STUDIES ON ANXIOLYTIC EFFECT OF Morus alba L.

LEAVES
EXTRACT IN RODENTS

SYNOPSIS FOR
M.PHARM DISSERTATION

SUBMITTED TO
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE.

BY
M.FAREED AHAMED
DEPARTMENT OF PHARMACOLOGY
NARGUND COLLEGE OF PHARMACY
BANGALORE-560085
(2010-2012)

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 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR PG DISSERTATION

1. Name of the candidate and address(in M. FAREED AHAMED

block letters) NARGUND COLLEGE OF PHARMACY,
DATTATREYA NAGAR, II MAIN,100 FT RING
ROAD,BSK III STAGE, BANGALORE-560 085

2. Name of the institution NARGUND COLLEGE OF PHARMACY

DATTATREYA NAGAR, II MAIN,100 FT RING
ROAD,BSK III STAGE, BANGALORE-560 085

3. Course of study and subject MASTER OF PHARMACY IN

PHARMACOLOGY

4. Date of the admission 22 JUNE 2010

5.
Title of the topic:

STUDIES ON ANXIOLYTIC EFFECT OF Morus alba L. LEAVES EXTRACT

IN RODENTS

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6. BRIEF RESUME OF THE INTENDED WORK:

6.1 NEED FOR THE STUDY:

Anxiety is a cardinal symptom of many psychiatric disorders and an almost inevitable component
of many medical and surgical conditions. Indeed, it is a universal human emotion, closely allied
with appropriate fear and often serving psycho biologically adaptive purposes. A most important
clinical generalization is that anxiety is rather infrequently a “disease” in itself. In addition
symptoms of anxiety are commonly associated with depression, especially with dysthymic
disorder, panic disorder, agrophobia, obsessive, compulsive disorder.1 Currently different
therapeutic regimens are employed to treat anxiety and depressive disorders; but their clinical uses
are limited by their side effects such as psychomotor impairment, potentiation of other central
depressant drugs and dependence liability. In the search for new therapeutics for the treatment of
neurological disorders-medicinal plant research has also contributed by demonstrating
pharmacological effectiveness of different herbs in various animal models.2
Mulberry tree, a plant of the family Moraceae and the genus Morus, has been widely cultivated to
feed silkworms. The leaves and the roots of Morus alba have also been used in traditional medicine
as a cathartic, analgesic, diuretic, antitussive, sedative, hypotensive, and antiphlogistic and for the
treatment of edema.3 A survey of the literature on M. alba revealed only a few pharmacological
reports on the plant. No major investigative reports were found pertaining to its CNS activity; 4
hence the study aimed to evaluate the anxiolytic activity of aqueous extract obtained from the
leaves of M. alba using different in vivo experimental models in rodents namely muricidal action
test and behavioural despair test.

6.2 REVIEW OF LITERATURE:

In traditional medicine the decoction of M. alba leaves is used as a gargle for relief of
inflammation of the throat. The plant contains flavonoids, coumarine, and stilbene, which have
hepatoprotective and free radical scavenging activity.5 The other uses of M. alba are as
hypoglycemic,6 cardioprotective,7 and neuroprotective agent.8 The mulberry fruit has been used as
a medicinal agent to nourish the blood and for the treatment of weakness, fatigue, anemia, and

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 premature greying of hair. In addition, some phenolic compounds from M. alba have been reported
to have antioxidant properties. A piperidine alkaloid and some glycoproteins were isolated from the
bark and leaves, which had antidiabetic effects.9 Phytochemical reports on M. alba L. indicates that
the plant contains flavonoids, tannins, triterpenes, anthocyanins, anthroquinones, phytosterols,
sitosterols, benzofuran derivatives, morusimic acid, oleanolic acid, alkaloids, steroids, saponins,
and phenolic compounds.7,10

Antiviral activity of flavonoids obtained from root bark of M. alba11

Study reported hypoglycaemic effect of M. alba leaf extract.12

Studies demonstrated melanin–concentrating hormone-1 receptor antagonism and
antidiabetic effects in diet induced obese mice from M. alba leaf extract.13

Results suggested that M. alba L. could improve high- fat diet-induced Hypotension,
hyperlipidemia, and vascular dysfunction through inhibition of cell adhesion molecules
expression and induction of vascular relaxation.14

The possibility of 5’-AMP-activated protein kinase is involved in metabolic enhancement
by M. alba leaf.15

6.3 OBJECTIVE OF THE STUDY:

Evaluation of the anxiolytic activity of aqueous extract obtained from the leaves of M. alba
using different in vivo experimental models in rodents namely muricidal action test and behavioural
despair test.
To estimate the superoxide dismutase content in rats brain and kidney.

7. MATERIAL AND METHOD

7.1 SOURCE OF DATA:
Data will be obtained from the experimental work, which includes laboratory based animals
studies and evaluation of various parameters.

7.2 METHOD OF COLLECTION OF DATA:

The data collected is based on animal experimentation as per the parameters studied under
each animal model. The sampling procedure and collection of data is as follows.

