To determine anti-anxiety of beta sitosterol , Chandan Chauhan M.S. Research Scholar, Dept. of Pharmacology & toxicology, National Institute of Pharmaceutical Education & Research (NIPER), Hajipur.

1. To determine the anti-anxiety activity of Beta sitosterol
Research Guide:-
Dr. Sugato Banerjee
Associate Professor
NIPER Kolkata
banerjeesugato1@gmail.com
Administrative Guide:-
Dr Sanjiv Singh
Assistant Professor
NIPER Hajipur
sanjivpg2006@gmail.com
Investigator:-
Mr. Chandan Chauhan
M.S. (Pharm)/2019-2021/PT/02
M.S. Research Scholar Dept. of Pharmacology & toxicology
NIPER Hajipur
Chauhan.niper@gmail.com

2. 1. Introduction
2. Review of literature
3. Rationale of project
4. Objectives
5. Plan of work
6. Time line
7. References

3.  Fear is an automatic neurophysiological state of alarm characterized by a fight or flight
response to a cognitive appraisal of present or imminent danger (real or perceived).
 Anxiety is linked to fear and manifests as a future-oriented mood state that consists of a
complex cognitive, affective, physiological, and behavioral response system associated
with preparation for the anticipated events or circumstances perceived as threatening.
Epdemiology:-
 Anxiety is one of the most common psychiatric disorders in the general population.
Specific phobia is the most common with a 12-month prevalence rate of 12.1%.
 Anxiety disorders are present in up to 13.3% of individuals in the U.S. and constitute
the most prevalent subgroup of mental disorder.
 Social anxiety disorder is the next most common, with a 12-month prevalence rate of
7.4%..
 Anxiety disorders are the most common class of emotional disorders and an important
cause of disability worldwide.
Peter. G., Alastair. F., 2018, American Academy of Neurology, 24, 893-919.
Suma P. Chand; Raman Marwaha, 2020, Stat pearls publishing,
https://www.ncbi.nlm.nih.gov/books/NBK470361.

4. The significant mediators of anxiety in the central nervous system are thought to be norepinephrine,
serotonin, dopamine, and gamma-aminobutyric acid (GABA). The autonomic nervous system,
especially the sympathetic nervous system, mediates most of the symptoms.
The prefrontal cortex, insula, and amygdala are key structures in the pathophysiology of anxiety.
Reduction in the activation of the prefrontal cortex–amygdala circuit to aversive stimuli may be a
common neurobiological mechanism underlying effective treatments for anxiety.
Patients with anxiety disorders have been found to show heightened amygdala response to anxiety
cues.
The amygdala and limbic system structures are connected to prefrontal cortex regions, and prefrontal-
limbic activation abnormalities may be reversed with psychological or pharmacologic interventions.
Alexander Bystrisky, Sahib S. Khalsa, Michael E. Cameron, Jason Schiffman, Pharmacy &
therapeutics, 2013, 38(1),30-38.
Suma P. Chand; Raman Marwaha, 2020, Stat pearls publishing,
https://www.ncbi.nlm.nih.gov/books/NBK470361

5. Characteristic Symptoms Pathological Anxiety
Anxiety Disorders as defined in the Diagnostic and Statistical Manual of
Mental Disorders (5th ed.; DSM–5; American Psychiatric Association,
2013):-
Separation Anxiety Disorder.
Selective Mutism.
Specific Phobia.
Social Anxiety Disorder.
Panic Disorder.
Agoraphobia.
Generalized Anxiety Disorder.
Substance/Medication-Induced Anxiety Disorder.
Anxiety Disorder Due to Other Medical Conditions.
Suma P. Chand; Raman Marwaha, 2020, Stat pearls publishing,
https://www.ncbi.nlm.nih.gov/books/NBK470361.

6. Beta-sitosterol (BS):-
•Beta-sitosterol (BS) is one of the several phytosterols with a chemical structure similar
to that of cholesterol.
•It is a natural micronutrient in higher plants and is found in the serum and tissues of
healthy individuals at a concentration 800–1000 times lower than that of endogenous
cholesterol. Its glycoside, sitosterolin, is also present in serum, but in lower
concentration.
•These molecules are synthesized in plants; whereas animals obtain them through diet.
•In a series of scientific publications by the European Food Safety Authority (EFSA),
BS was also not mentioned singly . BS is generally considered as a safe, natural, and
effective nutritional supplement and has been shown to have many potential benefits.
Figure 1. Beta-sitosterol(BS)

7. Neuropharmacological action of Beta-sitoseterol :-
BS containing plants show antinociceptive , anxiolytic, and sedative
effects in rats, but such findings in humans are not available. Neither the
brain region nor the pathway affected by BS has been studied extensively
yet.
It has been shown that the effect of BS is some what similar to
diazepam but whether the mechanism of action is similar or not has been
studied .
It has been proposed that BS is effectual by interacting with GABA
receptor, but there is no confirmatory evidence for this claim.
Studies is immortalized mouse hippocampal cell line HT22 showed
that BS prevents oxidative damage & neurotoxicity & a series of other
studies showed the beneficial effects in preventing neuronal damage.
M.S.B.Sayeed., S.M.R.Karim., T.Sarmin., M.M.Morshed., 2016, Medicines, 3(4), 1-25

8. 1. P. Giacobbe et al,.2018, Reported that anxiety disorders are the
most common class of emotional disorders and a leading cause of
disability worldwide.
2. Muhammad et al,. 2016, Reported that Clinical trials with Beta-
sitosterol have shown beneficial effects in different diseases .
Therefore, it is highly recommended extensive research regarding
its effect at cellular and molecular level in humans as well as
addressing the claims made by commercial manufacturers such as
the cholesterol lowering ability & immunological activity.
3. R. Mishra et al,.2016, Reported that 50% ethanolic extract of
Tinospora cordifolia (TCE) possesses anxiolytic properties and may
play important role in preventing anxiety induced learning and
memory impairments.
Giacobbe P, Flint A., 2018 Jun;24(3, BEHAVIORAL NEUROLOGY AND
PSYCHIATRY):893-919.
Bin Sayeed MS, Karim SMR, Sharmin T, Morshed MM., Medicines (Basel). 2016 Nov 15;3(4):29.
 Mishra R, Manchanda S, Gupta M, Kaur T, Saini V, Sharma A, Kaur G., Scientific Reports
2016 May 5;6:25564.

9. 4. Valitova JN, et al,. 2016, Reported that The main enzymes involved in plant sterol
biosynthesis are 3hydroxy3 methylglutarylCoA reductase, C24sterol
methyltransferase, and C22sterol desaturase. These enzymes are responsible for
maintaining the optimal balance between sterols. Regulation of the ratios between
the different types of sterols and sterols/sphingolipids can be of crucial importance
in the responses of plants to stresses.
5. Singh D., et al,. 2017, Reported that the compounds isolated from T. cordifolia
having antidiabetic, anti-anxiety, anti-inflammatory, anticancer and
immunomodulatory activity can be used as therapeutic agents against these
diseases either alone or in combination in a standardized form.
6. Saha A., et al,. 2013, Reported that Petroleum ether extract of A. paeoniifolius may
act as a potent anxiolytic agent in mice.
Valitova JN, Sulkarnayeva AG, Minibayeva FV., Biochemistry (Mosc). 2016 Aug;81(8):819-34.
 Singh, Deepika, and Prabir K. Chaudhuri., Natural Product Communications, Feb. 2017.
Anindita Saha, Sankhadip Bose, Sugato Banerjee,.Journal of Pharmacy Research,Volume 6,
Issue 7,2013,Pages 748-752,

10. Saha S, Ghosh S., Ancient Science of Life. 2012 Apr;31(4):151-9.
Lakhan SE, Vieira KF., Nutrition Journal, 2010 Oct 7;9:42.
E.A.Hernandez., Planta Medica(Journal of Medicinal Plant and Natural Product Research), 2007,
73:1148-1155.
7. Saha S., et al, 2012, Reported that Tinospora cordifolia is a versatile resource for all
forms of life. active compounds have immunomodulatory and physiological roles of
different types, thereby demonstrating the diverse versatility of the plant. review
remains in exploiting the biochemical and signaling pathways of the active components
of Tinospora thus, enabling effective disease targeting.
8. Lakhan S.E., et al, 2010, Reported that nutritional and herbal supplementation-
betasitosterol is an effective method for treating anxiety and anxiety-related conditions
without the risk of serious side effects. There is the possibility that any positive effects
seen could be due to a placebo effect, which may have a significant psychological
impact on participants with mental disorders.
9. E.A.Hernandez., et al, 2007, Reported that beta-sitosterol is one of the active
compound partly responsible for the central activety of Tilia americana ver mexicana .
Morever, administration of beta-sitosterol i.p. at low doses causes anxiolytic like effects
in mice without generating marked depressive actions on central nervous system.

11. 3. Rationale of projrct:-
• Tinospora steroids are responsible for its anti-anxiety activity .
• One of the primary steroids present in Tinospora Cardifolia is beta sitosterol
.
• Hence we determine the anti-anxiety activity of beta sitosterol
Mishra R, Manchanda S, Gupta M, Kaur T, Saini V, Sharma A, Kaur G. Tinospora cordifolia
ameliorates anxiety-like behavior and improves cognitive functions in acute sleep deprived rats.
Sci Rep. 2016 May 5;6:25564. doi: 10.1038/srep25564. PMID: 27146164; PMCID: PMC4857086.
Saha S, Ghosh S. Tinospora cordifolia: One plant, many roles. Anc Sci Life. 2012 Apr;31(4):151-
9. doi: 10.4103/0257-7941.107344. PMID: 23661861; PMCID: PMC3644751.

