Journal of Ethnopharmacology 136 (2011) 452456

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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 Institute of Pathology, Safdarjung Hospital Campus, New Delhi, India 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
b c

a r t i c l e

i n f o

a b s t r a c t
Ethnopharmacological relevance: 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. 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 completed 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 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. 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.

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

1. Introduction Tulsi or Holy Basil (Ocimum sanctum Linn.) is widely distributed 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: [email protected], [email protected] (S.C. Mahapatra). 0378-8741/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.05.012

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, cardioprotective 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 ethanolic (70%) extract in healthy volunteers through a double-blind randomized controlled trial.

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2. Materials and methods 2.1. Study design A double-blind randomized controlled trial in a cross-over format with a washout period was designed to study the immunomodulatory 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 criteria 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 having 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 interventional 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, Ministry of Health and Family Welfare, Government of India. Placebo that contained sucrose was supplied by the Dabur Pharmaceutical (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 [Interferongamma (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 RPMI1640 medium and was stimulated with Escherichia coli derived

Table 1 Comparison of basic parameters of volunteers at the baseline (0 weeks). Basic parameters Placebo-Tulsi sequence (n = 12) Age Height Weight (kg) BMI (kg/m2 ) Systolic BP (mmHg) Diastolic BP (mmHg) 27.5 166.16 62.5 22.36 109.16 74.16 4.75 7.62 10.52 2.52 7.50 4.62 Baseline Tulsi-Placebo sequence (n = 10) 26.5 166.1 65.8 23.80 113.2 76.2 3.43 5.40 6.05 1.13 7.37 3.45 0.585 0.981 0.391 0.111 0.220 0.265 p-Value

All values presented in mean SD. p-Value 0.05 was considered as signicant. Independent t-test applied. BMI = Body Mass Index, BP = Blood Pressure.

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 version 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 instructions. 2.7. Statistical analysis Two-way Analysis of Variance (ANOVA) for crossover design was used to test the statistical signicance of the results. Independent 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-

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

Allocation

Allocated to intervention (n= 11) Received allocated intervention (n= 11) Did not receive allocated intervention (n= 0)

Lost to follow-up (n= 0) Discontinued intervention (n=01) Reason- Feeling of nausea

Follow-Up

Lost to follow-up (n= 0) Discontinued intervention (n= 01) Reason- loose motions

Analyzed (n= 12) Excluded from analysis (n=01) Reason- not completed the study

Analysis

Analyzed (n=10) Excluded from analysis (n= 01) Reason-not completed the study

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 compliance rate was more than 95%.

4. Discussion There were no signicant period or carryover effects observed during the study period which indicates that the chosen washout period of three weeks were sufcient to undo the effects of rst intervention. INF- and IL-4 are clinically important because secreted levels of these cytokines polarize effective functions, either Th1 or Th2 type response. INF- is known to be secreted during infection due to intracellular pathogens and has potentially antiviral,

3.2. Effects on Th1 and Th2 cytokine release In in vitro culture of whole blood stimulated with PHA and LPS, there were no signicant differences in the IFN- (Th1) and IL4 (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 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).

Placebo (sucrose) group 130 120 110 100 90 80 70 60 50 40 30 20 10 0

At baseline

After 4 weeks

3.3. Effects on lymphocytes In the present study, we did not nd any signicant differences in the percentage of lymphocytes and NK-cell at the baseline examination in both the groups. There were also no signicant differences observed in lymphocyte and NK-cell percentages in placebo group (Fig. 4). However, a signicant increase in the Thelper cells (p = 0.001) in Tulsi extract intervention group (Fig. 5) was observed. Apparently healthy volunteers participated in this study did not show any signicant difference in the percentages 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 (p = 0.017) was noticed in the Tulsi extract group after 4 weeks of intervention (Fig. 5).

pg/ml in culture supernatent

Interferon-gamma

Interleukin-4

Th1 and Th2 Cytokines

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 signicant difference after 4 weeks of placebo intervention. Two-way ANOVA for cross-over design applied.

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150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

**

Tulsi extract group

At baseline

After 4 weeks

*
Interferon-gamma Interleukin-4

Th1 and Th2 Cytokine

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 signicant 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 55 50 45 40 35 30 25 20 15 10 5 0 CD3+CD4+ CD3+CD8+ At baseline After 4 weeks

CD19+

CD16+CD56+

Lymphocytes and NK Cells

Fig. 4. Intervention effects of placebo (sucrose) capsules on T-Lymphocytes, BLymphocytes and NK-cells after 4 weeks (n = 22). All values in mean SD. There was no signicant difference after 4 weeks of placebo intervention. Two-way ANOVA for cross-over design applied.

55 50 45

**

Tulsi extract group At baseline After 4 weeks

Percentage

40 35 30 25 20 15 10 5 0 CD3+CD4+ CD3+CD8+ CD19+ CD16+CD56+

Lymphocytes and NK-cells

Fig. 5. Intervention effects of Tulsi extract capsules on T-Lymphocytes, BLymphocytes 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 (CD16+ CD56+ ) (*p = 0.017) after 4 weeks of Tulsi extract intervention. Two-way ANOVA for crossover design applied.

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 supported by signicant increase in the percentages of T-helper cells (CD3+ CD4+ ) and NK-cells (CD16+ CD56+ ), however, no signicant 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, immunomodulatory 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, ranging 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 physiological and biochemical results [data not shown]. There were no signicant 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 signicant 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 reference ranges. When triglycerides levels were analyzed, it was found that the reduction was statistically not signicant. However, it was noticed that triglycerides levels were reduced in few subjects who had an initial elevated value. Thus, though not signicant statistically (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 signicant reduction in triglycerides, low density lipoprotein and very low density lipoprotein was reported (Rai et al., 1997). Animal 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 diseases in humans. Thus reduction in cholesterol and triglycerides seen in our study population is a very positive outcome. This reinforces the traditional claim (Ghosh, 1995) that it is good for heart. However caution should be taken in to consideration while translating these ndings as our study population was of normal healthy persons with no history of hypercholesterolemia or hypertriglyceridemia. 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 volunteers without any side effects and a potential benet for those with hypercholesterolemia. Conict 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 assistance in capsule preparation for the study.

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 activation of Th1 by inhibiting the actions of INF- (Jiang and Chess, 2004). Although levels of IL-4 were increased signicantly in the Tulsi extract group, the increase was not so high as compared

Percentage

pg/ml in culture supernatant

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S. Mondal et al. / Journal of Ethnopharmacology 136 (2011) 452456 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, 1520. 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, 291302. Mukherjee, R., Dash, P.K., Ram, G.C., 2005. Immunotherapeutic potential of Ocimum sanctum (L.) in bovine subclinical mastitis. Research in Vetenary Science 79, 3743. 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 noninsulin dependent diabetes-mellitus. Journal of Nutritional and Environmental Medicine 7, 113118. Singh, S, Majumdar, D.K., 1994. Toxicological studies of the xed oil of Ocimum sanctum Linn. (Tulsi). New Botanist 21, 139146. Singh, S., Majumdar, D.K., Singh, J.P., 1995. Studies of therapeutic efcacy of xed oil of Ocimum sanctum in bovine mastitis. Indian Vetenary Journal 72, 867869. 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, 189195. Wealth of India, 1991. New Delhi, CSIR. Publication and information directorate Vol. 7, 7989. Wong, C.K., Tse, P.S., Wong, E.L., Leung, P.C., Fung, K.P., Lam, C.W., 2004. Immunomodulatory effects of yun zhi and danshen capsules in health subjects a randomized, double-blind, placebo-controlled, crossover study. International Immunopharmacology 4, 201211.

This research was funded by Central Council for Research in Ayurveda and Siddha, Department of Ayurveda, Yoga & Naturopathy, 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. 34 , pp. 179204. Bhargava, K.P., Singh, N., 1981. Antistress activity of Ocimum sanctum Linn. Indian Journal of Medical Research 73, 443451. 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. 253281. Devi, P.U., Ganasoundari, A., 1995. Radioprotective effect of leaf extract of Indian medicinal plant Ocimum sanctum. Indian Journal of Experimental Biology 33, 205208. Ghosh, G.R., 1995. Tulasi (N.O. Labiatae, Genus-Ocimum). New Approaches to Medicine and Health (NAMAH) 3, 2329. Godhwani, S., Godhwani, J.L., Vyas, D.S., 1988. Ocimum sanctum a preliminary study evaluating its immunoregulatory prole in albino rats. Journal of Ethnopharmacology 24, 193198. Gupta, S., Mediratta, P.K., Singh, S., Sharma, K.K., Shukla, R., 2006. Antidiabetic, antihypercholestrolaemic and antioxidant effect of Ocimum sanctum (Linn) seed oil. Indian Journal of Experimental Biology 44, 300304. Jiang, H., Chess, L., 2004. Integrated view of suppressor T cell subsets in immunoregulation. Journal of Clinical Investigation 114, 11981208.