INTRODUCTION:

An analgesic, or painkiller, is any member of the group of drugs used to achieve analgesia-relief from pain. Analgesic drugs act in various ways on the peripheral and central nervous systems. They are distinct from anesthetics, which reversibly eliminate sensation, and include Ibuprofen, the non-steroidal anti-inflammatory drugs [NSAIDs] such as the salicylates, and opioid drugs such as morphine and opium. In choosing analgesics, the severity and response to other medication determines the choice of agent; the World Health Organization [WHO] pain ladder specifies mild analgesics as its first step. Analgesia/Pain is ill-defined unpleasant sensation evoked by stimulus [external/internal] – the most important symptom giving warning signal and primarily protective in nature.

Analgesia due to blockade of pain nerve sensitizing mechanism induced by bradykinin, TNFα, ILs. An analgesic is a drug that selectively relieves pain by acting in the CNS or on peripheral pain mechanisms, without significantly altering consciousness. Pain is a warning signal, primarily protective in nature, but causes discomfort and suffering; may even be unbearable and incapacitating. Excessive pain may produce other effects- sinking sensation, apprehension, sweating, nausea, palpitation, rise or fall in BP, tachypnoea. Analgesics relieve pain as symptoms, without affecting it’s causes. Herbal medicine is based on the fact that plants contain natural substances that can promote health and alleviate illness.Traditional Indian system of medicine such as Ayurveda is based on holistic treatment of diseases primarily relying on naturally occurring medicinal substances drug. Various sources of analgesic drugs synthetic analgesic and natural analgesic, natural analgesics like opoid analgesics, Aloevera Barbedensis, Glycyrrhiza glabra, Zingiber Officinale, Eugenia caryophyllata, Cinnamomum camphora, Matricaria pubescens etc²

Methods for Determination of Analgesic Activity:

1. Acetic Acid-Induced Writhing Test:

Acetic acid induced writhing model was used to evaluate analgesic activity of the synthesized compounds. Five groups of six Swiss albino mice, each 20–25g b. w, were used. 0.6% acetic acid [dose¼ 10ml/Kg] was injected intra-peritoneally. The numbers of writhes were counted for 20 min, after 5 min of injection of acetic acid into each mice. This reading was taken as a control. Next day, same groups of mice were used for evaluating analgesic activity. Each group was administered orally with the synthesized compounds. The dose of 100mg/kg of animal was given 1 hour before injection of acetic acid. After 5 min of acetic acid injection, mice were observed for the number of writhings for the duration of 20 min. The mean value for each group was calculated and compared with control.³

2. Hot Plate Test:

The hot plate test was carried out at a fixed temperature of 55±0.5^◦C. Animals were habituated twice to the hot plate in advance. Response was defined as licking or biting of a paw, or jumping [where all four paws leave the plate]. The time in seconds between the platform and reaction was recorded as the response latency. The mice exhibiting latency time greater than 30s or less than 5s were excluded. The latency time was determined at 30 min, 60 min, 90 min, and 120 min after administration of the test drugs, Meperidine hydrochloride, and saline. A latency period of 60 s was defined as complete analgesia, and if so, we will cut off its time to prevent damage to mice. After each testing, the hot plate was wiped clean with wet paper towels, removing urine and faeces. The female ICR mice were divided into seven groups, control group with i.g. isometrical physiological saline, and the mice were administered test drugs and positive drug for three days. The positive control group was received dolatin injection [25mg/kg] before placed on hot plate.

3. Tail Flick Test:

The apparatus used in the tail flick test consisted of a circulating immersion water heater. The thermostat was adjusted so that a constant temperature of 54±1^◦C was maintained in the water bath. Before treatment, the terminal 3 cm of each mouse’s tail was immersed in the water bath and the time in seconds taken to flick the tail was recorded. Only mice showing a pre-treatment reaction time less or equal to 3s were selected for the study. Immediately after basal latency assessment, the plant extracts, reference substance or solvent were administered by the oral route to groups of 5 mice and the reaction time was again measured 1 and 2 h after the injections. Cut-off time was 6 s for tail-flick measurements in order to minimize tissue injury.⁴

