Garlic Powder Has no Anti-Inflammatory, Hypolipidemic, or Blood Pressure Lowering Effects in Subjects with Risk Factors for Atherosclerosis
Sonia M.S. Espirito Santo*, MSc.; Martijn B.A. van Doorn*, MD; Piet Meijer, PhD; Ingrid Kamerling, PhD; Rik C. Schoemaker, PhD; Verena Dirsch, PhD; Angelika Vollmar, PhD; Thomas Haffner, PhD; Rolf Gebhardt, PhD; Adam F. Cohen, MD, PhD; Hans M.G. Princen, PhD; Jacobus Burggraaf, MD, PhD.
*Both authors contributed equally
TNO Prevention and Health, Gaubius Laboratory, Leiden, The Netherlands (S.M.S.E.S., H.M.G.P.); Department of General Internal Medicine, Leiden University Medical Center, Leiden, The Netherlands (S.M.S.E.S.); Centre for Human Drug Research, Leiden, The Netherlands (M.B.A.D., I.K., R.S., A.C., J.B.);Department of Pharmacy, University of Munich, Munich, Germany (V.D., A.V.); Lichtwer AG, Berlin, Germany (T.H.); Institute of Leipzig, University of Leipzig, Leipzig, Germany (R.G.).
All correspondence addressed to:
M.B.A. van Doorn, M.D.
Centre for Human Drug Research,
Zerrnikedreef 10
2333 CL Leiden, The Netherlands
Tel: + 31 71 5246419
Text word count: 4352
Abstract word count: 244
ABSTRACT
Context Epidemiological studies suggest that garlic may have beneficial effects on risk factors associated with cardiovascular disease (CVD). However, these findings are not unambiguously supported by randomized placebo controlled clinical trials.
Objective To investigate the effects of a well-characterized enteric coated garlic preparation on relevant biomarkers in subjects with risk factors for CVD. Efficacy parameters included: markers of inflammation, endothelial function, lipid metabolism, and haemodynamic parameters.
Design, Setting, Participants, and Interventions Double-blind, randomized placebo-controlled trial in 90 obese (BMI >24.5 kg.m-2) subjects aged 40-75 years, who smoked >10 cigarettes.day-1. Subjects were randomly assigned to 3 parallel treatment groups: the garlic powder Printanor (2.1 g.day-1), atorvastatin (40 mg.day-1) or placebo. Measurements were performed at baseline, after 1 month and 3 months of treatment. Treatments were compared with ANOVA and differences between treatments were reported as mean percentage difference and corresponding 95% confidence interval (95% CI).
Results None of the efficacy parameters showed significant changes between the garlic treated group versus placebo. In contrast, significant decreases were observed in the atorvastatin treated group in plasma concentrations of CRP (20%; 95%CI: 4-34%), total cholesterol (37%; 95%CI: 34-41%), LDL-cholesterol (52%; 95% CI: 49-57%), triglycerides (32%; 95%CI: 22-40%) and TNF (42%; 95%CI: 22-56%). In addition, atorvastatin increased the ratio of ex vivo whole blood LPS-stimulated over non-stimulated TNF concentrations by 110% (95%CI: 46-202%).
Conclusion Awell-characterized enteric coated garlic preparation has no significant effect on a comprehensive array of relevant biomarkers in subjects with risk factors for cardiovascular disease.
KEYWORDS: Garlic, C-reactive protein (CRP), lipids, blood pressure, and humans.
INTRODUCTION
Beneficial effects of garlic on human health have been claimed for decades. However, none of these claims are unambiguously supported by results from placebo controlled clinical trials. One of these health claims entails the beneficial effects of garlic on risk factors associated with atherosclerosis and consequently the occurrence of cardiovascular events. In support of this notion, a number of studies reported a lowering of plasma lipids, systolic blood pressure, and enhanced fibrinolytic activity associated with garlic use.1-7 Through these actions, garlic is believed to protect the vessel wall from progressive atherosclerotic changes and consequently reduce the incidence of cardiovascular events.1-3 However, a number of other studies do not support these observations and showed no significant effects on these parameters.1-3,8-16 These conflicting findings may be ascribed to the use of different study designs (e.g. lack of placebo group), different patient inclusion criteria, as well as to the use of different garlic derived-material, such as raw garlic, garlic powders, and garlic oil, all of which carry variation in production conditions and chemical composition.1-3 Hence, from these reports it remains difficult to conclude whether garlic can truly exert beneficial effects on clinical risk factors associated with atherosclerosis.
In order to adequately investigate the effects of garlic on various human health parameters, a major collaborative EU program entitled “Garlic and Health” was started. This program aimed to identify garlic species containing the highest content of the compounds believed to be responsible for the claimed health effects. Subsequently, this garlic preparation was used to perform pre-clinical experiments in order to better define the role of garlic on presumed pharmacological targets. Moreover, the program aimed to investigate the effects of the garlic preparation on a comprehensive array of cardiovascular biomarkers in a well-powered clinical trial (‘Human Intervention Study’) of which the results are described in the current article.
A growing body of evidence suggests that inflammation may play an important role in the pathophysiology of atherosclerosis. This is illustrated by the results from a number of recent studies, which have shown C-reactive protein (CRP) te be one of the strongest predictors for the risk of atherosclerosis and cardiovascular events in subjects with and without cardiovascular disease.17-18Remarkably, there are no studies reporting on CRP or other markers of inflammation in subjects treated with garlic. Only in vitro studies have shown that high concentrations of garlic can decrease cytokine production in endothelial cells suggesting anti-inflammatory properties.19-21 Hence, the importance of investigating the effects of garlic on markers of inflammation in addition to traditional markers (plasma lipids and blood pressure) is evident.
We investigated the effects of a chemically well-characterized and production-controlled garlic powder (Printanor) on plasma CRP concentrations (primary endpoint) in subjects with known risk factors for CVD, during 12 weeks of treatment. In addition, we determined the effects of Printanor on plasma markers of endothelial function (von Willebrand factor (vWF), soluble vascular cell adhesion molecule (s-VCAM), soluble intercellular adhesion molecule (s-ICAM), and s-selectine) and general inflammation (fibrinogen), sensitivity of leukocytes to an inflammatory stimulus, plasma lipid levels and blood pressure.
METHODS
Subjects
The European Union Garlic Intervention Study (EUGIS) was a double-blind, randomized placebo-controlled trial in which atorvastatin was used as positive control. The study protocol was approved by the Committee on Medical Ethics of Leiden University Medical Center (LUMC) and conducted in the Netherlands. Subjects were recruited by advertisements in local newspapers. The participants were of either gender in general good health with known risk factors for atherosclerosis, i.e. aged 40-75 years, smoking >10 cigarettes.day-1, and a body mass index of >24.5 kg.m-2. Subjects were excluded if they used chronic (i.e. hormone replacement therapy) or any other medication (i.e. aspirin or NSAIDs) interfering with the measures of the study. Eligibility was assessed using a general health questionnaire, by measurement of body weight, height, and routine laboratory parameters including a urine pregnancy test for female subjects. A total of 150 subjects were informed about the study of which 142 signed the informed consent. Ninety subjects were eligible and entered the study.
Study design
Eligible subjects started with a 2-week placebo run-in period after which blood samples for determination of liver enzymes, creatine phosphokinase (CPK), hemoglobin, viral serology (Hepatitis B/C, HIV), and a urine sample for pregnancy testing (for females) were collected. If all parameters were still within the inclusion range, subjects were randomized to one of the three treatment groups in this parallel design study. Characteristics of the study population are listed in Table 1. The treatments were given in a double-dummy design. Thus, the subjects received either the garlic preparation (daily dose of 2.1 g; three 300 mg garlic tablets in the morning and four 300 mg garlic tablets in the evening plus one Atorvastatin-matching placebo tablet), or atorvastatin (daily dose of 40 mg; 3 garlic-matching placebo tablets in the morning and 4 garlic-matching placebo tablets in the evening plus a 40 mg atorvastatin tablet), or placebo (3 garlic-matching placebo tablets in the morning and 4 garlic-matching placebo tablets in the evening plus one atorvastatin-matching placebo tablet in the evening). The total treatment period was 12 weeks with follow-up visits scheduled after 4, 5, 11, and 12 weeks. On these visits, fasting blood samples for safety measurements and plasma biomarkers were collected, adverse events were recorded, and study medication was counted. In addition, on each visit blood pressure, and heart rate were measured.
