Evaluation of anxiolytic effect of whole plant of cuscuta reflexa.

Abstract

The anxiolytic effect of methanol extract of whole plant of Cuscuta reflexa at doses of 200 and 400 mg/kg was evaluated by elevated plus maze and light and dark chamber in mice. Animals were treated orally with normal saline, extract and diazepam 5 mg/kg used as standard drug. The extract 400 mg /kg showed significant anxiolytic effect compared to 200 mg/kg in both models. The 400 mg/kg effect was comparable to standard. Thus methanol extract of Cuscuta reflexa could serve as good anxiolytic agents and seems to be promising for the development of phytomedicines for anxiety.

Full text

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Thomas et al. World Journal of Pharmacy and Pharmaceutical Sciences
EVALUATION OF ANXIOLTIC EFFECT OF WHOLE
PLANT OF “CUSCUTA REFLEXA”
Sujith Thomas*1, Sapna Shrikumar2, C Velmurugan3 and B.S. Ashok Kumar4
1Department of Pharmacology, Moulana College of Pharmacy, Kerala-679321.
2Department of Pharmacognosy, Moulana College of Pharmacy, Kerala-679321.
3Department of Pharmacology, Sri Krishna Chaithanya College of Pharmacy,
Madanapalle, AP-517325
4Department of Pharmacognosy, Sri K.V. College of Pharmacy, Chickballapur, Karnataka.
ABSTRACT
The anxiolytic effect of methanol extract of whole plant of Cuscuta
reflexa at doses of 200 and 400 mg/kg was evaluated by elevated plus
maze and light and dark chamber in mice. Animals were treated orally
with normal saline, extract and diazepam 5 mg/kg used as standard
drug. The extract 400 mg /kg showed significant anxiolytic effect
compared to 200 mg/kg in both models. The 400 mg/kg effect was
comparable to standard. Thus methanol extract of Cuscuta reflexa
could serve as good anxiolytic agents and seems to be promising for
the development of phytomedicines for anxiety.
KEYWORDS: Anxiolytic, Cuscuta reflexa, methanol, light and dark
chamber, elevated plus maze.
INTRODUCTION
The science and technology have contributed to an enormous improvement in the quality of
life of humankind. However, modern life stress and associated trails and tribulation are
responsible for the surge in incidence of variety of psychiatric disorders. Anxiety disorders,
the most prevalent psychiatric illnesses in the general community are present in 15 to 20
percent of medical clinic patients. The primary anxiety disorders are classified according to
their duration and course and the existence and nature of precipitants.[1] Approximately one-
third of medical patients presenting with anxiety have an organic etiology for their
psychiatric symptoms, but an anxiety disorder can also present with somatic symptoms in
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Article Received on
27 May 2015,
Revised on 22 June 2015,
Accepted on 15 July 2015
*Correspondence for
Author
Sujith Thomas
Department of
Pharmacology, Moulana
College of Pharmacy,
Kerala-679321.

