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STUDY OF ANTI-INFLAMMATORY ACTIVITY OF
DRUGS USING CARRAGEENAN INDUCED PAW
EDEMA MODEL
LEKKALA LITHIN KUMAR
GROUP-4 Y2031
SCHOOL OF PHARMACEUTICAL SCIENCES
LOVELY PROFESSIONAL UNIVERSITY, PHAGWARA, PUNJAB-
144411.
ABSTRACT:
Inflammation is defined as the local response of living mammalian tissues to
injury due to any agent. It is a body defence reaction in order to eliminate or
limit the spread of injurious agent, followed by removal of the necrosed cells
and tissues. Depending upon the defence capacity of the host and duration of
response, inflammation can be classified as acute and chronic. Carrageenan
will be administered to the rats into the plantar surface of the right hind limb
to induce paw oedema. The volume will be measured immediately and after 3
hours using plethysmometer. Acetyl salicylic acid 300mg/kg, Aspirin, an
acetylated salicylate (acetylsalicylic acid), is classified among the nonsteroidal
anti-inflammatory drugs (NSAIDs). These agents reduce the signs and
symptoms of inflammation and exhibit a broad range of pharmacologic
activities, including analgesic, antipyretic, and antiplatelet properties.
Decreased average paw volume in drug (Acetyl salicylic acid) treated animals
shows anti-inflammatory activity of drug (Acetyl salicylic acid).
KEYWORDS: Inflammation, Plethysmometer, Carrageenan, Oedema, Aspirin,
NSAID’S
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INTRODUCTION:
Inflammation is defined as the local response of living mammalian tissues to injury due to
any agent. It is a body defence reaction in order to eliminate or limit the spread of injurious
agent, followed by removal of the necrosed cells and tissues. The agents causing
inflammation may be as under 1. Infective agents like bacteria, viruses and their toxins,
fungi, parasites. 2. Immunological agents like cell-mediated and antigenantibody reactions.
3. Physical agents like heat, cold, radiation, mechanical trauma. 4. Chemical agents like
organic and inorganic poisons. 5. Inert materials such as foreign bodies.
TYPES OF INFLAMMATION. Depending upon the defence capacity of the host and duration
of response, inflammation can be classified as acute and chronic. Acute inflammation is of
short duration (lasting less than 2 weeks) and represents the early body reaction, resolves
quickly and is usually followed by healing. The main features of acute inflammation are:
1. accumulation of fluid and plasma at the affected site; 2. intravascular activation of
platelets; and 3. polymorphonuclear neutrophils as inflammatory cells.
Sometimes, the acute inflammatory response may be quite severe and is termed as
fulminant acute inflammation. Chronic inflammation is of longer duration and occurs either
after the causative agent of acute inflammation persists for a long time, or the stimulus is
such that it induces chronic inflammation from the beginning. A variant, chronic active
inflammation, is the type of chronic inflammation in which during the course of disease
there are acute exacerbations of activity. The characteristic feature of chronic inflammation
is presence of chronic inflammatory cells such as lymphocytes, plasma cells and
macrophages, granulation tissue formation, and in specific situations as granulomatous
inflammation. In some instances, the term subacute inflammation is used for the state of
inflammation between acute and chronic.
The Greek word oedema means swelling. Oedema may be defined as abnormal and
excessive accumulation of “free fluid” in the interstitial tissue spaces and serous cavities.
The presence of abnormal collection of fluid within the cell is sometimes called intracellular
oedema but should more appropriately be called hydropic degeneration. Free fluid in body
cavities: Depending upon the body cavity in which the fluid accumulates, it is
correspondingly known as ascites (if in the peritoneal cavity), hydrothorax or pleural
effusion (if in the pleural cavity), and hydropericardium or pericardial effusion (if in the
pericardial cavity). Free fluid in interstitial space: The oedema fluid lies free in the interstitial
space between the cells and can be displaced from one place to another. In the case of
oedema in the subcutaneous tissues, momentary pressure of finger produces a depression
known as pitting oedema. The other variety is non-pitting or solid oedema in which no
pitting is produced on pressure e.g., in myxoedema, elephantiasis. The oedema may be of 2
main types: Localised when limited to an organ or limb e.g., lymphatic oedema,
inflammatory oedema, allergic oedema. Generalised (anasarca or dropsy) when it is
systemic in distribution, particularly noticeable in the subcutaneous tissues e.g. renal
oedema, cardiac oedema, nutritional oedema. Besides, there are a few special forms of
oedema (e.g., pulmonary oedema, cerebral oedema) discussed later. Depending upon fluid
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composition, oedema fluid may be transudate which is more often the case, such as in
oedema of cardiac and renal disease; or exudate such as in inflammatory oedema.
