INTRODUCTION:

It is essential to do animal experiments in the modern scientific period because they provide insight into diseases and drug action, also reveal the harmful effects of pharmaceuticals by observing how animals respond to them behaviorally. Before administering any medicine to human, drug screening in lower animals is necessary for the safety and efficacy. In order to understand the effect of drug on living organisms, isolated tissues, distributed cells, and also to give an insight into where and how the drugs acts several animal studies have been developed. Animal experiments include pharmacological study and toxicological study.

The objective of pharmacological study is to provide a scientific foundation for better therapeutics. Toxicity studies deal with the study of harmful effects of chemicals and the drugs on living organisms¹.

Panchatikta Kashaya is a widely used formulation in clinical practice, owing to its efficacy in fever management. This particular formulation is taken from Cakradatta Jwara Chikitsa². The modified form of Panchatikta Kashaya Varti aims to bypass first-pass metabolism, thereby enhancing absorption rates and bioavailability. It also offers a solution for patients facing challenges with oral medication, such as those who are aged or children. The bitterness of Panchatikta Kashaya, from its five herbal components, can be overcome through the use of Varti dosage form.

The modified form of Panchatikta Kashaya as a Varti is taken here for the experimental study. The drug's efficacy in reducing pyrexia is assessed by recording rectal temperatures in experimental animals. The anti-pyretic effect is then compared with both a control group and a standard group. As this Kashaya helps in alleviating all types of Jwara, the modified form of Panchatikta Kashaya into a Guda Varti was taken up for the study. This research was undertaken due to the scarcity of studies related to the use of rectal suppositories in evaluating the Jwaraghna (fever-reducing) effect in Ayurveda.

OBJECTIVES:

Evaluation of anti-pyretic effect of Panchatikta Kashaya Varti in Wistar Albino rats.

MATERIALS AND METHODS:

Drug and Chemicals Used:

1. Test drug : Panchatikta Kashaya Varti

2. Standard drug: Paracetamol suppository

3. Distilled water

4. Brewer’s yeast solution

Equipment Used:

1. Digital tele thermometer

2. Glass beaker and stirrer

3. Disposable syringes

4. Weighing balance

5. Hand gloves

Experimental Animals:

Wistar strain albino rats were selected from animal house of SDM Center for Research in Ayurveda and Allied Science Udupi. The rats were maintained under strict laboratory conditions, controlled with environmental, temperature, humidity, and light dark cycles. Rats were fed with balanced pelleted diet commercially obtained from VRK nutritional solutions, Maharashtra and water ad libitum. The experimental protocol was approved by the institutional animal ethical committee.

Inclusion criteria:

a. Healthy albino rats of either sex

b. Weight of the rats will be within 300g

Exclusion criteria:

a. Pregnant and diseased rats

b. Less than 180 g and more than 300g

c. Rats which are under trial of other experiments

Dose fixation:^3,4

General dose of Varti explained in AFI is 12g, which was converted into rat dose by using the formula.

Animal dose (mg/kg) = Human Equalent dose × KM ratio

Km ratio = Average body weight ÷ Body surface area

Therefore, dosage of test drug Panchatiktaka Kashaya Varti is

=12×0.162

= 1.94 g

(To convert animal dose in mg/kg to HED in mg/kg, multiply the animal dose by 0.162)

Standard drug paracetamol suppository =175mg

Yeast injection dose: 1ml/100g body weight of rats.

The test drugs and standard drug were administered rectally, while the yeast suspension was injected subcutaneously. Animals were marked with saturated picric acid solution in water for proper identification. Within the cages, markings denoted areas such as Head (Rat1), Neck (Rat 2), Body (Rat 3), Tail Rat 4), Forelimb (Rat 5), and no mark (Rat 3). All the selected animals were kept under acclimatization for 7 days before dosing.

Table 1: Grouping of Wistar albino rats for experimentation

Group	Number of rats	Drug
Control	6	Standard diet and water
Test drug	6	Panchatikta Kashaya Varti
Standard drug	6	Paracetamol Suppository (Neomol- 175mg)

Preparation of brewer’s yeast ⁵

In a glass beaker, 6.250g of collected sample of brewer’s yeast was dissolved in 50ml of distilled water by constant stirring with a glass rod. This resulted in the preparation of a 12.5% yeast solution. The solution was then incubated at 37℃ for 48 hours for fermentation.

Procedure⁶

Eighteen Wistar albino rats were randomly divided into 3 groups, each containing 6 animals. All healthy rats selected for the experimental study underwent overnight fasting before the experiment. Initial normal rectal temperatures of all animals were recorded using a digital tele thermometer. Pyrexia was induced by administering 12.5% brewer’s yeast solution subcutaneously at a dose of 1ml/100mg of body weight to all rats, which were then placed in cages and kept overnight fasting. After 18 hours of brewer’s yeast injection, rectal temperatures of each rat were noted to confirm pyrexia. Only animals with a rectal temperature increase of 0.2 to 2℃ after yeast injection were included in the study. Corresponding standard and test drugs were administered to each group after 18 hours of yeast injection. Group 1 rats were administered distilled water as a control, group 2 received the Panchatikta Kashaya Varti (1.9g), and group 3 received paracetamol suppository at a dose of 175mg. After administering drugs to each group, rectal temperatures of each rat were recorded hourly for 5 hours and then again after 24 hours to obtain a sixth reading. The difference between the starting values and the obtained values was recorded. The maximum reduction in rectal temperature of the test drug was compared with the control group and the standard. The results were then analysed and compared. The data obtained was analysed by using one way analysis of variance (ANNOVA) followed by Dunnett multiple comparison T test as post hoc test for determination the level of significance of the observed effects. Graph Pad stat software was used for statistical analysis of the generated data.

