Bojjam Narasimhulu Pharmacy College For Women

Evaluation of neuroprotective and anti - Alzheimer activity of

Centratherum anthelminticum against Scopolamine induced
memory impairment in mice.

Presented by
N. Navya [20Z31R0038]
T.Nikitha [20Z31R0039] GUIDED BY:
S.Shashanka [20Z31R0049] P. Easwari
G. Shivani[20Z31R0053] Asst. Professor
S. Supriya[20Z31R0062] Pharmacology department
S. Swathi[20Z31R0063]
 INTRODUCTION
Aging is a major risk factor for neurodegenerative disorders, such as Alzheimer’s
disease (AD) and Parkinson’s disease (PD), and the number of people with these
conditions is increasing rapidly.
This incurable, degenerative, and terminal disease was first described by German
psychiatrist and neuropathologist named Alois Alzheimer in 1906, the disease that
bears his name largely remained an enigma until the twilight of the 20th century.
Generally, it is diagnosed in people over 65 years of age, although the less-
prevalent early-onset Alzheimer's can occur much earlier.
According to WHO in 2001, more than 24 million people had dementia, a number
that is expected to double every 20 years up to 81 million in 2040 because of the
anticipated increase in life expectancy.
 LITERATURE REVIEW
Li-Kai Huang et al, (2023) Alzheimer's disease (AD) is the leading cause of dementia,
presenting a significant unmet medical need worldwide.
Wenwen Lian et al, (2017) Deficiency of the cholinergic system is thought to play a vital role
in cognitive impairment of dementia. DL0410 was discovered as a dual inhibitor of acetyl
cholinesterase (AChE) and butyrylcholinestease (BuChE), with potent efficiency in in-vitro
experiments, but its in vivo effect on the cholinergic model has not been evaluated, and its
action mechanism has also not been illustrated.
Hari Kumar Cheedella et al, (2023) Nature is the best source of complementary and
alternative medicine. The plant Biophytum reinwardtii has been used traditionally in pain,
inflammatory and oxidative stress related disorders.
Khalid Bashir Dar et al., (2016),⁵⁸ To evaluate the phytochemical screening, antibacterial and antifungal
potential of aqueous and methanolic extracts of Rheum spiciformis, a traditionally used edible
medicinal plant.
Shahet al (2008)⁵⁶ reported similar observations using methanol extract of C. anthelminticum. These
studies indicated that treatment with C. Anthelminticum is beneficial with less side effects, compared to
the standard drug glibenclamide.
 RISK FACTORS OF ALZHEIMER’S DISEASE
a. Age: Most individuals with the illness are 65 and older. Developing of Alzheimer’s approximately
doubles every five years after age 65. After age 85, the risk reaches nearly 50 percent.
B. Family history and genetics: Research has shown that those who have a parent, brother or sister with
Alzheimer’s are two to three times more likely to develop the disease. The risk increases if more than
one family member has the illness. Scientists have so far identified one gene (Apolipoprotein E-e4
(APOE4)) that increases the risk of Alzheimer’s but does not guarantee an individual will develop the
disorder.
C. Other risk factors: The other risk factors include easily avoidable factors like severe head injury,
certain viral infections(herpes simplex virus), excessive consumption of alcohol and smoking, and
various heart diseases like stroke, diabetes, high blood pressure and high cholesterol. Oxidative stress
and life style modifications may play the major risk factor.
The
SYMPTOMS OF ALZHEIMER’S DISEASE
earliest observable symptoms are often mistakenly thought to be 'age-related' concerns, or

manifestations of stress. In the early stages, the most commonly recognized symptom is memory loss,

such as difficulty in remembering recently learned facts. When, AD is suspected, the diagnosis is

usually confirmed with behavioural assessments and cognitive tests, often followed by a brain scan if

available.

As the disease advances, symptoms include confusion, irritability and aggression, mood swings, language

breakdown, long-term memory loss, and the general withdrawal of the sufferer as their senses decline.

Gradually, bodily functions are lost, ultimately leading to death.

