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12
Pharmacological Screening of Drugs from Natural Sources
Jayesh D. Kadam1, Adaeze L. Onugwu2, Yogesh A. Kulkarni
1
Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKM’s Narsee Monjee Institute of Management Studies (NMIMS) Deemed to be University,
Mumbai, India
2
Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Nigeria Nsukka, Enugu State, Nigeria
1
12.1 Introduction
Herbal drugs have been used for millions of years for the
treatment and management of diseases all over the world
[1]. The plant kingdom contains a wide variety of compounds with varied structures. It also serves as a source of
novel chemical compounds that may be valuable due to
their pharmacological properties [2–5]. “Phytotherapy” is
the study of herbal plants for their use in treating particu
lar diseases. Standardization of plant extracts and pharmacological evaluation of these extracts is a crucial component
of phytotherapy. Plants synthesize a variety of secondary
metabolites, such as alkaloids, flavonoids, saponins, terpenoids, steroids, glycosides, tannins, and volatile oils, which
have significant therapeutic value [6].
Utilizing these compounds has lowered the chance of
developing various illnesses in humans, including diabetes,
cancer, cardiovascular disease, hepatitis, and brain disorders
(Figure 12.1). Furthermore, plants and natural compounds
possess various other pharmacological properties, such as
antioxidant, antiulcer, antiviral, expectorant, antibacterial,
anti-hemorrhagic, anti-inflammatory, astringent, antican
cer, antidiabetic, hemostatic, antimalarial, antiphlogistic,
analgesic, and antiparasitic properties [7–9]. This chapter
describes the screening methods for plant extracts and natural products to determine their pharmacological activity.
-
-
12.2 Pharmacological Approaches
There are three main approaches for screening of herbal
drugs from natural sources:
1. In vivo methods that employ various animal models.
2. Cell line studies that employ cultures of animal cells.
3. Clinical methods that involve an extract of a plant
with a long history of conventional use as a clinical
assay rather than a clinical evaluation of a pharmaco
logically defined compound, following appropriate
toxicological investigation [10].
12.2.1 Discovery of Biologically Active Compounds
The following process is typically followed to identify active
compounds from any medicinal plant (Figure 12.2) [11].
12.2.2 Pharmacological Screening Methods
12.2.2.1 In vivo Models
Drug effects on human diseases or organs are evaluated
using in vivo models. They are a safe way to carry out res
earch before a drug goes to clinica l trials on humans [12].
-
236 12 Pharmacological Screening of Drugs from Natural Sources
Digestive Disorders
Cardiovascular Disorders
Reproductive Disorders
Figure 12.1 Herbal drugs used in various disorders.
Gathering all Ethnobotanical data
Identifying the plants under investigation botanically
Preparing extracts
Testing plant extracts for pharmacological activity
Bioactivity-directed fractionation
The bioactive constituents pharmacological assessment
The bioactive constituents structural elucidation
Structural changes and synthesis
Herbal Drugs
approaches to enhance these diseases’ diagnosis and
treatment. Numerous models, including atherothrombotic and cardiac disorders, have been established to
address cardiovascular difficulties. The same pathology
has been effectively replicated in other species, including
small and large animal disease models. Animals, such as
rats, mice, rabbits, guinea pigs, and dogs are used to
screen cardiovascular diseases, such as aortic aneurysm,
arrhythmia, atrial fibrillation, cardiac arrest, cardiomyopathy, congenital heart defects, heart attack, heart failure,
heart murmurs, heart valve problems and disease, high
blood pressure, infective endocarditis, Kawasaki disease,
metabolic syndrome, myocarditis, pericarditis, peripheral
artery disease, stroke, and venous thromboembolism.
1. Fructose-induced Hypertension in Rats
Endocrine Disorders
Renal Disorders
CNS Disorders
Toxicological study of bioactive components
Clinical assessment for efcacy
Figure 12.2 Steps in the evaluation of the biological activity of
plant chemicals.
12.2.2.1.1 Screening Models for Cardiovascular System Diseases
In developed countries, cardiovascular diseases constitute the primary cause of mortality and morbidity.
The animal model use has enabled us to understand the
pathophysiology and led to the development of novel
In experimental rats, blood pressure can rise in response to
an increase in dietary carbohydrate intake [13, 14]. It has
been found that consuming more glucose or sucrose can
accelerate the development of spontaneous hypertension
in rats (Figure 12.3).
Procedure:
1. Healthy adult rats weighing 210–250 g are used.
2. A sufficient number of male rats per group is used.
Rats are housed in two rats in each cage with an unrestricted supply of laboratory fluid and diet.
3. A 10% fructose solution is used to replace the drinking
water for 12 weeks.
12.2 Pharmacological Approaches 237
4. During the treatment, the fluid, food, and body weights
of the rats are measured weekly.
5. Before starting treatment and each week after, using the
tail-cuff method, pulse rate and blood pressure can be
measured.
6. Blood is withdrawn to measure triglycerides, insulin,
and plasma glucose levels before beginning treatment
and every two weeks thereafter.
Evaluation: Recording of electrocardiograms (ECGs) with
data acquisition system, determination of inflammatory
biomarkers like TNF-α, IL-6, etc. determination of oxidative
biomarkers like glutathione reductase, superoxide dismutase, catalase, and histopathological analysis of the kidneys, heart, and aorta can be performed (Figure 12.4) [15].
2. Coronary Ligation-induced Myocardial Infarc-
tion in Rats
Left coronary artery ligation is the most used method that
causes clinical conditions similar to human myocardial
infarction (MI), where the heart’s inability to maintain normal function is due to an insufficient blood supply [14, 16].
The heart undergoes morphological and electrocardiographic changes with time, confirmed by myocardial
infarction and ST-segment elevation. Decrease in the pump
function and the dilation of the left ventricle occur in rats
when the left coronary artery ligation is done. This model
is used in the evaluation of drugs.
High fructose diet
Insulin resistance
Hyperinsulinemia
Sympathetic nervous system activity
Catecholamine
Vasoconstriction
Hypertension
Figure 12.3 Development of hypertension following a high
fructose diet.
Endothelial Dysfunction
Procedure:
Part I
1. Male rats weighing 200–300 g are used and anesthe-
1
tized with a suitable anesthetic agent. (60 mg kg
i.p.
pentobarbital, etc.)
2. A left thoracotomy is used to open the chest, and a
thread (Prolene® 6/0 thread, etc.) is placed across the
center of the wound’s lateral border and passed
through the left shoulder muscle tunnel to the half of
the cranial incision. Applying pressure to the abdomen
causes the heart to externalize softly.
3. Ligation of the left coronary artery is done, and it is
tightened near its origin.
4. The heart is repositioned within seconds, and the chest
wall is closed by tightening the thread ends, enabling
the rat to breathe spontaneously.
5. The rat does not require mechanical breathing.
6. The evaluation of the drug can be done by administer-
ing the rats with subcutaneous injection (propranolol
1
5 mg kg
) after five minutes and after 24 h post-
occlusion.
Part II
1. Forty-eight hours post-surgery, rats are anesthetized
with a suitable anesthetic agent, and the cannulation
of the right carotid artery is done using a catheter,
which is connected to a pressure transducer when connected to the data acquisition system.
2. After that, the fluid-filled catheter is pushed into the left
ventricle through the aortic valve in order to measure the
ventricular end-diastolic pressure and systolic pressure.
3. After assessing the cardiac output, arterial pressure,
etc. 2 mL potassium chloride (2.5 ) is injected to stop
the activity of the heart.
4. To preserve a full diastole, the heart is separated and
washed with 300 m KCl.
5. A double-lumen catheter is placed through the ascend-
ing aorta into the left ventricle, the right ventricle is
opened, and the left and right atria are tied with a ligature. Via the smaller of the two catheter lumens, a cryostatic freeze medium is pumped into the left ventricular
chamber. After that, this chamber is connected to a
hydrostatic pressure reservoir, the level is maintained
to equal the end-diastolic pressure.
0.6
0.5
0.4
0.3
0.2
0.1
0.0
–0.1
–0.2
Figure 12.4 Normal ECG of the rat.
238 12 Pharmacological Screening of Drugs from Natural Sources
6. To allow the fluid in each of the two lumens to equili-
brate, the bigger lumen is lifted to the same level as the
smaller one.
7. Using dry ice and hexane, the heart freezes quickly.
8. Using a cryostat, the heart tissue is cut into transverse
sections (40 µm-thick) oriented perpendicular to the
longitudinal axis and extending from the apex to the
base of the heart. Each heart is cut into eight sections,
which are then collected on glass slides covered with
gelatin at a predetermined spacing. After air drying,
the sections are treated for 30 minutes at 25 C with
490 µ nitro blue tetrazolium and 50 m succinic acid
in 0.2 phosphate buffer (pH 7.6). After that, they are
rinsed with chilled distilled water, dehydrated with
95% ethyl alcohol, cleaned with xylene, and mounted
using a synthetic glue medium. Necrotic tissue is
unstained, while viable tissue is dark blue.
Evaluation: Recording of ECGs with data acquisition system, determination of inflammatory biomarkers like TNFα, IL-6, etc. determination of oxidative biomarkers like
glutathione reductase, superoxide dismutase, catalase, and
histopathological analysis of kidneys, heart, and aorta can
be performed. Also, the thickness of an infarct can be
stated as a percent of the non-infarcted wall thickness of
the ventricle, and its size is calculated using planimetry
and evaluated as a percentage of the area of the left ventricle. A planimeter is a tool used for measuring the area of an
organ in an arbitrary two-dimensional shape.
3. DOCA-salt-induced Hypertension
TNF-α, IL-6, etc. determination of oxidative biomarkers
like glutathione reductase, superoxide dismutase, catalase,
and histopathological analysis of the kidneys, heart, and
aorta can be performed.
4. Isoprenaline-induced Myocardial Infraction
The synthetic nonselective β adrenoceptor agonist isopren-
aline (ISO) has been demonstrated to induce myocardial
infarction in rats when given at high dosages [18]. It disturbs the balance between producing free radicals and antioxidant defense systems.
Procedure:
1. The rats are divided into groups randomly, with 6–8
rats per group.
2. Rats are given a subcutaneous injection of ISO daily
for 10 days.
3. On day 11, the blood is withdrawn, and the rats are
sacrificed.
4. Heart tissue is isolated, washed with cold saline, and
dried.
5. The heart tissue is promptly cryopreserved for analysis
in liquid nitrogen and kept at 80 °C.
Evaluation: Recording ECGs with data acquisition system, determination of inflammatory biomarkers like TNFα, IL-6, etc. determination of oxidative biomarkers like
glutathione reductase, superoxide dismutase, catalase, and
histopathological analysis of the kidneys, heart, and aorta
can be performed. Other evaluation parameters are enzyme
markers like CK-MB, CPK, etc.
A synthetic mineralocorticoid derivative called deoxycorticosterone acetate (DOCA), together with a diet high in salt
is administered to the animals after surgically removing
one of their kidneys [17]. Rats develop hypertension with
cardiovascular remodeling that is similar to hypertension
caused by volume overload in humans. The condition can
also lead to endothelial dysfunction, hypertrophy, fibrosis,
and aberrant conduction.
Procedure:
1. Using a suitable anesthetic agent, male rats weighing
250–300 g are anesthetized.
2. The left kidney is removed by making a flank incision.
3. For four weeks, the rats receive two weekly DOCA
1
injections (15 mg kg
) in olive oil subcutaneously.
4. One percent NaCl solution is used instead of drinking
water.
5. After one week, blood pressure increases, and after four
weeks, it reaches systolic values of 160–180 mm Hg.
Evaluation: Recording of ECGs with the data acquisition
system, determination of inflammatory biomarkers like
12.2.2.1.2 Screening Models for Nervous System Diseases
Preclinical research on possible treatments and fundamental scientific investigations of disease causes are based on
animal models of human illness. Animal modeling has
made rapid progress in understanding the basic disease
mechanisms of many CNS diseases, including motor and
non-motor pathologies, initial cell death, subsequent repair
in stroke, Parkinson’s disease, axonal regeneration in optic
and peripheral nerve injury, etc. Animals, such as rats,
mice, and monkeys are used to screen for neurological
diseases.
5. Yohimbine-induced Convulsions in Mice
The potentiation of lethality in mice by yohimbine has
been utilized as a model to anticipate the effects of antidepressants. Yohimbine causes clonic convulsions before
death or at sublethal levels [19]. Antagonism against the
yohimbine-induced convulsions in mice serves as a
screening perspective for GABA-mimetic and anxiolytic
drugs.
12.2 Pharmacological Approaches 239
Procedure:
1. Male mice weighing 20–30 g are used and kept in
transparent plastic cages individually.