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METHODOLOGY:

7.2.1 Muricidal action test on rats:16

Model :
Observation cage
The observation cage consists of 30 cm x 30 cm x 25 cm with an open surface for maximal
ventilation illumination. The three sides were painted black to enhance easy observation of the
animals, behaviour and ensure that the animals were not distracted in the course of the experiment.
The base of the cage is not painted, but a known quantity of fine sawdust was placed there to
absorb any assault done on the cage by the animals, thereby ensuring the cleanlilness and dryness
of the cage. A modified small cage (6 cm x 6 cm x 15 cm) that is transparent all over with an open
top was introduced to study the muricidal effect of the extract.

Procedure:
The rats will be placed inside the observation cage (30 cm x 30 cm x 25 cm) inside which a smaller
cage (6 cm x 6 cm x 15 cm) that contain a mouse is placed in a definite corner of the bigger cage.
The number of aggressive attacks that the rats made on the mouse is recorded as a measure of
muricidal effect of the M. alba leaves extract.

Evaluation: Number of aggressive attacks on mouse.

Muricidal action of rats is observed.
Animals
Rats Male Wistar (8-10 weeks, 200-
250g)
Mice Swiss-Albino (4-6 weeks,18-
24 g)
No. of animal groups 7
No. of animals in each group 6
Vehicle of herbal drug Distilled water
Vehicle of standard drug Distilled water
Water and food Ad. Libitum

DRUGS AND TREATMENT:

Group I: Vehicle control

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Group II: Diazepam 2 mg/kg
Group III: Aqueous extract of M. alba leaves (Low dose- 100 mg/kg)
Group IV: Aqueous extract of M. alba leaves (High dose- 200 mg/kg)
Group V: Flumazenil 2 mg/kg i.p.
Group VI: Flumazenil + Aqueous extract of M. alba leaves (Low dose- 100 mg/kg)
Group VII: Flumazenil + Aqueous extract of M. alba leaves (High dose- 200 mg/kg)
No. of days of drug treatment: 21 days

7.2.2 Behavioural despair test:17

Procedure:
Rats will be forced to swim individually in glass jar (45 cm x 12 cm x 45 cm) containing
fresh water of height 35 cm and maintained at 25±3 oC. After an initial 2-3 min period of vigorous
activity, each animal assumed a typical immobile posture. A rat will be considered to be immobile
when it remained floating in the water without struggling, making only minimum movements of its
limbs necessary to keep its head above water. The total duration of immobility is recorded during
the next four min of a total 6 min test. Rats are taken then allowed to dry in a pre-warmed enclosure
(~32oC) before being returned to their home cage.

Evaluation: Floating time will be measured & Immobility of animals.

Animals:
Species Rat
Strain Wistar
Age and sex Two months, either sex
Body weight 160-175 g
No. of animals in each group 10
No. of groups 7
Vehicle of herbal drugs Distilled water
Vehicle of standard drugs Distilled water
Water and food Ad. Libitum

DRUGS AND TREATMENT:

Group I: Vehicle control
Group II: Diazepam 2 mg/kg
Group III: Aqueous extract of M. alba leaves (Low dose- 100 mg/kg)
Group IV: Aqueous extract of M. alba leaves (High dose- 200 mg/kg)

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 Group V: Flumazenil 2 mg/kg i.p.
Group VI: Flumazenil + Aqueous extract of M. alba leaves (Low dose- 100 mg/kg)
Group VII: Flumazenil + Aqueous extract of M. alba leaves (High dose- 200 mg/kg)
No. of days of drug treatment: 21 days

Estimation of superoxide dismutase (SOD) in rats brain and kidney :17

SOD estimation was performed based on its ability to spontaneously inhibit oxidation of adrenaline
to adrenochrome.18 2.78 ml of sodium carbonate buffer (0.05mM; pH 10.2), 100 µl of EDTA (1.0
mM) and 20 µl of the supernatant or sucrose (blank) were incubated at 30 oC for 45 min. Thereafter,
the reaction was initiated by adding 100 µl of adrenaline solution (9.0 mM). The change in the
absorbance was recorded at 480 nm for 8 min. Throughout the assay procedure temperature was
maintained at 30oC. One unit of SOD produced approximately 50% of auto-oxidation of adrenaline.
Results were expressed units/mg protein

STATISTICAL ANALYSIS:
The data will be analyzed using analysis of variance (ANOVA) followed by the student-
Newman-keuls test for multiple comparisons.

7.3 Does the study require any investigation to be conducted on patients

Or other humans or animals? If so please describe briefly
Yes, The above study requires investigation on animals. The effect of drug will be studying
on various parameters using rats and mice as animal model.

