EVALUATION OF ANTI-STRESS ACTIVITY OF HYDRO-
ALCOHOLIC EXTRACT OF SAUROPUS ANDROGYNUS LEAVES
MASTER OF PHARMACY DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA,
BANGALORE
By
DIVYA K V
Under The Guidance of
DR. KARUNAKAR HEGDE M.Pharm, Ph.D
DEPARTMENT OF PHARMACOLOGY
SRINIVAS COLLEGE OF PHARMACY
MANGALORE – 574143
2013 – 2015
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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1.0 NAME AND ADDRESS OF THE
CANDIDATE
DIVYA K V
VANI MANDIR
MAYIPPADI P O
KASARGOD DISTRICT
KERALA
PIN – 671124
2.0 NAME OF THE INSTITUTION SRINIVAS COLLEGE OF PHARMACY
VALACHIL, MANGALORE – 574143
3.0 COURSE OF STUDY AND
SUBJECT
MASTER OF PHARMACY IN
PHARMACOLOGY
4.0 DATE OF ADMISSION TO
COURSE
25/07/2013
5.0 TITLE OF THE TOPIC “EVALUATION OF ANTI-STRESS
ACTIVITY OF HYDRO-ALCOHOLIC
EXTRACT OF SAUROPUS ANDROGYNUS
LEAVES”
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6.0 BRIEF RESUME OF THE INTENDED WORK:
6.1. NEED FOR THE STUDY:
Stress can be termed as something that challenges our physical as well as mental well-
being. At certain times, stress motivates us and gets us going. Life can become boring and
purposeless if it is void of stress and everything goes on smoothly. But at most times,
stress affects both mental and physical health of a person in an adverse manner and is bad
for one's overall well being. In today’s world, every individual is likely to face stressful
situations in daily lives due to various factors such as insecurity, competition, job issues,
lack of sleep, illness, financial matters, etc1.
Stress is a reaction of the body to a stimulus which disturbs its physiological equilibrium
or in other word the homeostasis. When we feel stressed, the body’s defense system kicks
in an automatic process called as the “fight or flight” reaction or the stress response.
During a stress response, the hypothalamus secretes various hormones, namely the
corticotropin-releasing hormone. This stimulates the pituitary gland to release the
adrenocorticotropic hormone which in turn stimulates the cortex of the adrenal gland to
release cortisol in turn helps the body to regain homeostasis. It also suppresses the
immune-system for redistributing metabolic resources primarily to fight-or-flight organs.
Norepinephrine is a neuro-transmitter released by the locus ceruleans when stimulated by
the hypothalamus during stress response. It acts as a primary chemical messenger which
prepares the body for the flight or fight response.
Continued stress can result in a number of diseases like coronary heart diseases, liver
diseases, atherosclerosis, etc. as well as it can lead to conditions like hypertension,
headache, back pain, gastric ulcers, diabetes, depression, memory loss and can even
accelerate aging2, 3.
Drugs like benzodiazepines, amphetamines, caffeine and steroids are normally used to
fight against stress. But toxicity, undesired side effects and possibility of addiction is
reducing the attractiveness of these drugs. In this scenario, natural remedies to fight
against stress are gaining importance. In traditional medicines such as the Ayurveda,
several plants such as Glycyrrhiza glabra, Withania somnifera, Panax ginseng, etc. are
already recognized to have anti-stress activity. Scientific evaluation of these plants is
being conducted to confirm these properties4. There are such many plants considered as
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medicinally useful by the local tribes to ameliorate stressful conditions.
One such plant, Sauropus androgynus belongs to the family Euphorbiaceae, is a medicinal
herb having a long history of usage in Ayurveda. It has been used to fight headache, treat
urinary problems, act against fever, stimulate milk production and recover the womb after
birth. Sauropus androgynus is reported to contain a high content of vitamin K, provita-
min A carotenoids, vitamins B, vitamin C, protein and minerals5. The anti oxidative,
analgesic, anti inflammatory, anti-cancer and anti microbial effect of Sauropus
androgynus has been already studied6, 7. This plant has been found to contain high
concentrations of flavonoids, kaempferol and quercetin8. These flavonoids are well proven
to possess anxiolytic and anti depressant properties9, 10. The presence of high
concentrations of kaempferol and quercetin flavonoids in Sauropus androgynus leaves
makes it a possible candidate for screening its anti-stress potentials. However, no such
scientific data are available in published form to support anti-stress activity.
Keeping the above information in view, the present study has been designed to evaluate
the anti-stress activity of the hydro-alcoholic extract of the plant Sauropus androgynus
leaves.
6.2. REVIEW OF LITERATURE:
Botanical Name : Sauropus androgynus
Synonym : Katuk, Star Gooseberry or Sweet leaf
Vernacular names : Mani Cai (China), Amame Shiba (Japan), Pak Wan (Thai),
Rau Ngot (Vietnam) and Malay Cheera (Kerala, India)
Family : Euphorbiaceae
Description:
Sauropus androgynus is an erect perennial herb. It has upright main stems which grow up
to a height of 3.5 m. Leaves are dark-green, oval-shaped and 5-6 cm long. The leaves
usually have faint grayish spots. Leaves and stem tips are said to have pleasant taste. It has
orange/red flowers 1-2 cm in size. It bears fruits of the form of small capsules measuring
1.5 cm in diameter. Sauropus androgynus can tolerate high rainfall and also dry
conditions. The plant will grow in full sun, as well as in shade. Growth is rapid during the
warm months, slowing down in winter and even going dormant.
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Chemical constituents:
The main chemical constituents are kaempferol, quercetin, alkaloid papaverine,
provitamin A carotenoids, octadecatrienoic acid, ethyl ester, phytol, glycerin, 1-methyl-2-
pyrrolidineethanol, acetic acid, Pent-1-en-3-one, 1-(2-furyl)-5-dimethylamino,
Benzofuran, 2,3-dihydro-2-Acetylpyrrolidine, 4-O-methylmannose, N-Ethyl-2-
carbomethoxyazetidine etc5.
Uses:
In medicine, the leaves are used as tonic, for soothing lungs and for relieving from fever.
Leaves are also used for rejuvenating cells and for regular bowel elimination. It is also
used for treating headaches and urinary problems. It is also said to stimulate milk
production and also used for recovering the womb after birth. Sauropus androgynus is
highly nutritious and has many culinary uses. Fresh leaves are excellent source of
provitamin A carotenoids, vitamin B and C, proteins and minerals. Its shoot, fruit flowers
and leaves are eaten raw or cooked. Young shoots are sold as a delicacy in Malaysia and
fruits are candied. A green dye obtained from rubbing and squeezing the leaves is used as
food colour5, 6.
6.3. OBJECTIVES OF THE STUDY:
The main objective of the present study are as follows:
1. Authentication and collection of the plant Sauropus androgynus leaves.
2. Extraction of Sauropus androgynus leaves using 1:1 hydro-alcohol.
3. Preliminary phytochemical investigation.
4. Acute toxicity evaluation to derive the dose and safety.
5. To study the anti-stress activity of the hydro-alcoholic extract using following
animal models
a. Forced swimming endurance test
b. Chronic cold restraint stress test
6. To determine the following parameters
a. Plasma cortisol
b. Cholesterol
c. Triglycerides
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7. To evaluate histopathological examination of adrenal gland.
7.0 MATERIALS AND METHODS
7.1. SOURCE OF DATA
Experiment will be done in accordance with standard bibliography, literatures and text
books. The reputed journals and publications are obtained from college library and
through web search.