1
2. Yohimbine hydrochloride (45 mg kg
, s.c.) is administered, and animals are then observed in the beginning
and the frequency of seizures for one hour.
3. The drug is administered 30 minutes before adminis-
tering yohimbine hydrochloride.
Evaluation: The three observation periods (0: Absent, 1:
Slight, 2: Medium, and 3: Severe) summarize the convulsion scores for all mice in each group. The ratio of the treatment group scores to the vehicle group is calculated as a
percentage.
6. Scopolamine-induced Amnesia in Mice
Amnesia is the most prevalent type of dementia, which has
affected more than 44 million people globally [14]. It has
been demonstrated that scopolamine causes memory
impairment when administered to mice just before training in the dark avoidance test. Scopolamine, a tropane
alkaloid with muscarinic antagonistic effects when administered to rodents, causes central cholinergic blocking and
results in a well-documented, reversible deficit in the
maintenance of attention, information processing, and
learning new information.
Procedure:
1. In a single trial, passive avoidance paradigm, 10 male
mice weighing 25–30 g are used for the scopolamine
test.
2. After receiving an intraperitoneal injection of scopola-
mine hydrobromide, every animal is placed in the
bright section of the two-chambered training apparatus individually for five minutes.
3. Following a brief moment for orienting, the mouse
moves into the second, darker chamber. The door is
closed as the mouse enters the second chamber, and a
1 mA, 1-s shock is given to the foot through the grid
floor.
4. After that, the mouse is put back in its cage. The ani-
mal is tested again by placing it in the bright chamber
24 hours later.
5. An electronic timer assesses the delay in accessing the
second, darker chamber during a five-minute test session. In the second trial, untreated control animals
exhibit a latency of approximately 250 seconds to enter
the dark chamber; however, scopolamine treatment
reduces this latency to 50 seconds.
6. Ninety minutes before training, the herbal extract is
administered to the animals. An extended period of
inactivity suggests that the animal is aware of its punishment and does not avoid the dark chamber.
Evaluation: The efficacy of herbal drugs is evaluated
through behavioral tests like Y-maze to assess short-term
memory and Morris water maze tests to study spatial memory and learning. The acetylcholine (Ach) esterase level
and oxidative biomarkers are also evaluated [20].
7. Tremorine and Oxotremorine Antagonism
Tremor, ataxia, spasticity, salivation, lacrimation, and
hypothermia are parkinsonism-like symptoms brought on
by muscarinic agonists, oxotremorine and tremorine [14].
Anticholinergic drugs are antagonistic to these symptoms
and are used for the screening of anti-Parkinson medicines.
Procedure:
1. Male mice weighing 18–22 g are divided into groups
randomly. The herbal extract or the standard benztro-
1
pine mesylate (5 mg kg
) is orally administered to
them one hour before the 0.5 mg kg1 oxotremorine,
s.c. is administered.
2. Temperature is recorded through the rectal route at
one, two, and three hours following the injection of
oxotremorine and before the herbal extract administration (base value). After taking oxotremorine, tremors are scored in 10-second intervals every 15 minutes
for one hour.
3. Tremor is scored as 0: Absent, 1: Slight, 2: Medium,
3: Severe.
4. After 15 and 30 minutes of oxotremorine injection, lac-
rimation and salivation are evaluated and are scored as
0 = Absent, 1 = Slight, 2 = Medium, and 3 = Severe.
Evaluation: The body temperature differences after one,
two, and three hours are summarized against basal values
for each mouse in both groups and are compared using
statistics.
The ratio of the treatment group scores to the control
group is evaluated as a percentage.
The scores for salivation and lacrimation symptoms are
summarized for all mice per group. The ratio of the treatment group scores to the control group is evaluated as a
percentage.
8. Aluminum Chloride (AlCl
)-induced Alzheimer’s
3
Disease in Rats
Alzheimer’s disease (AD) is a progressive neurological
dysfunctional disorder primarily affecting speech, mobility, memory, and cognitive function [21]. The AD hallmark is the degeneration of neurons in the brain’s
amygdala, hippocampus, cerebral cortex, and basal ganglia. This results in decreased neurotransmitter production, Ach secretion, and amyloid beta (Aβ) deposition,
which causes dementia.
