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Methods Of Pharmacological Screening

Anti Oxidant Activity By DPPH:

Materials and Methods:

The antioxidant potential of the methanolic extract is determined on the basis of
their scavenging activity of the stable 2,2- diphenyl -1- picryl hydrazyl (DPPH)
free radical. DPPH is a stable free radical containing an odd electron in its
structure and usually utilized for detection of the free radical scavenging activity in
chemical analysis. The aliquots of the different concentrations (1-500 µg/ml) of
the extract are added to 3ml of a 0.004% w/v solution of DPPH. Absorbance at
517 nm is determined after 30 min.

Fig- : A typical free radical scavenging reaction of DPPH

Quantitative assay:

Stock solution of the plant extract was prepared in ethanol from which a serial
dilution was carried out to obtain concentration of 1, 5, 10, 50, 100, 500 µg/ml.
Diluted solutions (2ml) were added to 3 ml of a 0.004% ethanol solution of DPPH,
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mixed and allowed to stand for 30 minutes for reaction to occur. The absorbance
was determined at 517nm and from these values corresponding percentage of
inhibitions were calculated. Then % inhibitions were plotted against log
concentration and from the graph IC50 was calculated. The experiment was
performed 3 times and average absorption was noted for each concentration.

Reagents:

Ethanol

0.004% DPPH (1 , 1 - diphenyl - 2 – picrylhydrazyl - hydrate)

Preparation 0.004% DPPH solution:

4mg of DPPH was measured and dissolved in 100ml of ethanol thus 0.004%
DPPH solution was prepared.

Procedures:

At first 6 volumetric flasks are taken to make 6 different types of concentration (1,
5, 10, 50, 100 and 500 µg/ml)

Test tubes and volumetric flasks are rapped with foil paper.

In 6 volumetric flaks serial dilution of extract is done and marked them

respectively.

1ml of sample from each concentration and 3 ml of 0.004% DPPH solution is

taken with the help of pipette in 6 test tubes respectively.

Then solution is kept in dark place for 30 minutes with raping each test tube with
foil paper.
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In another test tube 3ml 0.004% DPPH & 1ml methanol is taken to prepare blank
solution.

Then absorbance is taken by UV Spectroscopy.

The percent of inhibition is calculated by using following formula

% inhibition= {( Blank absorbance – sample absorbance) /Blank absorbance} X

100

Cytotoxicity activity:

Materials and Methods:

Viability levels and/or proliferation rates of cells are good indicators of cell health.
Physical and chemical agents can affect cell health and metabolism. These agents
may cause toxicity on cells via different mechanisms such as destruction of cell
membranes, prevention of protein synthesis, irreversible binding to receptors,
inhibition of polydeoxynucleotide elongation, and enzymatic reactions. In order to
determine the cell death caused by these mechanisms, there is a need for cheap,
reliable and reproducible short-term cytotoxicity and cell viability assays.

In vitro cell viability and cytotoxicity assays with cultured cells are widely used
for cytotoxicity tests of chemicals and for drug screening. Application of these
assays has been of increasing interest over recent years. Currently, these assays
are also used in oncological researches to evaluate both compound toxicity and
tumor cell growth inhibition during drug development. Because, they are rapid,
inexpensive and do not require the use of animals. Furthermore, they are useful
for testing large number of samples. Cell viability and cytotoxicity assays are
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based on various cell functions such as cell membrane permeability, enzyme

activity, cell adherence, ATP production, co-enzyme production, and nucleotide
uptake activity.

In vitro cytotoxicity and/or cell viability assays have some advantages,such as

speed, reduced cost and potential for automation, and tests using human cells may
be more relevant than some in vivo animal tests. However, they have some
disadvantages because they are not technically advanced enough yet, to replace
animal tests.

It is important to know how many viable cells are remaining and/or how many
cells are dead at the end of the experiment. A broad spectrum of cytotoxicity and
cell viability assays is currently used in the fields of toxicology and pharmacology.
The choice of assay method is crucial in the assessment of the interaction type.

Working procedure and dose calculation of Cytotoxic activity (Brine Shrimp

method)

Preparation of sea water

38 gm sea salt (without iodine) was weighed, dissolved in one liter of
distilled water and filtered off to get clear solution.

