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© 2016 Pharmacognosy Magazine|Published by Wolters Kluwer‑Medknow 1
This is an open access article distributed under the terms of the Creative Commons
Attribution‑Non Commercial‑Share Alike 3.0 License, which allows others to remix,
tweak, and build upon the work non‑commercially, as long as the author is credited
and the new creations are licensed under the identical terms.
For reprints contact: reprints@medknow.com
Cite this article as: FPO
Submitted: 18‑08‑2016 Revised: 24‑10‑2016 Published: 00‑00‑0000
ABSTRACT
Alcohol addiction is a worldwide problem. It has mainly two components:
dependence and withdrawal. Characteristic properties of most anti-addictive
compounds include anti-anxiety, anticonvulsant, antidepressant, and
nootropic actions. Shankhpushpi (Convolvulus pluricaulis. Convolvulaceae),
known ethnopharmacologically as brain tonic, possess all the properties
mentioned above. Here, we screen shankhpushpi for possible anti-addictive
potential. Effect of shankhpushpi churna was measured on ethanol
withdrawal anxiety using elevated plus maze. The role of shankhpushpi
on chronic ethanol consumption (21 days) was measured using two bottle
choice protocol of voluntary drinking. We also measured the effect of
the above herb on cortico-hippocampal GABA levels. Shankhpushpi was
found to reduce alcohol withdrawal anxiety in a dose-dependent manner.
The herb also decreased ethanol intake and increased water intake
signicantly (P < 0.001) after 4 days of administration. Both these effects
were blocked (P < 0.001) by GABAA antagonist suggesting the role of
GABAA receptor. Chronic administration of shankhpushpi also signicantly
(P < 0.01) increased cortico-hippocampal GABA levels in mice. Shankhpushpi
reduced both alcohol dependence and withdrawal in a GABAA-dependent
manner, thus showing anti-addictive potential.
SUMMARY
• Shankhpushpi prevented ethanol withdrawal anxiety and alcohol addiction in
a GABAA-dependent manner.
Eect of Shankhpushpi on Alcohol Addiction in Mice
Mahi Heba, Sana Faraz, Sugato Banerjee
Department of Pharmaceutical Science and Technology, Birla Institute of Technology, Mesra, Ranchi, Jharkhand, India
INTRODUCTION
Alcohol addiction is a worldwide problem. Alcohol consumption
accounts for more than 3.3 and is the h leading risk factor for
premature death and disability. Alcohol contributes various diseases and
injury-related health conditions, most notably alcohol dependence, liver
cirrhosis, cancers, HIV, and injuries, primarily road trac accidents.[1,2]
Alcohol dependence is a complex and dynamic process involving
various neurobiologic and environmental factors.[3] Individual’s
propensity to alcohol consumption is primarily a balance between
alcohol’s rewarding eects and its withdrawal consequences. One
factor contributing to relapse is withdrawal related anxiety, which
likely reects adaptive changes in the brain in response to continued
alcohol exposure. Association of these pleasant/unpleasant feelings with
environmental clues may inuence alcohol intake.[4] Alcohol withdrawal
symptoms include irritability, agitation, anxiety, sleep disturbances,
and reduced pain threshold both in human and animals.[5] Disulram,
naltrexone, and acamprosate are US Food and Drug Administration
(US-FDA) approved medications for treatment of alcohol dependence.
However, they have various side eects including palpitation, ushing,
nausea, vomiting, headache, anxiety, sedation, and transient diarrhea,
which aect the quality of life of the addicted individual under
anti-addictive therapy.
Herbal medicines can be used for the development of new therapeutically
active compounds with higher potency and lower toxicity. Most modern
drugs have a natural origin and play a major role in drug development.[6]
Shankhpushpi (Convolvulus pluricaulis Choisy) is a perennial herb
referred to as morning glory. e plant is used locally in Indian and
Chinese system of medicine to treat various diseases. Its dierent
pharmacologic actions include cough suppressant, antihypertensive,
antiulcer, hypolipidemic, and against various neurologic disorders. It
may be used as a stress reliever, a mood stabilizer, and memory enhancer.
