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12.2 Pharmacological Approaches 241
Procedure:
1. Ach and histamine are administered as an aerosol
from the built-in nebulizer of the histamine chamber
at a 40 mm/Hg pressure.
2. Histamine and Ach have a notable effect on the guinea
pig, causing severe bronchoconstriction that results in
suffocation and convulsive dyspnea.
3. These symptoms can be postponed using bronchodila-
tors. The amount of time needed for the histamine and
Ach-induced preconvulsive dyspnea (PCD) to manifest is noted for every animal.
4. This paradigm assesses an herbal extract and/or stand-
ard drug bronchodilator efficacy against Ach and histamine-induced guinea pig bronchoconstriction.
Evaluation: The effect of herbal drugs on Ach and
histamine-induced bronchoconstriction is evaluated
through respiratory compliance (the expandability of the
lungs) and conductance (lung volume) [25]. It is calculated as a percentage of preconvulsive time increase compared to control. It is possible to find ED50 values or 50%
increases in preconvulsive times.
12.2.2.1.4 Screening Models for Urinary System Diseases
It is necessary to understand bladder function, which
includes physiology, pathology, and even psychology, to
treat urinary bladder dysfunction. Numerous animal models are available for the investigation of various bladder
conditions. These models range in animal species from
nonhuman primates like rabbits, cats, dogs, pigs, and minipigs to rodents like mice and rats.
1. Acute Renal Failure in Rats by Nephrectomy
Chronic renal failure is a frequent pathological condition
in humans [14]. As described, the animal model is useful
for testing novel diuretics.
Procedure:
1. Rats of 150–200 g weight are anesthetized with a suit-
able anesthetic.
2. The cecum and the small bowel are removed through
a midline (6 cm) abdominal wall incision and placed
on gauze sponges moistened in saline.
3. Then, dissection of the right kidney is done, and the
kidney is removed after the ureteric and vascular pedicles are sutured with silk sutures (2–0).
4. The three main renal arteries are visible after the left
kidney’s renal artery is dissected from the hilum.
5. There is no dissection of the kidney from the perito-
neum. The supplied volume of renal tissue is then
determined by ligating the artery’s anterior caudal
branch.
6. In 10 to 15 seconds, the ischemia-affected region is
defined. If this approximates one-third to one-fourth
of the kidney, permanent ligation is done.
7. After that, the abdomen’s viscera are restored, and the
dissected area is shut with a continuous stitch. With
stainless-steel clamps, the skin is sealed.
8. A retro-orbital puncture is used to collect blood under
anesthesia for the measurement of serum creatinine at
different intervals up to a year. Along with this, urine
samples are taken every 24 hours to test the levels of
creatinine, protein, and specific gravity.
Evaluation: The effect of herbal drugs on acute renal failure is evaluated by lipid peroxidation, malondialdehyde
(MDA) assay, and histopathology of the kidney [26]. After
a year, creatinine clearance falls while serum creatinine
rises to 500 µ/L. Significantly, higher urine volumes are
associated with lower urine-specific gravities, suggesting a
lower concentration capacity. A substantial increase in proteinuria has occurred. After 14–15 months, terminal uremia develops.
2. Allantoxanamide-induced Hypouricemia in Rats
It has been found that potassium oxonate and allantoxanamide are both effective reagents for blocking urate oxidase
(urate oxidase, also known as uricase, is a peroxisomal
enzyme that converts uric acid to allantoin in most mammals [27]. However, humans and some other primates
have lost the enzyme through an unknown mechanism),
but in rats, allantoxanamide has a longer-lasting hyperuricemic impact than potassium oxonate.
Procedure:
1. Male, non-fasting rats weighing 230–280 g are admin-
istered an allantoxanamide intraperitoneal injection
dissolved in sesame oil.
2. The herbal extract and standard allopurinol
1
(50 mg kg
p.o.) is administered.
3. A sufficient number of rats are utilized for every dos-
age of herbal extract and standard medicines. Every
animal is housed in a metabolic cage with unrestricted
food and water access.
4. Collection of urine is done at one, six, 12, and 24 hours.
5. A retroorbital puncture is used to withdraw blood
before two, six, and 24 hours after administering the
compound, and plasma is separated.
6. Determining uric acid in urine (mmol/L) and plasma
(mmol/L) is done using the Uric-aquant® method.
Evaluation: The test group’s mean uric acid excretion levels and the mean plasma uric acid concentrations at various time intervals after six and 24 hours are compared to
the control groups. The urinary proteins, urine volume,
and urine clearance are also evaluated.
242 12 Pharmacological Screening of Drugs from Natural Sources
3. Adenine-induced Chronic Kidney Disease in Rats
Chronic kidney disease (CKD) stands as a leading cause of
mortality globally. Its onset can be attributed to various factors, such as systemic angiopathy resulting from diabetes,
hypertension, congenital disorders, and glomerulonephritis [28]. As CKD progresses, fibrosis of the renal interstitium typically manifests, ultimately culminating in renal
failure characterized by a poor prognosis.
Procedure:
1. Wistar albino rats aged 9–10 weeks, initially weighing
approximately 150–200 g, are used.
2. The control group animals are administered with
saline for five weeks.
3. In the disease group, for five weeks, the rats are fed
with adenine (0.25% w/w in feed).