7.2. COLLECTION OF MATERIAL
7.2.1. COLLECTION OF PLANT MATERIAL AND EXTRACTION
Sauropus androgynus leaves will be collected from local region of Mangalore district, and
authenticated by a Taxonomist. Using 1:1 hydro-alcohol solvent system, the powdered
leaves will be extracted by hot percolation method (Soxhlet apparatus). The extract will be
filtered through a cotton plug followed by Whattman filter paper No.1 and then
concentrated by using a rotary evaporator at low temperature. The extract will be
preserved in airtight container and kept at 4-5°C until further use.
7.2.2. DRUGS AND CHEMICALS
All the drugs and chemicals will be of pure analytical grade and they will be obtained
from the local suppliers.
7.2.3. ANIMALS
Wistar rats (150-200 g) and Swiss albino mice (20-25g) of either sex will be used for
experimental study. They will be maintained under standard conditions (temperature 22 ±
2°C, relative humidity 50±5% and 12 hour light/dark cycle). The animals will be housed
in sanitized polypropylene cages containing sterile paddy husk as bedding. They will have
free access to standard pellet diet and water ad libitum. All the procedures will be
performed in accordance with Institutional Animal ethics committee constituted as per the
direction of the committee for the purpose of control and supervision of experiments on
animals (CPCSEA.
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7.3. EXPERIMENTAL METHODS
7.3.1. ASSESSMENT OF ACUTE TOXICITY
Acute toxicity study of the extract of will be performed as per the OECD guidelines 425 at
a limited dose of 2000 mg/kg or 5000 mg/kg. The doses will be administered by oral route
in mouse. Animals will observed individually at least once during the first 30 minutes
after dosing, periodically during the first 24 hours (with special attention given during the
first 4 hours), and daily thereafter, for a total of 14 days for sign of toxicity and/or
mortality if any. The LD50 will be calculated as per OECD guidelines11.
7.3.2. FORCED SWIMMING ENDURANCE TEST
Experimental Design
Wistar Rats (150 - 200g) of either sex will be randomly divided into four groups of six
animals each. The different groups will be assigned as below.
Group I : Vehicle control
Group II : Standard group (Diazepam)
Group III : Sauropus androgynus leaves extract (low dose)
Group IV : Sauropus androgynus leaves extract (high dose)
All the animals will be subjected for swimming on day 1, day 7, day 14 and day 21. The
animals will be allowed to swim till complete exhaustion and the endpoint will be taken
when they starts drowning. The mean swimming time for each group will be noted. All
the treatment will be given daily orally. On day 21, animals in each group will be
anaesthetized with anaesthetic ether and blood will be collected by retro-orbital puncture.
The anti-stress activity will be evaluated using parameters like plasma cortisol,
cholesterol, and triglycerides12.
7.3.3. CHRONIC COLD RESTRAINT STRESS TEST
Experimental Design
Wistar Rats (150-200g) of either sex will be randomly divided into four groups of six
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animals each. The different groups will be assigned as below.
Group I : Vehicle control
Group II : Standard group (Diazepam)
Group III : Sauropus androgynus leaves extract (low dose)
Group IV : Sauropus androgynus leaves extract (high dose)
All the animals will be exposed to a temperature of 4ºC for 2 h daily for a period of 10
days. All the treatment will be given daily orally. On the 11th day, the animals in each
group will be anaesthetized with anaesthetic ether and blood will be collected by retro-
orbital puncture. The anti-stress activity will be evaluated using parameters like plasma
cortisol, cholesterol and triglycerides. Further the adrenal gland will be collected by
sacrificing the animals and used for histopathological examinations13.
7.4. STATISTICAL ANALYSIS:
The data will be expressed as Mean value ±SEM and will be analyzed by the one-way
ANOVA.
7.5. DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR
INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER
HUMANS OR ANIMALS? IF SO PLEASE DESCRIBE BRIEFLY.
Yes. Study requires investigation on Wistar strain albino rats and mice.
7.6. HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR
INSTITUTION?
Yes. Ethical clearance has been obtained (Copy enclosed).