PATHOGENESIS OF OEDEMA: Oedema is caused by mechanisms that interfere with normal
fluid balance of plasma, interstitial fluid and lymph flow. The following mechanisms may be
operating singly or in combination to produce oedema: 1. Decreased plasma oncotic
pressure 2. Increased capillary hydrostatic pressure 3. Lymphatic obstruction 4. Tissue
factors (increased oncotic pressure of interstitial fluid, and decreased tissue tension) 5.
Increased capillary permeability 6. Sodium and water retention.
Carageenan induced paw oedema is the most commonly used method in experimental
pharmacology to study anti-inflammatory activity of the drug. Carageenan is a sulphated
polysaccharide obtained from sea wood (Rhodophyceae). It causes the release of release of
inflammatory mediators such as histamine, 5-HT, Bradykinin and Prostaglandin and
produces inflammation and oedema too. Most carrageenan is extracted from Kappaphycus
alvarezii and Eucheuma denticulatum. The original source of carrageenan was Chondrus
crispus, and this is still used to a limited extent.
MATERIALS AND METHOD:
Animal: rats(150-200g), Chemical/Drug: Carrageenan and Aspirin(300mg/kg), Normal saline.
Equipment and apparatus: Plethysmograph or Digital Plethysmometer, vernier callipers,
syringes, oral feeding tube.
Twelve healthy male albino rats weighing 100-200 gms will be selected and made into two
groups of six animals each. All the animals will be kept on fasting for 18 hours. The hind paw
of the rats will be marked at the level of tibio tarsal junction of hind leg, so that while
measuring the volume, the dipping will be done to the same 22 level. 0.1 ml of 1%
Carrageenan will be administered to the rats into the plantar surface of the right hind limb
to induce paw oedema. The volume will be measured immediately and after 3 hours using
plethysmometer. One group serve as control, 0.3 ml of Normal saline will be given orally.
Another group will receive the test drug, Acetyl salicylic acid 300 mg/Kg. After 30 minutes of
the administration of drug, The change in the paw volume was compared with the control
animals. The percentage of oedema compared to the control by the test drug.
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OBSERVATION:
Vehicle treated Drug treated
0.75 0.51
0.73 0.49
0.78 0.53
0.74 0.55
0.74 0.58
0.77 0.56
0.751 0.53 Average
The above values which are mentioned are the values of paw
swelling of rats which are divided into Vehicle (Normal saline) and
Drug treated(Aspirin) which is recorded in plethysmometer by the
mercury displacement.
RESULT: The carrageenan which is injected in the subplantar region
of the paw induces inflammatory reaction which leads to the paw
swelling causing inflammation and oedema. This inflammatory
condition is decreased by the action of aspirin. The average paw
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swelling is reduced by aspirin and other non-steroid anti-
inflammatory drugs (NSAIDs) which inhibits the activity of the
enzyme now called cyclooxygenase (COX) which leads to the
formation of prostaglandins (PGs) that cause inflammation, swelling,
pain and fever.
CONCLUSION: From this experiment we can conclude that
carrageenan induced paw has been an effective method for the
induction of inflammatory mediators and other promediators which
leads to inflammatory response which is very useful for the
evaluation of various anti-inflammatory drugs which basically are
NSAIDS class. Here the anti-inflammatory effects of aspirin is
evaluated and aspirin has shown promising results in reducing
inflammation.
REFERENCE:
1. Harsh Mohan Textbook of Pathology by Jaypee Brothers
Medical Publishers (P) Ltd, 6th edition, 2010. Pg:116-130.