4. The treatment group receives herbal extract for five
weeks.
5. Animals are euthanized, and parameters are evaluated.
Evaluation: The biochemical parameters, such as NF-κB,
total antioxidant capacity (TAC), MDA, serum creatinine,
serum urea, serum IL-6, and histopathological analysis are
done.
12.2.2.1.5 Screening Models for Musculoskeletal Diseases
Due to the significant social and economic burdens, musculoskeletal conditions, such as osteoporosis, osteoarthritis
(OA), and rheumatoid arthritis (RA) have left society with
significant morbidity and disability rates. In order to comprehend the pathophysiology of musculoskeletal illnesses
and to create efficacious therapeutics, preclinical models
are important.
1. Collagen Type-II-induced Arthritis in Rats
An inflammatory polyarthritis is produced by intradermal
injection of either heterologous or homologous type II collagen in incomplete Freund’s adjuvant in rats [14]. The
presence of collagen-specific antibodies in rheumatic polyarthritis patients raises the possibility that autoimmunity
plays a key role in synovitis and joint degradation pathogenesis. Test chemicals may have immunosuppressive and
anti-inflammatory qualities since the clinical manifestations in rats are similar to those of humans.
Procedure:
1. Nasal septum cartilage is used to make bovine type II
collagen.
2. It is chopped into tiny pieces, frozen in liquid nitrogen,
and then ground in a freezer mill.
3. To extract proteoglycans, pulverize 25 g of cartilage
and mix it in one liter of (0.2 N) NaOH overnight.
4. After centrifugation for 30 minutes at 20 000 g, then
the residue is vacuum-dried, washed with 250 mL of
pure ethanol, and the supernatant is vented.
5. To get a pepsin-to-cartilage of 1 : 10 (w/w), 100 mg of
pepsin is added to 150 mL of acetic acid (0.5 ).
6. Next, 1 g of cartilage is added. After 18 hours of stir-
ring at room temperature, the mixture is centrifuged
for one hour at 20 000 g.
7. The acid-soluble collagen in the supernatant is pre-
cipitated by centrifuging the mixture at 20 000 g for
one hour after adding NaCl to reach a concentration of
0.9 .
8. 100 mL of 1 N NaCl/0.005 Tris-HCl, pH 7.5, is used
to dissolve the precipitate from 1 g of cartilage and
stirred for three days.
9. After dialyzing the solution against 0.02 disodium
hydrogen phosphate (Na
HPO4) at pH 9.4, the precipi-
2
tate is collected by centrifuging it at 30 000 g for one
hour.
10. The pellet is dialyzed against 6 L of 0.01 m acetic acid
after being dissolved in 0.5 m acetic acid and then lyophilized. Unless otherwise specified, all operations are
carried out at 4 °C.
Test Procedure:
1
1. At 4 °C for the whole night, 2.0 mg mL
of collagen is
dissolved in 0.1 acetic acid.
2. This resulting solution is dropwise added to an equiva-
lent volume of cooled incomplete Freund’s adjuvant.
3. Six to 12 rats weighing around 120 gms are used for
each group.
4. On the first day, 0.5 mg of collagen in 0.5 mL is admin-
istered to each rat equally. Two intradermal injections
are administered: one at the nape of the neck and one
at the base of each appendage.
5. The animals get identical booster shots seven days
after vaccination.
6. Only the incomplete Freund’s adjuvant that is diluted
with (0.1 ) acetic acid is given to control animals.
7. On day 20, measurement of both the hind paw’s vol-
ume is done using a plethysmograph.
8. Only animals with paw volumes of 1.8 mL or more are
utilized in subsequent experiments.
9. The animals are given the herbal extract orally once a
day from days 20 to 40. The paw volumes are once
more observed on day 41.
Evaluation: The increase in row volume comparison to
day 20 is computed. The growth is contrasted with that of
animals given regular medication or as controls. On the
other hand, arthritis scores can be calculated as:
0 = Good health, one to two synovial membrane cell
layers, and no inflammatory infiltrates;
12.2 Pharmacological Approaches 243
1 = A synovial membrane with three to five layers, a
minor cellular infiltration into the synovium, and a
low-density exudate in the joint cavity;
2 = Many layers of synovial membranes, improved cell
infiltrations, and higher cell densities in the joints;
3 = Hyperplastic, highly densely packed synovial tissue
engulfing all joint cavities, representing maximally
enlarged inflammation.
2. Ovariectomy-induced Osteoporosis in Female
Rats
The most common health problem in the world linked to a
persistent deterioration of bones is post-menopausal osteoporosis, which affects over two billion individuals globally
[29]. The fragility of the bone rises with the severity of the
illness, increasing the possibility of fractures. Animal models of rats and mice are used for the screening of the agents
for osteoporosis. In rodents, ovariectomy induces oxidant
and estrogen imbalances, ultimately resulting in
osteoporosis.
Procedure:
1. Following a week of acclimation, the female rats (230–
250 g) are randomly split into six groups containing a
sufficient number of animals.
2. Animals are anesthetized with suitable anesthetic
agents.
3. Animals in five other groups undergo ovariectomies,
while Group I is the Sham operation group.
4. A recertified spirit is utilized to clean the surgical
instruments aseptically. Every animal is placed on its
ventral surface to undergo the procedure.
