis paniculata(50mg). Group 4 will receive aqueous extract of Andrographis paniculata(100mg).9 The dose is calculated according to the previous studies.

In acute study, animals will receive the drug or vehicle orally, 1 hour prior to placing the animal over the hot plate or before injecting 0.6% acetic acid. While in chronic study, each animal will receive the above treatment once daily for 10 days. On the 10th day, drugs/ vehicle will be administered 1 hour before placing the animal over the hot plate or injecting 0.6% acetic acid.

Procedure:

1. Thermal method of pain induction

Hot Plate Method (Eddy’s hot plate): The method originally described by Woolfe and

Mac Donald as modified by Eddy and Leimbach has been used frequently. The hot pate consists of an electrically heated surface. The temperature is controlled at 55°- 56° C. The animals are placed on the hot plate and the time until either paw licking or jumping occurs is recorded by a stopwatch. The latency is recorded before and after 20, 60 and 90 minutes following oral administration of the standard or the test compound. A cut off time of 30 seconds is followed to avoid any thermal injury to the paws.10

2. Chemical method of pain induction: Acetic acid induced writhing: Pain is induced by injection of acetic acid (0.1ml of 0.6% solution) into peritoneal cavity of mice. Test animals are administered the test compound and the standard 1 hour before acetic acid injection. The mice are placed individually into glass beakers and five minutes are allowed to elapse. The mice are then observed for a period of ten minutes and the number of writhes is recorded for each animal. A writhe is indicated by stretching of the abdomen with simultaneous stretching of at least one limb. The formula for computing percent inhibition is: average writhes in the control group minus writhes in the drug group divided by writhes in the control group times 100%.10

Statistical Analysis:

All data will be analysed using one way ANOVA.

HOT PLATE METHOD- ACUTE AND CHRONIC STUDY

Groups Treatments Acute Chronic Reaction time in seconds ( n= 6) ( Dose/ kg) Study Study 20 min 60 min 90 min I Normal saline Single Once a day Dose For ten days

II Diclofenac Single Once a day Sodium Dose For ten days ( 100mg/kg)

III Aq extract of Single Once a day AP Dose For ten days (50mg/kg)

IV Aq extract of Single Once a day AP Dose For ten days (100mg/kg) ACETIC ACID INDUCED WRITHING- ACUTE AND CHRONIC STUDY

Groups Treatments Acute Chronic Time of No. of writhes in ( n= 6) ( Dose/ kg) Study Study onset of 15 Writhing Minutes ( in min) Period ( in min) I Normal saline Single Once a day Dose For ten days

II Diclofenac Single Once a day Sodium Dose For ten days ( 100mg/kg) III Aq extract of AP Single Once a day (50mg/kg) Dose For ten days

IV Aq extract of AP Single Once a day (100mg/kg) Dose For ten days

7.3 Does the study require any investigation or interventions to be conducted on patients

or other human or animals? If so, please describe briefly.

7.4 Has the ethics clearance obtained from your institution?

8 LIST OF REFERENCES:

1) Pain & Temperature. In: Barrett KE, Barman SM, Boitano S, Brooks HL, editors. Ganong’s Review of Medical Physiology. TATA : McGraw–Hill, 2010:168. 2) Furst DE, Ulrich RW, Varkey-Altamirano C. Nonsteroidal Anti-Inflammatory Drugs, Disease-Modifying Antirheumatic Drugs, Nonopioid Analgesics, & Drugs Used in Gout. In: Katzung BG, Masters SB, Trevor AJ, editors. Basic and clinical Pharmacology. TATA : McGraw–Hill, 2009:626. 3) Burke A, Smyth E, FitzGerald GA. Analgesic-Antipyretic Agents; Pharmacotherapy Of Gout. In: Brunton LL, Lazo JS, Parker KL, editors. Goodman & Gilman’s – The Pharmacological Basis of Therapeutics. New York : McGraw-Hill, 2006:698. 4) Madav S, Tripathi HC, Tandan, Mishra SK. Analgesic, antipyretic and antiulcerogenic effects of andrographolide. Indian Journal of Pharmaceutical Sciences.1995;57(3):121-125. 5) Jarukamjorn K, Nemoto N. Pharmacological Aspects of Andrographis paniculata on Health and Its Major Diterpenoid Constituent Andrographolide. Journal of Health Science. 2008;54(4):370-381. 6) Shruthi, Sekhar S S, Shetty N, Gopalakrishna H N, Pai MRSW. A preliminary study on the analgesic activity of ethanolic extract of Andrographis paniculata. Indian Journal of Physiology and Pharmacology. 2008;52(5): 152 7) Levita J, Nawawi A, Mutalib A, Ibrahim S. Andrographolide: A review of its anti- inflammatory activity via inhibition of NF-kappaB activation from computational chemistry aspects. International Journal of Pharmacology. 2010;6:569-576. 8) Ojewole JAO. Analgesic, antiinflammatory and hypoglycaemic effects of ethanol extract of Zingiber officinale (roscoe) rhizomes (zingiberaceae) in mice and rats. Phytotherapy research. 2006;20(9):764-772 9) Lin FL, Wu SJ, Lee SC, Ng LT. Antioxidant, antioedema and analgesic activities of Andrographis paniculata extracts and their active constituent andrographolide. Phytotherapy research. 2009;23(7):958-964. 10) Analgesic, anti-inflammatory, and anti-pyretic activity. In: Vogel HG, Vogel WH, Scholkens BA, Sandow J, Muller G, Vogel WF, editors. Drug Discovery and Evaluation– Pharmacological Assays. New York : Springer-Verlag. 2002:696–716.

9 SIGNATURE OF CANDIDATE:

10 REMARKS OF THE GUIDE:

11 NAME AND DESIGNATION OF:

11.1. GUIDE DR MOHANDAS RAI

PROFESSOR & HOD DEPT OF PHARMACOLOGY A.J.INSTITUTE OF MEDICAL SCIENCES MANGALORE- 575004

11.2. SIGNATURE

11.3. COGUIDE: ----

11.4. SIGNATURE:----

11.5. HEAD OF THE OF THE DR. MOHANDAS RAI DEPARTMENT:

11.6. SIGNATURE:

12 12.1.REMARKS OF THE CHAIRMAN AND PRINCIPAL:

12.2.SIGNATURE OF THE PRINCIPAL: