BIOLOGICAL SCREENING OF HERBAL DRUGS

SEMINAR ON

SCREENING OF ALZHEIMER’S DRUGS

Submitted By:
Inder
makhija
M. Pharm-I
Roll No.
090605015
 Pharmacognos
y
MCOPS,
Manipal

1. INTRODUCTION

First described by German psychiatrist and neuropathologist, Alois Alzheimer in 1906.

Alzheimer’s disease (AD) is the age- related dementia, which does not have any previous
cause such as stroke, brain trauma or alcohol toxicity & also distinct from vascular
dementia which is associated with brain infarction. Alzheimer’s disease (AD) is a steadily
progressive, neuropsychiatric condition that is mainly characterized by cognitive deficit
and debility. There is a disturbance of many higher cortical functions such as memory,
learning, thought, orientation and judgement. Consciousness is not affected.

As it progresses the person may:

- Routinely forget recent events, appointments, names and faces
- Difficulty in understanding what is being said,
In advanced cases people may also:
- Adopt unsettling behaviour
- like getting up in the middle of the night,
- or wandering off from their home and becoming lost.

2. PATHOPHYSIOLOGY
a) Amyloid plaque, consisting of extracellular deposits of β-amyloid protein (Aβ) in
selective areas of brain such as cortex, hippocampus and subcortical nuclei etc.
neurotoxicity in AD results from excessive formation of Aβ. Fig1: Formation of β-amyloid
plaque
Amyloid precursor protein (APP) is the precursor to amyloid plaque.
1. APP sticks through the neuron membrane.
2. Enzymes cut the APP into fragments of protein, including beta-amyloid.
3. Beta-amyloid fragments come together in clumps to form plaques.

b) Neurofibrillary tangles comprise aggregates of a highly phosphoraylated form of a

normal protein (Tau). The relationship of these structures to neurodegradation is not
known.
Neurons have an internal support structure partly made up of microtubules. A protein
called tau helps stabilize microtubules. In AD, tau changes, causing microtubules to
collapse, and tau proteins clump together to form neurofibrillary tangles. Fig 2: Formation
of Neurofibrillary tangles.

The Changing Brain in Alzheimer’s Disease

Fig3: Pet Scan of Normal Brain & Alzheimer’s Disease Brain

Preclinical AD

• Signs of AD are first noticed in the entorhinal cortex, then proceed to the
hippocampus.
• Affected regions begin to shrink as nerve cells die.
• Changes can begin 10-20 years before symptoms appear.
• Memory loss is the first sign of AD.

Mild to Moderate AD

• AD spreads through the brain. The cerebral cortex begins to shrink as more and more
neurons stop working and die.
• Mild AD signs can include memory loss, confusion, trouble handling money, poor
judgment, mood changes, and increased anxiety.
 • Moderate AD signs can include increased memory loss and confusion, problems
recognizing people, difficulty with language and thoughts, restlessness, agitation,
wandering, and repetitive statements.

Severe AD

• In severe AD, extreme shrinkage occurs in the brain. Patients are completely
dependent on others for care.
• Symptoms can include weight loss, seizures, skin infections, groaning, moaning, or
grunting, increased sleeping, loss of bladder and bowel control.

3) AD Research: the Search for Causes

• AD develops when genetic, lifestyle, and environmental factors work together to cause the
disease process to start.
• In recent years, scientists have discovered genetic links to AD. They are also investigating
other factors that may play a role in causing AD. NIA-funded Alzheimer’s Disease Centers
(ADCs) across the country are leading the research efforts looking into causes, diagnosis,
and treatment of AD.

Genetic Studies

• The two main types of AD are early-onset and late-onset: Early-onset AD is rare, usually
affecting people aged 30 to 60 and usually running in families. Researchers have identified
mutations in three genes that cause early-onset AD. Late-onset AD is more common. It
usually affects people over age 65. Researchers have identified a gene that produces a
protein called apolipoprotein E (ApoE). Scientists believe this protein is involved in the
formation of beta-amyloid plaques.

Studies at the Cellular and Molecular Level

• Oxidative damage from free radical molecules can injure neurons.
• Homocysteine, an amino acid, is a risk factor for heart disease. A study shows that an
elevated level of homocysteine is associated with increased risk of AD.
• Scientists are also looking at inflammation in certain regions of the brain and strokes as risk
factors for AD.

