Shankar Mondal, PhD, B.R. Mirdha, MD, M.M. Padhi, MD, S.C. Mahapatra, MD

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Dried leaf extract of Tulsi (Ocimum sanctum Linn) reduces cardiovascular disease risk factors: Results of a double blinded randomized controlled trial in healthy volunteers.

1 Dried leaf extract of Tulsi (Ocimum sanctum Linn) reduces cardiovascular disease risk factors: Results of a double blinded randomized controlled trial in healthy volunteers Shankar Mondal, PhD, B.R. Mirdha, MD, M.M. Padhi, MD, S.C. Mahapatra, MD 1 Departments of Physiology and Microbiology, All India Institute of Medical Sciences (AIIMS), New Delhi, INDIA 3 Central Council for Research in Ayurveda and Siddha, Department of Ayurveda, Yoga & Naturopathy, Unani and Sidhha (AYUSH), Janakpuri, New Delhi, India Abstract Herb Tulsi (Ocimum sanctum Linn.) is traditionally used for various ailments and believed to prevent many diseases if taken regularly on an empty stomach. Ethanolic extract of dried leaf of Tulsi [Tulsi extract] have been found to have immunomodulatory properties in many animal experiments and few clinical trials. This study was designed to evaluate the of Tulsi extract on biochemical parameters of healthy volunteers, having no known history of biochemical or immunological dysfunctions, through a double blinded randomized controlled trial. Biochemical parameters of healthy volunteers such as lipid profile, liver and kidney function tests were monitored in 4 subjects. One capsule of 300 mg Tulsi extract or placebo (sucrose) was administered on empty stomach once daily for 4 weeks followed by a washout period of 3 weeks. Then the subjects were crossover to other group and continued for another 4 weeks of. Blood samples were obtained; 1) before starting of capsule intake ) after completion of 4 weeks 3) after 3 weeks of washout and 4) after completion of 4 weeks in the crossover group. It was found that some of the subjects who had higher levels of triglycerides and cholesterols than the normal physiological values at beginning significantly reduced their cholesterol levels. There was also a reducing trend found for triglycerides levels. There were no significant changes in the other parameters tested. In conclusion it can be said that Tulsi extract have shown cardioprotective properties in the study population by significantly reducing higher levels of cholesterol. Key Words Cardioprotective Cholesterol Triglycerides Medicinal plant Introduction Plant Tulsi (Ocimum sanctum Linn.) belongs to the Lamiacae family which is known for its specific aromatic fragrance and medicinal values in traditional medicine system. Tulsi considered as the sacred plant in Indian subcontinent. Its medicinal properties have been mentioned in the ancient medicinal texts of various 1-3 civilizations of the world. Traditionally, it is believed that regular intake of Tulsi leaves on empty stomach keeps the person healthy and provides longevity. Scientific evidences that are available on various medicinal aspects of this holy plant i.e. antimicrobial, adaptogenic, anti-diabetic, hepatoprotective, anti-inflammatory, anti-carcinogenic, radioprotective, immunomodulatory, neuro-protective, cardioprotective, mosquito repellent etc. to name a few. Most of these evidences are based on in-vitro, experimental 4 and a few human studies. It is very well known fact that Tulsi have no side and experimental studies shown that LD 50 (Lethal Dose-50) of ethanolic extract is ranging Received: ; Revised: 7-0-1; Accepted: Disclosures: This article has not received any funding and has no vested commercial interest Acknowledgements: Indian Council of Medical Research [ICMR], New Delhi for Research Associate Fellowship to S. Mondal. Central Council for Research in Ayurveda and Siddha [CCRAS], Department of AYUSH, Ministry of Health and Family Welfare, Government of India for proving Tulsi extract and funds. Dabur Pharmaceuticals [P] Ltd, Ghaziabad, U.P., India for providing placebo and capsuling facility. 177