5. After using rectified spirit to clean the fur on the rat’s
abdomen, a blade removes all of the fur.
6. The midline ventral skin incision (bilateral), measur-
ing 3 cm in length and situated almost halfway between
the body’s midline and the base of the tail, is performed before the ovariectomy.
7. Adipose tissue is removed once the peritoneal cavity is
reached to reveal the ovary and the uterine tube, encircled by varying amounts of fat.
8. Before removing the ovary, a ligation with an absorb-
able suture is done around the distal uterine horn to
stop bleeding. The ovary is removed, and the fallopian
tube and uterine horn are disconnected.
9. Rats with sham operations, however, have their ova-
ries exposed but left intact.
10. After the ovaries are removed, the uterine horn is
repositioned into the peritoneal cavity in all ovariectomized groups.
11. Both skin incisions are shut with absorbable sutures,
and the muscle incision is stitched up with absorbable
1
sutures. Amoxicillin (25 mg kg
, i.p.) is administered to
the operated animals once for a duration of five days.
12. Povidone iodine solution is applied topically, and ani-
mals are monitored carefully.
13. After two days of surgery, two rats are housed in each
polyurethane cage for a period of one week to allow
recovery and also observed for coprophagy.
14. They are also observed for normal behavior and then
regrouped in their home cages.
15. After recovery, rats are administered with herbal
extract and standard drugs for 45 days.
16. After terminating the experiment, various biochemi-
cal parameters are performed.
Evaluation: Measurement of the Femur Bone’s Length, Thickness,
and Weight Using a vernier caliper (Sando), the length is
measured as the separation between the greater trochanter
and the medial condyle, and the thickness at the femoral
midshaft is also measured. Afterward, the bones are dried
at 100 ˚C in an oven, and their weights are calculated using
a digital weighing balance.
Body Weight and Uterine Weight Measurements For 105
days, the body weight of every rat in every group is recorded
once a week. At the conclusion of the study, the uterus is
taken out and weighed using a digital weighing balance
(Metler).
Biochemical Markers in Serum The amounts of calcium,
phosphorus, and alkaline 348 phosphatase in the serum
are measured using diagnostic kits.
Bone Mineral Density Analysis Using µ-CT (Tri-foil imag-
ing) with the following parameters, femurs are scanned at
a resolution of 21 µ:
60 kV X-ray voltage and 130 µA electric current. Micro
View v. 2.0 362 Software analyzes bone mineral density,
content, trabecular thickness, and space.
Femur Bone Biomechanical Strength (Force at Rupture) The
rats’ right femurs are used in this experiment. The digital
hardness tester determines the fracture force of the specified femur sample. After isolating the right femur, the tissues around it are cleansed. A digital hardness tester is
used to pressure the new bone until it is shattered, and the
2
reading is recorded in Newtons (kg cm
12.2.2.1.6 Screening Models for Digestive System Diseases
).
Acute and chronic inflammatory bowel disorders significantly and negatively impact the health and well-being of
both nonhuman and human mammals. Comprehending
244 12 Pharmacological Screening of Drugs from Natural Sources
the fundamental causes of inflammatory diseases is
essential to create efficient preventative and therapeutic
plans. Since the causes of inflammatory diseases are complex, it is crucial to employ suitable animal models and
related disease metrics. Research on rats and mice has
focused on the basic physiological mechanisms underlying
cellular adaptation. These mechanisms include the control
of mucosal proliferation, apoptosis, transport, and the
expression of digestive enzymes. Additionally, these mechanisms readily permit genetic or exogenous manipulation
of growth factors and their receptors.
1. Carbon Tetrachloride-induced Liver Fibrosis
in Rats
Carbon tetrachloride causes inflammation and damage
to the liver, which eventually results in the development
of hepatic fibrosis [14]. The fibrotic reaction and connective tissue proliferation are closely linked to the pathological course of liver fibrosis. Rats given tetrachloride
for an extended period develop severe liver function
impairments as well as histologically detectable liver
fibrosis.
Procedure:
1. Groups of 20 female rats weighing between 100 and
150 g are used in the study.
2. For eight weeks, the animals receive carbon tetrachlo-
ride (CCl4) orally twice a week in a solution of 1 : 1
olive oil.
3. Twenty animals are used as controls, which receive
only olive oil; 40–60 animals receive only CCl4, and
groups of 20 animals receive the herbal extract and are
administered twice daily by gavage in different doses
based on actual body weight.
4. Every week, the animal’s weight is monitored.
5. The animals are sacrificed, and evaluations are done.
Histological Analysis:
Three to five liver pieces weighing roughly 1 g are preserved in formalin and Carnoy solution for histological
examination. Each liver is divided into three to five parts,
embedded, sliced, and stained using the azocarmine aniline blue dye (AZAN). The livers are then scored from 0 to
IV to determine whether fibrosis has developed.
Grade 0: hepatic histology is normal.
Grade I: tiny, short connective tissue septa that do not
affect the hepatic lobules’ structural makeup.
Grade II: extensive connective tissue septa penetrating
the parenchyma and flowing together.
The propensity for nodules to form.
Grade III: hepatic lobule structure and nodular altera-
tions to the liver’s architecture are lost.
Grade IV: subdivision of the regenerating lobules, excessive connective tissue production and deposition, and the
development of scars.
2. Pylorus Ligation-induced Peptic Ulcers in Rats
Pylorus ligature that causes stomach ulcers in rats [30].
The buildup of acidic gastric fluid in the stomach causes
the ulceration. Because of mucosal digesting and pyloric
blockage, there is a rise in acid-pepsin buildup that leads to
pylorus-ligation-produced gastric ulcers. When there is
damage, mucus is released in a large volume, which facilitates repair. Stomach acid production is thought to be
increased in the hypersecretion model of pylorus ligation
by stimulating pressure receptors present in the gastric
mucosa, which activates the vagus-vagal reflux mechanism. The gastric ulcers in animals with pyloric ligation are
thought to be caused by stress-induced gastric HCl secretion and acid stasis elevation. The secretion volume also
plays a significant role in ulcer formation, as it exposes the
stomach’s exposed lumen to building up acid.
Evaluation: The unpaired t-test is used to identify signifi-
cant differences. The Chi-squared test is used to compare
the scores obtained from the histopathological evaluation.
The parameters observed in the serum are:
Procollagen III N-peptide, aspartate aminotransferase,
total bilirubin, alanine aminotransferase, total bile acids,
alkaline phosphatase, and the 7S segment of type IV
collagen.
The organs listed below are prepared for the hydroxyproline measurement:
The tendons in the tail, aortic wall, kidneys, and liver.
The weight of the organ specimens is recorded and thoroughly hydrolyzed in 6 N HCl. Using HPLC to measure it,
hydroxyproline is expressed in mg/mg of the organs’
weight (wet weight).
Procedure:
1. The rats are randomly grouped.
2. The vehicle control group is given a suspension of 1 or
1
0.5% carboxymethyl cellulose (CMC) in 10 mL kg
of
distilled water.
3. The standard group is given omeprazole (8 mg kg
1
oral). The herbal extract is given to other treatment
groups.
4. One hour following treatment, the pylorus is ligated.
5. The rats are euthanized, and the stomach is isolated
for six hours following the ligation.
6. The supernatant is quantified after collecting and cen-
trifuging the stomach contents.
7. The measurement and scoring of the stomach mucosal
ulcer are done.
12.2 Pharmacological Approaches 245
8. Estimates are made for the ulcer index, the ulcerated
surface percentage, and the rate of inhibition.
9. A pH meter is used to titrate against a 0.01 N solution
of NaOH to determine the hydrogen ion concentration
of 1 mL of the entire centrifuged stomach contents
from each rat.
10. The readings are taken in triplicate.
Evaluation: The ulcer number is noted, and the severity is
assessed with the following scores:
0: no ulcer; 1: superficial ulcers; 2: deep ulcers;
3: perforation.
The ulcer index (UI) is calculated as:
UI = UN + US + UP × 10
UN = The mean quantity of ulcers in each animal; US =
–1
The average of the severity score; and UP = The proportion of ulcer-ridden animals.
Animals receiving treatment have ulcer index and gastric
content acidity compared to controls. Dose-response curves
for stomach acid secretion and ulcer formation can be
generated using different dosages. Using probit analysis,
ID50 values can be computed, where 0% denotes no stimulated stomach acid output and 100% denotes maximal
stimulation.
12.2.2.1.7 Screening Models for Metabolic Diseases
A disease that adversely affects how the body processes and
distributes macronutrients like carbs, proteins, and lipids
is known as a metabolic disorder. When aberrant chemical
interactions within the body change the regular metabolic
process, metabolic diseases may result. Metabolic problems can manifest as tiredness, weight loss, jaundice, and
seizures, among other symptoms. The symptoms displayed
would depend on the type of metabolic disease. Acute,
gradual, late-onset, general, and persistent symptoms are
the types of symptoms. Globally, metabolic illnesses like
cancer, diabetes type 2, obesity, and polycystic ovarian syndrome are common.
1. Streptozotocin-induced Diabetes in Rats
Globally, diabetes is a chronic disorder that is rather prevalent [31]. Globally, around 150 million individuals have
diabetes, according to figures from the World Health
Organization, and humankind has not yet found a solution
to this issue. Streptozotocin is frequently used to induce
experimental diabetes in mice because it specifically
destroys beta cells.
Procedure:
1. To induce diabetes, 250–300 g of weight Sprague-
Dawley rats are used.
2. Animals are randomly grouped, and intravenous
streptozotocin injections at a concentration of body
weight are given to the animals.
3. Streptozotocin should be weighed and processed in a
dark environment to prevent light exposure.
4. Streptozotocin kills beta cells to cause diabetes in three
days.
5. Individual and separate metabolic cages with feeding
and metabolism controls should be used to house diabetic animals and a control group of nondiabetic
animals.
6. Rats with diabetes have higher blood glucose levels
1
>250 mg dl
than the nondiabetic control group.
Evaluation: Urine volume should be assessed daily in
terms of mL, food consumption should be measured in
grams, and water intake should be measured in milliliters.
C-peptide, NF-κB, insulin, AST, ALT, and glucose concentrations in serum are also monitored every two to four
weeks for 80 days to confirm diabetes in rats given streptozotocin injections. Histopathological evaluation of the pancreas is used to check the drug’s pharmacological effect.
2. Letrozole-induced Polycystic Ovarian Syndrome
in Female Rats (Figure 12.5)
In women of reproductive age, polycystic ovarian syndrome, or PCOS, is a common, intricate, and varied
Letrozole Induction
Figure 12.5 Letrozole-induced PCOS.
Rat Ovaries
Polycystic Ovarian Syndrome
246 12 Pharmacological Screening of Drugs from Natural Sources
Normal cells
Cancer cells
NMU Induction
Figure 12.6 NMU-induced breast cancer.
Rats with Breast Cancer
endocrine-metabolic network disorder that accounts for
6–21% of infertility cases [32]. According to the research,
persons with PCOS have a 15-fold higher chance of infertility than those without PCOS. Various chemicals, including
letrozole, testosterone, dihydrotestosterone, and surgical
models like ovariectomy, are used for screening drugs for
PCOS. Letrozole blocks the aromatase enzyme, which leads
to an increase in testosterone and a decrease in the concentration of estradiol.
Procedure:
1. Rats weighing between 150 and 200 g have full access
to water and standard laboratory food.
1
2. The vehicle control group is given 10 mL kg
of 1%
CMC solution orally for 21 days.
3. The disease control group receives oral letrozole for 21
days.
4. A 1% CMC solution is used to dissolve the letrozole.
5. The rats are euthanized on day 22 when the severity of
PCOS is assessed.
6. The PCOS-induced female rats are treated with herbal
extract and standard drug for 14 days.
7. The degree of PCOS is assessed by comparing it with a
vehicle control group.
Evaluation: The herbal extract is evaluated by determining blood glucose level, plasma insulin level, and inflammatory biomarkers (TNF-α, IL-6). Estimation of hormones
like FSH, LH, and estradiol and histopathology of the ovaries to assess the severity of PCOS.
12.2.2.1.8 Screening Models for Cancer
Worldwide, one of the primary reasons for death is cancer,
a group of diseases characterized by unchecked cell proliferation. The inappropriate or selective application of preclinical methods in anticancer drug screening is the cause
of this high attrition rate. Every preclinical screening technique has advantages and disadvantages, including genetically modified mice, in vivo tumor xenograft models, and in
vitro human cancer cell lines [33].
Breast Carcinoma
1. N-nitroso-N-methylurea-induced Breast Cancer
in Female Rats (Figure 12.6)
Breast cancer is the most prevalent cancer diagnosed in
women and the primary cause of cancer-related deaths in
women [34]. It is a heterogeneous illness with a wide range
of pathological, molecular, and clinical characteristics.
Therefore, research is necessary to comprehend the pathophysiology of breast cancer and to find specific therapies for
the disease’s identification and treatment. Various chemicals, including N-nitroso-N-methylurea (NMU),
7,12-Dimethylbenz(a)anthracene (DMBA), cell lineinduced animal models, and radiation-induced models are
used for screening of drugs for breast cancer. DNA alkylation is directly caused by N-nitroso-N-methylurea, which
also interferes with DNA synthesis and repair. It exhibits
the potential to be teratogenic, mutagenic, and carcinogenic
according to the age, dose, rate, and administration route.
Procedure:
1. Female rats of 21 days of age are used and are ran-
domly divided into groups consisting of sufficient
numbers of animals.
2. To prepare the NMU solution, it is dissolved in 0.9%
normal saline. Subsequently, the solution is gently
heated in a water bath and vigorously agitated.
3. Two intraperitoneal injections of the NMU
1
(70 mg kg
) are administered according to the body
weight. The first NMU injection is given, and then the
second injection on alternate days.
4. Twice a week, the rats are weighed and palpated to
check for breast tumors.
5. The growths in the mammary lesions are examined,
and a vernier caliper is used to measure the tumor
diameter.
6. Any disease signs or side effects that could result from
NMU toxicity are also monitored.
7. After four weeks of administration of NMU, the treat-
ment of herbal extract and the standard is started for
four weeks.
12.2 Pharmacological Approaches 247
8. The mammary tissue is isolated from the rats after
they are sacrificed at the end of the study.
Evaluation: Body weight, latency period, tumor volume,
and number of tumors per rat are assessed. To identify the
alterations, inflammatory biomarkers (TNF-α, IL-6) and
hematological analysis are carried out. Breast cancer biomarkers like levels of plasma adiponectin, platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular
endothelial growth factor (VEGF), and proliferating cell
nuclear antigen (PCNA) are evaluated. Histopathology of
the mammary glands is also done.
12.2.2.1.9 Screening Models for Immunomodulatory Diseases
In Western countries, most of the population suffers from
autoimmune illnesses. Type I diabetes, RA, systemic lupus
erythematosus (SLE), and multiple sclerosis are among the
most prevalent of the many that have been described.
1. Acute-systemic Anaphylaxis in Rats
Rats receive adjuvant immunizations with Bordetella pertussis suspension and ovalbumin [14]. The animals are
given an intravenous dose of ovalbumin after 11 days.
Intravenous disodium cromoglycate and corticosteroids
can both reduce the symptoms of shock.
Procedure:
1
1. Highly purified ovalbumin (10 mg kg
) is injected
intraperitoneally (i.m.) into 120 g female SpragueDawley rats.
2. Intraperitoneally injecting 1 mL of B. pertussis suspen-
10
sion (2 × 10
organisms) co-occurs.
3. Basophilic granulocytes and mast cells produce and
cling to IgE antibodies.
4. The mice are given a challenge 11 days later: an intra-
1
venous dose of 25 mg kg
of ovalbumin (highly
purified).
5. As a result, antigen-antibody complexes form on the
basophilic granulocytes and mast cells surface in the
blood and throughout all organs, causing the production of various anaphylaxis mediators rapidly, including prostaglandins, histamine, serotonin, and SRS-A,
as well as shock symptoms and 80% fatality.
6. Corticosteroids, such as dexamethasone 1–10 mg kg
1
s.c. or 30 mg kg1 disodium cromoglycate i.v. Ovalbumin
injection is administered 18 hours before the challenge.
7. For every group, 10 to 20 animals are employed.
Evaluation: The heart rate and the blood pressure are
recorded. The concentration of nitric oxide (NO) and the
accumulation of mast cells and eosinophil in the lung,
small bowel, and spleen are also evaluated [35]. Mortality
rates are calculated, and shock symptoms are graded.
Following therapy, outcomes are contrasted with
untreated controls. Pretreatment with disodium cromoglycate or corticosteroids can lessen shock symptoms
and prevent death. To do statistical calculations, the
χ2-test is employed.
12.2.2.1.10 Screening Models for Ophthalmic Diseases
1. Alpha-chymotrypsin-induced Glaucoma
Alpha-chymotrypsin injections into the eyes caused glaucoma in rabbits [14]. The general mechanism of αchymotrypsin glaucoma is the obstruction of the outflow
channels by zonular fragments. In addition, there aren’t
many reports of animal models that mimic human
glaucoma.
Procedure:
1. To avoid the sudden beginning of inflammation, male
1
rabbits weighing around 2 kg are given 10 mg kg
of
pretreatment intraperitoneal indomethacin.
2. After this, they are given mild anesthesia with pento-
barbital to prevent any nystagmus. A 2% lidocaine is
used topically to anesthetize the right eye.
3. A 30-gauge needle connected to a pressure-tested res-
ervoir to 25 mm Hg cannulates the anterior chamber.
4. Subsequently, a 32-gauge cannula is inserted into the
anterior chamber near the limbus and guided toward
the posterior chamber via the pupil.
5. A 0.5 mL sterile isotonic saline solution containing
150 units of alpha-chymotrypsin through the cannula
is irrigated into the posterior chamber.
6. All injections of enzymes into the corneal stroma are
carefully avoided.
7. Subsequently, both cannulae are extracted with mini-
mal aqueous humor loss. During the first week of the
trials, the eyes are checked daily.
8. After that, they are checked every other day for the
second week and finally once a week for the rest of the
study.
9. A tonometer designed specifically for rabbit eyes
measures intraocular pressure.
10. The herbal extract and standard are used both before
and after surgery through the ocular route.
Evaluation: The effect of herbal drug treatment on alphachymotrypsin-induced glaucoma is evaluated by analyzing
the cornea (degree of opacity), iris, and conjunctivae (redness) [36]. Animals are administered alpha-chymotrypsin,
and those given extra medication have their intraocular
pressure, which is then statistically compared to untreated
controls.
248 12 Pharmacological Screening of Drugs from Natural Sources
2. Scopolamine-induced Dry Eye Disease in Rats
Clinical indications of dry eyes, altered conjunctiva, production of inflammatory cytokines in the conjunctiva and
exorbital lacrimal gland, and changes in the fatty acid composition of the exorbital lacrimal gland are the hallmarks
of the scopolamine-induced model [37].
Procedure:
1. Scopolamine is used for inducing dry eye disease in
animals.
2. The herbal extract is injected subcutaneously into the
animals’ osmotic pump, allowing for the drug’s constant, systemic delivery.
3. In the first trial, scopolamine in three doses is given for
28 days.
4. In a second series of studies, pumps are used to admin-
ister a scopolamine solution at 12.5 mg per day for 1, 2,
3, 7, 10, 17, or 28 days to determine the duration of
inflammation.
5. The control group’s animals are also given anesthesia,
even if pumps are not implanted, and it was confirmed
that outcomes from anesthetized animals are comparable to those from animals injected with 0.9% sodium
chloride-filled pumps as a control.
Evaluation: The assessment of clinical indicators of dry
eyes, changes in the conjunctiva, inflammatory cytokines
expression in the conjunctiva and exorbital lacrimal glands,
and fatty acid composition changes in the exorbital lacrimal glands. Polyunsaturated fatty acids (PUFAs) are recognized to be implicated in inflammatory processes in various
illnesses as precursors of inflammatory mediators.
12.2.2.1.11 Screening Models for Anti-inflammatory Activity
One can assess novel compounds’ anti-inflammatory properties through various preclinical screening techniques.
Phlogistic agents such as egg albumin, carrageenan, kaolin, Brewer’s yeast, croton oil, dextran, cotton wool, and
aerosol cause inflammation and quantify the degree of
reduction in the inciting properties.
1. Croton Oil-induced Ear Edema in Rats and Mice
The primary irritating agents in croton oil are 12-otetracanoilphorbol-13-acetate (TPA) and other phorbol
esters [38]. Platelet activation factors (PAF) and arachidonic acid (AA) are released when TPA stimulates protein
kinase C (PKC), which further triggers other enzymatic
cascades like mitogen-activated protein kinases (MAPK),
and phospholipase A2 (PLA2) and vasodilation, polymorphonuclear leukocyte movement, serotonin and histamine
release, and the modest synthesis of eicosanoids by the
enzymes 5-lipoxygenase (5-LOX) and cyclooxygenase
(COX) are all stimulated by this series of events. The
method is mainly created as a bioassay for simultaneously
evaluating topically administered steroids’ thymolytic and
antiphlogistic properties.