Treatments
The garlic powder (Printanor) was produced under high sulfur-fertilization levels during the cultivation procedures as a part of European Union research program entitled “Garlic and Health” carried out by a consortium of 15 independent research groups from six countries. This garlic powder was produced and supplied by one of the participants (INRA, Dijon, France) and analyzed by standard high performance liquid chromatography (HPLC) procedure for the content of garlic sulfur-containing compounds, i.e. alliin content and allicin liberation capacity.22 The garlic powder contained 313.413.6 nmol of alliin per mg of garlic powder with the capacity to liberate 4.5 µg of allicin per mg of garlic powder. This means that in this study the subjects had a daily intake of 107.3 mg of alliin in the form of garlic powder. Subsequently, 300 mg tablets of this garlic powder and matching placebo tablets were produced under good manufacturing practice (GMP) standards by Lichtwer Pharma AG, Berlin, Germany.These tablets were coated such that the tablets were (gastric) acid resistant for at least 2 hours.
Atorvastatin (Lipitor®40 mg) tablets were purchased by the LUMC pharmacy and the matching placebo tablets were produced and supplied by Katwijk Pharma, The Netherlands.
All study medication was packed, labeled, and dispensed by the LUMC pharmacy.
Measurements
Tolerability assessment consisted of adverse event assessment, measurements of vital signs, 12-lead ECG recordings (Cardiofax V equipped with ECAPS12 analysis program, Nihon Kohden, Tokyo, Japan) and routine laboratory safety measurements (aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (ALKPHOS), -glutamyl transpeptidase (GT), lactate dehydrogenase (LDH), CPK, total bilirubin and conjugated bilirubin)on all visits. At these time points automated measurements of blood pressure and heart rate were made.
Blood sampling and sample handling
All blood samples collected at screening, randomization, after 4 and 5 weeks, and after 11 and 12 weeks of treatment, were taken after an overnight fast and at least 10 minutes of supine rest. Blood samples for haemoglobin concentration (at screening) were collected in tubes containing EDTA and analysed by the Central Clinical Haematology Laboratory (CKHL) of LUMC using a standard automated assay. Blood samples for ASAT, ALAT, ALKPHOS,GT, LDH, CPK, bilirubin, cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides (at screening and during study) were collected in plain serum tubes and analysed by the Central Clinical Chemistry Laboratory (CCCL) of LUMC using a standard automated assay.5Blood samples for C-reactive protein23, von Willebrand factor24, and fibrinogen (STA fibrinogen methods, Roche Diagnostics, Mannheim, Germany) were collected on ice in tubes containing 0.105 M citrate and processed within 60 minutes. Blood samples for tumor necrosis factor-α (TNFα) were collected in EDTA-tubes, stored at 37ºC and processed within 30 minutes. TNFα was measured using the Quantikine TNFα elisa (R&D systems, Abington, United Kingdom) in EDTA plasma ofa whole blood lipopolysaccharide (LPS)-stimulation test using 0 and 10 ng/mL LPS (final concentration) and overnight incubation at 37°C in a 5% CO2 incubator.25Blood samplesformarkers of vessel wall activation (soluble vascular cell adhesion molecule, soluble intercellular adhesion molecule, and s-selectine) were collected on ice in Li-Heparin tubes, processed within 60 minutes, and analysed by the department of Pharmacy at the University of Munich as previously described.26
Statistics
The a priori power of the study was calculated using data from an earlier experiment investigating the effects of atorvastatin on plasma CRP concentrations.23 Based upon these data this study had a power of 0.72/0.80 to detect a 25/30% decrease in CRP (at a 2-sided alpha level of 0.05) in each treatment group of 30 subjects.