2 Mondal S, et al from 4600 mg/kg/bw (body weight) to 600 mg/kg/bw in 5, 6 experimental mice. Tulsi extract has been found to be cardioprotective in experimental animal studies. Long term oral administration of fresh Tulsi leaves augments cardiac endogenous antioxidants and prevents myocardial necrosis models in rats. Suanarunsawat et al (011), conducted animal experiments to investigate the lipid-lowering and antioxidative activities of Ocimum sanctum L. (OS) leaf extracts in liver and heart of rats fed with high-cholesterol (HC) diet for seven weeks. Their results showed that OS suppressed the high levels of serum lipid profile and hepatic lipid content without significant on fecal lipid excretion. Fecal bile acids excretion was increased in HC rats treated with OS. The high serum levels of TBARS [Thiobarbituric Acid Reactive Substances] as well as AST [Aspartate Aminotransferase], ALT [Alanine Aminotransferase], AP [Alkaline Phosphatase], LDH [Lactate Dehydrogenase], CK-MB [Creatine Kinase MB subunit] significantly decreased in HC rats treated with OS. OS treatment also suppressed the high level of TBARS and raised the low activities of GPx [Glutathione Peroxides] and CAT [Catalase] without any impact on SOD [Superoxide Dismutase] in the liver. Histopathological results also confirmed that OS preserved the liver and myocardial tissues. Present study was undertaken to validate the traditional claims of Tulsi regarding its usefulness in general health protection and to monitor various biochemical parameters of healthy volunteers after administration of 300 mg of Tulsi extract or placebo on empty stomach for a period of 4 weeks. Material and methods Design of the study This was a double blind randomized controlled trial of total 11 weeks in a crossover format which included a 3 weeks wash-out period, to study the of ethanolic extract of Tulsi leaf on clinical biochemistry parameters of healthy volunteers. The study was approved by the institutional ethics committee on research involving human subjects and the trial was registered with Clinical Trial Registry of India (No. CTRI/009/091/000350). Screening of Volunteers Forty five volunteers were screened to enroll in the study. Twenty four healthy volunteers, who met the inclusion criteria and gave their consent, were enrolled. Following 178 were the inclusion criteria for the enrollment of healthy volunteers; a) age years, either sex, b) not on any regular medication and c) devoid of any medication at least during last one month. Subjects suffering from different diseases/disorders and/or having any kind of allergy, undergone surgery during last one year, organ transplant recipients, chronic smokers, pregnant and 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 al capsules. Allocation of group was concealed in opaque envelope. Tulsi extract and placebo administration Ethanolic (70%) 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 Family Health and Welfare, Government of India. Placebo (sucrose, nutrition grade) was supplied by the Dabur Pharmaceutical (India) Ltd, Ghaziabad (U.P.), India. Capsulation facilities were provided by the Dabur Pharmaceutical to make 300 mg capsules using Tulsi extract or placebo. Packaging of both, Tulsi extract and placebo, were similar to avoid identification. The was allocated as per the randomized sequence once the volunteer met the inclusion criteria and given a written informed consent. Total period of trial was of 11 weeks in which two active administration period of 4 weeks each separated with a three weeks washout period in between. One capsule [code named, having Tulsi extract or placebo] of 300 mg was administered once daily, as per randomization, on empty stomach for four weeks followed by a wash out period of three weeks before the volunteer was crossover for the next. Collection of blood samples Venous blood samples of 5 ml were collected at four different time points i.e. i) before administration of capsule, ii) after administration of placebo or Tulsi extract capsules for four weeks iii) after the end of three weeks washout period and finally iv) after completion of four weeks in crossover period. The sampling was done in fasting state [at least 1h] between 8:30 am to 10:00 am at Department of Physiology, AIIMS, New Delhi. Subjects were contacted personally or telephonically twice a week for better compliance and unused capsules were taken back from the subjects. Two volunteers did not continue the due to their personal choice. Clinical biochemical parameters were studied using commercially available kits [Transasia, India; Randox, United Kingdom] and semi-automatic biochemistry analyzer [Labmate-0, Invitron, Bangalore, India].