Procedure:
1. 25–35 g adult Swiss albino male mice are utilized.
2. On the right ear’s inner surface, each animal is given
20 µL of freshly prepared croton oil (2.5%, 1 mg/ear)
dissolved in acetone.
3. As a control, the left ear is exposed to the same amount
of acetone.
4. Using a hand-held hair dryer, both ears are quickly
dried.
5. Six hours after the animals are given the croton oil,
they are euthanized by cervical dislocation, and from
each ear, a 6 mm diameter metal punch is used to
remove a disc.
6. The variation in weight between the ear croton oil-
induced and the one vehicle-treated is used to assess
edema.
7. The herbal extract (dissolved in acetone or ethanol) is
administered 30–60 minutes before or 180 minutes
following the administration of croton oil.
Evaluation: The increased weight of the induced ear as a
percentage of the control ear’s weight can be used to calculate the antiphlogistic effect. The contralateral ear’s weight
multiplied by 100 is used to divide the difference between
the two weights. Otherwise, the average results for the
treated and control groups are used to compute the difference between the two ears or excised discs, and statistical
techniques are used to assess the impact. It has been demonstrated that hydrocortisone at concentrations of
1
0.5–1 mg mL
is beneficial.
2. Carrageenan-induced Paw Edema
One of the most widely utilized approaches for screening
anti-inflammatory drug candidates is predicated on the
agents’ capacity to prevent the edema that results from
injecting a phlogistic agent into a rat’s hind paw [39].
Numerous phlogistic substances, or irritants, have been
employed, including formaldehyde, dextran, kaolin, egg
albumin, Brewer’s yeast, and sulfated polysaccharides
like naphthoylheparamine or carrageenan. Paw edema
caused by carrageenan develops in two phases in the natural world. The first stage, which occurs one-to-two hours
after the carrageenan injection, is triggered by the release
of serotonin, bradykinins, and histamine into the injured
tissues around the mast cells. The production of numerous cytokines, such as Interleukin - 1β, Tumor Necrosis
12.2 Pharmacological Approaches 249
Factor - α, Interleukin - 6, Interleukin - 10, and arachidonate metabolites, such as prostaglandins and leukotrienes, is linked to the second phase of the inflammatory
reaction, which occurs three-to-six hours after the carrageenan injection. The volume of the paw injected with
carrageenan is typically evaluated both before and after
the irritant is applied, and the treated animals’ paw volumes are compared with those of the negative controls.
Procedure:
1. Animals are given herbal extract at the proper time
before receiving carrageenan injections, if necessary.
The compound’s pharmacological profile will define
the timing, or time-course studies may be used to
ascertain it.
2. Impacts of herbal extract are typically contrasted with
reference compounds, the pharmacology and MOA of
which are established. Nonsteroidal anti-inflammatory
drugs (NSAIDs) are a good example, as is indometha-
cin (5 mg kg
1 per oral).
3. The preinjection paw’s volume is measured before the
carrageenan injection. Subcutaneous injection of a 1%
carrageenan solution in 0.9% saline (100 µL for rats,
25 µL for mice) is administered subcutaneously into
the plantar region of the left hind paw.
4. The injection site is located in the plantar region’s
center, which is significant because the mouse lacks a
clearly defined region of the pad on the bottom of its
hind paw.
5. When necessary, volumes of the paw’s injected with
carrageenan and the control paw are measured hourly
between 1 and 6 hours and one more at 24 hours.
Evaluation: Plethysmometry is the most practical, quick,
and precise assessment method and the most relevant to
the paw. The plethysmometer, which consists of two vertical Perspex tubes connected by water and filled with water,
monitors changes in paw volume directly. For every period,
the average difference between the control groups and the
treated animals is computed and statistically assessed.
Following the last examination and euthanasia, the paws
of the rats are amputated at the tibiotarsal level.
Then, paws can be utilized for various tests connected
to the inflammatory response. Paw tissue can be homogenized and extracted to measure things like prostaglandin E2, elastase activity, and cyclooxygenases. It can also
be examined using immunohistochemistry or in situ
hybridization to find specific proteins within cells and
measure them, like cytokines, growth factors, and tachykinins. Additionally, edema fluid can be eliminated by
centrifuging the entire paw and measuring particular
chemicals directly.
12.2.2.1.12 Screening Models for Analgesic Activity
For treating many painful illnesses, primarily chronic
pain, the medical community still requires strong, safe
analgesics, notwithstanding recent advancements in pain
therapy. Thousands of people who suffer from extreme
pain, for example, that is brought on by any severe injury or
cancer, are dependent on the current regimes, which
include nonsteroidal anti-inflammatory medications, aspirin, and morphine, which can act peripherally or centrally.
Research has demonstrated that whereas NSAIDs typically
result in gastrointestinal problems, opiates promote physical dependency, tolerance, and addiction. Finding different
ways to manage pain is essential for that. An intriguing
treatment option for opioid abuse and withdrawal may be
herbal therapy.