4. Tail Pressure Test:

In the present study, the tail pressure test was performed according to the procedures with minor modification. Different alkaloids dissolved in hydrogel were topically administered to the tail of the mice at a dose of 6mg/kg. The threshold of the motor response to pressure applied to the tail by an Analgesy Meter was measured. Using this device, the distal part of the tail was supported by a plinth while linearly increasing pressure applied with a cone-shaped pusher. The mechanical nociceptive threshold was defined as the force at which the mice withdrew its tail or struggled 60 min after administration. The cut-off pressure was 500gm to avoid tissue damage. order

5. Tail immersion method:

Analgesic activity was also checked in Wister albino rats by the caudal immersion. Tail immersion method is very much similar to the tail flick method as both involve heat stimuli for causation of pain, but differ in type of heat. In tail, flick heat source is coil and in tail immersion, hot water is used as stimulus. Rests of the procedure are same. The experimental animal were kept in cage and only one third of tail is allowed to come out side and then deepen in 51-55°C hot water bath until rat withdraw its tail, this is reaction time to stimuli and it is noted down. The cut out time is about 180 sec to prevent injury.⁵

6. Tail Formalin Test:

A modified formalin test was used. The different extracts at 5% [w/v] concentration were applied by topical administration as described in the tail flick test, and mice were immediately intradermically injected 20uL of a formaldehyde solution to 10% into dorsal surface of their tail, using a tuberculin syringe. Then the mouse was located into a chamber, with mirrors walls to enable clear observation of animal tails for 5 min. The nociceptive behavior is directly proportional to the licking time of the tail, which is a monophasic process. The time-course observation was restricted to 5 min, the length of time during which pain occurs. Antinociceptive activity [A] was expressed according to the following formula: A [%] = 100−[[T1×100]/T2], where T1 stands for the mean licking time post extract and T2 is the mean licking time control. For each concentration of the different extracts, the topic analgesic activity was evaluated using groups of 8 animals for a single dose and groups of 16 control animals were similarly treated, but they did not receive the extracts. Another one group of eight animals were pretreated with ibuprofen, reference drug at 5, 2.5, 1.2 and 0.6% [w/v] concentration.

7. Formalin Test:

Control group received 5% formalin. Twenty microliter of 5% formalin was injected into the dorsal surface of the right hind paw 60 min after administration of WET [0.1, 0.5, 1.0 and 2.0g/kg, p.o.] and 30 min after administration of Indo [10mg/kg ip] the mice were observed for 30 min after the injection of formalin, and the amount of time spent licking the injected hind paw was recorded. The first 5 min post formalin injection is referred to as the early phase and the period between 15 and 40 min as the late phase. The total time spent licking or biting the injured paw [pain behavior] was measured with a stop watch. The activity was recorded in 5 min intervals

8. Haffner’s Tail Clip Methods:

Haffner gave this method of analgesic evaluation in around 1929. According to this procedure if the base of tail is clipped with any object and tightly than there will be a generation of pain in tail, thus mice will start biting that portion of its tail. By using this simple yet important phenomenon, we may apply drug to be evaluated and record the response weather it bite tail quickly or in latency. If given drugs have analgesic potential than rat will not bite its tail so frequently. Mice that do not show any response within 15 seconds will reject from experiment.

9. Grid Shock Test:

In this model, mice are used for the evaluation purpose. They were place in the chamber before the experiment so that they may become familiar to the assembly. This assembly is made of a plastic chamber, which is equipped with the wired mesh at bottom. The stimulus was given in the form of electrical photons, on 30-32 cycles per second bases for maximum of 02 minutes to avoid any injury. After initiation of current flow, mice try to escape or jump from that surface. Then this activity may be record either by using oscilloscope or by simply slow motion video recorder. The same activity is repeated after the injection of drug at every 15 minutes interval.

10. Electrical Stimulation in Tail:

Stimulation on tail also gives satisfactory result in evaluation of analgesic drugs. When an electrode is inserted subcutaneously in tail of rat and connected to the electric source which supply very nominal current of about 40-50 V. when current is supplied rat initiate the reflex action, this reflex initiation time is recorded and calculated for the analgesic potency when analgesic drug is injected in rat. This process has one disadvantage that animal may feel more pain than usual and some time death may be possible. ⁶

MATERIALS AND METHODS:

Materials:

Ibuprofen tablets was purchased from the Pharmacy Unit. The drug was powdered and mixed with distilled water in a glass mortar and administered as aqueous suspension by oral gavage. The drug was continuously agitated during administration in order to deliver the drug homogeneously to the animals

Animal:

