experimental study

Experimental study

“The experiment should be set up to open as many windows as possible
on the unfore seen”

-Frederic Joliot _ curie


“Observation, hypothesis, experimentation, discovery & law “all
these words are the part of the one procedure and i.e. “To know the truth”,

Observation: To see the nature (Natural process)

Hypothesis: To imagine the nature.

Experimentation: To investigate the nature.

Discover: To understand the nature.

Law: To put the nature in practise.

Everyone has to possess a new drug formulation for the natural
diseases in a natural process in order to find a new drug with its
authentication to prove that the drug possess on that particular organ without
affecting the other organs.

Due to certain limitation in the clinical studies mainly ethical
consideration, every new formulations cannot be tested in human beings
directly. Therefore to prove and to get authentication of new drug for
particular disease, animal studies should be performed initially.

In experimental studies laboratory animals will be used to find the
exact result of the action of drug observing the activities of animals give us
some ides related to disease and drug etc.

The present study were carried out in order to assess the induction
and formation of gall stone with lithogenic diet and reduction of gallstone
with the help of MAKKAYA PANEEYAKSHARA for cholelithiasis.an attempt was made
to ascertain the nature of stone formation and reduction in  gallbladder with different bio-chemical and
histological methods.


Aim and objectives:

The present study was divided into two phases

To observe the
formation of Gallbladder stone by inducing with Lithogenic Diet.

To evaluate the
efficacy of MAKKAYA PANEEYAKSHARA in Gallstones.


Materials and Methods


Animals :

The experimental animals used were Swiss Albino Mice of either sex,
weighing between 10-15gms were obtained from the animal house attached to
pharmacology laboratory of PIA, Vadodara, Gujarat. They were maintained on
branded rat pellet food and tap water. The animals were well maintained under
the control of natural humidity day and night cycles.

Total 24 Swiss Albino Mice were procured to this study for 50days.


Two chemicals were used to the formations of gallstones in mice.
Chemicals were brought from Hi-Media brand from Aatur Instru-Chem, Vadodara,

Crystal chemical powders are,

Cholesterol 250Gm

Cholic Acid 125Gm

Dose Fixation;

HED in mg/kg=Animal dose (mg/kg) * Animal kg/human kg.

HED (Human Equilant dose)


Experimental Procedure:

Before starting the procedure all the animals were weighed with
weighing machine and approximately weight was 10-15gms. Each animals were
marked with ethylene marker for better findings. These 24 animals was divided
into four groups which contains 6 animals in each, was kept separately in
4different rat cage.

According to the dose fixation formula, 10gm Cholesterol and 5gm
Cholic acid powder was well mixed with 500gm of fine powder of rat pellet added
with 250ml of Water to make bolus form. Bolus was divided into four equal parts
and provided to those animals with free drinking water.

For first 15days the consumption of food was more after 40th
day the consumption of food was less compared to initial days. On every 10th
day the weight of the mice was noted and recorded. On 50th day
feeding of lithogenic diet was stopped. Randomly one animal from each
group  was selected and made to sacrifice
in order to note the formation of Gallstones in the mice.

After dissecting the mice, carefully gallbladder was removed along
with the liver. Deposition of white colour as well as increased size of liver
were noted. While dissecting the gallbladder crystal clear size of many stones
were formed.

Histo-Pathological study:

After dissection specimen of liver and gallbladder was collected in
a container containing normal saline in it. Then the collected sample was send
Laboratory of Parul Sevashram Hospital, Vadodara, Gujarat. Under the
microscope, gallbladder was viewed and formation of white coloured Gallstones
were noted clearly.


Treatment procedure:

After the confirmation of gallstones through microscopic studies,
remaining 20 animals were weighted separately and kept in their cage safely.20
animals were divided into 4 groups as follows

Group 1-Normal animals

Group 2-Diseases controlling(Lithogenic diet)

Group 3-Lithogenic diet + ursodeoxycholic acid

Group 4-Lithogenic diet + Alkaline product(Paneeya

Ursodeoxycholic acid 300mg flim coated
tablets of marc laboratories pvt.ltd was taken and made into fine powder form
and it was made into solution form by adding water in it.

Prepared makkaya paneeyakshara (powder
form) was made into solution form by adding water and was kept ready.


HED in mg/kg=Animal dose (mg/kg) * Animal kg/human kg.

HED (Human Equilant dose)

Route of administartion:

1ml insulin syringe was taken at the tip
of the needle was attached wih the adjstable needle.

Without injuring the mouth ,the prepared
medicine solution was given directly to the osephagus of the mic for 15days.

On regular basis of 7 days interval the
weight of the mice was noted.


Histo-Pathological :

After 15days of administration of drugs ,2
animals from each group was selected and made into sacrifies to note the
efficacy of the ursodeoxycholic acid as well as makkaya paneeyakshara.

After dissecting, liver and gallbladder
was collected in white container containing normal saline in it and it was send
to laboratory for evualtion.

Analysis of serum lipids and hepatic

Plasma cholesterol was measured by an
enzymic method (Cholesterol esterase – Cholesterol oxidase –
peroxidase:Boehringer Mannheim) and triacylglycerols were assayed by an enzymic
method without correction of free glycerol( Lipase – Glycerol kinase-glycerol
phosphate oxidase-per oxidase).The concentration of total and free Chlesterol
in liver homogenates and Microomal fractions were determained by isotope
dilutation mass spectrometry , after the addition of deuterium-labelled
Cholesterol as an internal standard.Free cholesterol was determined by same
method except that saponification with alcoholic KOH was omitted.The
concentration of osterified cholesterol was calculated as the difference
between total and free cholesterol in the same sample.




Conclusion and Discussion: