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

1

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”

2

3

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

4

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.

5

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

6

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.

7

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

8

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

9

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.

10

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

11