1. Analgesic Activity Using the Hot Plate Method
(Figure 12.7)
A basic behavioral screen called the hot plate test, developed by Eddy and Leimbach in 1953, is used to estimate the
impact of NCEs on the threshold for pain detection [40].
The principle behind it is that when rodents are placed on
a heated surface, they will first lick their paws to show that
the thermal stimulus is unpleasant, and then they will
make overt attempts to jump or flee the area. The latency to
jumping or licking can be increased (analgesic effect) or
decreased (hyperalgesia impact) by substances that modify
the nociceptive threshold. In this test, analgesics with an
anti-inflammatory character, such as paracetamol, aspirin,
and ibuprofen, are less effective than more potent analgesics, like opioids. Mice and rats have extremely sensitive
paws to heat, even at 38–45 ° temperature. The reactions
include paw withdrawal and paw licking. Peripheral analgesics of the phenyl-acetic acid type or acetylsalicylic acid
typically do not impact these responses, but administering
centrally acting analgesics prolongs the period until these
responses occur.
Hot Plate Test
Figure 12.7 Hot plate test for analgesic activity.
250 12 Pharmacological Screening of Drugs from Natural Sources
Procedure:
1. For every dosage, groups of 10 mice of each sex, weigh-
ing between 18 and 22 g at baseline, are employed.
Rats can also be used.
2. The hot plate is made up of an electrically heated sur-
face (55–56 °C).
3. This could be a heated glass surface or a copper plate.
A stopwatch is used to time how long the animals
jump or lick the hot plate.
4. Before and after the standard or test substance is
administered orally or subcutaneously, the latency is
measured for 20, 60, and 90 minutes. The time points
can also be modified based on a drug.
Evaluation: The t-test can be used to statistically compare
the values obtained from the extension of the latency times
between the control and experimental groups or between
the values obtained before and after the herbal extract is
administered. Alternatively, readings that are 50% or 100%
higher than the pre-administration value can be considered positive, and ED50 values can be computed. The percentage protection after treatment of herbal extract therapy
against thermal pain stimulus is calculated according to
the following formula [41]:
ntage protection against thermal stimulus
(Ta)
Test mean
_______________________________
=
12.2.2.1.13 Screening Models for Antipyretic Activity
− Control mean(Tb)
Control mean(Tb
X 100
)
In the days before antibiotics, antipyretic therapy is crucial.
However, antipyretics are still required to reduce raised
body temperature in cases of acute viral illnesses and protozoal infections, such as malaria. An antipyretic action for
anti-inflammatory drugs is considered a favorable side
effect. To assess these characteristics, lipopolysaccharide or
Brewer’s yeast injections are used to cause fever in rats or
rabbits.
1. Antipyretic Activity Using Brewer’s Yeast in Rats
It is well known that injecting Brewer’s yeast suspension
subcutaneously can cause fever [14]. Brewer’s yeast
induces both prostaglandin and TNF-α production.
Therefore, the test drug’s prevention of a higher amount of
prostaglandin E2 production lowers the raised body temperature and keeps it steady. Antipyretic substances can be
administered to lower the body temperature.
Procedure:
1. Brewer’s yeast is suspended at a concentration of 15%
in 0.9% saline.
2. Six male or female rats weighing between 150 and
200 g are used.
3. The first rectal temperatures are measured by inserting
a thermocouple 2 cm into the rectum.
4. Brewer’s yeast suspension is subcutaneously injected
beneath the rear nape of the neck in the animals to
induce fever.
5. The injection site is rubbed to distribute the suspen-
sion under the skin. The room is maintained between
22 and 24 °C.
6. Food is stopped right away after yeast injection. The
increase in rectal temperature is noted 18 hours after
the challenge. After 30 minutes, the measurement is
done again.
7. The test is only conducted on animals whose body
temperatures are at least 38 °C. The test substance or
the regular medication is given orally to the animals.
8. After taking a dose, rectal temperatures are taken
again 30, 60, 120, and 180 minutes later.
Evaluation: The variations between the beginning and
actual values are recorded for every time interval. It is calculated to find the highest decline in rectal temperature
relative to the control group. The outcomes are compared
with the effects of common medications, such as
1
100 mg kg
of p.o. amino phenazone or 100 mg kg1 p.o. of
phenacetin.
12.2.2.1.14 Screening Models for Dermal Diseases
In general practice, 7% of appointments involve dermatological problems. Most skin conditions related to dermatology are immune- and inflammatory-based. Atopic
dermatitis, psoriasis, acne, and contact eczema are some
of the most common and well-known dermal conditions.
The use of dermatological disease models in in vivo
pharmacology to find treatments for psoriasis and other
conditions.
1. Mouse Tail Model for Psoriasis
The concept is predicated on the production of orthokeratosis in adult mouse tail sections that typically differentiate
into parakeratotic structures [14]. Orthokeratosis is quantified using a semi-automated evaluation unit to assist in the
morphometric assessment of the scales.
Procedure:
1. Mice weighing 25–27 g are used for the study.
2. The proximal portion of the tails receives a topical
treatment of 0.1 mL ointment.
3. A plastic cylinder is placed over the tail and secured
with adhesive tape for a two-hour contact period.
4. The tails are cleaned after removing the cylinders after
the contact period.