Materials and Methods

SECTION 3

MATERIALS AND METHODS

3.1 Collection of Trayodasanga guggulu (TDNm)

For this research work for toxicological study, Trayodasanga guggulu
(TDNm) was collected from Sri KundeswariAushadhalaya Ltd,
Chittagong. (Anonymous, 1968; Anonymous, 1978a; Anonymous, 1978b;
Anonymous, 2005; Anonymous, 2010; Anonymous, 2011a; Anonymous,
2011b; Pandey, 2005).

3.2 Preparation of the suspension

The suspension was prepared from ground tablet according to the

procedure mentioned in Bangladesh National Ayurvedic Formulary
(BNAF), 1992.

3.3 Dose of administration

For the toxicological experiment, the liquid was administered at a volume

such that it would permit optimal dosage accuracy without contributing
much to the total increase in the body fluid. For the toxicological studies
the drug was administered per oral route at a dose of 200 mg/kg of the
body weight (Chatterjee, 1993; Tedeschi and Tedeschi, 1968)

3.4 Route of Administration

For the toxicological studies, the suspension was administered orally.

[Per oral (p.o.) route]. Ketamine were administered intra-peritoneailly
(500 mg/kg i.p.).

3.5 Experimental Animal

For this toxicological research work, healthy Albino rats
(Rattusnovergicus: Sprague-Dawley strain,) of eight-week old of both
sexes were used. These animals were weighed about 50-70 g. The rats

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are maintained at the Animal House of the Department of Pharmacy,

Jahangirnagar University. Prior to the experiment, Rats were randomly
divided into 2 groups of 8 male animals. Thus eight rats were taken for
each group for both control and the experimental group.

3.6 Control Groups

A group of the same number of rat as the drug treated group was
concurrently employed in the experiment and this group served as the
control They were administered with distilled water as placebo as par the
same volume as the drug treated group for the same number of days.

3.7 Animal Care

All of the rats were kept in plastic cages having dimensions of 30 x 20 x
13 cm and soft wood shavings were employed as bedding in the cages.
Feeding of animals was done ad libitum, along with drinking water and
maintained at natural day night cycle. The animals were housed in a
well-ventilated hygienic experimental animal house.

Constant environmental parameters with adequate nutritional conditions

were maintained. The rat ware fed with “mouse chow” (prepared
according to the formula developed at BCSIR, Dhaka). All experiments on
rats were carried out in absolute compliance with the ethical guide for
care and use of laboratory animals.The experiment animals were marked
carefully on the tail which helped to identify a particular animal.By using
identification mark response were noted separately for a particular rat
prior to and after the administration.

3.8 Toxicological Experiment

Intra-gastric syringe has been used for the administration of the
Ayurvedic medicinal preparation. Administration of drug has been
carried out between the hours of 10 AM and noon(Gad, 1992; Weingand
et al., 1996; Kinter et al., 1994; ICH S7A., 2000; Seuter, 1996)

3.9 Doses Used In Different Experiments

Dose: 200 mg/kg body weight.

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3.10 Animal Treatment

At the end of the 45-day treatment period, the animals were fasted for 18
hours and also twenty-four hours after the last administration. Ketamine
(500 mg/kg i.p.) was administered for the purpose of anesthesia (Garner,
1957; Clarke et al., 1981; Sikula, 1981; Coles, 1974; Duncan et al.,
1994; Levine, 1995; Knoll, 1998; Matsuzawa et al., 1997; Tomaszewski,
1993)

3.11 Blood Samples Collection:

Whole blood samples were collected from post vena cava and transferred
to EDTA-added tubes immediately. All analyses were completed within
12 hours of sample collection. (Kelly, 1984).

3.12Preparation of Serum

Blood was left to clot and thus the serum was recovered. Serum samples
were separated and were collected using dry Pasteur pipette and stored
in the refrigerator for analyses. All analyses were completed within 24 h
of sample collection.

3.13 Chemicals and Reagents

All other reagents and chemicals that were used in this work were of
analytical grade and were prepared in all glass-distilled water.

3.14 Determination of the Pituitary Hormone Profile:

3.14.1 Test Procedure for Serum circulating Luteining Hormone (LH)

level (Beastall GH, et al, 1987)

Reagent : Architect LH Reagent Kit

Origin : Abbott Laboratories, USA

Method : ChemiluminescentMicroparticle Immunoassay (CMIA)

Requirements:

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A) Microparticles:Anti-β LH coated Microparticles in HEPES buffer

with(mouse, monoclonal) sucrose stabilizers.

B) Conjugate:Anti-LH (mouse, monoclonal) acridinium labeled Conjugate

in MES buffer with protein (bovine) stabilizers.

C) Pre-Trigger Solution:Pre-Trigger Solution containing 1.32% (w/v)

hydrogen peroxide.

D) Trigger Solution:Trigger Solution containing 0.35N sodium

hydroxide.

