Double-blinded randomized controlled trial for immunomodulatory effects of Tulsi (Ocimum sanctum Linn.) leaf extract on healthy volunteers

Abstract

Tulsi (Ocimum sanctum Linn.) is considered as a sacred herb and traditionally it is believed that consumption of Tulsi leaf on empty stomach increases immunity. Experimental studies have shown that alcoholic extract of Tulsi modulates immunity.
The present study was designed to evaluate the immunomodulatory effects of ethanolic extract of Tulsi leaves through a double-blinded randomized controlled cross-over trial on healthy volunteers. Three hundred milligrams capsules of ethanolic extracts of leaves of Tulsi or placebo were administered to 24 healthy volunteers on empty stomach and the results of 22 subjects who completed the study were analyzed. The primary objective was to study the levels of Th1 and Th2 cytokines (interferon-γ and interleukin-4) during both pre and post intervention period in blood culture supernatants following stimulation with lipopolysaccharide and phytohaemagglutinin. Other immunological parameters such as T-helper and T-cytotoxic cells, B-cells and NK-cells also were analyzed using Flowcytometry.
Statistically significant increase in the levels of IFN-γ (p=0.039), IL-4 (p=0.001) and percentages of T-helper cells (p=0.001) and NK-cells (p=0.017) were observed after 4 weeks in the Tulsi extract intervention group in contrast to the placebo group.
These observations clearly ascertain the immunomodulatory role of Tulsi leaves extract on healthy volunteers.

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Journal
of
Ethnopharmacology
136 (2011) 452–
456
Contents
lists
available
at
ScienceDirect
Journal
of
Ethnopharmacology
jo
ur
nal
homep
age
:
www.elsevier.com/locate/jethpharm
Double-blinded
randomized
controlled
trial
for
immunomodulatory
effects
of
Tulsi
(Ocimum
sanctum
Linn.)
leaf
extract
on
healthy
volunteers
Shankar
Mondala,
Saurabh
Varmab,
Vishwa
Deepak
Bamolaa,
Satya
Narayan
Naikc,
Bijay
Ranjan
Mirdhad,
Madan
Mohan
Padhie,
Nalin
Mehtaa,
Sushil
Chandra
Mahapatraa,∗
aDepartment
of
Physiology,
All
India
Institute
of
Medical
Sciences,
New
Delhi
110029,
India
bInstitute
of
Pathology,
Safdarjung
Hospital
Campus,
New
Delhi,
India
cCentre
for
Rural
Development
and
Technology,
Indian
Institute
of
Technology
Delhi,
New
Delhi,
India
dDepartment
of
Microbiology,
All
India
Institute
of
Medical
Sciences,
New
Delhi
110029,
India
eCentral
Council
for
Research
in
Ayurveda
and
Siddha,
Department
of
Ayurveda,
Yoga
and
Naturopathy,
Unani
and
Sidhha
(AYUSH),
Janakpuri,
New
Delhi,
India
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
20
January
2011
Accepted
10
May
2011
Available online 17 May 2011
Keywords:
Cytokine
Flowcytometry
Medicinal
plant
NK-cells
T-helper
cells
a
b
s
t
r
a
c
t
Ethnopharmacological
relevance:
Tulsi
(Ocimum
sanctum
Linn.)
is
considered
as
a
sacred
herb
and
tradi-
tionally
it
is
believed
that
consumption
of
Tulsi
leaf
on
empty
stomach
increases
immunity.
Experimental
studies
have
shown
that
alcoholic
extract
of
Tulsi
modulates
immunity.
Materials
and
Methods:
The
present
study
was
designed
to
evaluate
the
immunomodulatory
effects
of
ethanolic
extract
of
Tulsi
leaves
through
a
double-blinded
randomized
controlled
cross-over
trial
on
healthy
volunteers.
Three
hundred
milligrams
capsules
of
ethanolic
extracts
of
leaves
of
Tulsi
or
placebo
were
administered
to
24
healthy
volunteers
on
empty
stomach
and
the
results
of
22
subjects
who
com-
pleted
the
study
were
analyzed.
The
primary
objective
was
to
study
the
levels
of
Th1
and
Th2
cytokines
(interferon-␥
and
interleukin-4)
during
both
pre
and
post
intervention
period
in
blood
culture
super-
natants
following
stimulation
with
lipopolysaccharide
and
phytohaemagglutinin.