2. Handbook of Experimental Pharmacology by Kulkarni S.K,
Vallabh Prakashan, 5th edition, 2015. Pg: 87-88.
3. Essential of Medical Pharmacology by K.D. Tripathi, Jaypee
Brothers Medical Publishers (P) Ltd, 8th edition, 2019. Pg: 212-
213.
ACKNOWLEDGEMENT: We are thankful to the authorities of
School of Pharmaceutical sciences, LOVELY PROFESSIONAL
UNIVERSITY for providing the lab facilities.
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STUDY OF ANALGESIC ACTIVITY OF DRUGS
USING CENTRAL AND PERIPHERAL METHODS
LEKKALA LITHIN KUMAR
GROUP-4 Y2031
SCHOOL OF PHARMACEUTICAL SCIENCES
LOVELY PROFESSIONAL UNIVERSITY, PHAGWARA, PUNJAB-
144411.
ABSTRACT:
Pain is a warning signal, primarily protective in nature, but causes discomfort
and suffering; may even be unbearable and incapacitating. It is the most
important symptom that brings the patient to the physician. Excessive pain
may produce other effects—sinking sensation, apprehension, sweating,
nausea, palpitation, rise or fall in BP, tachypnoea. Analgesics relieve pain as a
symptom, without affecting its cause. They are used when the noxious
stimulus (evoking the pain) cannot be removed or as adjuvants to more
etiological approach to pain. Eddy's hot-plate is a device used to give heat
stimulus to the paws of animal. A metal plate at the top of hot plate apparatus
can be heated in the controlled manner and maintained at the required
temperature for the duration of an experiment. Generally, in testing of the
analgesic activity of drug in animal, 55* C temperature is used as a thermal
stimulus. The time taken by the animal to reveal pain sensation through
vocalization or paw licking or effort to escape the painful stimulus (withdrawal
of the body part on which pain is inflicted-paw or tail) is determined. The
analgesics (Pentazocine 10mg/kg) increase the time taken by the treated
animal to reveal pain sensation.
KEYWORDS: Pain, Eddy’s hot plate, Pentazocine, Analgesics, Thermal stimulus,
Tachypnoea.
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INTRODUCTION:
Pain is a warning signal, primarily protective in nature, but causes discomfort
and suffering; may even be unbearable and incapacitating. It is the most
important symptom that brings the patient to the physician. Excessive pain
may produce other effects—sinking sensation, apprehension, sweating,
nausea, palpitation, rise or fall in BP, tachypnoea. Analgesics relieve pain as a
symptom, without affecting its cause. They are used when the noxious
stimulus (evoking the pain) cannot be removed or as adjuvants to more
etiological approach to pain. Analgesics can be divided into three groups, viz.
A. Opioid/narcotic/morphine-like analgesics.
B. Nonopioid/non-narcotic/aspirin-like/antipyretic or anti-inflammatory
analgesics
C. Adjuvant analgesics: Anticonvulsants, viz. gabapentin/pregabalin,
carbamazepine, lamotrigine; antidepressants, viz. amitriptyline, duloxetine.
Algesia (pain) is an unpleasant bodily sensation perceived as suffering, usually
evoked by an external or internal noxious stimulus. Analgesic A drug that
selectively relieves pain by acting in the CNS or on peripheral pain
mechanisms, without significantly altering consciousness.
κ receptor This receptor is defined by its high affinity for ketocyclazocine and
dynorphin A; the latter is considered to be its endogenous ligand.
Norbinaltorphimine is a selective κ antagonist. Two subtypes of κ receptor κ1
and κ3 are functionally important. Analgesia caused by κ agonists is primarily
spinal (through κ1receptor). However, κ3 receptors mediate lower ceiling
supraspinal analgesia.
Pentazocine It is the first agonist-antagonist to be used as an analgesic. Along
with being a weak µ antagonist, it has more marked κ agonistic actions.