Hatching of Brine Shrimp

Artemia salina leach (brine shrimp eggs) collected from pet shops was used
as the test organism. Seawater was taken in the small tank and shrimp eggs were
added to one side of the tank and then this side was covered. Two days were
allowed to hatch the shrimp and to be matured as nauplii. Constant oxygen supply
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was carried out through the hatching time. The hatched shrimps are attracted to the
light (phototaxis) and so nauplii free from egg shell was collected from the
illuminated part of the tank. The nauplii was taken from the fish tank by a pipette
and diluted in fresh clear sea water to increase visibility and 10 nauplii were
taken carefully by micropipette.

Preparation of test solutions with samples of experimental plants 32 mg of

each of the test samples were taken and dissolved in 200μl of pure
dimethyl sulfoxide (DMSO) and finally the volume was made to 20 ml with
sea water. Thus the concentration of the stock solution was 1600μg/ml. Then the
solution was serial diluted to 800, 400, 200, 100, 50, 25, 12.5, 6.25 µg/ml
with sea water. Then 2.5 ml of plant extract solution was added to 2.5 ml
of sea water containing 10 nauplii: Concentration (µg/ml) Extract Solution Sea
water containing 10 nauplii

Final volume

800 µg/ml = 2.5 ml (1600µg/ml) + 2.5 ml = 5 ml

400 µg/ml = 2.5 ml (800µg/ml) + 2.5 ml = 5 ml
200 µg/ml = 2.5 ml (400µg/ml) + 2.5 ml = 5 ml
100 µg/ml = 2.5 ml (200µg/ml) + 2.5 ml = 5 ml
50 µg/ml = 2.5 ml (100µg/ml) + 2.5 ml = 5 ml
25 µg/ml = 2.5 ml (50µg/ml) + 2.5 ml = 5 ml
12.5 µg/ml = 2.5 ml (25µg/ml) + 2.5 ml = 5 ml
6.25 µg/ml = 2.5 ml (12.5µg/ml) + 2.5 ml = 5 ml
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Preparation of control group

Control groups were used in cytotoxicity study to validate the test method and
ensure that the results obtained were only due to the activity of the test agent and
the effects of the other possible factors were nullified. Two types of control groups
were used
i) Positive control
ii) Negative control

Preparation of the positive control group

Positive control in a cytotoxicity study is a widely accepted cytotoxic agent and
the result of the test agent is compared with the result obtained for the positive
control. In the present study vincristine sulfate was used. As vincristine is a very
cytotoxic alkaloid it was evaluated at very low concentration (10, 5, 1, 0.5, 0.25,
0.125 and 0.06 μg/ml)

Preparation of the negative control group

50 µl of DMSO was added to each of three premarked test tubes
containing 4.95 ml of simulated sea water and 10 shrimp nauplii to use as control
groups. If the brine shrimps in these vials show a rapid mortality rate, then the test
is considered as invalid as the nauplii died due to some reason other than the
cytotoxicity of the compounds.

Counting of nauplii
After 24 hours, the test tube were inspected using a magnifying glass
against a black background and the number of survived nauplii in each tube was
counted. From this data, the percent (%) of lethality of the brine shrimp
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nauplii was calculated for each concentration. The mortality was corrected using
Abott’s formula (Abott W. S., 1925)
Pt= [(Po-Pc)/ (100-Pc)] × 100
Where, Po= Observed mortality
Pc= Control mortality

The effectiveness or the concentration-mortality relationship of plant product

is usually expressed as a median lethal concentration (LC50). This represents the
concentration of the chemical that produces death in half of the test subjects after a
certain exposure time and determined by linear regression method from
plotting % mortality against correspondent log of concentration.