Other
immunological
parameters
such
as
T-helper
and
T-cytotoxic
cells,
B-cells
and
NK-cells
also
were
analyzed
using
Flow-
cytometry.
Results:
Statistically
significant
increase
in
the
levels
of
IFN-␥
(p
=
0.039),
IL-4
(p
=
0.001)
and
percentages
of
T-helper
cells
(p
=
0.001)
and
NK-cells
(p
=
0.017)
were
observed
after
4
weeks
in
the
Tulsi
extract
intervention
group
in
contrast
to
the
placebo
group.
Conclusions:
These
observations
clearly
ascertain
the
immunomodulatory
role
of
Tulsi
leaves
extract
on
healthy
volunteers.
© 2011 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
Tulsi
or
Holy
Basil
(Ocimum
sanctum
Linn.)
is
widely
dis-
tributed
in
India
from
the
sea
level
to
up
to
1800
m
altitude
in
the
Himalayas
(Wealth
of
India,
1991).
Its
medicinal
properties
have
been
described
in
the
Indian
medicinal
text
Ayurveda
(The
science
of
Life)
which
is
believed
to
be
about
5000
years
old.
Traditionally,
various
parts
of
this
plant
have
been
used
for
different
ailments
such
as
cough
and
cold,
asthma,
bronchitis,
digestive
disorders,
skin
problems,
eye
and
ear
infections,
undifferentiated
fever,
snake
and
scorpion
bites
(Ghosh,
1995).
Scientific
explorations
of
traditional
medicinal
claims
of
Tulsi
got
the
momentum
in
the
middle
part
of
the
20th
century.
Most
of
the
scientific
evidences
of
medicinal
properties
of
this
plant
were
observed
largely
in
∗Corresponding
author.
Tel.:
+91
11
26594812;
fax:
+91
11
26588643.
E-mail
addresses:
scm@aiims.ac.in,
scmahapatra@gmail.com
(S.C.
Mahapatra).
experimental
animal
studies
with
only
a
few
human
studies.
These
studies
have
shown
notable
properties
such
as
antimicrobial,
adaptogenic,
anti-diabetic,
hepato-protective,
anti-inflammatory,
anti-carcinogenic,
radio-protective,
neuro-protective,
cardio-
protective
and
larvicidal/mosquito
repellent
of
different
parts
of
the
Tulsi
plant
(Mondal
et
al.,
2009).
Experimental
animal
studies
have
clearly
shown
immunomodulatory
properties
in
the
extract
of
Tulsi
leaves
(Godhwani
et
al.,
1988;
Singh
et
al.,
1995;
Mediratta
et
al.,
2002;
Mukherjee
et
al.,
2005).
Immune
system
of
human
is
very
complex
and
there
lies
a
delicate
balance
between
health
and
disease.
Any
substance,
synthetic
or
biological,
which
can
enhance,
suppress
or
modulate
the
immune
system,
is
called
an
immunomodulator
(Agarwal
and
Singh,
1999).
It
is
often
believed
in
India
that
taking
Tulsi
leaves
on
empty
stomach
is
beneficial
and
improves
immunity,
thus
the
present
study
was
designed
to
determine
the
immunomodulatory
properties
of
Tulsi
leaf
ethano-
lic
(70%)
extract
in
healthy
volunteers
through
a
double-blind
randomized
controlled
trial.
0378-8741/$
–
see
front
matter ©
2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2011.05.012

Author's personal copy
S.
Mondal
et
al.
/
Journal
of
Ethnopharmacology
136 (2011) 452–
456 453
2.
Materials
and
methods
2.1.
Study
design
A
double-blind
randomized
controlled
trial
in
a
cross-over
for-
mat
with
a
washout
period
was
designed
to
study
the
immunomod-
ulatory
effects
of
ethanolic
extract
of
Tulsi
leaf
in
healthy
volunteers.
The
study
was
approved
by
the
institutional
ethics
committee
on
research
involving
human
subjects
and
registered
with
clinical
trial
registry
of
India
(no.
CTRI/2009/091/000350).
2.2.
Recruitment
of
volunteers
Twenty
four
healthy
volunteers
were
enrolled
in
the
study
after
initial
screening
of
45
subjects.
Following
were
the
inclusion
crite-
ria
for
the
enrollment
of
healthy
volunteers,
i.e.,
(a)
age
18–60
years,
either
sex,
(b)
devoid
of
any
medication
during
last
one
month.
Subjects
suffering
from
different
diseases/disorders
and/or
hav-
ing
any
kind
of
allergy,
undergone
surgery
during
last
one
year,
received
organ
transplant,
chronic
smokers,
underlying
conditions
which
might
affect
immunity
and
pregnant/lactating
women
were
excluded
from
the
study.
Enrolled
individuals
were
randomized
into
two
groups.
Subjects
and
the
staff
directly
involved
with
the
study
subjects
and
data
analysis
were
blinded
about
the
interven-
tional
capsules.
Allocation
of
Tulsi
extract
or
placebo
was
concealed
in
opaque
envelop.
2.3.
Intervention
Interventional
drug
(70%
ethanolic
extract
of
Ocimum
sanctum
Linn.
leaves,
‘Tulsi
extract’)
was
supplied
by
the
Central
Council
for
Research
in
Ayurveda
and
Siddha,
Department
of
AYUSH,
Min-
istry
of
Health
and
Family
Welfare,
Government
of
India.
Placebo
that
contained
sucrose
was
supplied
by
the
Dabur
Pharmaceuti-
cal
(India)
Ltd.,
Ghaziabad
(U.P.),
India.
Three
hundred
milligram
capsules
of
Tulsi
extract
or
placebo
(sucrose)
were
prepared
by
the
Dabur
Pharmaceutical
(India)
Ltd.
To
avoid
identification,
the
shape,
size,
color
and
packaging
of
both,
Tulsi
extract
and
placebo,
were
similar.
Once
the
volunteer
met
the
inclusion
criteria,
a
written
informed
consent
was
obtained
and
the
intervention
was
allocated
as
per
the
randomized
sequence.
The
capsules
were
administered
on
empty
stomach
for
four
weeks
followed
by
a
wash
out
period
of
three
weeks
before
the
volunteer
cross-over
to
the
next
intervention.
2.4.
Blood
sampling
Venous
blood
samples
were
collected
through
vene
puncture
of
antecubital
vein
at
four
different
time
points,
i.e.,
(i)
at
baseline,
(ii)
after
four
weeks
of
placebo
or
Tulsi
extract,
(iii)
after
washout
period
of
three
weeks
and
lastly,
(iv)
after
completion
of
four
weeks
in
crossover
intervention
period.
The
compliance
of
the
capsule
intake
was
monitored
by
reminding
the
volunteers
personally
or
telephonically
twice
a
week
and
also
from
the
numbers
of
unused
capsules
returned
by
the
subjects.
Two
(n
=
2)
volunteers
were
lost
during
follow-up.
2.5.
Whole
blood
culture
and
cytokine
assay
Mitogen
induced
secretion
of
Th1
and
Th2
cytokines
[Interferon-
gamma
(INF-␥)
and
Interleukin-4
(IL-4)]
levels
in
whole
blood
culture
were
monitored
at
four
different
time
points
using
ELISA.
Secreted
levels
of
cytokines
(IFN-␥
and
IL-4)
were
studied
as
per
Viallard
et
al.
(1999),
in
the
whole
blood
culture.
Briefly,
venous
blood
obtained
from
volunteers
was
cultured
in
RPMI-
1640
medium
and
was
stimulated
with
Escherichia
coli
derived
Table
1
Comparison
of
basic
parameters
of
volunteers
at
the
baseline
(0
weeks).
Basic
parameters
Baseline
p-Value
Placebo-Tulsi
sequence
(n
=
12)
Tulsi-Placebo
sequence
(n
=
10)
Age 27.5 ±
4.75
26.5 ±
3.43
0.585
Height 166.16 ±
7.62
166.1
±
5.40
0.981
Weight
(kg) 62.5
±
10.52
65.8
±
6.05
0.391
BMI
(kg/m2)
22.36
±
2.52
23.80
±
1.13
0.111
Systolic
BP
(mmHg)
109.16
±
7.50
113.2
±
7.37
0.220
Diastolic
BP
(mmHg)
74.16
±
4.62
76.2
±
3.45
0.265
All
values
presented
in
mean
±
SD.
p-Value
≤
0.05
was
considered
as
significant.