3 Tulsi and CVD Statistical analysis of results Two-way ANOVA (Analysis of Variance) for crossover design was used to test the statistical significance of the results. Independent t-test was applied to compare the baseline of two groups. Wilcoxon singed-rank test was applied to analyze triglycerides levels. The results were considered significant if the p-value was 0.05 for period and. However, p-value of for carryover was considered significant. All statistical analysis was carried out using Stata 9.0 software (Statacorp, Texas, USA). The blinding was decoded only after the statistical analysis. Results The volunteers of two groups were comparable on basic anthropometric and physiological parameters. We had a wide range of age in inclusion criteria but subjects in relatively young age ranging from to 37 years participated in the study. The subjects had a healthy BMI ranging from 17.3 to 5.64 kg/m. Clinical biochemistry parameters of the subjects were monitored at both pre and post periods during the study. Total cholesterol (TC), triglycerides (TG), fasting blood glucose levels (FBG), liver function test, renal function tests were recorded. Table 1 shows the comparison of basic and biochemical parameters of placebo and Tulsi extract groups at baseline. Independent t-test applied. Fasting blood glucose There was a difference of about 1.56 mg/dl decrease in the Tulsi extract treated groups (Table ). When two-way ANOVA for crossover design was applied a statistically significant (intrasubject p=0.057) changes was seen in Tulsi extract treated groups during pre and post periods. However, normally the fasting blood glucose levels does not remain constant and it varies with durations of overnight fasting, type of last meal taken along with several other factors. Considering the dynamic behavior of blood glucose levels under physiological conditions this 1.56 mg/dl change may be ignored clinically. Liver Function Tests (LFT) and Renal Function Tests (RFT) Liver function tests were monitored by measuring SGOT [Serum glutamate oxaloacetate transferase], SGPT [Serum glutamate pyruvate transferase] and also total bilirubin levels of serum in few subjects. It was found that there were no significant differences during pre and post periods in either group. There were no significant changes in the blood urea nitrogen (p=0.511) serum protein (p=0.838) and creatinine (p=0.766) levels in either the placebo or the Tulsi extract groups (Table ). Triglycerides and total Cholesterol There was no significant (p=0.18 & p=0.3) difference at the baseline in triglycerides and cholesterol in both the groups respectively (Table, 3). However it was found that few individual who otherwise had an apparently normal medical history had high cholesterol and triglycerides levels. It was observed that there was a significant decrease in the elevated levels of cholesterol (intra-subject p=0.003) of these individuals (n=6) in the Tulsi extract group and this decrease did not continue when subjects crossed-over to the placebo group. Basic parameters Table 1: Comparison of Anthropometric and Physiological Parameters at (before administration of capsules) of Placebo-Tulsi sequence (n=1) of Tulsi- Placebo sequence (n=10) Age (years) (-37) (3-35) Height (cm) ( ) ( ) Weight (kg) (50-80) (54-78) P-Value BMI (kg/m ).36.5( ) ( ) Systolic BP (mm Hg) (100-10) (100-14) 0.0 Diastolic BP (mm Hg) (68-80) (70-80) 0.65 All values presented in mean SD (range) was considered as significant. 179

4 Mondal S, et al Table : Intervention Effects of Tulsi Extract or Placebo (Sucrose) Capsules on Biochemical Parameters After 4 Weeks Biochemical parameters Sequence of s 4 weeks after first 4 weeks after second comparison Period Carryover Intervention First Second (at 0 week) th (4 week) th (11 Week) # a b c Fasting blood glucose Cholesterol Total protein (g/ml) Blood Urea Nitrogen Creatinine P (n=1) TE (n=10) TE P P TE TE P P TE TE P P TE TE P P TE TE P * All values presented in mean #ac b p-value and were considered as significant. *Significant decrease [Intra-subject, n=6]. # abc SD. P- Placebo, TE- Tulsi extract, independent t-tests used. Two-way ANOVA for crossover design. Discussion All the subjects were relatively young, average age of both groups were years, ranging from -37 years. They had a normal BMI, (ranging from kg/m ). There was no significant period or carryover observed (Table ).Thus, the washout period of Table 3: Effects of Tulsi Extract or Placebo (Sucrose) Capsules on Triglycerides After 4 Weeks of Intervention Triglycerides After 4 weeks of Placebo (n=1) 57.5 (5-194) 61.5 (39-190) Tulsi extract (10) 78.5 (5-350) 74 (31-8) Value presented in median (range) was considered as significant. Wilcoxon singed-rank test applied. three weeks was sufficient to undo the of first. 5,6,1 Tulsi is a non-toxic plant and its LD50 value is very high however, for any side of Tulsi, biochemical parameters were evaluated. It was observed that with 300 mg capsules of Tulsi extract for 8 days on empty stomach did not show any toxic as evidenced by basic biochemical results. There were no significant changes observed in BMI, BP, fasting blood sugar, LFT and RFT. However, some of the subjects (n=6) who had higher than the normal physiological ranges of cholesterol showed significant reduction (intra-subject, p= 0.003) in the elevated levels after taking capsules of Tulsi extract for 4 weeks. However, no significant reduction was observed in the subjects who had cholesterol levels within normal physiological reference ranges. The reduction were also observed in the subjects who had higher than the normal physiological ranges of TGs levels, however when tested for normal distribution of the data, it showed the carryover after washout period. Thus, 180

5 Tulsi and CVD 11 according to Jones and Kenward (003), second was not taken into consideration and results were analyzed based on first only. When TGs levels were analyzed, it was found that the reduction was statistically not significant (Table 3). However, it was noticed that TGs levels were reduced in few subjects who had elevated levels. Thus, though not significant statistically, a reduction trend was observed in persons with higher than the normal physiological reference ranges of TGs (Table 3). Therefore, the observed reduction in elevated levels of TGs and cholesterol in Tulsi extract treated group was due to treatment only. Earlier studies have noted significant reduction in triglycerides, LDL [low density lipoprotein] and VLDL [very low density lipoprotein] in the clinical trial of Tulsi leaves on NIDDM 13,14 [Non-insulin dependent diabetes mellitus] patients. Animal studies had also showed that feeding of Tulsi leaves 15 reduces cholesterol levels. Present study also in consent with the findings of these studies. Higher levels of TGs and cholesterol are one of the risk 15,16 factors of CAD in humans. Thus, reduction in cholesterol and TGs seen in our study population is a very positive outcome for reduction of dyslipidemia related 17 CVD risks. This reinforces the traditional claim that Tulsi is good for heart. However caution should be taken in to consideration while translating these findings in patients that our study population was of normal healthy person with no history of hypercholesterolemia or hypertriglyceriglycemia. Thus, in patients with history of hypercholesterolemia or hyper-triglyceriglycemia this property may be further explored. References 1. Wealth of India. CSIR, Publication and information directorate, New Delhi, 1991; VII: WHO Monogram on selected medicinal plants. Dr Xiaorui Zhang, Acting Coordinator, Traditional Medicine, Department of Essential Drugs and Medicines Policy, World Health Organization. Geneva, 00; Vol : Gupta SK, Jai Prakash and Srivastava S. Validation of traditional claim of Tulsi, Ocimum sanctum Linn. as a medicinal plant. Indian J Exp Biol, 00; 40: Mondal S, Mirdha BR, Mahapatra SC. The science behind sacredness of Tulsi (Ocimum sanctum Linn). Indian J Physio Pharmacol, 009; 53: Bhargava KP, Singh N. Antistress activity of Ocimum sanctum Lin. Indian J Med. Res, 1981; 73: Devi PU, Ganasoundari A. Radioprotective effect of leaf extract of Indian medicinal plant Ocimum sanctum. Indian J Exp Biol, 1995; 33: Sharma M, Kishore K, Gupta SK, Joshi S, Arya DS. Cardioprotective potential of Ocimum sanctum in isoproterenol induced myocardial infarction in rats. Mol Cell Biochem, 001; 5(9): Sood S, Narang D, Dinda, AK, Maulik SK. Chronic oral administration of Ocimum sanctum Linn. augments cardiac endogenous antioxidants and prevents isoproterenol-induced myocardial necrosis in rats. J Pharmac Pharmacol, 005; 57: Arya D S, Nandave M, Ojha S K, Kumari S, Joshi S, Mohanty I. Myocardial salvaging of Ocimum sanctum in experimental model of myocardial necrosis: a haemodynamic, biochemical and histoarchitectural assessment. Current Science, 006; 91: Suanarunsawat T, Ayutthaya WD, Songsak T, Thirawarapan S, Poungshompoo S. Lipid-lowering and antioxidative activities of aqueous extracts of Ocimum sanctum L. leaves in rats fed with a high-cholesterol diet. Oxid Med Cell Longev, 011; doi: /011/ Jones B, Kenward MG. Design and analysis of cross-over trials. nd Ed. Chapman & Hall/CRC. New York. US Singh S, Majumdar DK, Yadav MR. Chemical and pharmacological studies of Ocimum sanctum fixed oil. Indian J Exp Biol, 1996; 34: Rai V, Mani UV, Iyer UM. Effect of Ocimum sanctum leaf powder on blood lipoproteins, glycated protein and total amino acids in patients with non-insulin-dependent diabetes mellitus. J Nutr Environ Med. 1997;7: Agrawal P, Rai V, Singh RB. Randomized placebo-controlled, single blind trial of holy basil leaves in patients with noninsulindependent diabetes mellitus. Int J Clin Pharmacol Ther. 1996;34: Gupta S, Mediratta PK, Singh S, Sharma KK, Shukla R. Antidiabetic, antihypocholestrolaemic and antioxidant effect of Ocimum sanctum (Linn) seed oil. Indian J Exp Biol, 006; 44: Talayero BG, Sacks FM. The role of triglycerides in atherosclerosis. Curr Cardiol Rep. 011; 13(6): Saggini A, Anogeianaki A, Angelucci D, et al. Cholesterol and vitamins: revisited study. J Biol Regul Homeost Agents. 011; 5(4): Address for correspondence Dr. Sushil Chandra Mahapatra: scmahapatra@gmail.com 181

Chemistry Reference Ranges and Critical Values

Chemistry Reference Ranges and Critical Values Alanine Aminotransferase (ALT, SGPT) 3-9 years 9-18 years 1-9 years 9-18 years 10-25 U/L 10-35 U/L 10-30 U/L 10-25 U/L 10-30 U/L 10-35 U/L 10-25 U/L 10-35 U/L 10-25 U/L 10-20 U/L 10-35 U/L Albumin 0-6