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Thomas et al. World Journal of Pharmacy and Pharmaceutical Sciences
absence of diagnosable medical condition.[2] Path breaking research in psychopharmacology
has floded the market place with drugs for specification. For instance, benzodiazepines
(diazepam, nitrazepam, lorazepam and alprazolam etc.) are the most frequently prescribed
synthetic drugs for variety of condition particularly anxiety, depression, epilepsy and
insomnia. But these psychoneural drugs have very serious side effects like chronic use of
benzodiazepines causes detioriation of cognitive function, physical dependence and
tolerance. Besides addiction-liabilities, benzodiazepines adversely affect the respiratory,
digestive and immune system of body. Since the chronic treatment with benzodiazepines
often prove more harmful in the longer run.[3]
Cuscuta reflexa Roxb. is a rootless, leafless perennial parasitic twining herb of
Convolvulaceae family, commonly known as Akashvalli or Dodder. The plant is distributed
worldwide and in India about 6 species are found. It has no chlorophyll and cannot make its
own food by photosynthesis. It grows on thorny or other shrubs, sometimes completely
covering the bushes and trees.[4] The Cuscuta reflexa is investigated for antitumor[4],
antimicrobial[5], hepatoprotective[6], anticonvulsant[7], antioxidant[8], induced alopecia[9]
activities. Many chemical constituents have been isolated from Cuscuta reflexa such as
cuscutin, amarbelin, beta-sterol, stigmasterol, myricetin, qurecetin, cuscutamine, luteolin,
bergenin[10]etc. So this is medicinal important plant having numerous pharmacological
activities. So the present study designed to evaluate the anxiolytic effect of whole plant of
Cuscuta reflexa.
MATERIAL AND METHODS
Collection and authentication of the plant material
The whole plant of Cuscuta reflexa had been collected from the medicinal garden of sri
Krishna Chaithanya college of Pharmacy, Chittoor District, Andhra pradesh, India. The plant
was identified and authenticated by the Botanist Dr. K. Madhava Chetty, Assistant Professor,
Department of botany, Sri Venkateswara University, Tirupathi.
Extraction procedure
The fresh whole plant of Cuscuta reflexa were collected and dried under shade and ground
into powder with mechanical grinder. The powder was passed through sieve no. 40. The
250gm of dried powder of Cuscuta reflexa was defatted with petroleum ether. The defatted
powder material (marc) thus obtained was extracted with methanol by maceration.[11]

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Thomas et al. World Journal of Pharmacy and Pharmaceutical Sciences
Table: Data showing the % yield of dried powder of Cuscuta reflexa
Plant name
Part used
Method of
extraction
Colour of extract
Nature of
extract
% yield
of extract
Cuscuta reflexa
Whole plant
Maceration
greenish Brown
Semi solid
12.56%
Preliminary Phytochemical Investigation
Methanol extract of Cuscuta reflexa was subjected to qualitative analysis for the various
phytoconstituents.[12]
Acute oral toxicity study[13]
Acute oral toxicity studies was performed as per OECD-423 guidelines (acute toxic class
method), with methanol extract of Cuscuta reflexa. Swiss albino mice with weight ranging
(20-25 gm female) were taken for the experiment. The animals were made into a group of
third each, dose of methanol extract was given according to the body weight (mg/kg), starting
dose of 5 mg /kg was given to the first individual animal, no death was occurred, and higher
doses were given to next group of animals.
Table: Acute toxicity study of Methanol extract of Cuscuta reflexa based on OECD 423
guidelines.
S. No
Number of animals
Dose in mg/kg
Report
1
3
5mg/kg
No death
2
3
50mg/kg
No death
3
3
500mg/kg
No death
4
3
2000mg/kg
No death
Table: Study period and observation parameters
Initial once observation
First 30 min and periodically 24 hr
Special attention
First 1-4 hr after drug administration
Long term observation
Up to 14 days
Direct observation parameters
Tremors,
convulsions,
salivation,
diarrhea,
lethargy,
sleep and coma.
Additional observation parameters
Skin and fur,
eyes and mucous membrane,
respiratory, circulatory,
autonomic and central nervous systems,
somatomotor activity and behavior pattern etc.
From the observation the methanol extract of Cuscuta reflexa was does not cause any
mortality and morbidity up to 2000mg/kg. Hence 2000mg/kg consider as LD50. Therefore its