E) Wash Buffer

Assay Procedure:

LH assay is a two-step immunoassay. In the first step, sample and anti-β

LH coated paramagnetic micro-particles are combined. LH present in the
sample binds to the anti-β LH coated micro-particles. After washing,
anti- LH acridinium labeled conjugate is added in the second step. Pre-
Trigger and Trigger Solutions are then added to the reaction mixture; the
resulting chemiluminescent reaction is measured as relative light units
(RLUs). A direct relationship exists between the amount of LH in the
sample and the RLUs detected by the System optics of the instrument .

3.14.2 Test Procedure for Serum circulating Follicle Stimulating

Hormone (FSH) level (Beastall GH, et al, 1987)

Reagent : Architect FSH Reagent Kit

Origin : Abbott Laboratories, USA

Method : ChemiluminescentMicroparticle Immunoassay (CMIA)

Requirements:
A) Microparticles:Anti-f FSH coated Microparticles in MES buffer with
protein (murine and caprine) stabilizers.

B) Conjugate:Anti-a FSH (mouse, monoclonal) acridinium labeled

Conjugate in MES buffer with protein (bovine) stabilizers.

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C) Pre-Trigger Solution:Pre-Trigger Solution containing 1.32% (w/v)

hydrogen peroxide.

D) Trigger Solution: Trigger Solution containing 0.35N sodium

hydroxide.

E) Wash Buffer

Assay Procedure:

FSH assay is a two-step immunoassay. In the first step, sample and anti-
f FSH coated paramagnetic micro particles are combined. FSH present in
the sample binds to the anti-f FSH coated micro particles. After washing,
anti-a FSH acridinium labeled conjugate is added in the second step.
Pre-Trigger and Trigger Solutions are then added to the reaction mixture;
the resulting chemiluminescent reaction is measured as relative light
units (RLUs). A direct relationship exists between the amount of FSH in
the sample and the RLUs detected by the System optics of the
instrument.

3.14.3 Test Procedure for Serum circulating Prolactin (PRL) level

(Friesen HH, et al, 1972)

Reagent : Architect Prolactin Reagent Kit

Origin : Abbott Laboratories, USA

Method : ChemiluminescentMicroparticle Immunoassay (CMIA)

Requirements:

A) Microparticles:Anti-Prolactin coated Microparticles in TRIS buffer

with protein (bovine and murine) stabilizers.

B) Conjugate: Anti-Prolactin (mouse, monoclonal) acridinium labeled

Conjugate in phosphate buffer with protein (piscine and bovine)
stabilizers.

C) Pre-Trigger Solution:Pre-Trigger Solution containing 1.32% (w/v)

hydrogen peroxide.

D) Trigger Solution:Trigger Solution containing 0.35N sodium

hydroxide.

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E) Wash Buffer

Assay Procedure:

Prolactin assay is a two-step immunoassay. In the first step, sample and

anti-Prolactin coated paramagnetic micro-particles are combined.
Prolactin present in the sample binds to the anti-Prolactin coated micro-
particles. After washing, anti-Prolactin acridinium labeled conjugate is
added in the second step. Pre-Trigger and Trigger Solutions are then
added to the reaction mixture; the resulting chemiluminescent reaction
is measured as relative light units (RLUs). A direct relationship exists
between the amount of Prolactin in the sample and the RLUs detected by
the System optics of the instrument.

3.15 Determination of the Cortical Hormone:

3.15.1 Test Procedure for Serum circulating Cortisol (CORT-F) level

(Kehlet H and Binder C.,1973)

Reagent : Architect Cortisol Reagent Kit

Origin : Abbott Laboratories, USA

Method : ChemiluminescentMicroparticle Immunoassay (CMIA)

Requirements:

A) Microparticles:Anti-Cortisol (mouse, monoclonal) coated

Microparticles in TRIS/BIS-TRIS buffer with protein (bovine)
stabilizers.

B) Conjugate: Cortisol acridinium labeled Conjugate in citrate buffer

with surfactant stabilizer.

C) Pre-Trigger Solution:Pre-Trigger Solution containing 1.32% (w/v)

hydrogen peroxide.

D) Trigger Solution:Trigger Solution containing 0.35N sodium

hydroxide.

E) Wash Buffer

Assay Procedure:

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Cortisol assay is a delayed one-step immunoassay.

Sample and anti-cortisol coated paramagnetic microparticles are

combined to create a reaction mixture. Cortisol present in the sample
binds to the anti-cortisol coated microparticles. After incubation, cortisol
acridinium-labeled conjugate is added to the reaction mixture. The
cortisol acridinium-labeled conjugate competes for the available binding
sites on the anti-cortisol coated microparticles. Following a second
incubation, the microparticles are washed, and pre-trigger and trigger
solutions are added to the reaction mixture. The resulting
chemiluminescent reaction is measured as relative light units (RLUs). An
inverse relationship exists between the amount of cortisol in the sample
and the RLUs detected by the System optics of the instrument.

 Statistical Analysis

The group data are expressed as Mean ± SEM (Standard Error of the
Mean). Unpaired "t" tests were done for statistical significance tests.
SPSS (Statistical Package for Social Science) for WINDOWS (Ver. 11) was
applied for the analysis of data. Differences between groups were
considered significant at p < 0.05, 0.01 and 0.001.

 Significance of p Value

Value Significance

0.001 Very highly significant

0.01 Highly significant

0.05 Significant

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