AD develops for an indeterminate period of time before becoming fully apparent, and it can progress

undiagnosed for years. The mean life expectancy following diagnosis is approximately seven years.
 VARIOUS STAGES OF ALZHEIMER’S DISEASE
A. Pre-dementia: The first symptoms are often mistaken as related to ageing or stress. The most noticeable
deficit is memory loss, which shows up as difficulty in remembering recently learned facts and inability to
acquire new information .
B. Early dementia: Symptoms include difficulties with language, executive functions, perception (agnosia),
or executions of movements (apraxia) are more prominent than memory problems. Older memories of the
person's life, facts learned, and implicit memory are affected to a lesser degree than new facts or memories .
Language problems are mainly characterized. While performing fine motor tasks such as writing, drawing
or dressing, certain movement coordination and planning difficulties may be present but they are
commonly unnoticed .
C. Moderate dementia: subjects being unable to perform most common activities of daily living. Reading
and writing skills are also progressively lost. During this phase, memory problems worsen, and the person
may fail to recognize close relatives. Long-term memory, which was previously intact, becomes impaired.
D. Advanced dementia: During this last stage of AD, the patient is completely dependent upon caregivers .
Language is reduced to simple phrases or even single words, eventually leading to complete loss of speech.
Despite the loss of verbal language abilities, patients can often understand and return emotional signals .
 DIAGNOSIS OF ALZHEIMER’S DISEASE
Alzheimer's disease is usually diagnosed clinically from the patient history, collateral history from relatives,
and clinical observations, based on the presence of characteristic neurological and neuropsychological
features and the absence of alternative conditions .
Approaches to early diagnostic marker discovery for Mild cognitive impairment (MCI) and AD include
neuroimaging, genetic testing and neurochemical testing for body fluid, such as cerebrospinal fluid (CSF),
plasma, serum, urine and blood cells.
Advanced medical imaging with computed tomography (CT) or magnetic resonance imaging (MRI), and
with single photon emission computed tomography (SPECT) or positron emission tomography (PET) can be
used to help exclude other cerebral pathology or subtypes of dementia.
Assessment of intellectual functioning including memory testing can further characterized the state of the
disease .
Changes in the cholinergic, serotonergic, noradrenergic, dopaminergic, GABAergic and
somatostatinergic neurons were investigated to determine their roles in Alzheimer's disease (AD).
The cholinergic abnormality is the most severe and most closely related to the severity of the disease.
Therefore, in pharmacotherapy of AD, attempts to restore deficits of the transmitter systems should be
directed foremost to the cholinergic system.
 PATHOPHYSIOLOGY
There are two signature lesions in Alzheimer's disease. They are:
1. Neuritic plaques/ B-amyloid plaques, which are dense deposits of protein and cellular
material that accumulate outside and around nerve cells.
2. Neurofibrillary tangles (NFT's), which are twisted fibers that build up inside the nerve cell.
1. NEURITIC PLAQUES:
Deposits of a protein fragment called ẞ-amyloid, that accumulates in the spaces between the
nerve cells (neurons).
APP (amyloid precursor protein) is the precursor of amyloid plaque.
a) APP sticks through the neuron membrane.
b) Enzymes like ẞ-secretase and t-secretase cut the APP into fragments of protein (neurotoxic
Aẞ4 fragment), including ẞ-amyloid.
c) B-amyloid fragments come together in clumps to form plaques.
In AD, many of these clumps form, disrupting the work of neurons. This affects the
hippocampus and other areas of the cerebral cortex.
 APP (amyloid precursor protein)

Cleaved by ẞ-secretase and a-secretase

Mutation of amyloid precursor protein on chromosome no. 21

Increased production of amyloid precursor protein

Produces of amyloid protein

Accumulation of ẞ-amyloid protein (due to increased production of APP)

Directly neurotoxin

Alzheimer's disease
 2. NEUROFIBRILLARY TANGLES (NFT'S):
Neurons have an internal support structure partly made up of microtubules. A protein called
"tau" helps to stabilize microtubules. In AD, "tau" changes, causing microtubules to collapse
and "tau" proteins clump together to form neurofibrillary tangles.
Although autopsy studies show that most people develop some plaques & tangles as they age
Those with AD tend to develop them far more & in a predictable pattern
They develop in the areas important for memory before spreading to other regions
Plaques & tangles disable/block communication among neurons
Gradually spread to other areas of brain
Causes symptoms of Alzheimer's disease
AIM: To study and evaluate neuroprotective and anti - alzheimer activity of
Centratherum anthelminticum against scopolamine induced memory
impairment in mice.

OBJECTIVE:
1. To evaluate the mechanism of action of Centratherum anthelminticum in
scopolamine induced dementia.
2. To carry out Pharamacognostical study of C. anthelminticum in scopolamine
induced dementia.
3. The main purpose of the present study is to compare the neuroprotective and
anti-alzheimer effect of C. anthelminticum with the standard drug donepezil on
scopolamine induced memory impairment in Wistar albino rats.
 PLAN OF WORK
1. Collection and authentication of plant.
2. Preparation of extract and preliminary phytochemical screening.
3. Acute toxicity study of the plant extract and Drugs and Doses selection.
4. Scopolamine induced Alzheimer’ disease in animal
5. Treatment of animal with standardized dose of plant extract
COMMON NAMES

Language Common Names

Ayurvedic Vanyajiraka, Aranya-Jiraka

Siddha Kaattuseerakam

Unani Kali zeeri, Kamoonbarri

Hindi Kaalijeeree, Karajiri

Telugu Adavijilakaroa, Garetikamma

English Stone wart, Purple Fleabane

SCIENTIFIC CLASSIFICATION
Level Classification

Kingdom Plantae

Division Magnoliophyta

Class Magnoliopsida

Order Asterales

Family Asteraceae

Genus Centratherum Cass.