It may also reduce peripheral nerve hyperexcitability, as well as used as an
adjuvant to anticonvulsant therapy.[7,8] Its primary chemical constituent
pm_376_16
Key words: Alcohol, addiction, Convolvulus pluricaulis, shankhpushpi,
mice Correspondence:
Dr. Sugato Banerjee,
Department of Pharmaceutical Sciences and
Technology, Birla Institute of Technology,
Mesra, Ranchi, Jharkhand, India.
E-mail: sbanerjee@bitmesra.ac.in
Pharmacogn. Mag.
A multifaceted peer r
eviewed journal in the eld of Pharmacognosy and Natural Products
www
.phcog.com|www.phcog.net
Abbreviations used: GABA: Gamma-Aminobutyric Acid, HIV: Human
Immunodeciency Virus, CNS: Central Nervous System
ORIGINAL ARTICLE
MAHI HEBA, et al.: Eect of Shankhpushpi on Alcohol Addiction
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2 Pharmacognosy Magazine, Apr‑Jun 0000, Vol 00, Issue 00 (Supplement 2)
includes alkaloids like shankhpushpine, convolamine, convoline,
convolidine, convolvine, confoline, convosine, and so on, found in
dierent species from this family. e fresh plant contains volatile oils,
fatty acids, fatty alcohols, and hydrocarbons like myristic acid, palmitic
acid, linoleic acid, and straight-chain hydrocarbons like hextriacontane.
e avonoid kampferol and steroids phytosterol, β-sitosterol have also
been extracted.[7,9] Many of these active constituents isolated from aerial
parts of C. pluricaulisy like shankhpushpine, scopoletin, kaempferol
phytosterol, and β-sitosterol have been shown to act as GABAA agonists,
which could be attributed to the anxiolytic and CNS-depressant actions
of shankhpushpi.[10]
Here, we report that acute administration of shankhpushpi may
prevent alcohol withdrawal anxiety, whereas its chronic administration
may reduce alcohol consumption in mice. Chronic administration of
shankhpushpi extract led to change in cortico-hippocampal GABA
levels, whereas GABAA blocker completely reversed its anti-addictive
properties. us, above results suggest that C. pluricaulis may prevent
alcohol addiction in a GABAA-dependent manner.
MATERIALS AND METHODS
Animals
Swiss albino mice (20–30 g) were used in this study. Animals were issued
from the Institutional Animal House (Reg. No. 621/02/AC/CPCSEA) of
Birla Institute of Technology, Mesra. All animals were kept in polyacrylic
cages and maintained under standard conditions (room temperature
24–27°C and humidity 60–65% with 12:12 light:dark cycles). e food
was provided in the form of dry pellets and water ad libitum. e animals
were allowed to get acclimatized to the laboratory conditions for 7 days
before the commencement of the experiment. All experiments involving
animals complied with the ethical standards of animal handling and
approved by the Institutional Animal Ethics Committee (BIT/PH/
IAEC/28/2013).
Estimation of blood alcohol levels
Blood was collected by retro-orbital bleeding with animals in light ether
anesthesia aer 20 min of ethanol administration. Ethanol levels were
measured using ultraviolet (UV) assay kit for alcohol estimation based
on manufacturer’s protocol (NZY Tech Genes and Enzymes Portugal).
Development of conditioned place preference
model
Apparatus
e conditioned place preference (CPP) apparatus contain three
compartments. e two end compartments (30.5 cm × 26.5 cm × 37 cm)
were connected by a central corridor (12.75 cm × 23 cm × 15.25 cm). e
compartment on the le had black walls with a perforated stainless steel
oor with round holes on staggered centers. e central corridor was
transparent with a smooth plexiglass oor, and the right compartment
had white walls with a stainless steel mesh oor.
CPP was performed as described previously,[11,12] with slight modications.
It mainly consists of three phases:
(1) Preconditioning phase: (rst and second day) e animals were
placed in the middle chamber and allowed to explore both the chambers
for 30 min.
