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Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Double-blinded randomized controlled trial for immunomodulatory effects of

Tulsi (Ocimum sanctum Linn.) leaf extract on healthy volunteers
Shankar Mondal a , Saurabh Varma b , Vishwa Deepak Bamola a , Satya Narayan Naik c ,
Bijay Ranjan Mirdha d , Madan Mohan Padhi e , Nalin Mehta a , Sushil Chandra Mahapatra a,
a
Department of Physiology, All India Institute of Medical Sciences, New Delhi 110029, India
b
Institute of Pathology, Safdarjung Hospital Campus, New Delhi, India
c
Centre for Rural Development and Technology, Indian Institute of Technology Delhi, New Delhi, India
d
Department of Microbiology, All India Institute of Medical Sciences, New Delhi 110029, India
e
Central 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 a b s t r a c t

Article history: Ethnopharmacological relevance: Tulsi (Ocimum sanctum Linn.) is considered as a sacred herb and tradi-
Received 20 January 2011 tionally it is believed that consumption of Tulsi leaf on empty stomach increases immunity. Experimental
Accepted 10 May 2011 studies have shown that alcoholic extract of Tulsi modulates immunity.
Available online xxx
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
Keywords:
healthy volunteers. Three hundred milligrams capsules of ethanolic extracts of leaves of Tulsi or placebo
Cytokine
were administered to 24 healthy volunteers on empty stomach and the results of 22 subjects who com-
Flowcytometry
Medicinal plant
pleted the study were analyzed. The primary objective was to study the levels of Th1 and Th2 cytokines
NK-cells (interferon- and interleukin-4) during both pre and post intervention period in blood culture super-
T-helper cells 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 signicant 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). Scientic explorations
of traditional medicinal claims of Tulsi got the momentum in the
middle part of the 20th century. Most of the scientic 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-inammatory,
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 benecial
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

Please cite this article in press as: Mondal, S., et al., Double-blinded randomized controlled trial for immunomodulatory effects of Tulsi (Ocimum
sanctum Linn.) leaf extract on healthy volunteers. J. Ethnopharmacol. (2011), doi:10.1016/j.jep.2011.05.012
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2. Materials and methods Table 1

Comparison of basic parameters of volunteers at the baseline (0 weeks).
2.1. Study design Basic parameters Baseline p-Value

A double-blind randomized controlled trial in a cross-over for- Placebo-Tulsi Tulsi-Placebo

sequence sequence
mat with a washout period was designed to study the immunomod-
(n = 12) (n = 10)
ulatory effects of ethanolic extract of Tulsi leaf in healthy volunteers.
Age 27.5 4.75 26.5 3.43 0.585
The study was approved by the institutional ethics committee on
Height 166.16 7.62 166.1 5.40 0.981
research involving human subjects and registered with clinical trial Weight (kg) 62.5 10.52 65.8 6.05 0.391
registry of India (no. CTRI/2009/091/000350). 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
2.2. Recruitment of volunteers
All values presented in mean SD. p-Value 0.05 was considered as signicant.
Twenty four healthy volunteers were enrolled in the study after Independent t-test applied. BMI = Body Mass Index, BP = Blood Pressure.

initial screening of 45 subjects. Following were the inclusion crite-

ria for the enrollment of healthy volunteers, i.e., (a) age 1860 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 identication, 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. Briey,
venous blood obtained from volunteers was cultured in RPMI-
1640 medium and was stimulated with Escherichia coli derived
lipopolysaccharide (LPS) at a nal concentration of 25 g/ml and
phytohaemagglutinin (PHA) at a nal concentration of 5 g/ml
(Calbiochem, Germany). Culture supernatant were harvested after
24 h of incubation at 37 C in 5% CO2 humidied chamber and
cytokine levels were measured using commercial ELISA kits
(Thermo Scientic, IL, USA) with a sensitivity of less than 2 pg/ml.
Samples for ELISA test was performed as per the manufacturers
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 manufactures instruc-
tions.
2.7. Statistical analysis
Two-way Analysis of Variance (ANOVA) for crossover design
was used to test the statistical signicance of the results. Inde-
pendent t-test was applied to compare the baseline of two groups.
The results were considered signicant if the p-value was 0.05
for period effects and intervention effects. However, p-value of
0.10 for carryover effects was considered signicant (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 signicant differences were noted
(Table 1). No signicant 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 rst 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-

Please cite this article in press as: Mondal, S., et al., Double-blinded randomized controlled trial for immunomodulatory effects of Tulsi (Ocimum
sanctum Linn.) leaf extract on healthy volunteers. J. Ethnopharmacol. (2011), doi:10.1016/j.jep.2011.05.012
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S. Mondal et al. / Journal of Ethnopharmacology xxx (2011) xxxxxx 3

Assessed for eligibility

(n= 45)

Excluded (n= 21)

Not meeting inclusion criteria (n= 9)
Refused to participate (n=12)
Randomized (n=24)

Group-A Group-B

Allocated to intervention (n=13) Allocated to intervention (n= 11)

Received allocated intervention (n= 13) Received allocated intervention (n= 11)
Did not receive allocated Allocation
Did not receive allocated
intervention (n= 0 ) intervention (n= 0)

Lost to follow-up (n= 0) Lost to follow-up (n= 0)

Discontinued intervention (n=01) Discontinued intervention (n= 01)
Reason- Feeling of nausea Follow-Up
Reason- loose motions

Analyzed (n= 12) Analyzed (n=10)

Excluded from analysis (n=01) Analysis Excluded from analysis (n= 01)
Reason- not completed the study Reason-not completed the study

Fig. 1. Flowchart of recruitment of healthy volunteers.

over were grouped together and similarly placebo treated subjects 4. Discussion
were also grouped together for analysis and interpretation. The
compliance of the capsule intake was very satisfactory and com- There were no signicant period or carryover effects observed
pliance rate was more than 95%. during the study period which indicates that the chosen washout
period of three weeks were sufcient to undo the effects of rst
intervention.
3.2. Effects on Th1 and Th2 cytokine release INF- and IL-4 are clinically important because secreted lev-
els of these cytokines polarize effective functions, either Th1 or
In in vitro culture of whole blood stimulated with PHA and LPS, Th2 type response. INF- is known to be secreted during infec-
there were no signicant differences in the IFN- (Th1) and IL- tion due to intracellular pathogens and has potentially antiviral,
4 (Th2) cytokines at the baseline of both the groups. However, it
observed that there were signicant increase in the levels of both
IFN- and IL-4 (p = 0.039 and p = 0.001, respectively) in the blood Placebo (sucrose) group
samples of Tulsi extract intervention group. This increase did not 130 At baseline After 4 weeks
continue when subjects were crossed-over to placebo intervention 120
pg/ml in culture supernatent

after the wash out period (Figs. 2 and 3). 110

100
90
3.3. Effects on lymphocytes 80
70
60
In the present study, we did not nd any signicant differ-
50
ences in the percentage of lymphocytes and NK-cell at the baseline 40
examination in both the groups. There were also no signicant 30
differences observed in lymphocyte and NK-cell percentages in 20
placebo group (Fig. 4). However, a signicant increase in the T- 10
helper cells (p = 0.001) in Tulsi extract intervention group (Fig. 5) 0
Interferon-gamma Interleukin-4
was observed. Apparently healthy volunteers participated in this
study did not show any signicant difference in the percentages Th1 and Th2 Cytokines
of T-cytotoxic and B-cells even after 4 weeks of intervention in
both the groups (Figs. 4 and 5). Signicant increase in the NK-cells 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-
(p = 0.017) was noticed in the Tulsi extract group after 4 weeks of nicant difference after 4 weeks of placebo intervention. Two-way ANOVA for
intervention (Fig. 5). cross-over design applied.

Please cite this article in press as: Mondal, S., et al., Double-blinded randomized controlled trial for immunomodulatory effects of Tulsi (Ocimum
sanctum Linn.) leaf extract on healthy volunteers. J. Ethnopharmacol. (2011), doi:10.1016/j.jep.2011.05.012
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** Tulsi extract group to the increase in the levels of INF-. This indicates that in Tulsi
150
140 extract treated group, there was initial polarization of Th1 type
pg/ml in culture supernatant