Species Centratherum anthelminticum

Synonym Vernonia anthelmintica Willd

 PLANT DESCRIPTION

Feature Description

Growth Habit Erect, pubescent, annual herb

Height Up to 90 cm
Elliptic-lanceolate, 5 to 9 cm long, 2.5
Leaves
to 3.2 cm wide
Leaf Apex Acute

Leaf Base Tapering into the petiole

Leaf Margins Coarsely serrate

Leaf Surfaces Pubescent on both surfaces

Homogamous purple florets, solitary,

Inflorescence
axillary, or terminal heads
Brownish color, hot sharp taste,
Seeds
astringent properties
Chemical Constituent Percentage/Content Other Information
Glycosides Not specified
Carbohydrates 14.7%
Phenolic Compounds Not specified
Tannins Not specified
Flavonoids Not specified
Proteins 22.5%
Saponins Not specified
Lipids and Fats 21.4% fat Fixed oil: 18%, volatile oil: 0.02%
Fatty acids: linoleic acid (50%),
palmitic acid, palmitoleic acid,
stearic acid, oleic acid
Delta-7-avenasterol, demanolide
Other Active Principles lactone, a bitter principle, essential
oil, resins
Moisture Content 4.9%
Fiber 29.3%
IMPORTANT MEDICINAL PROPERTIES
It is widely used as folk medicine for diabetes in Rayalaseema, India and a popular ingredient in
Ayurvedic medicine. In other places, C.anthelminticum has been traditionally applied as
anthelmintic, stomachic, digestive, diuretic, tonic, alterative, anti-phlegmatic, anti-asthmatic,
anti-phlegmatic treatment, as well as a therapeutic agent for cough, diarrhea, helminth, skin
diseases, ulcers, leukoderma and fevers. It has medicinal properties like Antimicrobial,
Antidiabetic, Anthelmintic, Astringents, Anti-inflammatory, Antiseptic, Antioxidant, Antiulcer,
Diuretic, Digestive, Insecticide, Stomachic and Smooth muscle relaxant.
COLLECTION OF PLANT MATERIALS
Seeds of C. anthelminticum were collected from the local herbal market Ayurvedic raw material
shop at Ranga Reddy dist., Hyderabad. and authenticated by Botanical Survey of India,
Hyderabad.
PREPARATION OF ETHANOLIC EXTRACT
500 gm Fresh, healthy seeds of the selected plant were shade dried and ground with the help of
an electrical grinder to get a free-flowing powder. This powder was subjected to extraction with
methanol at room temperature for 24hours. The extract obtained was filtered through
Whatman filter paper and vacuum dried at 40-50°C to get a blackish green semisolid mass,
which was dissolved in saline (0.9% NaCl) solution for final use.⁷⁰
 QUALITATIVE PHYTOCHEMICAL ANALYSIS
A) Test for Alkaloids Few mg of each extract was taken separately in 5mL of 1.5% v/v HCl and filtered. These
filtrates were then subjected to following tests-

Test Procedure Result

Spread the extract on Whatman filter paper and dry.

Make the test filter paper alkaline with ammonia.
Dragendroff’s test Extract the filter paper with chloroform. Apply the
chloroform extract on filter paper impregnated with
Dragendroff’s reagent.
Mayer’s reagent Treat the extract with Mayer’s reagent.
Wagner’s Reagent Treat the extract with Wagner’s reagent.
Hager’s reagent Treat the extract with Hager’s reagent.
Development of orange-red color on
the filter paper impregnated with
Dragendroff’s reagent indicates
alkaloids.
Formation of cream-colored
precipitate indicates the presence of
alkaloids
Formation of reddish-yellow
precipitate indicates the presence of
alkaloids.
Formation of yellow precipitate
indicates the presence of alkaloids.
 Test for Glycosides