240 12 Pharmacological Screening of Drugs from Natural Sources
In several animal models, aluminum chloride (AlCl3)
has been widely used to induce dementia. One well-known
neurotoxin that is linked to the abnormal development of
many neurologic conditions is aluminum. Aluminum can
cross-link amyloid β-protein, causing oligomerization that
increases neurotoxicity. Moreover, it has previously been
documented that prolonged exposure to AlCl
may result
3
in dementia in rats.
Procedure:
1. Sprague-Dawley rats of 150–200 g weight are randomly
divided into groups.
2. A 0.1% NaCl solution is administered to the control
group rats.
3. The disease-induced group rats are administered AlCl
(175 mg kg1 oral) for 25 days.
4. From day 25 to 36, the animals are supplemented with
herbal extract and standard compound.
5. Every day, the diet intake and weight fluctuations of
rats are monitored.
6. Behavioral analysis of the rats using behavioral
parameters is done.
7. Blood is withdrawn, and after sacrificing the animals,
the brain samples are isolated for pathological and
biochemical examination.
Evaluation: The elevated plus maze test and open
field maze test are used in behavioral analysis to examine
rats’ capacity for learning and memory. Also, the AchE
level, oxidative biomarkers, inflammatory biomarkers, and
brain histopathological analysis are done.
9. Rotenone-induced Parkinson’s Disease in Rats
PD is the second most common neurodegenerative illness
globally and a movement disorder affecting the central
nervous system (CNS) [22]. Glial cell neuroinflammatory
activation, phosphorylated-alpha synuclein aggregation,
loss of striatal dopamine (DA), and decrease of dopaminergic
neurons within the substantia nigra pars compacta are
among the clinical signs of PD. Rotenone significantly
inhibits the mitochondrial electron transport chain’s
Complex I (NADH: ubiquinone oxidoreductase).
Procedure:
1. Rats weighing 150–200 g are randomly divided into
groups.
1
2. Rotenone (1 mg kg
) is administered to animals
intraperitoneally by making its emulsion in a sterile
oil (sunflower oil) for 60 days.
3. The drugs are given to the animals one hour before
rotenone administration.
4. The animals are sacrificed after the completion of the
protocol, and behavioral, biochemical, and
histopathological analyses are done.
Evaluation: The rotarod test, an object recognition test, is
used in behavioral analysis to examine the capacity of the
animals for muscle strength, learning, and memory. Also,
the alpha-synuclein level, oxidative biomarkers, inflammatory biomarkers, and brain histopathology are done.
10. Chronic Unpredictable Stress-induced Depression
To create depression in an animal model, one experimental
technique known as chronic unpredictive stress (CUS)
exposes animals to a variety of unpredictable stimuli that
lead to depression [23]. The CUS method effectively
changes adult hippocampus neurogenesis and causes
behavioral alterations.
3
Procedure:
1. The following stressors are included in the depression
model, in random sequence, to maximize their unexpected nature: A 30-minute cage rotation, a five-minute forced swimming session, a reversal of the light/
dark cycle, a 40-hour food and drink fasting, and a
five-minute exposure to a 40 °C hot environment are
all included.
2. The animals undergo the CUS technique for five weeks.
The treatment groups are supplemented through herbal
extract and standard drugs for 35 days (five weeks).
3. Except for essential tasks like routine cage cleaning,
the unstressed animals in the control group are left
without exposing them to stress.
Evaluation: The open-field test is used in behavioral
research to examine stress. Also, the monoamine oxidase
level, oxidative stress biomarkers, and inflammatory biomarkers are estimated, and a brain histopathology study is
performed.
12.2.2.1.3 Screening Models for Respiratory System Diseases
Various pathogenic mechanisms are included in the occurrence and progression of respiratory disorders, which have
a complex etiology. Current research results cannot accurately represent the development stage and function in vivo
since the methods have trouble mimicking the disease’s
natural developing state in the body.
1. Acetylcholine and Histamine-induced Broncho
constriction in Guinea Pigs
This is the conventional immunological model of airway
blockage caused by antigens [24]. In guinea pigs, inhaling
histamine or other sympathogens can cause symptoms,
such as asphyctic convulsions that resemble bronchial
asthma. Inhaled histamine and Ach-induced hypoxia and
convulsions in guinea pigs. Muscular smooth muscle
contraction, severe hypotension, and cardiovascular system capillary dilatation are caused by histamine.