Independent
t-test
applied.
BMI
=
Body
Mass
Index,
BP
=
Blood
Pressure.
lipopolysaccharide
(LPS)
at
a
final
concentration
of
25
␮g/ml
and
phytohaemagglutinin
(PHA)
at
a
final
concentration
of
5
␮g/ml
(Calbiochem,
Germany).
Culture
supernatant
were
harvested
after
24
h
of
incubation
at
37 ◦C
in
5%
CO2humidified
chamber
and
cytokine
levels
were
measured
using
commercial
ELISA
kits
(Thermo
Scientific,
IL,
USA)
with
a
sensitivity
of
less
than
2
pg/ml.
Samples
for
ELISA
test
was
performed
as
per
the
manufacturer’s
instructions
and
reading
was
taken
using
Bio-Rad
ELISA
reader
(Benchmark
Plus,
CA,
USA)
and
Microplate
manager
software
ver-
sion
5.2.1.
2.6.
Flowcytometry
for
phenotyping
of
lymphocytes
Phenotyping
of
T-cells
(CD3+CD4+,
CD3+CD8+),
B-cells
(CD19+)
and
NK-cells
(CD16+CD56+)
was
carried
out
using
Flowcytometer
(FACSCaliber,
Beckton
Dikinson)
and
CELLQuest®software.
CD3
labeled
with
Phycoerythrin
Cyanin
5
(PE
Cy
5),
CD4
labeled
with
Phycoerythrin
(PE),
CD8
labeled
with
Fluorescein
isothiocyanate
(FITC),
CD19
labeled
with
FITC,
CD16
labeled
with
FITC
and
CD56
labeled
with
PE
were
procured
commercially
(BD-Pharmigen,
USA)
and
the
samples
were
processed
as
per
the
manufacture’s
instruc-
tions.
2.7.
Statistical
analysis
Two-way
Analysis
of
Variance
(ANOVA)
for
crossover
design
was
used
to
test
the
statistical
significance
of
the
results.
Inde-
pendent
t-test
was
applied
to
compare
the
baseline
of
two
groups.
The
results
were
considered
significant
if
the
p-value
was
≤0.05
for
period
effects
and
intervention
effects.
However,
p-value
of
≤0.10
for
carryover
effects
was
considered
significant
(Jones
and
Kenward,
2003).
All
statistical
analysis
was
carried
out
using
Stata
9.0
software
(Statacorp,
TX,
USA).
The
blinding
was
decoded
only
after
the
statistical
analysis.
3.
Results
3.1.
Study
population,
compliance
and
carry
over
effects
All
baseline
parameters
of
both
placebo
and
Tulsi
extract
groups
were
comparable
and
no
significant
differences
were
noted
(Table
1).
No
significant
adverse
effects
of
the
intervention
were
noted
amongst
study
individuals
during
the
study
period
of
11
week
except
in
two
subjects,
one
of
the
subjects
complained
of
nausea
while
the
other
had
loose
motions,
after
first
visit
to
the
laboratory.
These
two
subjects
could
not
complete
the
study
and
their
data
were
excluded
from
the
analysis
(Fig.
1).
Further,
there
were
no
carry
over
or
sequence
of
effect
(i.e.,
whether
the
placebo
or
Tulsi
extract
administered
initially
or
later)
of
the
placebo
or
Tulsi
extract
intervention.
Thus,
the
data
of
the
subjects
who
received
Tulsi
extract
intervention
either
before
cross-over
or
after
cross-

Author's personal copy
454 S.
Mondal
et
al.
/
Journal
of
Ethnopharmacology
136 (2011) 452–
456
Randomized (n=24)
Excluded (n= 21)
Not meeting inclusion criteria (n= 9)
Refused to participate (n=12)
Allocation
Follow-Up
Lost to follow-up (n= 0)
Discontinued intervention (n= 01)
Reason- loose motions
Allocated to intervention (n= 11)
Received allocated intervention (n= 11)
Did not receive allocated
intervention (n=
0)
Analysis
Group-A
Group-B
Analyzed (n=10)
Excluded from analysis (n= 01)
Reason-not completed the study
Assessed for eligibility
(n= 45)
Lost to follow-up (n= 0)
Discontinued intervention (n=01)
Reason-
Feeling of nausea
Analyzed (n= 12)
Excluded from analysis (n=01)
Reason-
not completed
the study
Allocated to intervention (n=13)
Received allocated intervention (n= 13)
Did not receive allocated
intervention (n= 0 )
Fig.
1.
Flowchart
of
recruitment
of
healthy
volunteers.
over
were
grouped
together
and
similarly
placebo
treated
subjects
were
also
grouped
together
for
analysis
and
interpretation.
The
compliance
of
the
capsule
intake
was
very
satisfactory
and
com-
pliance
rate
was
more
than
95%.
3.2.
Effects
on
Th1
and
Th2
cytokine
release
In
in
vitro
culture
of
whole
blood
stimulated
with
PHA
and
LPS,
there
were
no
significant
differences
in
the
IFN-␥(Th1)
and
IL-
4
(Th2)
cytokines
at
the
baseline
of
both
the
groups.
However,
it
observed
that
there
were
significant
increase
in
the
levels
of
both
IFN-␥
and
IL-4
(p
=
0.039
and
p
=
0.001,
respectively)
in
the
blood
samples
of
Tulsi
extract
intervention
group.
This
increase
did
not
continue
when
subjects
were
crossed-over
to
placebo
intervention
after
the
wash
out
period
(Figs.
2
and
3).
3.3.
Effects
on
lymphocytes
In
the
present
study,
we
did
not
find
any
significant
differ-
ences
in
the
percentage
of
lymphocytes
and
NK-cell
at
the
baseline
examination
in
both
the
groups.
There
were
also
no
significant
differences
observed
in
lymphocyte
and
NK-cell
percentages
in
placebo
group
(Fig.
4).
However,
a
significant
increase
in
the
T-
helper
cells
(p
=
0.001)
in
Tulsi
extract
intervention
group
(Fig.
5)
was
observed.
Apparently
healthy
volunteers
participated
in
this
study
did
not
show
any
significant
difference
in
the
percentages
of
T-cytotoxic
and
B-cells
even
after
4
weeks
of
intervention
in
both
the
groups
(Figs.
4
and
5).
Significant
increase
in
the
NK-cells
(p
=
0.017)
was
noticed
in
the
Tulsi
extract
group
after
4
weeks
of
intervention
(Fig.
5).
4.
Discussion
There
were
no
significant
period
or
carryover
effects
observed
during
the
study
period
which
indicates
that
the
chosen
washout
period
of
three
weeks
were
sufficient
to
undo
the
effects
of
first
intervention.
INF-␥
and
IL-4
are
clinically
important
because
secreted
lev-
els
of
these
cytokines
polarize
effective
functions,
either
Th1
or
Th2
type
response.
INF-␥
is
known
to
be
secreted
during
infec-
tion
due
to
intracellular
pathogens
and
has
potentially
antiviral,
Placebo (sucrose) group
0
10
20
30
40
50
60
70
80
90
100
110
120
130
Interferon-gamma Interleukin-4
Th1 and Th2 Cytokines
pg/ml in culture supernatent
At baseline
After 4 weeks
Fig.
2.
Effects
of
placebo
(sucrose)
capsules
on
IFN-␥(Th1)
and
IL-4
(Th2)
cytokines
after
4
weeks
of
intervention
(n
=
22).
All
values
in
mean
±
SD.
There
was
no
sig-
nificant
difference
after
4
weeks
of
placebo
intervention.
Two-way
ANOVA
for
cross-over
design
applied.

Author's personal copy
S.
Mondal
et
al.
/
Journal
of
Ethnopharmacology
136 (2011) 452–
456 455
Tulsi extract group
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
Interferon-gamma Interleukin-4
Th1 and Th2 Cytokine
pg/ml in culture supernatant
At baseline After 4 weeks
**
*
Fig.
3.
Effects
of
Tulsi
extract
on
Th1
and
Th2
cytokines
after
4
weeks
of
intervention
(n
=
22).
All
values
in
mean
±
SD.
There
was
a
significant
increase
in
IFN-␥
(Th1)
(**p
=
0.039)
and
IL-4
(Th2)
(*p
=
0.001)
levels
in
culture
supernatant
of
whole
blood
of
Tulsi
extract
group
after
4
weeks
of
intervention.
Two-way
ANOVA
for
cross-over
design
applied.
Placebo (sucrose) group
0
5
10
15
20
25
30
35
40
45
50
55
CD3+CD4+
CD3+CD8+ CD19+ CD16+CD56+
Lymphocytes and NK Cells
Percentage
At baseline After 4 weeks
Fig.
4.
Intervention
effects
of
placebo
(sucrose)
capsules
on
T-Lymphocytes,
B-
Lymphocytes
and
NK-cells
after
4
weeks
(n
=
22).
All
values
in
mean
±
SD.
There
was
no
significant
difference
after
4
weeks
of
placebo
intervention.
Two-way
ANOVA
for
cross-over
design
applied.
Tulsi extract group
0
5
10
15
20
25
30
35
40
45
50
55
CD3+CD4+ CD3+CD8+ CD19+
CD16+CD56+
Lymphocytes and NK-cells
Percentage
At baseline After 4 weeks
**
*
Fig.
5.
Intervention
effects
of
Tulsi
extract
capsules
on
T-Lymphocytes,
B-
Lymphocytes
and
NK-cells
after
4
weeks
(n
=
22).
All
values
in
mean
±
SD.
There
was
a
significant
increase
in
T-helper
cells
(CD3+CD4+)
(**p
=
0.001)
and
NK-Cells
(CD16+CD56+)
(*p
=
0.017)
after
4
weeks
of
Tulsi
extract
intervention.
Two-way
ANOVA
for
crossover
design
applied.
antibacterial,
anti-proliferative,
anti-tumor
and
anti-allergic
effects
(Dafny
et
al.,
2007).
INF-␥
acts
as
one
of
the
inhibitors
of
Th2
type
response
due
to
IL-4,
and
IL-4
secretion
in
turn
limits
over
acti-
vation
of
Th1
by
inhibiting
the
actions
of
INF-␥
(Jiang
and
Chess,
2004).
Although
levels
of
IL-4
were
increased
significantly
in
the
Tulsi
extract
group,
the
increase
was
not
so
high
as
compared
to
the
increase
in
the
levels
of
INF-␥.
This
indicates
that
in
Tulsi
extract
treated
group,
there
was
initial
polarization
of
Th1
type
of
response
(INF-␥)
followed
by
Th2
type
(IL-4).
Thus,
it
can
be
inferred
from
these
results
that
when
immune
challenge
was
given
in
the
form
of
PHA
and
LPS
in
the
whole
blood
culture,
Tulsi
extract
intervention
group
had
mounted
an
effective
immune
response
of
Th1
type
(INF-␥).
This
increase
in
cytokine
level
is
also
sup-
ported
by
significant
increase
in
the
percentages
of
T-helper
cells
(CD3+CD4+)
and
NK-cells
(CD16+CD56+),
however,
no
significant
changes
were
found
in
T-cytotoxic
cells
(CD3+CD4+)
and
B-cells
(CD19+).
Flavonoids
present
in
the
extracts
of
Tulsi
leaves
have
been
found
to
be
responsible
for
the
immunomodulatory
properties
(Mukherjee
et
al.,
2005).
In
a
similar
clinical
trial,
immunomodula-
tory
effects
of
traditional
Chinese
medicinal
plant
Yun
Zhi
(Coriolus
versicolor)
and
Danshen
(Salvia
miltiorrhiza)
was
observed
after
six
months
of
intake
(Wong
et
al.,
2004).
Tulsi
is
a
non-toxic
plant
and
its
LD50 value
is
very
high,
rang-
ing
from
4600
to
6400
mg/kg
body
weight
of
experimental
murine
models
(Bhargava
and
Singh,
1981;
Devi
and
Ganasoundari,
1995;
Singh
and
Majumdar,
1994).
To
determine
any
side
effects
of
Tulsi,
biochemical
parameters
were
also
evaluated
at
the
same
time
points.
It
was
observed
that
intervention
with
300
mg
capsules
of
Tulsi
extract
did
not
show
any
toxic
effects
as
evident
by
basic
phys-
iological
and
biochemical
results
[data
not
shown].