In vitro methods

1) In vitro inhibition of acetylcholine-esterase activity in rat striatum

Purpose and Rationale

The purpose of this assay is to screen drugs for inhibition of acetylcholine-esterase activity.
Inhibitors of this enzyme may be useful for the treatment of Alzheimer’s disease.
Procedure
Reagents
1. 0.05 M Phosphate buffer, pH 7.2
2. Substrate in buffer
3. DTNB in buffer
4. Stock solution of the test drug

Tissue preparation

Male Wistar rats are decapitated, brains rapidly removed, corpora striata dissected free,
weighed and homogenized in 0.05 M NaH2PO4, pH 7.2.
A 25 l aliquot of this suspension is added to 1 ml of the vehicle or various concentrations
of the test drug and reincubated for 10 min at 37 °C.

Assay

Enzyme activity is measured with the Beckman DU-50 spectrophotometer. This method can
be used for IC50 determinations and for measuring kinetic constants.
Reagents are added to the blank and sample cuvettes as follows:
Blank: PO4 buffer/DTNB/Substrate
Control: PO4 buffer/DTNB/Substrate/Enzyme
Drug: PO4 buffer/DTNB/Substrate/Enzyme/Drug
Blank values are determined for each run to control for non-enzymatic hydrolysis of
substrate.
Evaluation: The percent inhibition at each dose is calculated by comparison with the
enzyme activity of the vehicle control group.

2) In vitro inhibition of butyrylcholine-esterase activity in human serum

Purpose and Rationale

This assay can be used in conjunction with the acetylcholine-esterase assay to determine
the enzyme selectivity of various cholinesterase inhibitors.
Procedure
Reagents
1. 0.05 M phosphate buffer, pH 7.2
2. Substrate in buffer
3. DTNB in buffer
4. Stock solution of the test drug

Enzyme Preparation
A vial of lyophilized human serum is reconstituted in 3 ml of distilled water. A 25 ml aliquot of
this suspension is added to 1 ml of the vehicle or various concentrations of the test drug and
pre-incubated for 10 min at 37 °C.

Assay
Enzyme activity is measured with the Beckman DU-50 spectrophotometer. This method can
be used for IC50 determinations and for measuring kinetic constants.
Reagents are added to the blank and sample cuvettes as follows:
Blank: PO4 buffer/DTNB/Substrate
Control: PO4 buffer/DTNB/Substrate/Enzyme
Drug: PO4 buffer/DTNB/Substrate/Enzyme/Drug
Blank values are determined for each run to control for non-enzymatic hydrolysis of
substrate.

Evaluation: The percent inhibition at each dose is calculated by comparison with the
enzyme activity of the vehicle control group.

3. ex vivo cholinesterase inhibition

Purpose and Rationale

This assay is used to determine the dose-response relationship and duration of action of
cholinesterase inhibitors in vivo.
Procedure
Reagents
1. 0.05 M Phosphate buffer, pH 7.2
2. DTNB in buffer
3. Substrate in buffer
Drug treatment
Groups of four male Wistar rats are dosed i.p. or p.o. with vehicle or the test drug. For the
initial dose response study, the rats are given varying doses of drug based on toxicity criteria
and sacrificed at either 30 min or 1 h after dosing. The animals are observed and the
occurrence of cholinergic signs is noted (piloerection, tremors, convulsions, salivation,
diarrhoea).
For the Time-course study, a dose of the test drug is given which gave significant inhibition
of cholinesterase activity.

Tissue preparation
Male Wistar rats are decapitated, brains rapidly removed, corpora striata dissected free,
weighed and ho-mogenized in 0.05 M phosphate buffer, pH 7.2 .a 12.5 ml aliquot of the
homogenate is added to 1 ml 0.05 M phosphate buffer, pH 7.2/DTNB (reagent 2).

Assay
Enzyme activity is measured with the Beckman DU-50 spectrophotometer.
Reagents are added to the blank and sample cuvettes as follows:
Blank: PO4 buffer/DTNB (reagent 2)/Substrate (reagent 3)
Control: PO4 buffer/DTNB/Substrate/Enzyme from control animal
Drug: PO4 buffer/DTNB/Substrate/Enzyme from treated animal/Drug
Blank values are determined for each run to control for non-enzymatic hydrolysis of
substrate

Evaluation
The percent inhibition at each dose or time is calculated by comparison with the enzyme
activity of the vehicle control group.

In vivo methods

1. Scopolamine-induced amnesia in mice

Purpose and Rationale

The scopolamine amnesia test is widely used as primary screening test for anti-Alzheimer
drugs.The administration of the antimuscarinic agent scopolamine to young human
volunteers produces transient memory deficits. Analogously, scopolamine has been shown
to impair memory retention when given to mice shortly before training in a dark avoidance
task.
The ability of a range of different cholinergic agonist drugs to reverse the amnesic effects of
scopolamine is now well documented in animals and human volunteers.