7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Applied for the IAEC of Nargund college of pharmacy

REFERENCE:

1. Baldessarini RJ, Hardman JG, Limbird LE, Gilman AG. Goodman & Gilman The
Pharmacological Basis of Therapeutics. 10th ed. McGraw-Hill; 2001. P. 472-3.
2. Samhita C (Chikitsa stana-1), Sharma PV, Orientalia C.Varanasi.1996

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 3. Taro Nomura. The chemistry and biosynthesis of isoprenylated flavonoids from moraceous
8. plants. Pure Appl Chem 1999;71:1115-8.
4. Yadav AV, Kawale LA, Nade VS. Effect of Morus alba L. (mulberry) leaves on anxiety in
mice. Indian J Pharmacol 2008;32-6.
5. Oh H, Ko EK, Jun JY, Oh MH, Park SU, Kang KH, et al . Hepatoprotective and radical
scavenging activities of preflavonoids, Coumarin and stilbene from Morus alba. Planta Med
2002;68:932-4.
6. Hesham A, Abdel NB, Jari S, Kalevi P. Hypolipidemic and antioxidant effect of Morus alba
L (Egyptian mulberry) root bark fractions supplementation in cholesterol- fed rats. J
Ethnopharmacol 2005;78:2724-33.
7. Enkhmaa B, Shiwaku K, Katsube T, Kitajima K, Anuurad E, Yamasaki M, et al . Mulberry (
Morus alba L.) leaves and Their major flavonal Quercetin 3-(6-malonylglucaside) attenuate
Atherosclerotic Lesion development in LDL receptor-deficient mice. J Nutr 2005;135:729-
34.
8. Kang TH, Oh HR, Jung SM, Ryu JH, Park MW, Park YK, et al . Enhancement of
Neuroprotection of Mulberry leaves ( Morus alba L.) prepared by the Anaerobic treatment
against ischemic damage. Biol Pharm Bull 2006;29:270-4.
9. Abdel NB, Hesham A, Makiko Y, Taro N, Toshio F. Hypoglycemic effect of Egyptian
Morus alba root bark extract: Effect on diabetes and lipid peroxidation of streptozotocin
-induced diabetic rats. J Ethnopharmacol 2005;100:333-8.
10. Kusano G, Orihara S, Tsukamato D, Shibano M, Coskun M, Guvenc A, et al . Five new
nortropane alkaloids and six new amino acids from the fruit of Morus alba L. growing in
Turkey. Chem Pharm Bull 2002;50:185-92.
11. Jiang DU, Zhen-Dan He, Ren-Wang Jiang, Wen-Caiye, Hong-Xixu, Paul-Hay But. Antiviral
flavonoids from the root bark of Morus alba L. Phytochemistry 2003;62(8):1235-8.
12. Mohammadi J, Naik PR. Evaluation of hypoglycemic effect of Morus alba in an animal
model. Indian J Pharmacol 2008;40:15-8
13. Kwang-seok Oh, Shiyong Ryu, Snghou Lee, Howon Seo, Byung Koo Oh, Young Sup Kim,
Byung Ho Lee. Melanin-concentrating hormone -1 receptor antagonism and anti-obesity
effects of ethanolic extract from Morus alba leaves in diet induced obese mice. J
Ethnopharmacol 2009;122(2):216-20 .
14. Yun Jung Lee, Deok Ho Choi, Sun Mi Hwang, You Mee Ahn, Jin Sook Kim, et

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 al.Hypotensive, hypolipidemic and vascular protective effects of Morus alba L.In rats fed a
high fat diet. FASEB J 2010;24:986-3.
15. Xiao Ma, Iwanaka N, Masuda S, Karaike K et al. Morus alba leaf extract stimulates 5-
AMP-activated protein kinase in isolated rat skeletal muscle. J Ethnopharmacol
2009;122(1):54-9.
16. Ayoka AO, Akomolafe RO, Iwalewa EO, Ukponmwan EO. Studies on Anxiolytic effect of
Spondias mombin L.(Anacardiaceae) extracts. Afr. J.Trad. CAM 2005;2(2):153-65.

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17. Nimal J, Babu CS, Harisudhan T, Ramanathan M. Evaluation of behavioral and anti
oxidant activity of Cytisus scoparius Link in rats exposed to chronic unpredictable mild
stress . BMC Complement Alter Med 2008;8:15.
18. Saggu H, Cooksey J, Dexter JJ.A selective increase in particulate superoxide dismutase
activity in parkinsonian substantia nigra. J Neurochemistry 1989;53:692-7.

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9 SIGNATURE OF THE CANDIDATE

10 REMARKS OF THE GUIDE

RECOMMENDED

11 NAME AND DESIGNATION

11.1:- GUIDE Dr. H J. HRISHIKESHAVAN

PROFESSOR AND HEAD
DEPT. OF PHARMACOLOGY
NARGUND COLLEGE OF
PHARMACY,
Bangalore-560085.

11.2:- SIGNATURE

11.3:- CO-GUIDE

11.4:- SIGNATURE

11.5:- HEAD OF THE DEPT. Dr. H J. HRISHIKESHAVAN

PROFESSOR AND HEAD
DEPT. OF PHARMACOLOGY
NARGUND COLLEGE OF
PHARMACY,
Bangalore-560085.

11.6:- SIGNATURE

12 REMARKS OF THE PRINCIPAL

RECOMMENDED

12.1 :- SIGNATURE

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