(2) Conditioning phase: (3rd - 10th day) Each mouse was treated for eight
consecutive sessions with the alternate oral administration of alcohol
and saline. On days 3, 5, 7, and 9, the animals were administered ethanol
(2 g/kg body weight; i.p. 10% [v/v]) and placed in one compartment for
30 min. Besides, on days 4, 6, 8, and 10, the animals were administered
saline and placed in opposite compartment.
(3) Postconditioning phase: (11th–12th day) Mice were placed in the
middle chamber and allowed free access to both chambers for 30 min.
Time spent in ethanol and saline-paired chamber was measured.
(4) Treatment protocol: Aer development of withdrawal (15th day), the
following treatment schedule was followed:
Group 1: saline
Group 2: ethanol
Group 3: ethanol + shankhpushpi (Vyas Pharmaceuticals, Haridwar; 100
mg/kg)
Group 4: ethanol + shankhpushpi (200 mg/kg)
Group 5: ethanol + diazepam (1 mg/kg)
Group 6: ethanol + GABAA antagonist
Group 7: ethanol + GABAB agonist
Group 8: ethanol + shankhpushpi + GABAA antagonist
Group 9: ethanol + shankhpushpi + GABAB agonist
e behavioral tests were performed 60 min aer oral drug administration
and 30 min aer intraperitoneal administration.
Behavioral studies to measure alcohol withdrawal
anxiety
Elevated plus maze
e model has been validated pharmacologically and currently
considered the “gold standard” test of anxiety-related behavior. Elevated
plus maze (EPM) was performed as described by Kokare et al.[13] In
summary, aer drug treatment, individual mice were placed at the center
of the maze, head facing an open arm. During the 5 min test period,
the number of entries and time spent on the open arm were recorded
automatically (Medicra Electromedical, Lucknow, India).
Chronic‑treatment study to measure alcohol intake
Two‑bottle choice ethanol drinking
We used the standard two-bottle choice protocol, which is a widely used
animal model to capture aspects of voluntary alcohol consumption in
humans.[14] Following 7 days of acclimatization, animals were subjected
to an ethanol drinking acquisition regimen. e animals remained in
their home cages at all times throughout the study but had their water
bottles removed during a 4 h and ethanol presentation period. During this
time, animals were exposed to a free choice between ethanol (15% v/v)
and water for 20 days but with no drug pretreatment.
Aer 20 days of ethanol administration, animals were divided into
dierent groups for 10 days of treatment. Each day, the bottles were
weighed before and aer 4 h of limited access period and the dierences
were used to calculate the water and ethanol intake. e mean intake was
expressed as g/kg body weight/day of water and g/ kg body weight/day of
ethanol intake. All animals were given unrestricted food access. Every 2
days, the bottles were switched to eliminate place preference.[15] Aer 20
days of pretreatment with ethanol (15% v/v), the animals were divided
into dierent treatment groups (n = 7 per group) as follows:
Group 1: (control) received saline 30 days
Group 2: received free choice ethanol (15% v/v)/water 30 days
Group 3: received free choice ethanol (15% v/v)/water and shankhpushpi
(200 mg/kg) 21st–30th day
Group 4: received free choice ethanol (15% v/v)/water and diazepam
21st–30th day.
Group 5: received free choice ethanol (15% v/v)/water, GABAA antagonist
and shankhpushpi 21st–30th day.
(Thermo Fisher Scientific (India) Pvt. Ltd)
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Pharmacognosy Magazine, Apr‑Jun 0000, Vol 00, Issue 00 (Supplement 2) 3
Aer the above experimental protocol of 30 days, ve animals per
group were sacriced under ether anesthesia by cervical dislocation for
biochemical estimation.