There
were
no
significant
changes
observed
in
body
mass
index,
blood
pressure,
fasting
blood
sugar,
liver
and
renal
function
tests.
However,
some
of
the
subjects
who
had
higher
than
the
normal
physiological
ranges
of
total
cholesterol
showed
a
significant
reduction
(intrasubject
p
=
0.003,
in
6
subjects)
after
taking
capsules
of
Tulsi
extract
for
4
weeks.
However,
the
reduction
was
not
observed
in
the
subjects
who
had
total
cholesterol
levels
within
normal
physiological
refer-
ence
ranges.
When
triglycerides
levels
were
analyzed,
it
was
found
that
the
reduction
was
statistically
not
significant.
However,
it
was
noticed
that
triglycerides
levels
were
reduced
in
few
subjects
who
had
an
initial
elevated
value.
Thus,
though
not
significant
statisti-
cally
(p
=
0.350),
a
reduction
trend
was
observed
in
persons
with
higher
than
normal
physiological
reference
ranges
of
triglycerides.
In
an
earlier
clinical
trial
of
Tulsi
leaf
on
type-II
diabetic
patients
a
significant
reduction
in
triglycerides,
low
density
lipoprotein
and
very
low
density
lipoprotein
was
reported
(Rai
et
al.,
1997).
Ani-
mal
studies
have
also
shown
that
feeding
of
Tulsi
leaves
reduces
cholesterol
levels
(Gupta
et
al.,
2006).
Higher
levels
of
triglycerides
and
cholesterol
are
one
of
the
risk
factors
of
coronary
artery
dis-
eases
in
humans.
Thus
reduction
in
cholesterol
and
triglycerides
seen
in
our
study
population
is
a
very
positive
outcome.
This
rein-
forces
the
traditional
claim
(Ghosh,
1995)
that
it
is
good
for
heart.
However
caution
should
be
taken
in
to
consideration
while
trans-
lating
these
findings
as
our
study
population
was
of
normal
healthy
persons
with
no
history
of
hypercholesterolemia
or
hypertriglyc-
eridemia.
Thus,
in
patients
with
history
of
hypercholesterolemia
or
hypertriglyceridemia
this
property
may
be
further
investigated.
In
conclusion,
it
can
be
inferred
from
the
results
of
this
study
that,
Tulsi
extract
have
immunomodulatory
effects
in
healthy
vol-
unteers
without
any
side
effects
and
a
potential
benefit
for
those
with
hypercholesterolemia.
Conflict
of
interest
Declared
none.
Acknowledgements
We
acknowledge
CSIR
and
ICMR
India,
for
awarding
Senior
Research
Fellowship
to
S.
Mondal
and
Dabur
India
(P)
Ltd.
for
assis-
tance
in
capsule
preparation
for
the
study.

Author's personal copy
456 S.
Mondal
et
al.
/
Journal
of
Ethnopharmacology
136 (2011) 452–
456
This
research
was
funded
by
Central
Council
for
Research
in
Ayurveda
and
Siddha,
Department
of
Ayurveda,
Yoga
&
Natur-
opathy,
Unani
and
Sidhha
(AYUSH),
Ministry
of
Health
&
Family
Welfare,
Govt.
of
India,
New
Delhi
110065.
References
Agarwal,
S.S.,
Singh,
V.K.,
1999.
Immunomodulators:
a
review
of
studies
on
Indian
medicinal
plants
and
synthetic
peptides.
Part
I.
Medicinal
plants.
In:
Proceedings
of
Indian
National
Science
Academy,
vol.
3–4
,
pp.
179–204.
Bhargava,
K.P.,
Singh,
N.,
1981.
Antistress
activity
of
Ocimum
sanctum
Linn.
Indian
Journal
of
Medical
Research
73,
443–451.
Dafny,
N.,
Yang,
P.B.,
Brod,
S.A.,
2007.
Interferon
in
health
and
disease.
In:
Plontikoff,
N.P.,
Faith,
R.E.,
Good,
R.A.
(Eds.),
Cytokines:
Stress
and
Immunity.
CRC
Press,
Florida,
pp.
253–281.
Devi,
P.U.,
Ganasoundari,
A.,
1995.
Radioprotective
effect
of
leaf
extract
of
Indian
medicinal
plant
Ocimum
sanctum.
