Screening Methods for Analgesic Agents

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Chapter: Pharmacognosy and Phytochemistry : Biological Screening of Herbal Drugs

The paws of mice and rats are very sensitive to heat even at temperatures which do not damage the skin. They respond by jumping, withdrawal of paws, and licking of paws. The time until these responses occur can be prolonged after administration of centrally acting analgesics, whereas peripheral analgesics of the acetyl salicylic acid or phenyl acetic acid type do not generally affect these responses.




Centrally Acting Analgesics


Hot plate method


The paws of mice and rats are very sensitive to heat even at temperatures which do not damage the skin. They respond by jumping, withdrawal of paws, and licking of paws. The time until these responses occur can be prolonged after administration of centrally acting analgesics, whereas peripheral analgesics of the acetyl salicylic acid or phenyl acetic acid type do not generally affect these responses.


The hot plate consists of an electrically heated surface. The temperature is controlled for 55°–56°C. Adult albino rats are used for the test. The animals are placed on the hot plate, and the time until either licking or jumping occurs is recorded by a stopwatch. The delay in response is recorded after administration of the standard or the test compound.

Haffner’s tail clip method


In this method, the raised tail phenomena in mice are observed. Six mice per group are used. A clip is applied to the base of the tail of mice, and the reaction time is noted. The test compounds are administered orally to fasted animals. The animal quickly responds to the stimuli by biting the clip or the tail near the location of the clip. The time between stimulation onset and response is measured by a stopwatch.

Tail immersion test


This method is based on the observation that morphine-like drugs are selectively capable of prolonging the reaction time of the typical tail-withdrawal reflex in rats induced by immersing the end of the tail in warm water of 55°C.


Adult albino rats are used for the test. They are placed in individual cages leaving the tail hanging out freely. The animals are allowed to adapt to the cages for 30 min. before testing. The lower 5 cm portion of the tail is marked. This part of the tail is immersed in a cup of freshly filled water of exactly 55°C. Within a few seconds, the rat reacts by withdrawing the tail. A stopwatch records the reaction time. The reaction time is determined before and periodically after either oral or subcutaneous administration of the test substance. A withdrawal time of more than 6 s is regarded as a positive response.


Radiant heat method


This test is useful for quantitative measurements of pain threshold against thermal radiation in man and for evaluation of analgesic activity. It is very useful for discriminating between centrally acting morphine-like analgesics and nonopiate analgesics.


The animal is put into a small cage with an opening for the tail at the rear wall. The investigator holds the tail gently. By the opening of a shutter, a light beam exerting radiant heat is directed at the end of the tail. For about 6 s, the reaction of the animal is observed. The mouse tries to pull the tail away and turns the head. The shutter is closed with a switch as soon as the investigator notices this reaction. Mice with a reaction time of more than 6 s are not used in the test. The escape reaction is the end point of this test. Before administration of the test compound or the standard, the normal reaction time is determined. The test compounds and the standard are administered either orally or subcutaneously. The analgesiometer can also be used to measure analgesic activities.


Formalin test in rats


Rats weighing 180–300 g are administered 0.05 ml of 10% formalin into the lower surface of the front paw. The test drug is administered simultaneously either subcutaneously or orally. Each individual rat is placed in a clear plastic cage for observation. Readings are taken and scored according to a pain scale. Pain responses are indicated by elevation of the paw or excessive licking and biting of the paw. Analgesic response or protection is indicated if both paws are resting on the floor with no elevation of the injected paw.


Tooth pulp stimulation


This test is based on the fact that stimulation of the tooth pulp induces characteristic reactions, such as licking, biting, chewing, and head flick which can be observed easily.


Adult healthy rabbits are used for the test. Rabbits are anaesthetized and their pulp chambers exposed with a high-speed dental drill. On the day of the experiment, clamping electrodes are placed into the drilled holes. After an accommodation period of 30 min, stimulation is started to determine the threshold value. The stimulus is applied with a frequency of 50 Hz and duration of 1 s. The electrical current is started at 0.2 mA and increased until the phenomenon of licking occurs. The test substance is either injected intravenously or given orally. The animals serve as their own controls.


Grid shock test


This test measures the analgesic properties by the ‘flinch-jump’ procedure in rats. The floor of the box used is wired with stainless steel wire, spaced about 1 mm apart. The stimulus is given in the form of an electric current, 30 cycles per second with duration of 2 ms per pulse. With increasing shock intensities, the mice flinch, exhibit a startling reaction, increase locomotion, or attempt to jump. The behaviour is accurately reflected on the oscilloscope by marked fluctuations of the pulse and defined as the pain threshold response. The current as measured in milliamperes is recorded for each animal before and after administration of the drug.

Electrical stimulation of the tail


This method is based on the fact that since the tail of mice is known to be sensitive to any stimulus, the stimulus can be varied either by the duration of the electric shock or by an increase in the electric current to check the efficacy of the analgesic agent.


Male mice weighing 20 g are placed in special cages. A pair of clips is attached to the tail, and the positive electrode is placed at the end of the tail. Electric current at an intensity of 40–50 V is applied. The frequency of the stimulation is 1 shock/second, and the pulse duration is 2.5 ms. The normal response time range of the stimuli is 3–4 sec. Following administration of the drug, the response time is registered at 15 min intervals until the reaction time returns to control levels.


Peripherally Acting Analgesics

Pain in inflamed tissue (Randall-Selitto test)


This method is based on the principle that inflammation increases the peripheral analgesic sensitivity to pain. Inflammation decreases the pain reaction threshold, but the threshold is readily elevated by nonnarcotic analgesics of the salicylate-amidopyrine type as well as by the narcotic analgesics.


Groups of healthy albino rats (130–175 g) are used. The animals are starved 18 to 24 h before administration. To induce inflammation, 0.1 ml of a 20% suspension of Brewer’s yeast in distilled water is injected subcutaneously into the plantar surface of the left hind paw of the rat. After three hours, pressure is applied through a tip to the plantar surface of the rat’s foot at a constant rate to the point when the animal struggles, squeals, or attempts to bite. Each animal is tested for its control pain threshold. Any animal with a control pain threshold greater than 80 g is eliminated and replaced. The mean applied force is determined for each time interval.


Writhing test


Pain is induced by injection of irritants into the peritoneal cavity of mice. The animals react with a characteristic stretching behaviour which is called writhing. The test is suitable to detect analgesic activity. An irritating agent such as phenyl quinone or acetic acid is injected intraperitoneally to mice, and the stretching reaction is evaluated.


Mice of either sex of weight 20–25 g are used. Phenyl quinone in a concentration of 0.02% is suspended in a 1% suspension of carboxy methyl cellulose. About 0.25 ml of this suspension is injected intraperitoneally. The mice are placed individually into glass beakers and are observed for a period often one minute. The number of writhes is recorded for each animal. A writhe is indicated by a stretching of the abdomen with simultaneous stretching of at least one hind limb.


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