Anti-Microbial activity by disc diffusion method:

Materials and Methods:

Media Preparation:
Nutrient agar media
Concentration: 28gm/L
That means, 1L or 1000ml distilled water contain 28gm Nutrient agar
Disc preparation:
Each petri disc contain 10ml Nutrient agar media
For Example:
Preparation 60ml nutrient agar media
1000ml distilled water contain 28gm Nutrient agar
60ml distilled water contain (28X60)/1000gm
= 1.68gm
That means, 1.68gm Nutrient agar dissolved in 60ml distilled water
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Stock solution of Plant Extract:

Concentration: 50µg /µl
That means, 1µl solvent contain 50µg extract
Preparations 1ml or 1000µl extract solution:
1µl solvent contain 50µg extract
1000µl or 1ml solvent contain (50X1000) µg
= 50000µg
= 50mg [1000µg = 1mg]
= 0.05gm [100mg = 1gm]

Dose of Plant Extract:

500µg/disc
That means,
50µg contain 1µl stock solution
500µg contain (500/50) µl stock solution
= 10µl stock solution per blank or filter paper disc
Standard:
Kanamycin 30µg/disc
Ciprofloxacin 30µg/disc etc.
Autoclave Condition: 121oC Temperature and 15Ib or 15atm pressure for 20 min
Refrigerator or Freezing: 2 hours
Incubation: 18 to 24 hours at 37oC Temperature
 Page |9

Determination of zone of inhibition:

ANALGESIC ACTIVITY:

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 temporarily affect, and in some instances completely eliminate,
sensation. Analgesics include paracetamol (known in North America as
acetaminophen or simply APAP), the nonsteroidal anti-inflammatory drugs
(NSAIDs) such as the salicylates, and opioid drugs such as morphine and
oxycodone.

Working procedure and dose calculation of analgesic activity

1% Acetic acid induced in the mice on dose 10ml/kg body weight because of
produced pain
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Dose:
Standard: Diclofenac Na 10mg/kg body weight (inject in the intra-peritoneal
route)
Control: 0.5% methyl cellulose or water (Oral route)
Extract: 300mg or 500mg/kg body weight (Oral route)
Standard:
∑ Injected Di-clofenac Na 10mg/kg body weight in the mice on the intra-
peritonea route
∑ After 15 minutes
∑ Then injected 1% acetic acid 10ml/kg body weight in the mice on the intra-
peritoneal route
∑ After 5 minutes Observation writhing in 10 minutes
Control:
∑ 0.5% methyl cellulose or water 10ml/kg body weight are put on oral route
∑ After 15 minutes
∑ Then injected 1% acetic acid 10ml/kg body weight in the mice on the intra-
peritoneal route
∑ After 5 minutes Observation writhing in 10 minutes
Plant Extract:
∑ 300mg or 500mg/kg body weight extract are put on oral route
∑ After 30 minutes
∑ Then injected 1% acetic acid 10ml/kg body weight in the mice on the intra-
peritoneal route
∑ After 5 minutes Observation writhing in 10 minutes
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ANTI-INFLAMMATORY EFFECTS BY RAT PAW EDEMA:

Definition

Inflammation (Latin, īnflammō, "I ignite, set alight") is part of the complex
biological response of vascular tissues to harmful stimuli, such as pathogens,
damaged cells, or irritants.[18] Inflammation is a protective attempt by the organism
to remove the injurious stimuli and to initiate the healing process. Inflammation is
not a synonym for infection, even in cases where inflammation is caused by
infection. Although infection is caused by a microorganism, inflammation is one of
the responses of the organism to the pathogen. However, inflammation is a
stereotyped response, and therefore it is considered as a mechanism of innate
immunity, as compared to adaptive immunity, which is specific for each pathogen

Drugs and Chemicals

The standard drug, Metformin hydrochloride was the generous gift samples from
Beximco Pharmaceuticals Ltd of Bangladesh. Alloxan monohydrate was purchased
from Loba Chemie, India. Carrageenan was purchased from Otto Chemika, India.
Blood samples analyzed for blood glucose content by using OK meter Match
glucose test meter (Hsinchu, Taiwan). Acetic acid was collected from laboratory.
The standard drug Diclofenac-Na was purchased from Square Pharmaceuticals
Limited of Bangladesh.

Experimental Animals

Eight week-old Swiss albino mice (27-30g) purchased from Jahangirnagar

University, Dhaka, Bangladesh and were housed in animals cages under standard
environmental conditions (22-25°C, humidity 60-70%, 12 hr light: 12 hr dark
 P a g e | 12

cycle). The mice were feed with standard pellet diet taken from, Jahangirnagar
University Dhaka. The animals used in this study were cared in accordance with
the guidelines on animal experimentation of our institute.