Estimation of gamma amino butyric acid levels from brain tissue
Brain tissue was homogenized in 5 mL of 0.01 M HCl. In this homogenate,
8 mL of ice cold ethanol was added and kept for 1 h at 0°C. e contents
were centrifuged for 10 min at 16,000 rpm and supernatant was collected
in a petri dish. e precipitate was washed three times with 5 mL of 75%
ethanol. e washes were combined with supernatant and evaporated
to dryness. To the dry mass, 1 mL water and 2 mL chloroform were
added and centrifuged at 2000 rpm. Upper phase containing GABA was
separated, and 10 μL of it was applied as spot on Whatman lter paper.
e mobile phase consisted of n-butanol, acetic acid, and water in 4:1:5
ratios. e chamber was saturated for half an hour with mobile phase.
e paper chromatogram was developed with ascending technique. e
paper was dried in a hot air oven and then sprayed with 0.5% ninhydrin
solution in 95% ethanol. e paper was dried. Blue spot developed on
paper, which was cut and heated with 2 mL ninhydrin solution on a
water bath at 60–65°C. Water was added to the solution and kept for 1
h and supernatant was used. Absorbance was measured at 570 nm on a
UV-visible spectrophotometer.
RESULTS
CPP
In CPP, the animal’s choice to spend more time in either environment
provides a direct measure of the conditioned reinforcing eect of a
drug. Animals were found to prefer the ethanol-paired chamber over
the saline-paired chamber. In our study on day 11, the ethanol-treated
animals spent signicantly more time in the ethanol-paired chamber
as compared to the saline-paired chamber (P < 0.001). Animals
spent about 66% of total time in ethanol-paired chamber versus the
saline-paired. For the control group (saline-treated), the time spent in
both the chambers were comparable [Figure 1]. Similar results were
observed on day 12 (data not shown). e CPP results suggested that the
animals got addicted to alcohol. Ethanol levels in the blood samples were
found to be 47 mg/dL (n = 6).
Eect of shankhpushpi on withdrawal anxiety
In the present study, according to the CPP model described by anos
et al.,[11] with minor modications, acute ethanol withdrawal anxiety
was developed and measured using elevated plus maze test. Five days
of abstinence followed 10 days of conditioning phase in which alternate
dose of ethanol and saline was given for 10 days. Aer 5 days of abstinence
from ethanol, animals showed a signicant decrease in time spent
(P < 0.01) in the open arm of the elevated plus maze as compared with the
control, suggesting withdrawal anxiety. Shankhpushpi (100 and 200 mg/kg)
administration led to a dose-dependent reversal of withdrawal anxiety
as evidenced by signicant increase in time spent in the open arm
(P < 0.01).
Both 200 mg/kg shankhpushpi and diazepam showed comparable
anxiolytic potential against ethanol withdrawal anxiety. However,
pretreatment with GABAA antagonist prevented shankhpushpi-mediated
reversal of withdrawal anxiety (P < 0.001) [Figure 2]. Treatment with
GABAB agonist and shankhpushpi did not show any signicant change
in withdrawal anxiety compared with shankhpushpi-treated animals
(data not shown). e above results suggest that shankhpushpi may
Figure 1: CPP on ethanol administration. Time spent in alcohol and saline
administration chamber on day 11 by control and ethanol‑treated group.
Ethanol‑treated animals showed signicant increase in (P < 0.001) time
spent in ethanol‑paired alcohol compared with saline‑paired chamber.
Values represent mean ± standard error of the mean n = 7
Figure 2: Eect of shankhpushpi on ethanol withdrawal anxiety using
elevated plus maze. Ethanol abstinence signicantly decreased (P < 0.001)
time spend in open arm compared with ethanol‑treated animals.
Shankhpushpi treatment to abstinent animals signicantly (100 mg/kg,
P < 0.01 and 200 mg/kg, P < 0.001) increased time spend in open arm
compared with abstinent animals. Diazepam also signicantly increased
time spend in open arm over abstinent and ethanol‑treated groups
(P < 0.001). Animals treated with shankhpushpi in the presence of GABAA
spent signicantly less time in the open arm (P < 0.001) compared with
abstinent animals.