The Albino rats weighing between 122g, obtained from the Animal House of the Department of Pharmacology, were used for the study. The animals were fed with standard rodent chow (Topfeeds Ltd, Sapele, Nigeria) and allowed free access to tap water ad libitum. They were maintained at a room temperature of 28.0±2.0^oC under natural lighting condition and handled in accordance with international guidelines for Care and Use of Laboratory Animals as promulgated by the Canadian Council of Animal Care.⁷

Experimental design:

Tail immersion method:

Analgesic activity was also checked in wistar albino rats by the caudal immersion. Tail immersion method is very much similar to the tail flick method as both involve heat stimuli for causation of pain, but differ in type of heat. In tail, flick heat source is coil and in tail immersion, hot water is used as stimulus. Rests of the procedure are same. The experimental animal were kept in cage and only one third of tail is allowed to come out side and then deepen in 51-55°C hot water bath until rat withdraw its tail, this is reaction time to stimuli and it is noted down. The cut out time is about 180 sec to prevent injury.

Assay:

Procedure:

1. Weigh and number the mice/rat used for experiment.

2. Dived animals into three groups- (1) Reference (2) Control (3) Experimental Groups.

3. Note the reaction time of rat by dipping its tail in 51-55°C warm water.

4. A cut off time will be about 120 sec to avoid unnecessary pain and damage.

5. Inject the drug (Plant extract) on experimental animal and allow the drug to be absorbed, and again place them to thermal stimulus source and note down the basal reaction time.

6. Compare response time before and after medicine insertion. 8

Dosage Calculation:

Required dose for 122 g rat - Weight of the animal (g). × Standard dose (mg).⁹

1000

- 122 × 100mg/kg

1000

- 12.2 mg

12.2mg Ibuprofen drug mixed in 1 ml of distilled water and mix it properly. 0.5 ml drug give to albino rat orally.

Mean:

Control: 1.21+1.50+1.23

= 1.31

Standard: 4.12+5.15+3.84

= 4.37

% Analgesia: standard – Control × 100

Cut off - Control

= 4.37 – 1.31. × 100

10- 1.31

= 3.06. × 100

8.69

= 0.352 ×100

= 35.2 %

Percentage analgesia in rat was found to be 35.2%. [10]

Graph:

In X axis – Time in min

In Y- axis - % analgesia in sec,

RESULT:

Percentage analgesia in rat by using tail immersion method was found to be 35.2%

CONCLUSION:

Tail immersion method can be concluded that, the drug inhibits pain stimulus and with increase in time after administration which has analgesic property.

REFERENCE:

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4. Veena, M. 2016. Analgesic activity of Cryptocarya stocksii plant by hot plate method International journal of herbal medicine, 2016; 4(1): 39-41.

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6. Verma, A. Et Al. 2014. Synthesis, Characterization And Anticancer Activity Of Novel N-(Sugar Pyranosyl) Thienopyrimidine 4-Amine Derivatives. International Research Journal of Pharmacy, 2014; 5(12): 922-925.

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8. Rezazadeh S, Abbas K A, Morteza. Anti-inflammatory and analgesic activity of methanolic extracts of aerial parts of stachys schtschegleevii sosn and stachys balansae boiss.and kotschy ex boiss in rats. DARU. 2005; 13(4): 165-169.

9. Krishnaveni M, Suja V, Vasanth S, Shyamaladevi CS. Antiinflammatory and analgesic actions of 4’,5,6-trihydroxy- 3’7-dimethoxy flavone-from vicoa indica d. Indian Journal of Pharmacology 1997; 29: 178-181.

10. Qiang Jia,Weiwei Su,Wei Peng, Peibo Li, YonggangWang. Anti-diarrhoea and analgesic activities of the methanol extract and its fractions of Jasminum amplexicaule Buch.-Ham. (Oleaceae), Journal of Ethnopharmacology 2008;119: 299–304.

Received on 28.07.2022 Modified on 14.08.2022

Asian J. Research Chem. 2022; 15(6):429-432.

DOI: 10.52711/0974-4150.2022.00075

Group	Treatment	Dose (mg)	0 min	15 min	30 min	Mean
Control	Normal Saline	-	1.21 sec	1.50 sec	1.23 sec	1.31
Standard	Ibuprofen	12.2 mg/ml	4.12 sec	5.15 sec	3.84 sec	4.37