Induction of Inflammation

Inflammation in mice was inducing by injecting 0.1ml of 1% carrageenan in

physiological saline into the sub plantar tissue of the left hind paw of each mouse.
Paw edema was induced by injecting 0.1ml of 1% Carrageenan in physiological
saline into the subplantar tissues of the left hind paw of each mouse.

The methanol extract of Leaves 500 mg/kg were administered orally 30 min prior
to Carrageenan administration. The paw volume was measured at 60, 120, 180,
240 minutes by using a micrometer screw gouge. The percentage inhibition of paw
volume in drug treated group was compared with the control group. Diclofenac
sodium (10 mg/kg p.o.) was used as reference standard.

Anti-Diabetic effect:

Definition

Diabetes mellitus (DM) is a group of metabolic diseases characterized by high

blood sugar (glucose) levels that result from defects in insulin secretion, or action,
or both. The term diabetes, without qualification, usually refers to diabetes
mellitus, which roughly translates to excessive sweet urine (known as
"glycosuria") and excessive muscle loss in the ancient world. Elevated levels of
blood glucose (hyperglycemia) lead to spillage of glucose into the urine, hence the
term sweet urine. When our food is digested the glucose makes its way into our
bloodstream. Our cells use the glucose for energy and growth. However, glucose
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cannot enter our cells without insulin being present – insulin makes it possible for
our cells to take in the glucose. Insulin is a hormone that is produced by the
pancreas. After eating, the pancreas automatically releases an adequate quantity of
insulin to move the glucose present in our blood into the cells, and lowers the
blood sugar level.

MATERIALS AND METHODS

Drugs and Chemicals

The standard drug, Metformin hydrochloride was the generous gift samples from
Beximco Pharmaceuticals Ltd of Bangladesh. Alloxan monohydrate was purchased
from LobaChemie, India. Carrageenan was purchased from Otto Chemika, India.
Blood samples analyzed for blood glucose content by using OK meter Match
glucose test meter (Hsinchu, Taiwan). Acetic acid was collected from laboratory of
Bangladesh University. The standard drug Diclofenac-Na was purchased from
Square Pharmaceuticals Limited of Bangladesh.

Experimental Animals

Eight week-old Swiss albino mice (27-30g) purchased from Jahangirnagar

University, Dhaka, Bangladesh and were housed in animals cages under standard
environmental conditions (22-25°C, humidity 60-70%, 12 hr light: 12 hr dark
cycle). The mice were feed with standard pellet diet taken from, Jahangirnagar
University Dhaka. The animals used in this study were cared in accordance with
the guidelines on animal experimentation of our institute.

Induction of Diabetes

After fasting 16 hr, diabetes was induced into mice by in intra-peritoneal injection
(i. p.) of alloxanmonohydrade (90 mg/kg), dissolved in saline (100 µl/mice, ip.).
 P a g e | 14

After 48 hour, plasma glucose levels were measured by OK meter Match

glucometer (Hsinchu, Taiwan) using a blood sample from tail-vein of mice. Mice
with blood sugar level higher than 08.5-11.5 mmol/l are considered as diabetic.
Age-matched healthy mice were used to examine the effects of extract on normal
mice.

Experimental Protocols

After induction of diabetes 16 mice were divided into four groups for the oral
administration either-

ÿ Normal Control ( Normal Group, Vehicle 0.5% methyl cellulose, n = 4)

ÿ Diabetic Control (Control Group, Vehicle 0.5% MC, n = 4)

ÿ Diabetic Standard (Standard Group, Metformin HCl, 100 mg/kg, op. n = 4)

ÿ Diabetic Extract (Extract Group 300 mg/kg , n = 4)

For analgesic test all mice were divided into three groups (Control Group, Standard
Group and Extract Group). Each group comprises 4 mice. Control group (received
0.5% Methyl cellulose), Standard Group (received Diclofenac sodium10 mg/kg),
and Extract Group (received 300 mg/kg extract).