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4 Pharmacognosy Magazine, Apr‑Jun 0000, Vol 00, Issue 00 (Supplement 2)
chamber, thus conrming the development of addiction. Ethanol
consumption followed by withdrawal results in the development of
abstinence syndrome.[14–18] Common and prominent feature of alcohol
withdrawal is anxiety, which is also considered to be the most important
negative motivation to revert to alcohol use.[19] e above sign of ethanol
withdrawal (EW) have been attributed to upregulation of NMDA
receptors[20] and downregulation of GABAA receptors.[21] erefore, a
drug that either facilitates the action of GABA or decreases glutamate
activity may be eective in EW-induced anxiety behavior. e elevated
plus maze is the most commonly employed tests for assessing anxiety-
like behavior aer alcohol withdrawal, which is a measure of psychologic
dependency.[22] Typical anxiolytic drugs increase the proportion of
entries, time spent, and rearing in the open arms. e anxiolytic potential
of shankhpushpi churna and diazepam were studied on acute ethanol
withdrawal anxiety using elevated plus maze. Abstinence to ethanol
led to precipitation of withdrawal anxiety as revealed by the signicant
reduction in time spent in open arm of EPM. Shankhpushpi churna and
diazepam increased the time spent in open arm, thus reducing ethanol
withdrawal anxiety, one of the primary reasons for alcohol addiction.
Benzodiazepines are a positive allosteric modulator of GABAA. ey
act by potentiating the eect of GABA on its receptor.[23] It has been
suggested that downregulation of GABAA receptor and/or decrease in
the GABAergic transmission may be responsible for ethanol withdrawal.
Diazepam acts by potentiating the eect of GABA at the receptor site
and shows its anxiolytic property. We also found a signicant increase
in GABA levels upon administration of shankhpushpi churna and
diazepam. Treating animals with GABAA antagonist successfully
reversed the anxiolytic property of shankhpushpi, thus pointing toward
the role of GABAA receptor in shankhpushpi-mediated reduction in
withdrawal anxiety.
Next, we determined the eect of shankhpushpi on alcohol consumption
in mice. e two-bottle choice protocol is a widely used model that
captures aspects of voluntary alcohol consumption in humans.[24] Aer
30 days of chronic ethanol intake, animals showed increased cortico-
hippocampal levels of GABA as we reported previously.[25] Decrease
in GABAergic function aer chronic administration of alcohol in
experimental animals has been widely attributed to decrease in
GABAA receptor expression and function.[26] Shankhpushpi treatment
signicantly increased GABA levels in the cortico-hippocampal lysates
over ethanol-treated animals. Diazepam, a standard anxiolytic drug also
showed increased GABA levels. Others have also reported the role of
GABA in anxiolytic property of aerial parts of C. pluricaulis.[27] Alcohol
is an indirect GABA agonist. Plasma and CSF level of GABA have been
found to remain high aer initial withdrawal than aer longer periods of
abstinence.[28] Evidence showed a decrease in GABAergic function aer
chronic administration of alcohol in experimental animals, which has
been attributed to decrease in the GABAA receptors or changes in the
composition of the receptor.[26]
Ethanol intake by mice was found to increase till the 20th day. Aer day
24, there was a signicant decrease in ethanol intake and increase in
water intake for shankhpushpi and diazepam, treated animal which was
completely blocked upon administration of GABAA blocker e above
results suggest that shankhpushpi may prevent ethanol intake in a GABAA-
dependent manner. Alcohol’s action on GABAA receptors strongly
depends on its subunit composition. GABAA receptors are composed of
α, β, γ, and δ subunits forming a pentameric ligand-gated ion channel
receptor. While most subunit compositions of GABAA receptors display
responses to alcohol only at high concentrations (460 mM), it has been
found that lower concentrations (1—3 mM) of alcohol may only alter
the activity the δ subunit.[29] Pharmacologic manipulation of GABAA
receptors has been studied in mice. Negative allosteric modulators of
reverse ethanol withdrawal anxiety in a GABAA-dependent manner.
Eect of shankhpushpi on cortico‑hippocampal
GABA levels
Aer 30 days of alcohol treatment, animals showed a signicant increase
(P < 0.01, n = 5) in cortico-hippocampal GABA as compared with
control group. Shankhpushpi (200 mg/kg) treatment (10 days) showed a
signicant (P < 0.01, n = 5) increase in GABA as compared with alcohol-
consuming animals. is signicance further increased (P < 0.001, n = 5)
in diazepam-treated group as compared with alcohol group [Figure 3].