Indian
Journal
of
Experimental
Biology
33,
205–208.
Ghosh,
G.R.,
1995.
Tulasi
(N.O.
Labiatae,
Genus-Ocimum).
New
Approaches
to
Medicine
and
Health
(NAMAH)
3,
23–29.
Godhwani,
S.,
Godhwani,
J.L.,
Vyas,
D.S.,
1988.
Ocimum
sanctum
–
a
preliminary
study
evaluating
its
immunoregulatory
profile
in
albino
rats.
Journal
of
Ethnopharma-
cology
24,
193–198.
Gupta,
S.,
Mediratta,
P.K.,
Singh,
S.,
Sharma,
K.K.,
Shukla,
R.,
2006.
Antidiabetic,
anti-
hypercholestrolaemic
and
antioxidant
effect
of
Ocimum
sanctum
(Linn)
seed
oil.
Indian
Journal
of
Experimental
Biology
44,
300–304.
Jiang,
H.,
Chess,
L.,
2004.
Integrated
view
of
suppressor
T
cell
subsets
in
immunoreg-
ulation.
Journal
of
Clinical
Investigation
114,
1198–1208.
Jones,
B.,
Kenward,
M.G.,
2003.
Design
and
Analysis
of
Cross-Over
Trials,
2nd
ed.
Chapman
&
Hall/CRC,
New
York.
Mediratta,
P.K.,
Sharma,
K.K.,
Singh,
S.,
2002.
Evaluation
of
immunomodulatory
potential
of
Ocimum
sanctum
seed
oil
and
its
possible
mechanism
of
action.
Journal
of
Ethnopharmacology
80,
15–20.
Mondal,
S.,
Mirdha,
B.R.,
Mahapatra,
S.C.,
2009.
The
science
behind
sacredness
of
Tulsi
(Ocimum
sanctum
Linn.).
Indian
Journal
of
Physiology
and
Pharmacology
53,
291–302.
Mukherjee,
R.,
Dash,
P.K.,
Ram,
G.C.,
2005.
Immunotherapeutic
potential
of
Ocimum
sanctum
(L.)
in
bovine
subclinical
mastitis.
Research
in
Vetenary
Science
79,
37–43.
Rai,
V.,
Ficn,
U.,
Mani,
V.,
Iyer,
U.M.,
1997.
Effect
of
Ocimum
sanctum
leaf
powder
on
blood
lipoproteins,
glycated
proteins
and
total
amino
acids
in
patients
with
non-
insulin
dependent
diabetes-mellitus.
Journal
of
Nutritional
and
Environmental
Medicine
7,
113–118.
Singh,
S,
Majumdar,
D.K.,
1994.
Toxicological
studies
of
the
fixed
oil
of
Ocimum
sanctum
Linn.
(Tulsi).
New
Botanist
21,
139–146.
Singh,
S.,
Majumdar,
D.K.,
Singh,
J.P.,
1995.
Studies
of
therapeutic
efficacy
of
fixed
oil
of
Ocimum
sanctum
in
bovine
mastitis.
Indian
Vetenary
Journal
72,
867–869.
Viallard,
J.F.,
Pellegrin,
J.L.,
Ranchin,
V.,
Schaeverbeke,
T.,
Dehais,
J.,
Longy-Boursier,
M.,
Ragnaud,
J.M.,
Leng,
B.,
Moreu,
J.F.,
1999.
Th1
(IL-2,
interferon-gamma
(IFN-
␥))
and
Th2
(IL-10,
IL-4)
cytokine
production
by
peripheral
blood
mononuclear
cells
(PBMC)
from
patients
with
systemic
lupus
erythematosus
(SLE).
Clinical
and
Experimental
Immunology
115,
189–195.
Wealth
of
India,
1991.
New
Delhi,
CSIR.
Publication
and
information
directorate
Vol.
7,
79–89.
Wong,
C.K.,
Tse,
P.S.,
Wong,
E.L.,
Leung,
P.C.,
Fung,
K.P.,
Lam,
C.W.,
2004.
Immunomod-
ulatory
effects
of
yun
zhi
and
danshen
capsules
in
health
subjects
–
a
randomized,
double-blind,
placebo-controlled,
crossover
study.
International
Immunopharmacology
4,
201–211.