The study was conducted after obtaining permission from the Institutional Animal Ethics Committee (IAEC) under approval number: SDMCRA /IAECISHR-10

Observations and results:

Initially normal body temperature of all rats was recorded. After the administration of Brewer’s yeast all the animals were observed for their behaviour changes. All the symptoms mentioned below confirmed that the rats are suffering from fever. The temperature of all albino rats increased, and trembling was noted after one hour of Brewer’s yeast injection. Additionally, their fur was erected, and the faces of all the animals were bent down. They were also not very active.

Observations and results of the study to evaluate the antipyretic activity of Panchatikta Kashaya Varti are presented in the following tables 2 to5.

Data showed that there was an increase in rectal temperature of control group at 1,2,3,4,5^th and 24^th hours compared to basal rectal temperature of the same group. The standard drug (paracetamol) administered group has shown significant reduction in rectal temperature measured during 3,4 hour and non-significant in 1^st,2^nd,5^th ,24^th hours after drug administration. The observed changes were not statistically significant while compared to initial rectal temperature after Brewer’s yeast injection.

The test drug Panchatikta Kashaya Varti administered group has shown significant reduction in rectal temperature measured during 1,2,3,4,5^th hours after drug administration and non- significant in 24^th hr. But the rectal temperature measured during 1,2,3,4,5, and 24^th hr. was found to be decreased while compared to initial rectal temperature after brewer’s yeast injection.

Table 2: The comparison of temperature (℃) from initial temperature to 24^th hour (control group)

Rat identification	Weight (gm)	Basal temperature	Yeast dose	Temperature after 18hrs	1^st hr	2^nd hr	3^rd hr	4^th hr	5^th hr	24^th hr
Head (Rat 1)	286	38.2	2.8ml	38.5	39.4	39.5	39.6	39.6	40.0	38.9
Neck (Rat 2)	264	38.2	2.6ml	38.8	40.4	39.1	39.7	40.1	39.9	39.2
Body (Rat 3)	274	38.2	2.7ml	38.5	39.2	38.9	39.6	40	39.3	38.9
Tail (Rat 4)	266	36.2	2.6ml	38.5	39.3	39.4	38.9	39.9	39.9	40.6
Forelimb (Rat 5)	237	38.6	2.3ml	38.7	38.9	39.4	38.8	40.0	40.2	38.7
Normal (Rat 6)	273	38.5	2.7ml	38.8	39.3	39.3	39.6	40.0	38.1	39.4

Table 3: The comparison of temperature (℃) from initial temperature to 24^th hour (Standard group)

Rat identification	Weight (gm)	Basal temperature	Yeast dose	Temperature after 18hrs	1^st hr	2^nd hr	3^rd hr	4^th hr	5^th hr	24^th hr
Head (Rat 1)	210	38.5	2.1ml	39	38.9	38.8	37.7	40.7	-	-
Neck (Rat 2)	198	38.1	1.9ml	39.2	38.2	37.3	37.7	38.4	38.3	-
Body (Rat 3)	210	38.1	2.1ml	39.9	39.1	39.0	38.1	38.7	39.1	39.1
Tail (Rat 4)	209	38.2	2.0ml	39.6	40.3	39.6	39.4	39.6	39.6	39.8
Forelimb (Rat 5)	233	38.8	2.3ml	39.5	38.9	38.3	38.1	39.0	38.6	40.4
Normal (Rat 6)	228	38.6	2.2ml	39.6	39.6	39.0	39	39.6	39.4	39.1

Table 4: The comparison of temperature (℃) from initial temperature to 24^th hour (Test group)

Rat identification	Weight (gm)	Basal temperature	Yeast dose	Temperature after 18hrs	1^st hr	2^nd hr	3^rd hr	4^th hr	5^th hr	24^th hr
Head (Rat 1)	240	38.4	2.4ml	39.2	38.7	37.9	38.0	38.9	39.1	39.0
Neck (Rat 2)	220	38.0	2.2ml	40.3	39.1	38.8	38.6	39.0	38.6	38.3
Body (Rat 3)	255	38.1	2.5ml	39.5	39.0	38.9	38.5	38.3	38.7	39.3
Tail (Rat 4)	207	38.6	2.0ml	39.6	37.9	37.6	37.6	38.0	38.0	38.0
Forelimb (Rat 5)	218	38.3	2.1ml	40.1	37.7	37.1	37.0	37.4	37.0	36.1
Normal (Rat 6)	212	38.8	2.1ml	39.6	38.3	37.7	37.1	37.1	-	-

Table 5: Effect of test group, standard and control group in wistar albino rats within the groups

Group	Rectal temperature measured at the different time intervals (℃)
Group	1^st	2^nd	3^rd	4^th	5^th	24^th
Control	39.41±0.20	39.26±0.09	39.36±0.16	39.93±0.07	39.56±0.77	39.28±0.28
Standard	39.22±0.35	38.64±0.39	38.46±0.31*	39.06±0.24*	39±0.54	39.6±0.31
Test drug	38.45±0.23*	38±0.28**	37.8±0.27**	38.11±0.31**	38.28±0.81*	38.14±0.56

Data expressed in Mean ±SEM, *P<0.05, **P<0.01 in comparison to rectal temperature (℃) 18 hrs after yeast induced pyrexia.

Table 6: Effect of Panchatikta Kashaya Varti on 1^st hour of temperature

Group	1^st hour	% changes
Control	39.41±0.20	-
Paracetamol	39.22 ±0.35	0.48↓
Panchatikta Kashaya Varti	38.45±0.23*	2.43↓