12. To determine the anti anxiety activity of beta sitosterol
by using Swiss albino mice.

13. 5.Plan of work
Animals are divided into 4 groups
A. Control
B. Anxiety induced animals
C. Anxiety induced animals treated with beta-sitosterol (TCL Chemicals)
for 2 weeks
D. Control animals treated with beta-sitosterol.
Techniques used
Passive avoidance test
Marble burying test
Water avoidance test
Oral administration
and blood collection
ELISA using plasma

14. Number Name of group Number of Animals
1 Control 6
2 Anxiety control 6
3 Anxiety group + Beta sitosterol
(25mg/kg) [1]
6
4 Beta sitosterol (25mg/kg) 6
5 Anxiety group + Diazepam
(0.5mg/kg)
6
Total 30
Detail study plan to justify the use of
animals:

15. Method description:-
Animals required = C57BL6 Male Mice.
PASSIVE AVOIDENCE TEST- Animals were placed in the
passive avoidance apparatus for 5min to explore the apparatus. Now,
after 24 hours the same animal is placed in the apparatus the mild
electric shock (1mA for 1sec) is administered to the animal. The
animal is placed in the apparatus for 5 min. After 24 hours of
administered shock, the animal is placed in the apparatus and the
time it takes to go to the grid was measured.
MORRIS WATER TEST- Animals are given training to reach a
hidden platform placed in a specific quadrant for 4 days. The time
to find the hidden platform was measured. Tracking was

16. Marble burying test- Rodents are placed for thirty minutes in a
standard cage filled with 5 cm depth of wood chip bedding with 10
marbles evenly spaced. After thirty minutes the amount of marbles
buried is measured. In this procedure a marble is considered buried
if 2/3 of the marble is covered with bedding. Within the study there
should be two groups of animals, one group which has been injected
with saline and another group which has been injected with the
agent being tested.
Retro orbital method for blood collection –
Animal should be anesthetized prior to performing this procedure.
Inhalant anesthetic is the performing method. It is imperative that

17. ELISA:-
PROCEDURE
 The cortisol competitive ELISA research use only kit is
designed to quantitatively measure cortisol present in plasma.
 A cortisol standard is provided to generate a standard curve for
the assay and all samples are read off a user generated standard
curve.
 Standard or diluted samples are pipetted in to a clear microtiter
plate coated with an antibody to capture mouse antibody.
 A cortisol-peroxidase conjugate is added to the wells.
 The binding reaction is initiated by the addition of a
monoclonal antibody to cortisol. After a 1-hour incubation the

18.  The substrate reacts with the bound cortisol-peroxidase
conjugate.
 After a short incubation, the reaction is stopped and the
intensity of the generated color is detected in a microtiter
plate reader at 450 nm

19. 6.TIME- LINE:-
Work to be done Nov- Jan Feb- April May – June
Literature survey
Skills development
handling
technique
Animal
Experimentation
Data Analysis and
thesis writing

20. 1. Giacobbe P, Flint A. Diagnosis and Management of Anxiety Disorders. Continuum (Minneap Minn).
2018 Jun;24(3, BEHAVIORAL NEUROLOGYAND PSYCHIATRY):893-919. doi:
10.1212/CON.0000000000000607. PMID: 29851884.
2. Mishra R, Manchanda S, Gupta M, Kaur T, Saini V, Sharma A, Kaur G. Tinospora cordifolia
ameliorates anxiety-like behavior and improves cognitive functions in acute sleep deprived rats. Sci
Rep. 2016 May 5;6:25564. doi: 10.1038/srep25564. PMID: 27146164; PMCID: PMC4857086.
3. Bin Sayeed MS, Karim SMR, Sharmin T, Morshed MM. Critical Analysis on Characterization,
Systemic Effect, and Therapeutic Potential of Beta-Sitosterol: A Plant-Derived Orphan Phytosterol.
Medicines (Basel). 2016 Nov 15;3(4):29. doi: 10.3390/medicines3040029. PMID: 28930139; PMCID:
PMC5456237.
4. Valitova JN, Sulkarnayeva AG, Minibayeva FV. Plant Sterols: Diversity, Biosynthesis, and
Physiological Functions. Biochemistry (Mosc). 2016 Aug;81(8):819-34. doi:
10.1134/S0006297916080046. PMID: 27677551.
5. Singh D, Chaudhuri PK. Chemistry and Pharmacology of Tinospora cordifolia. Natural Product
Communications. February 2017. doi:10.1177/1934578X1701200240.
6. Anindita Saha, Sankhadip Bose, Sugato Banerjee,Anti-anxiety activity of Amorphophallus
paeoniifolius tuber in mice,Journal of Pharmacy Research,Volume 6, Issue 7,2013,Pages 748-752,ISSN
0974-6943, https://doi.org/10.1016/j.jopr.2013.07.018.
7. Saha S, Ghosh S. Tinospora cordifolia: One plant, many roles. Anc Sci Life. 2012 Apr;31(4):151-9.
doi: 10.4103/0257-7941.107344. PMID: 23661861; PMCID: PMC3644751.
8. Lakhan SE, Vieira KF. Nutritional and herbal supplements for anxiety and anxiety-related disorders:
systematic review. Nutr J. 2010 Oct 7;9:42. doi: 10.1186/1475-2891-9-42. PMID: 20929532; PMCID:
PMC2959081.