To anti-inflammatory test all mice were divided into three groups (Control Group,
Standard Group and Extract Group). Each group comprises 4 mice. Control group
(received 3 ml/kg of 1% carboxyl methyl cellulose), Standard Group (received
Diclofenac 10 ml/kg), and Extract Group (received 400 mg/kg extract).

Anti-diabetic test in alloxan-induced diabetic mice

The mice were fasted over-night and next day blood samples were taken from all
groups of animals to estimate fasting blood glucose level (0 min). All mice
 P a g e | 15

received after half an hour of feeding of extract and/ drug and four time blood
samples were collected at 60, 120, 180 and 240 minutes intervals and blood
glucose level was estimated in all the experiments by using OK meter Match
glucose test meter (Hsinchu, Taiwan).

ANTI-DIARRHEAL ACTIVITY:

Diarrhea is an alteration in the normal intestinal motility as well as bowel

movement, characterized by increased frequency of bowel sound and movement,
wet stool with or without blood, abdominal pain and discomfort with weakness.
Clinically it is used to describe increased liquidity of stool, usually associated with
increased frequency (Suleiman et al., 2008). Regardless of the understanding
causes, treatment and prevention of diarrheal diseases, an estimated 4.6 million
people, with 2.5 million children, die from diarrhea every year, particularly in
developing countries (Kosek et al., 2000). Diarrhea may be acute or chronic. Acute
diarrhea being the most common is usually caused by an infectious agent, even
though drugs, poisons or acute inflammatory reactions can contribute a lot (Thapar
et al., 2004). Now a days, rotavirus is the major causative agent for infectious
diarrhea, particularly in young children, however, other viral (adenovirus,
enterovirus and norovirus), bacterial (Escherichia coli, Salmonella sp., Shigella
sp., Camphylobacter and Vibrio cholerae) and parasitic (Cryptosporidium and
Giardia) agents are important pathogens (Allen et al., 2004).

Principle

Diarrhea was considered by the presence of stool or any fluid material that stained
the blotting paper placed in every cage lined with the floor. Time taken before the
first defecation was considered as the ‘Latent period’. The latent period and stool
count after 4 hours of test group were compared with control group to evaluate
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antidiarrheal activity. Antidiarrheal agents increase latent period and decrease total
stool count.
Study design

Experimental animals were randomly selected and divided into four groups
denoted as group-I, group-II, group-III, group- IV consisting of 5 mice in each
group. These groups received a particular treatment respectively control, standard
and the two doses of the extract. Each mouse was weighed properly and the doses
of the test samples and control materials were adjusted accordingly.

Preparation of sample

To prepare the test samples at the doses of 250 and 500 mg/kg body weight, 500 &
250 mg of samples were measured respectively. The extract was triturated in
unidirectional manner by the addition of small amount of tween-80. After proper
mixing of extract and tween-80, the distilled water was slowly added. The final
volume was made 10 ml.

For the preparation of standard loperamide at the dose of 3 mg/kg body weight, 3
mg equivalent loperamide was triturated with small amount of tween-80 and then
adjusted with distilled water to make final volume 10 ml.

Methodology

Group-I considered as the control group received only distilled water containing
1% tween-80. Group-II considered as standard group received standard loperamide
at dose of 3 mg/kg body weight as oral suspension. Group-III and Group-IV were
treated with ethanol extract of plant. at the oral doses of 250 mg/kg body weight
and 500 mg/kg body weight.
 P a g e | 17

The mice were fed with the samples 1 hour prior to the oral administration of
castor oil at a dose of 0.5ml per mouse. Individual mouse of each group was placed
in separate cage having blotting paper placed in every cage lined with the floor to
examine for the presence of diarrhea every hour for 4 hours after the castor oil
administration.

Number of stools or any fluid material that stained the blotting paper were counted
at each successive hour during the 4 hours period and was noted for each mouse.

The latent period of each mouse was also counted. At the beginning of each hour
new blotting papers were placed for the old ones.

Oral admin. of
sample/ Oral admn.
loperamide castor oil

First
defecation

1 hr. x min 4 hr.

Time allowed
to ensure Latent period Total observn
proper absorption period

Fig. 10.1: Schematic representation of study of anti-diarrheal activity observation in test mice with
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