8.0 REFERENCES:
1. McEwen BS. Physiology and neurobiology of stress and adaptation: central role of the
brain. Physiol Rev 2007;87:873–904.
2. Lata H, Ahuja G, Narang A, Walia A. Effect of immobilisation stress on lipid
peroxidation and lipid profile in rabbits. J Pharmacol Online 2004;19:1-4
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3. Uresin Y, Erbas B, Mehmet O, Ozkok E, Gurol AO. Losartan may prevent the
elevation of plasma glucose, corticosterone and catecholamine levels induced by
chronic stress. JRASS 2004;5(2):93-6.
4. Vinod SP, Shivakumar H. A current status of adaptogens: natural remedy to stress.
Asian Pacific J Trop Disease 2012;2:S480.
5. Christi VEI, Perumal GN, Karpagavalli M, Malarkodi SA. Pharmacognostical,
physio-chemical and antimicrobial studies of Sauropus androgynus leaf. Herbal Tech
Industry 2011; 8(2):12-6.
6. Wei LS, Wee W, Siong JYF, Syamsumir DF. Characterization of antimicrobial,
antioxidant, anticancer properties and chemical composition of Sauropus androgynus
stem extract. Acta Medica Lituanica 2011;18(1):12–6.
7. Selvi VS, Bhaskar A. Characterization of anti-inflammatory activities and
antinociceptive effects of papaverine from Sauropus androgynus (L.) Merr. Global J
Pharmacol 2012;6(3):186-92.
8. Nuri A, Ratna B, Diny A S, Bradley B, Hanny W. Flavonoid content and antioxidant
activity of vegetables from Indonesia. Food Chem 2010;121:1231-5.
9. Hossein H, Vahidehsadat M, Farzin H. Antidepressant effect of kaempferol, a
constituent of saffron (Crocus sativus) petal, in mice and rats. Pharmacologyonline
2007;2:367-70.
10. Tong-Un T, Wannanon P, Wattanathorn J, Phachonpai W. Quercetin Liposomes via
Nasal Administration Reduce Anxiety and Depression-Like Behaviors and Enhance
Cognitive Performances in Rats. American J Pharmacol Toxicol 2010;5(2):80-8.
11. OECD, Guidelines for testing of chemicals, Acute oral toxicity, Environmental Health
and Safety Monograph Series on Testing and Adjustment No. 425, 2001, 1.
12. Anju. Adaptogenic and anti-stress activity of Ocimum sanctum in mice. Res J
Pharmac Biol Chem Sci 2011;2(3):670-8.
13. Ishola IO, Ashorobi RB. Anti-stress potential of aqueous root extract of Cnestis
ferruginea. Int J Pharmacol 2007;3(3):295-8.
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9.0 SIGNATURE OF THE CANDIDATE
10.0 REMARKS OF THE GUIDE Recommended and Forwarded.
11.0 NAME AND DESIGNATION
11.1 GUIDE DR. KARUNAKAR HEGDEASST. PROFESSORDEPARTMENT OF PHARMACOLOGYSRINIVAS COLLEGE OF PHARMACY MANGALORE – 574143
11.2 SIGNATURE
11.3 CO-GUIDE (IF ANY) Not Applicable
11.4 SIGNATURE CO-GUIDE -
11.5 HEAD OF THE
DEPARTMENT
DR. KARUNAKAR HEGDEHEAD OF DEPARTMENTDEPARTMENT OF PHARMACOLOGYSRINIVAS COLLEGE OF PHARMACYMANGALORE – 574 143
11.6 SIGNATURE
12.0 12.1 REMARKS OF THE PRINCIPAL
Strongly recommended and forwarded for favourable action.
12.2 NAME & SIGNATURE OF PRINCIPAL
DR. A. R. SHABARAYAPRINCIPALSRINIVAS COLLAGE OF PHARMACYMANGALORE- 574 143
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