Distinctive features of pentazocine are: (a) Analgesia caused by pentazocine
has a lower ceiling and is primarily spinal (κ1). It has a different character than
that caused by morphine. (b) Sedation and respiratory depression is 1/3 to 1/2
of morphine at lower doses, and has a lower ceiling, does not increase much
beyond 60 mg dose. (c) Tachycardia and rise in BP are produced at higher
doses due to sympathetic stimulation. This may increase cardiac work.
Pentazocine is better avoided in coronary ischaemia and myocardial infarction.
(d) Biliary spasm and constipation are less marked.
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(e) Vomiting is less frequent. Other side effects are sweating and light-
headedness. (f) Subjective effects are pleasurable (morphine like) at lower
doses. These are recognised by post-addicts to be opiate. However, as dose is
increased, effects become unpleasant (nalorphinelike at > 60 mg i.m.) and
psychotomimetic effects (κ, σ mediated) appear.
Pentazocine is effective orally. though considerable first pass metabolism
occurs; oral: parenteral ratio is 1: 3. It is oxidized and glucuronide conjugated
in liver and excreted in urine. Plasma t½ is 3–4 hours, duration of action of a
single dose is 3–5 hours. Oral dose: 50–100 mg. Parenteral dose: 30–60 mg
i.m., s.c., may cause local fibrosis on repeated injection due to irritant
property. FORTWIN 25 mg tab., 30 mg/ml inj., FORTSTAR, SUSEVIN 30 mg/ml
inj; FORTAGESIC pentazocine 15 mg+paracetamol 500 mg tab.
Eddy’s Hot Plate Analgesia or Analgesiometer performs rapid & precise
screening of analgesic drug properties on small laboratory animals. Eddy’s Hot
Plate Analgesiometer has been developed for hot plate tests pain test. Pain
sensitivity to heat is assessed by placing the animal on the top of aluminium
plate and starting the timer. The operator stops the timer at the instant the
animal lifts its paws from the plate, reacting from discomfort. The front panel
timer displays the latency to react, a measurement of the animal’s resistance
to pain.
MATERIALS AND METHOD:
Animals: Mice (20-25 g) or Rat (150-180 g), Drugs: Pentazocine (20 mg/kg, i.p.)
Equipment: Eddy’s hot plate (55 °C heat for painful stimulus) analgesiometer
Animals are divided into two groups (6 animals in each). Administer one group
with the drug (Pentazocine 10mg/kg) to be tested and other with vehicle by
intraperitoneal route. After 60 minutes put the mice on the Hot Plate
maintained at 55 C. Record the response time at which the mouse licks its fore
paws or jumps.
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OBSERVATION:
Vehicle Treated Drug Treated
21 30
20 28
17 26
17 27
19 26
18 29
18.66 27.66 Average
The above mentioned table represents the time taken by the
rats or mice (Vehicle Treated & Drug Treated) to lick their
paws and jump from the hot plate analgesiometer.
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RESULT:
The analgesic activity of pentazocine was studied carefully
and the observations are noted. Pentazocine(10mg/kg) which
is an analgesic drug proved to increase the time taken by the
rats or mice to jump from hot plate analgesiometer and
reveal the pain sensation.
CONCLUSION:
From this experiment it is concluded that the Hot Plate
Analgesiometer is one of the main method in the evaluation
of the analgesic activity of the drugs by the principle of heat
stimulus. Pentazocine which is marked κ agonist is the which
belongs to the class of complex action opioids which has
shown good analgesic effect in response to heat stimulus.
REFERENCE:
1. Essential of Medical Pharmacology by K.D. Tripathi, Jaypee
Brothers Medical Publishers (P) Ltd, 8th edition, 2019. Pg: 497-
500.
2. Handbook of Experimental Pharmacology by Kulkarni S.K, Vallabh
Prakashan, 5th edition, 2015. Pg: 91-92.
3. Rang and Dale’s Pharmacology by James M. Ritter, Graeme
Henderson, Humphrey P. Rang, Elsevier Ltd, 9 th edition, 2020. Pg:
542-543.
ACKNOWLEDGEMENT: We are thankful to the authorities of
School of Pharmaceutical sciences, LOVELY PROFESSIONAL
UNIVERSITY for providing the lab facilities.
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