Test Procedure Interpretation

To 5 mg of extract, add 5 mL of dil. H2SO4 and warm on a water bath. If test A contains more
Filter the solution. Neutralize the filtrate with 5% NaOH solution. Add 0.5 precipitate than test B, it
Test A
mL Fehling’s solution A and 0.5 mL Fehling’s solution B. Heat the mixture indicates the presence of
on a water bath for 2 minutes. glycosides.
the solution. Neutralize the filtrate with 5% NaOH solution. Add 0.5 mL
Test
TestB for Triterpenoids B, it indicates the absence of
Fehling’s solution A and 0 5 mL Fehling’s solution B Heat the mixture on
l id

Test Procedure
g Interpretation
at the junction of two liquids indicates
Burchard test acetic anhydride was added to it followed by conc.H2SO4
presence of triterpenoids
Test for Naphthoquinones

Test Procedure Interpretation

Test for anthraquinones

Appearance of reddish-pink color
1. To the 5 mg extract, add 2 mL solvent ether and an
Juglone test indicates the presence of
equal volume of dil. ammonia solution.
naphthoquinones.

Test Procedure Interpretation

To the 5 mg extract, add 5 mL dil. HCl and boil.

Brontager’s test
Modified Brontager’s test
Appearance of a pink color in the
Cool the solution and add 2 mL solvent ether.
ammonical layer indicates the
Separate the ether layer. Add strong ammonia
presence of anthraquinones.
solution to the separated ether layer.
pp p
few drops of FeCl3. Boil the solution and cool it.
ammonical layer indicates the
Add 2 mL solvent ether. Separate the ether
presence of C-type anthraquinone
layer Add strong ammonia solution to the
 Test for Coumarin glycosides
Test Procedure Interpretation
Add a few drops of the extract solution onto paper Development of fluorescence indicates
With ammonia
previously impregnated with ammonia. the presence of coumarins.
Development of blue or green
Dissolve the extract in alcohol. Make the solution alkaline
fluorescence indicates the presence of
With alkali solution by adding KOH solution.
coumarins.
Test for Sterols
Transfer the extract into a flask stoppered with strips of
The sodium picrate paper turning red
Test for Cyanogenetic sodium picrate paper. Ensure that no paper touches the
indicates the presence of cyanogenetic
glycosides inner side of the test tube. Warm the content for 30
glycosides.
minutes.

Test Procedure g Interpretation

p
Salkowski test 2 mL of concentrated
g H2SO4 to the test tube
y from the chloroform layer indicates the
Development of a blue color indicates
Liebermann test tube. Heat the mixture gently. Add a few drops of
the presence of sterols
 Test for Flavonoids
Test Procedure Interpretation
Development of pink, magenta and
A small quantity of the extract was dissolved in 5mL of
Shinoda test crimson colour indicates presence of
ethanol (95%v/v) and treated with few drops of conc. HCl
flavonoids.
and 0.5 g of magnesium turnings.
Test for Tannins

Test Procedure Interpretation

g
A small quantity of the extract is dissolved in 5 mL of
FeCl3 Test indicates presence of hydrolysable
ethanol (95% is
A matchstick v/v) thenintreated
dipped with FeCl3
the methanolic solution
solution of the magenta/pink coloration on the
Matchstick Test
extract dried then dipped in concentrated HCl matchstick Presence of catechins
 Tests for Carotenoids

Test Procedure Interpretation

development of blue colour indicates
Carr-price reaction
Extract was treated with antimony trichloride solution presence of carotenoids.
Test for carbohydrates

Test Procedure
g , g Interpretation
junction of two liquids indicates
Molisch’s
Test fortest
Proteins added, Mixed it. 2mL of Conc, H2SO4 was added from
presence of carbohydrates

Test Procedure Interpretation

The 5mg of extract was dissolved in 2 mL of alcohol. In
Development of violet colour
Biuret test alcoholic solution, 2 mL Biuret reagent was added.
indicates presences of protein.
PHARMACOLOGICAL INVESTIGATION
ANIMALS
Mus musculus Swiss of 2 months old weighing between 25 and 30g were used in this study. The mice were housed
and raised in the animal house of the Department of Pharmacology, with a 12 hr light and 12 hr dark cycle. They
were housed 3per cage in standard cages (29 cm X 22 cm X 14 cm) and were maintained on rat feed pellets and
tap water available in water bottles with stainless steel nozzles. The cages were cleaned every day and water and
feed were provided as per diet. The experiments were conducted strictly in accordance with the approved
guidelines of the “Institute Animal Ethical Committee” regulated by the Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPCSEA),Ministry of Social justice and Empowerment,
Government of India.
ORAL TOXICITY STUDY
Acute oral toxicity of the extracts of C. anthelmintic seed was carried out by the up and down procedure (UDP).
Principal value of UDP is to minimizing the number of animals required to estimate the acute oral toxicity of
chemical and estimating the median lethal dose. The LD50 can be estimated using 6-10 animals of 1 sex. Animals
are dosed, one at a time, at 24 H intervals. Depending on outcome the dose for the next animals is adjusted up or
down. The dose is increased if the animal survives and the dose is decreased if animal dies. After reaching the
reversal on initial outcome, i.e., point where an increasing or decreasing dose pattern is reversed by giving a
smaller or higher dose. Four additional animals are dosed the same UDP. In absence of any information about
the substance, the starting dose may be 200 or 500 mg/kg body weight. For further doses, a dose progression
factor of 1:3 is used. The next dose was administered according to mortality of the animal.
Equipment and chemicals
Electronic balance, elevated plus Maze, Y-maze, novel object apparatus, syringes, and needles, ethanol, CMC,
and scopolamine. Donepezil tablet, 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), acetylcholine, thiobarbituric
acid (TBA). Nicotinamide adenine dinucleotide phosphate (NADP), and NADPH. Sodium bicarbonate
glutathione reductase, and sodium azide.
EXPERIMENTAL DESIGN
The experimental animals were divided into six groups and each group consisted of seven animals. The different
groups are
Group I: Normal control (Saline treated)
Group II: Scopolamine control
Group III: C. anthelmintic control
Group IV: C. anthelmintic + Scopolamine
Group V: Donepezil control
Group VI: Donepezil + Scopolamine
PROCEDURE-
An individual animal was administered with 250mg/kg body weight dose one at a time orally. The animal
was observed for 24 hours. The animal was survived; the 750mg/kg body weight dose one at a time orally was
given to next animal. Again, animal was observed for 24 hours. The animal was survived; the 2000mg/kg
body weight dose one at a time orally was given to next animal. Again, animal was observed for 24 hours.
The animal was survived, four additional animals were dosed using the same dose. No deaths occur, 3
animals of the other sex were tested at the same dose level. If mortality was not registered again, the test was
terminated.
DESIGN OF BEHAVIORAL EXPERIMENTS: (1) T-MAZE
The T-Maze apparatus was kept in a sound attenuated area. The animal was left at the tail end. In one of the
arms of the T-maze, animal feed was placed in such a way that the rats were not able to see it, as soon as it
reaches the end of the longarm. The animal has to move forward and turn left or right of the T maze to reach
the food. When the animal reaches the food, that move was taken as a positive response. When it moves to
the other end, that move was taken as a negative response.
The T-maze test was carried out on the 1st, 8th and 15th day of the study. A total of 10 trials were given for a
set with a gap of five minutes per set. The trials were continuous till a set gets 90 % of positive response was
achieved. Then the total number of positive responses was added in all the sets and divided by the total
number of trials given in all the sets to get the percentage of positive response⁷⁵. The whole experiment was
performed in a silent dark room after 9.00 pm under a dim red light. Before doing this experiment, the
animals were left starving for 24 hours with only water to drink. This makes the animal more active to find
food during the experiment.
The values obtained as percentage of positive response in different animal groups were statistically analysed for significance
using post hoc test (LSD – Least Significant Difference) Statistical Package for Social Sciences (SPSS) version 16.
(2) ELEVATED PLUS MAZE
The elevated plus maze served as the exteroceptive behavioral model (wherein the stimulus existed outside the
body) to evaluate learning and memory in mice. The apparatus consisted of two open arms and two covered
arms.
The arms extended from a central platform (5 cm x 5 cm), and maze was elevated to a height of 25 cm from the
floor. On the first day, each animal was placed at the end of open arm, facing away from the central platform.
Transfer latency (TL) was taken as the time taken by animal to move into one of the covered arm with all its four
legs. TL was recorded on the first day. If the animal did not enter into one of the covered arm within 90 s, it was
gently pushed into one of the two covered arms and the TL was assigned as 90 s. The animal was allowed to
explore the maze for 10 s and then returned to its home cage. Memory retention was examined 24 h after the first
day trial on the second day⁸⁰.
PHARMACOLOGICAL DRUG ADMINISTRATION
Scopolamine butyl bromide manufactured by Kemwell biopharma Pvt. Ltd. and Donep-10 tablets manufactured by
Alkem laboratories Ltd were used in the present study. Scopolamine was administered to the mice as intraperitoneal
injection at a dosage of 1 mg/kg body weight for 15 days 30 minutes prior to the start of behavioural experiments.
Each Donep-10 tablets contained Donepezil hydrochloride 10 mg and excipients and the tablets were powdered and
mixed with sterile 0.9 % W/V normal saline. It was administered to the mice with orogastric feeding tube at a dosage
of 3 mg/kg body weight for 15 days and two hours prior to the start of the behavioural experiments.
COLLECTION OF BRAIN SAMPLE
Following the behavioural test, the animals were sacrificed by cervical dislocation and the brains were quickly
removed, cleaned with 0.9 % ice cold saline. The tissues were weighed, homogenized with 10times (W/V) ice cold 0.1
M phosphate buffer (pH 7.4) using a pestle and mortar and the homogenate was centrifuged using Sigma Aldrich
cooling centrifuge at 5400 rpm for 20 minutes at 4ᵒC to separate the supernatant. The supernatant was used for the
quantification of acetylcholinesterase, SOD and GST. A portion of the supernatant was mixed with 10 % TCA,
shaken well and centrifuged at 4000 rpm for 10 minutes at 4ᵒC and the precipitate was taken and re-dissolved in
0.1NNaOH. An aliquot was used to estimate soluble protein.
 BIOCHEMICAL ESTIMATION

Method Lowry et al. (1951)

Principle Color reaction of amino acids with Folin phenol reagent, spectrophotometric estimation
1. Sodium carbonate 2% (w/v) in 0.1 M NaOH 2. Copper sulphate 1% (w/v) 3. Sodium potassium
Reagents
tartarate 2% (w/v) 4. BSA: 1 mg/ml 5. Folin phenol reagent 0.1N
ESTIMATION OF-ACETYLCHOLINESTERASE
Prepare samples, BSA standard, ACTIVITY:
and blank - Adjust volume to 0.5 ml with 0.1 N NaOH - Add
Procedure reagent C and mix - Allow to stand for 10min at room temperature, then add Folin Ciocalteau’s
reagent - Measure absorbance at 660 nm - Calculate protein content using standard curve

Method Ellman et al. (1961)

Measurement of acetylcholinesterase activity by the increase in yellow color produced from

Principle
thiocholine reacting with DTNB
air and the absorbance was measured at 412nm in a spectrophotometer. When the
Procedure absorbance reaches a stable value, it was recorded as the basal reading. After that, 20 μl of
the substrate (acetyl thiocholine iodide) was added and the change in absorbance was
 PEROXIDATION

Method Niehaus (1968)

Measurement of lipid peroxide formation by the development of pink color with TCA-TBA-HCl
Principle g malondialdehyde (MDA) g g
reagent indicating
Procedure flocculent precipitate was removed by centrifugation at 3000 rpm for10 minutes. The

ANTIOXIDANT DEFENSE STATUS - SUPEROXIDE DISMUTASE (SOD):

Method Kono (1978)

Principle Measurement of SOD activity by inhibition of NBT reduction, indicating enzyme activity
-The reaction mixture contained 1.3 ml of 50 mM sodiumcarbonate solution with 0.1 mM EDTA
(pH 10.0), 0.5 ml of 96 μM ofNBT and 0.1 ml of 0.6 % triton-X-100. Reaction was initiated by
theaddition of 0.1 ml of 20 mM hydroxylamine hydrochloride (pH 6.0) tothe reaction mixture
Procedure and the rate of NBT reduction in the absence ofthe enzyme source was recorded for about 30
seconds. Following unit of enzyme was expressed as inverse of the amount ofprotein (mg)
required for inhibiting the reduction rate of NBT by 50 %.
 GLUTATHIONE-S-TRANSFERASE

Method Habig et al. (1974)

Measurement of GST activity by catalyzing the formation of glutathione and 1-chloro-2,4
Principle p p p
dinitrobenzene conjugate read at(p
340 nm) yp p
Procedure freshly prepared GSH and samples - Measure absorbance at 340 nm - Record absorbance
STATISTICAL ANALYSIS: The data were analysed using the computer software, Graph pad prism 9.0.
HISTOLOGY
The test samples were dehydrated using a series of alcohol dilutions ranging from 50% to 100%, followed
by immersion in xylene. Subsequently, the tissues were infiltrated with liquid paraffin wax overnight at
60°C in a wax melter. After embedding the tissues in paraffin wax blocks, they were trimmed into
pyramids and affixed to a wooden block for sectioning on a microtome. Sections of 2 μm thickness were
obtained and spread in a water bath at 40°C. These sections were then mounted on glass slides using
egg albumin glue and dried in an oven at 60°C for one hour. Clearing of the wax was achieved by
immersion in xylene, followed by rehydration through a series of alcohol dilutions.
REHYDRATION
The slides are passed through a series of alcohols of decreasing concentration.
Xylene: 2-3 minutes; Absolute alcohol: 2-3 minutes; Alcohol 95 %: 2-3 minutes; Alcohol 70 %: 2-3 minutes; Alcohol
60 %: 2-3 minutes; Running water: 3-5 minutes.
GORE'S EHRLICH HEMATOXYLIN-EOSIN STAINING

Staining Solution Ingredients

Hematoxylin: 2 grams dissolved in 100 ml ethanol, filtered, then mixed with 3 grams
Hematoxylin Stain ammonium alum dissolved in 100 ml distilled water. 100 ml glycerine added after
mixing.

Eosin: 2 grams dissolved in 70% alcohol, diluted to 1000 ml with 0.9% saline. Diluted
Eosin Stain
further with equal volume of 70% alcohol, and 2-3 drops of acetic acid added.
Equal portions of egg white and glycerine mixed well. Thymol crystals added as
Mayer’s Egg Albumin
preservative.
PROCEDURE
The rats were euthanized by cervical dislocation, followed by transcardial perfusion with 0.9% saline and 10% formalin.
After dissecting out the brain, it was post-fixed in 10% formalin for 24 hours. Tissue dehydration occurred via an
alcohol series and clearing with xylene. Subsequently, the tissue was embedded in paraffin wax and sectioned into two-
micron thick slices. These sections were mounted on glass slides with Mayer's egg albumin glue and incubated at 60°C
for one hour. Following incubation, the sections underwent rehydration in a graded alcohol series for 3 minutes,
followed by washing in running water. Haematoxylin stain was applied to the sections, checked after 1 minute, then
washed and counter-stained with eosin for 3 minutes. Slides were dehydrated in alcohol, placed in xylene, and mounted
in DPX mountant with a cover slip for microscopic examination.
ANALYSIS: The histological preparations of all the groups were photographed and compared.
RESULTS
Extract and extractive value: The yield obtained was about 10%. i.e. from 500 gm C. anthelminticum seeds 50gms of
extract was obtained. It is of blackish green in colour.
Acute toxicity and dose determination: Acute oral toxicity test was carried out according to the OECD guideline No. 423.
Wistar Albino mice were kept for overnight fasting prior to drug administration. A total of three animals were used,
which received a single oral dose in 2000 mg/kg, body weight of methanol extracts of C. anthelminticum seeds. The
animals were observed for a period of 24 hr for the changes in behavior, hypersensitivity reactions etc. Mortality, if any,
was determined over a period of 2 weeks.
Phytochemical analysis The preliminary phytochemical analysis of methanol extract of C. anthelminticum
seeds was given in table 1.

Sl. No. Phytoconstituents Methanol extract

1 Alkaloid -
2 Glycosides +
2 Flavonoid +
3 Triterpenoid -
4 Phytosterol +
5 Phenolic compound &Tannin +
6 Saponin +
7 Free anthraquinone -
8 Coumarin -
9 Carbohydrate +
10 Protein/Amino aci +
11 Lipid &Fat +
Behavioral studies: Table 2: Effect of C. anthelminticum on T-maze performance on dementia-induced mice
(n=7).

T-maze performance
Sl. No
1ˢᵗ day 8ᵗʰ day 15ᵗʰ day

Group I
1 73.14 ± 4.91 77.71 ± 2.92 77.42 ± 3.50
(Normal control)

Group II
2 37.43 ± 4.96** 29.00 ± 4.12 21.71 ± 4.54
(Scopolamine control)

Group III
3 (C.anthelminticum methanolic 74.57 ± 3.36* 78.85 ± 3.34* 80.57 ± 2.88*
extract control) 250 mg/kg

Group IV
4 (C.anthelminticum methanolic 71.43 ± 5.16** 73.00 ± 3.11** 75.57 ± 4.06**
extract 250 mg/kg +Scopolamine)

Group V
5 77.28 ± 1.70 79.86 ± 3.76 85.57 ± 3.15
(Donepezil control)

Group VI
6 71.71 ± 2.43* 74.00 ± 3.41* 77.71 ± 3.54*
(Donepezil +Scopolamine)
 Effect of C. anthelminticum on dementia induced mice in different groups in Elevated plus maze test
The impact of all the drug-treated groups was evaluated at the top of 14th day. TL was recorded. It absolutely
was seen that TL for all the drug-treated groups was less on the 15th day as compared to the 14th day.

Groups IR TL Day: 14 TL Day: 15

Group I
0.580 ±0.065 15.220 9.1
(Normal control)

Group II
0.479±0.113 35.43 26.67
(Scopolamine control)

Group III
(C.anthelminticummethanolic extract
0.685±0.071 23.25 13.11
control)
250 mg/kg

Group IV
(C.anthelminticum methanolic extract
0.493±0.042 26.44 16.3
250 mg/kg +
Scopolamine)

Group V
0.520±0.119 23 13.11
(Donepezil control)
Pharmacological study: Biochemical estimation

Biochemical parameter
Sl. No
AChE SOD GST MDA

Group I
1 126 ± 41.59 103 ± 10.23 17.59 ± 4.24 0.3847 ± 0.061
(Normal control)

Group II
2 196 ± 44.12** 88.9 ± 13.4** 11.53 ± 3.94** 0.04735 ± 0.068**
(Scopolamine control)

Group III
3 (C.anthelminticummethanolic extract 114 ± 22.18** 127 ± 25.96** 19.74 ± 2.16** 0.3526 ± 0.039**
control)250 mg/kg

Group IV
(C.anthelminticummethanolic extract
4 139 ± 26.78** 110 ± 24.83** 18.93 ± 1.75** 0.3708 ± 0.0522**
250 mg/kg +
Scopolamine)

Group V
5 88.4 ± 18.24 161 ± 5.008 22.06 ± 1.32 0.2584 ± 0.026
(Donepezil control)

Group VI
6 (Donepezil + 125 ± 40.97** 122 ± 25.53 18.98 ± 3.78** 0.3704 ± 0.0708**
Scopolamine)
HISTOLOGY
Histology of hippocampal neurons showed that the normalcontrol group with a row of normal hippocampal cells
(CA1, CA2 andCA3). But in scopolamine induced dementia group there were clearprominent white patches or
vaculation around the neuronal cells. Inthe case of control groups of C.anthelminticumandDonepezil it showed
almost normal hippocampal cells when comparedwith their corresponding scopolamine treated group

Normal Control Scopolamine Control C.anthelminticum control C.anthelminticum + Scopolamine

Donepezil control Donepezil + Scopolamine

CONCLUSION
The present study reveals that an aqueous preparation of C.anthelminticum has a significant role in the management of
cognitive impairment and oxidative stress.
Scopolamine-induced groups showed a decline in memory performance in the classical T-maze. The C.anthelminticum
treated groups showed a significant increase in performance level, which is equal to the normal control group and is better
than the C.anthelminticum + scopolamine groups. When the results are compared with the standard drug donepezil, the
sample treated groups showed a better performance.
Scopolamine-induced groups showed a drastic elevation in AChE level. When the scopolamine-induced group was treated
with moringa or vitex, a significant reduction in AChE level was observed. In the case of control groups of
C.anthelminticum, it was found that there is a decline in AChE level than the normal control group. The standard drug
donepezil treated group showed a better result when compared to the morning-treated group, which is better than the vitex-
treated group and the corresponding scopolamine-treated groups.
The change in behaviour of animals in scopolamine inducedamnesia model correlated with the histological changes
inhippocampal morphology. Scopolamine treatment showedvacuolation around the neurons and the morphology of
neuronsin CA1, CA2 and CA3 regions was shrunken and it improvedafter the administration of C.anthelminticum.
 REFERENCES
1. Tulving E: Memory and consciousness. Canadian Psychology/Psychologie Canadienne 1985, 26(1):1-15.
2. Suresh Kumar, Greeshma C, Sreekumaran E: Study of dementia in diabetic patients with particular reference to Cerad and Trail
Making Test. International Journal ofScientific and Engineering Research 2014, 5(10):74-77.
3. Rahmath A, Rajan N, Shahal MA, Seena T, Sreekumaran E: Neuroprotective effect of Moringa oleifera in scopolamineinduced
cognitive impairment and oxidative stress in Wistaralbino rats. Research Journal of Pharmaceutical, Biologicaland Chemical Sciences
2015, 6(4):1736-17444.
4. Bear MF, Connors BW, Paradiso MA: Neuroscience, 3rd Ed., Library of Congress cataloging-in - Publication data, vol. 2:
Lippincott Williams & Wilkins USA; 2007.148-159.
5. Srikumar B, Bindu B, Priya V, Ramkumar K, Shankaranarayana Rao B, Raju T, Kutty B: Methods ofassessment of learning and
memory in rodents. Brain andBehavior 2004:145-151.
6. Shiksharthi AR, Mittal S, Ramana J: Systematic review of herbals as potential memory enhancers. InternationalJournal of Research
in Pharmaceutical and BiomedicalSciences 2011, 2(3):918-925.
7. Grant WB: Dietary links to Alzheimer’s disease. Alzheimer Disease Review 1997, 2:42-55.
8. Coyle JT, Puttfarcken P: Oxidative stress, glutamate and neurodegenerative disorders. Science 1993, 262(5134):689-695.