Procedure
• The scopolamine test is performed in groups of 10 male NMRI mice weighing 26–32
g in a one-trial, passive avoidance paradigm. Five min after i.p. administration of 3
mg/kg scopolamine hydro bromide, each mouse is individually placed in the bright
part of a two-chambered apparatus for training.
 • After a brief orientation period, the mouse enters the second, darker chamber. Once
inside the second chamber, the door is closed which prevents the mouse from
escaping, and a 1 mA, 1-s foot shock is applied through the grid floor.
• The mouse is then returned to the home cage. Twenty four hours later, testing is
performed by placing the animal again in the bright chamber. The latency in entering
the second darker chamber within a 5 min test session is measured electronically.
• Whereas untreated control animals enter the darker chamber in the second trial with
a latency of about 250 s, treatment with scopolamine reduces the latency to 50 s.
• The test compounds are administered 90 min before training.
• A prolonged latency indicates that the animal remembers that it has been punished
and, therefore, does avoid the darker chamber.

Evaluation
Using various doses latencies after treatment with test compounds are expressed as
percentage of latencies in mice treated with scopolamine only.

2 Step-through passive avoidance method

Purpose and Rationale

This test uses normal behavior of mice and rats. These animals avoid bright light and prefer
dim illumination. When placed into a brightly illuminated space connected to a dark
enclosure, they rapidly enter the dark compartment and remain there.

Procedure
Amnesia can be produced by electroconvulsive shock, scopolamine, alcohol & co2.
Mice and rats of either sex are used. The test apparatus consists of a small chamber
connected to a larger dark chamber via a guillotine door.
• The small chamber is illuminated with a 7 W/12 V bulb. The test animals are given an
acquisition trial followed by a retention trial 24 h later.
• In the acquisition trial the animal is placed in the illuminated compartment at a
maximal distance
• From the guillotine door, and the latency to enter the dark compartment is measured.
• Animals that do not step through the door within a cut-off time: 90 s (mice) or 180 s
(rats) are not used. Immediately after the animal enters the dark compartment, the
door is shut automatically and an unavoidable footshock is delivered.
• The animal is then quickly removed (within 10 s) from the apparatus and put back into
its home cage.
• The test procedure is repeated with or without drug.

Evaluation
• The time to step-through during the learning phase is measured and the time during
the retention test is measured.
• In this test a prolongation of the step-through latencies is specific to the experimental
situation. An increase of the step-through latency is defined as learning.

3. Step-down passive avoidance method

Purpose and Rationale

An animal (mouse or rat) in an open field spends most of the time close to the walls and in
the corners. When placed on an elevated platform in the center of a rectangular
compartment, it steps down almost immediately to the floor to explore the enclosure and to
approach the wall.

Procedure
• Mice or rats of either sex are used. A rectangular box with electrifiable grid floor and
35 cm fits over the block. The grid floor is connected to a shock device which delivers
scrambled foot shocks.
• The actual experiments can be performed in different ways.

A typical paradigm consists of three phases:

(1.) Familiarization: The animal is placed on the platform, released after raising the cylinder,
and the latency to descend is measured. After 10 s of exploration, it is returned to the home
cage.

(2.) Learning: Immediately after the animal has descended from the platform an unavoidable
footshock is applied and the animal is returned to the home cage,

(3.) Retention Test: 24 h after the learning trial the animal is again placed on the platform
and the step-down latency is measured.The test procedure is repeated with or without drug.
The test is finished when the animal steps down or remains on the platform (cut-off time: 60
s).

Evaluation
The time of descent during the learning phase and the time during the retention test are
measured. A prolongation of the step-down latency is defined as learning.

References:

1) Vogel H. Gerhard, Drug Discovery and Evaluation, Pharmacological assays, second

edition, Springer, page no. 595-644.
2) Rang H. P., Dale M. M., Ritter J. M., Moore P. K.; “Pharmacology”; fifth edition; Churchill
Livingstone publication; page no. 446-455, 494-496.
3) Satoskar R. S., Bhandarkar S. D., Rege Nirmala N.; PHARMACOLOGY &
PHARMACOTHERAPEUTICS; nineteenth edition; popular prakashan; page no. 978-992,
223.
4) Sharma HL, Sharma KK, Principles of PHARMACOLOGY, first edition, Paras publication,
Delhi, page no. 551-552.
5) Dissertation work by Rashmi G. Prabhu on Pharmacognostical, Phytochemical and
Antiosteoporotic activity of Wedelia caladulacea less.
6) Dissertation work by Saleemulla Khan on Pharmacognostical, Phytochemical and
Antiosteoporotic activity of Ciassus quadrangularis linn.