Eect of shankhpushpi on alcohol consumption
Shankhpushpi-treated animals showed a signicant (P < 0.001, n = 7)
decrease in ethanol and water intake as compared with the control group
aer day 24 or 4 days postshankhpushpi therapy. is was comparable
with diazepam-treated animals, who also showed a signicant decrease
(P < 0.001, n = 7) in ethanol intake. However, animals’ administration
with GABAA blocker followed by shankhpushpi failed to show a decrease
in ethanol and increase in water intake till day 30. e above results
suggest that shankhpushpi prevented chronic ethanol intake, which may
be mediated by GABAA receptors [Figure 4].
DISCUSSION
In the present work, we study the eect of shankhpushpi in both acute
alcohol withdrawal and chronic alcoholism. Addiction model used in
the alcohol-withdrawal study was CPP. e CPP results suggest that
when given a choice aer administration of alcohol for 11 days, the
animals preferred the ethanol-paired chamber to that of saline-paired
Figure 3: Eect of shankhpushpi on cortico‑hippocampal GABA levels.
Shankhpushpi (200 mg/kg) treatment (10 days) led to signicant increase
(P < 0.01) in cortico‑hippocampal GABA levels compared with untreated
animals on ethanol (30 days). Diazepam treatment also showed a
signicant increase (P < 0.001) increase in cortico‑hippocampal gamma
amino butyric acid levels. Values represent mean ± standard error of the
mean n = 5
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Pharmacognosy Magazine, Apr‑Jun 0000, Vol 00, Issue 00 (Supplement 2) 5
Figure 4: Eect of shankhpushpi on ethanol intake. (a) Changes in the ethanol intake before and after treatment. Shankhpushpi‑ and diazepam‑treated
animals showed a signicant decrease in ethanol intake compared with untreated ethanol consuming animals from day 24 (P < 0.001, n = 7) or after 4 days of
treatment. In presence of GABAA blocker shankhpushpi showed little decrease in (P > 0.5) ethanol intake compared with ethanol‑consuming animals till day
30. (b) Changes in ethanol intake on day 24 of the 30 days study. All treatment groups other than GABAA blocker + shankhpushpi group showed signicant
(P < 0.001) decrease in alcohol intake. (c) Change in the water intake before and after treatment. Shankhpushpi (200 mg/kg) and diazepam‑treated animals
showed a signicant increase in water intake compared with untreated ethanol‑consuming animals from day 24 (P < 0.001, n = 7) or after 4 days of treatment.
In presence of GABAA blocker shankhpushpi did not show an increase (P > 0.5) in water intake compared with ethanol‑consuming animals. (d) Change in
alcohol intake on day 24 of the 30 days study. All treatment groups other than GABAA blocker + shankhpushpi group showed signicant (P < 0.001) increase
in water intake. Values represent mean ± standard error of the mean, n = 7
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the GABAA receptor have been shown to reduce alcohol consumption in
several alcohol-preferring mice.[21] Various phytoconstituents identied
from the aerial parts of C. pluricaulisy Choisy may act as GABAA agonist;
this may be attributed to its CNS-depressant actions.[30,31]
CONCLUSION
Hence, in the present study, we report anti-addictive potential of
shankhpushpi churna in alcohol addiction in mice. It not only reversed
ethanol withdrawal anxiety but also decreased chronic alcohol
consumption in these animals in a GABAA receptor-dependent manner.
Financial support and sponsorship
Nil.
Conicts of interest
ere are no conicts of interest.
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ABOUT AUTHOR
Dr. Sugato Banerjee, is Assistant Professor at Department of
Pharmaceutical Sciences and Technology, BIT, Mesra, Ranchi. His research
interests include screening of natural compounds with anti-addictive
potential and to study the molecular mechanisms involved in the process.
Sugato Banerjee