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Thomas et al. World Journal of Pharmacy and Pharmaceutical Sciences
ED50 was found to be 1/10th of LD50 as 200 mg/kg. The present study doses was fixed by
ED50 as 200 & double the dose of ED50 as 400 mg/kg of whole plant of methanol extract of
Cuscuta reflexa
ANTI-ANXIETY ACTIVITY
Experimental design
Swiss albino mice (20-25gm) were taken and divided into four groups, each group comprised
of 6 animals.
Group I : Negative control, administer normal saline as vehicle orally.
Group II : Received Cuscuta reflexa 200 mg/kg orally.
Group III : Received Cuscuta reflexa 400 mg/kg orally.
Group IV : Received standard drug Diazepam 5mg/kg orally.
Elevated plus maze[14]
The apparatus consist of two open arms (5x10cm) and two closed arms (5x10x15cm)
radiating from a platform (5x5cm) to form a plus-sign figure. The apparatus was situated 40
cm above the floor. The open-arm edges were 0.5 cm in height to keep the mice from falling,
and the closed-arm edges were 15 cm in height. Drugs and vehicle were administered to the
animals 30 min prior to study as per experimental design. Then the animal was placed at the
center of the maze, facing one of the closed arms and observed for 5 min. During 5 min test
period the number of entries into open and closed arms and time spent in the open arms and
closed arms were noted. Arm entry was counted when the animal has placed all of its four
paws on it. The procedure was conducted preferably in a sound attenuated room, with
observations made from an adjacent room.
Light and Dark chamber[15]
The apparatus consisted of a light and dark unit, consisting of a box with two distinct
chambers, black and white, connected by a small open door way. The dark compartment
(15x20cm) had its sidewalls and floor covered by a smooth black colored sunmica material.
The bright compartment (20x20cm) had its side walls and floor covered by white colored
sunmica material; it was brightly illuminated with a 60 W milky white bulb located 28 cm
above the floor on the partition wall. An opening of (7x9cm) was located at a floor level in
the center of the partition to allow access between two compartments for the animal.
Drugs and vehicle were administered to the animals 30 min prior to study as per experimental
design. At the start of the test, a mouse is placed in the middle of the illuminated part of the

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cage. The number of crossing into light and dark chamber is counted and time spent in light
and dark chamber during 10 min period were noted.
STATISTICAL ANALYSIS
Results were expressed as Mean±SEM .The differences between experimental groups were
compared by one-way Analysis of Variance (ANOVA) followed by Dunnett’s test and were
considered statistically significant when *p<0.05&**p<0.01.
RESULTS
Preliminary Phytochemical Screening
Qualitative analysis of the methanol extract of Cuscuta reflexa contains Flavonoids,
Alkaloids, sterols, carbohydrates, Aminoacids, Terpenoids & Saponins, Tannins, proteins,
phenolic compounds.
Table: Preliminary Phytochemical screening of the methanol extract of Cuscuta reflexa.
S.No
Constituents
Tests
Methanol
1
Alkaloids
Mayer’s test
Dragondraff’s test
Hager’s test
Wagner’s test
+
+
+
+
2
Sterols
Burchard test
Salkowski’s
+
+
3
Carbohydrates
Molisch’s test
Fehling’s test
Benedict’s test
Borntragers’s test
+
+
+
+
4
Glycosides
Legal test
Kellerkiallani test
-
-
5
Fixed oils & Fats
Spot test
Saponification test
-
-
6
Phenolic Compounds
Ferric chloride
+
7
Proteins & amino acids
Biuret test
Ninhydri test
Millon’s test
+
+
+
8
Terpenoids& Saponins
Foam test
Haemolysis test
+
+
9
Tannins
Gelatin test
Fecl3 test
+
+
10
Gums & mucilage
Precipitation to
90%alcohol
-
11
Flavonoids
Shinoda test
Lead acetate test
Ferric chloride test
Zinc HCL test
+
+
+
+
Where + =present, - =absent

