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Where, A0 = Absorbance of DPPH alone

As = Absorbance of sample
The values for the samples were calculated by extrapolating from standard graph

2.5. Test Animal
Eight male Albino Wister rats weighing between 120-140 gms were obtained from the animal house

of PBRI, Bhopal. The experiment were conducted as per permission of institutional animal ethical
common (IAEC) of PBRI.

2.5.1. Experimental design
All the rats were randomly divided into two groups of four rats each. Group first was used to
determine the maximum tolerated dose for the extract in the animal model and second group was used

for evaluating in vivo hepatoprotective activity of Cassia tora seed extract. The second group was sub-
divided into four groups having one rat each. The subgroup had three classification 1). Given only
normal saline (positive control group), 2). given only CCl4, 3). Given extract for seven days and given

inducer i.e. CCl4 on the eighth day(experimental group). The experimental group was divided into two
parts both were given different concentrations of extract. All the doses were administered orally. The

rats were kept in a fasted condition for 24 hours after the inducer was given on eighth day, however
free access to water was provided. After 24 hours the blood samples of rats were collected by
puncturing retro-orbital plexus and the rats were sacrificed to collect liver tissues.

2.5.2. Dose toxicity test
Four rats were selected for the toxicity test and weight of each rat was recorded. The rats were divided

into four groups each group containing one animal. The groups were orally administered with four
different doses i.e. 5mg/kg, 50mg/kg, 300mg/kg and 2000mg/kg of body weight. This was done to
test the maximum tolerated dose of extract by the animal. Any type of adverse effect and death of rats

was observed for 36 hours and it was concluded that 2000mg/kg of body weight was a tolerable dose
th
th
of extract. 1/10 and 1/20 part of this dose was used for the treatment groups.

2.6. Lipid peroxidation test
The process by which the lipids in the liver gets degenerated due to the generation of oxidative stress.
The lipid peroxidation produces several products such as malondialdehyde (MDA) which in reaction

mixture reacts with TBA in acidic conditions to produce TBARS which is a pink colour compound
and gives absorbance at 532nm (Aquino et. al. 2001). The test was done as described by Tang et.al.

(2019) . To perform the test liver tissue homogenate (obtained earlier from animal) was prepared in
0.15 M tris HCL buffer at pH 7.4. This tissue homogenate was then mixed with 0.2 ml of 8.1% SDS
o
and 1.5 ml of 8% TBA. This mixture was then heated on water bath for one hour at 95 C using glass
ball as condenser. After one hour the sample was taken out, cooled and a mixture of butanol : pyridine
in a ratio of 15:1 was added and vortexed for 2 minutes. The sample was then centrifuged at 3000

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