130 of response (INF-) followed by Th2 type (IL-4). Thus, it can be

120 inferred from these results that when immune challenge was given
110
At baseline After 4 weeks in the form of PHA and LPS in the whole blood culture, Tulsi extract
100
90 intervention group had mounted an effective immune response
80 of Th1 type (INF-). This increase in cytokine level is also sup-
70 ported by signicant increase in the percentages of T-helper cells
60
(CD3+ CD4+ ) and NK-cells (CD16+ CD56+ ), however, no signicant
50
40 changes were found in T-cytotoxic cells (CD3+ CD4+ ) and B-cells
30 (CD19+ ). Flavonoids present in the extracts of Tulsi leaves have
20 * been found to be responsible for the immunomodulatory properties
10
(Mukherjee et al., 2005). In a similar clinical trial, immunomodula-
0
Interferon-gamma Interleukin-4 tory effects of traditional Chinese medicinal plant Yun Zhi (Coriolus
versicolor) and Danshen (Salvia miltiorrhiza) was observed after six
Th1 and Th2 Cytokine
months of intake (Wong et al., 2004).
Fig. 3. Effects of Tulsi extract on Th1 and Th2 cytokines after 4 weeks of intervention Tulsi is a non-toxic plant and its LD50 value is very high, rang-
(n = 22). All values in mean SD. There was a signicant increase in IFN- (Th1) ing from 4600 to 6400 mg/kg body weight of experimental murine
(**p = 0.039) and IL-4 (Th2) (*p = 0.001) levels in culture supernatant of whole blood models (Bhargava and Singh, 1981; Devi and Ganasoundari, 1995;
of Tulsi extract group after 4 weeks of intervention. Two-way ANOVA for cross-over
Singh and Majumdar, 1994). To determine any side effects of Tulsi,
design applied.
biochemical parameters were also evaluated at the same time
points. It was observed that intervention with 300 mg capsules of
Placebo (sucrose) group
Tulsi extract did not show any toxic effects as evident by basic phys-
55
iological and biochemical results [data not shown]. There were no
50 At baseline After 4 weeks
45 signicant changes observed in body mass index, blood pressure,
fasting blood sugar, liver and renal function tests. However, some of
Percentage

40
35 the subjects who had higher than the normal physiological ranges
30 of total cholesterol showed a signicant reduction (intrasubject
25
20 p = 0.003, in 6 subjects) after taking capsules of Tulsi extract for 4
15 weeks. However, the reduction was not observed in the subjects
10 who had total cholesterol levels within normal physiological refer-
5 ence ranges. When triglycerides levels were analyzed, it was found
0
CD3+CD4+ CD3+CD8+ CD19+ CD16+CD56+ that the reduction was statistically not signicant. However, it was
noticed that triglycerides levels were reduced in few subjects who
Lymphocytes and NK Cells had an initial elevated value. Thus, though not signicant statisti-
Fig. 4. Intervention effects of placebo (sucrose) capsules on T-Lymphocytes, B-
cally (p = 0.350), a reduction trend was observed in persons with
Lymphocytes and NK-cells after 4 weeks (n = 22). All values in mean SD. There was higher than normal physiological reference ranges of triglycerides.
no signicant difference after 4 weeks of placebo intervention. Two-way ANOVA for In an earlier clinical trial of Tulsi leaf on type-II diabetic patients a
cross-over design applied. signicant reduction in triglycerides, low density lipoprotein and
very low density lipoprotein was reported (Rai et al., 1997). Ani-
55 Tulsi extract group
** mal studies have also shown that feeding of Tulsi leaves reduces
50 cholesterol levels (Gupta et al., 2006). Higher levels of triglycerides
45 At baseline After 4 weeks and cholesterol are one of the risk factors of coronary artery dis-
40 eases in humans. Thus reduction in cholesterol and triglycerides
Percentage

35 seen in our study population is a very positive outcome. This rein-

30 forces the traditional claim (Ghosh, 1995) that it is good for heart.
25
However caution should be taken in to consideration while trans-
20
lating these ndings as our study population was of normal healthy
15
persons with no history of hypercholesterolemia or hypertriglyc-
10 *
eridemia. Thus, in patients with history of hypercholesterolemia or
5
hypertriglyceridemia this property may be further investigated.
0
In conclusion, it can be inferred from the results of this study
CD3+CD4+ CD3+CD8+ CD19+ CD16+CD56+
that, Tulsi extract have immunomodulatory effects in healthy vol-
Lymphocytes and NK-cells unteers without any side effects and a potential benet for those
with hypercholesterolemia.
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 signicant increase in T-helper cells (CD3+ CD4+ ) (**p = 0.001) and NK-Cells Conict of interest
(CD16+ CD56+ ) (*p = 0.017) after 4 weeks of Tulsi extract intervention. Two-way
ANOVA for crossover design applied.
Declared none.
antibacterial, anti-proliferative, anti-tumor and anti-allergic effects
(Dafny et al., 2007). INF- acts as one of the inhibitors of Th2 type Acknowledgements
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, We acknowledge CSIR and ICMR India, for awarding Senior
2004). Although levels of IL-4 were increased signicantly in the Research Fellowship to S. Mondal and Dabur India (P) Ltd. for assis-
Tulsi extract group, the increase was not so high as compared tance in capsule preparation for the study.

Please cite this article in press as: Mondal, S., et al., Double-blinded randomized controlled trial for immunomodulatory effects of Tulsi (Ocimum
sanctum Linn.) leaf extract on healthy volunteers. J. Ethnopharmacol. (2011), doi:10.1016/j.jep.2011.05.012
 ARTICLE IN PRESS
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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.
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