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Elevated plus maze
High dose of methanol extract of whole plant of Cuscuta reflexa (400 mg/kg, p.o) and
standard drug (diazepam 5 mg/kg, p.o) showed a significant (p<0.01)decrease in the number
of entries into closed arms and increase in the number of entries open arms and also
significantly increase the time spent in open arms when compared with control. Low dose of
methanol extract of Cuscuta reflexa (200 mg/kg, p.o) shows non-significant when compared
to control except entry in closed arms shows less significant (p<0.05) whereas 400 mg/kg and
standard drug shows more significant (p<0.01) variation.
Table: Effects of methanolic extract of Cuscuta reflexa in elevated plus-maze test
Groups
No of entry into
Time spent in sec
Closed arms
Open arms
Closed arms
Open arms
Group I
13.33+1.11
4.83+1.35
155.24±6.54
78.26±8.46
Group II
9.83+1.35*
6.26+0.80ns
132.67±4.25ns
89.83±7.58ns
Group III
7.66+1.20**
8.83+1.66**
78.28±3.58**
152.58±8.25**
Group IV
7.16+0.95**
9.66+1.3**
64.19±4.63**
169.52±8.14**
All values are Mean+SEM, n = 6, ns- non-significant, *P<0.05, **P<0.01 When compared
with control.
Light and dark area
Standard drug (diazepam 4 mg/kg, p.o) and high dose of methanol extract of Cuscuta reflexa
(400 mg/kg, p.o) significantly increased the time spent in light chamber and also significantly
(p<0.01) increased the number of crossing into light and dark chamber compared to control.
Low dose of methanol extract of Cuscuta reflexa (200 mg/kg, p.o) showed significant
(p<0.05) differences in light and dark chamber activity when compared to control.
Table: Effects of methanol extract of Cuscuta reflexa in light and dark chamber test
Groups
No of crossing into
Time spent in
Light chamber
Dark
chamber
Light
Chamber
Dark chamber
Group I
21.3+3.84
22.33+1.97
149.5+9.29
229.5+18.43
Group II
29.16+1.24*
28.5+1.58*
197.33+9.6*
199+15.33*
Group III
36.83+1.24**
31.1+1.35**
278.33+16.2**
133.16+19.02**
Group IV
39.5+1.56**
39.1+1.35**
282+11.91**
153.1+12.4**
All values are Mean+SEM, n = 6, *P<0.05, **P<0.01 When compared with control
DISCUSSION
The present investigation successfully detected the anxiolytic-like effects of methanol extract
of Cuscuta reflexa. Light and dark chamber & Elevated plus-maze test was used to evaluate

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anxiety aspects of rodents.[16] The present study showed that methanol extract of Cuscuta
reflexa 400 mg/kg significantly increased the time spent on the open arms and decreased the
number of entries into closed arms. Diazepam also showed similar type of effect but the
extract 200 mg/kg effect as like as negative control. The light and dark chamber model
showed that methanol extract of Cuscuta reflexa increase the time spent in light chamber and
number of entries into light chamber significantly similar to the actions produced by
diazepam. Earlier studies showed that a reduction of anxiety-related behavior is observed
after administration of 5-HT1B receptor antagonists in different animal models. In general,
this data support the assumption that 5-HT1B receptor antagonist can exert anxiolytic
activity. High level of these receptors has been found in brain regions that are implicated in
the modulation of 5-HT1B auto receptor in the hippocampus and amygdala. Furthermore
study showed that participation of GABA/BZD receptors and 5-HT1A receptors also play a
role in anxiolytic activity. GABA/BZD and 5-HT1A agonists seem to be related to the
anxiolytic action.[17]
Earlier investigation of the phytoconstituents of medicinal plants reported that many
flavonoids are ligands for GABA-A receptors in the central nervous system which suggests
that they can act as benzodiazepine-like agents.[18] It has also been reported that some
flavanoids exhibit high affinity binding to the benzodiazepine site of GABA-A receptors.[19]
The studies reflected that methanol extract of Cuscuta reflexa (400 mg/kg) was effective in
both models of anti-anxiety activity compare to extract 200mg/kg. The Anxiolytic activity
might be due to the phytoconstituents, which is flavanoid in the methanol extract of the
Cuscuta reflexa.
CONCLUSION
The methanol extract of Cuscuta reflexa 400 mg/kg increased the number of crossing in open
field model, increase the time spent in open arm, entry in open arm (in elevated plus model)
as well as increase the time spent in light field (in Light Dark field) than the 200 mg/kg dose
thus we can conclude that Cuscuta reflexa shows anxiolytic action as dose dependent manner.
Conversely, it is very difficult to draw any definite conclusion about the mechanism of
anxiolytic activity of the extract. Further study is needed to identify the active constituent and
elucidate the possible mechanism.

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