The endpoints were analysed separately by mixed model analyses of variance (using SAS PROC MIXED with an unstructured covariance matrix) with subject as random effect and treatment, time and treatment by time as fixed effects, with baseline as covariate. Additionally, for graphical purposes, an analysis was performed with change from baseline values using baseline as covariate. This leads to statistically identical results compared with the alternate analysis, but allows graphs of change from baseline to be generated. Data were analyzed log-transformed and the analysis results were back-transformed and presented as geometric mean and percentage difference and the corresponding 95% confidence interval (95% CI) for the treatment contrasts. Calculation of time and treatment by time effects was performed for graphical presentation purposes only. All calculations were performed using SAS for Windows V8.2 (SAS Institute, Inc., Cary, NC).
RESULTS
Adverse events and safety parameters
Ten subjects (3 subjects in statin group, 5 subjects in garlic group and 2 subjects in placebo group) did not complete the study because of personal reasons (n=3) or adverse events (n=7). Two of these adverse events were possibly related to the study medication (abdominal discomfort (garlic group) and severe garlic odor (garlic group)),while the remaining five events were considered unrelated. Four subjects were replaced (2 subjects in statin arm and 2 subjects in garlic arm) with newly recruited volunteers. Therefore, the final study population consisted of 84 subjects; 29 subjects in the statin group, 27 subjects in the garlic group and 28 subjects in the placebo group. All other adverse events reported in the course of this study were mild or moderate and considered unrelated to study drug administration. In addition, vital signs, electrocardiogram (ECG), and routine laboratory parameters were virtually unchanged apart from a mild rise in liver enzyme levels noted in the atorvastatin treated group. This rise in liver enzyme levels is commonly seen in patients treated with statins and was considered not clinically significant.
Compliance
The compliance data showed only minor differences between subjects in tablet counts of returned study medication on each visit.The average daily intake of garlic was 7.1 ± 0.1 tablets (range 6.9-7.2) and the average daily intake of atorvastatin was 1.0 ± 0.1 tablets (range: 0.9-1.2).
Inflammation markers
After 12 weeks of garlic treatment, the mean plasma C-reactive protein (CRP) concentration increased by approximately 18% (95% CI: -2, -42%) compared with placebo. Atorvastatin treatment resulted in a 20% decrease (95% CI: 4, 34%) in mean CRP concentrations compared with placebo. Results are summarized in Table 2 and Figure 1.
After garlic treatment, plasma (non-stimulated) tumor necrosis factor- (TNF) concentrations and the ratio between lipopolysaccharide (LPS)-stimulated over non-stimulated TNF concentrations remained virtually unchanged. In contrast, atorvastatin treatment significantly decreased the mean plasma (non-stimulated) TNF concentration and increased the ratio of the LPS-stimulated over non-stimulated mean TNF concentration (Figure 2).The plasma fibrinogen concentrations were unaffected in both garlic and atorvastatin treatment groups.
Endothelial function markers
Both garlic and atorvastatin treatments did not affect the mean plasma concentrations of von Willebrand factor (vWF), soluble vascular cell adhesion molecule (s-VCAM), soluble intercellular adhesion molecule (s-ICAM), and soluble selectin (s-selectin). However, in the atorvastatin treated group the mean s-selectin concentration decreased by 9.8% (95% CI: -0.1, 18.7%) nearly reaching statistical significance (p=0.051). Results are summarized in Table 2.
Lipids and Lipoproteins
Garlic treatment did not influence the mean plasma cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol and triglyceride concentrations when compared with placebo treatment. In contrast, atorvastatin treatment resulted in a decrease of total cholesterol (37%; 95%CI: 34, 41%), LDL-cholesterol (52%; 95% CI: 49, 57%) and triglycerides (32%; 95%CI: 22, 40%) when compared with placebo, while HDL-cholesterol concentrations remained unaffected (p=0.32). Results are summarized in Table 2 and Figure 3.
Blood pressure and heart rate
Neither treatment affected blood pressure or heart rate at one and three months.
DISCUSSION
This clinical study investigated the effects of a chemically well-characterized and production-controlled garlic powder (2.1 g.day-1) and atorvastatin as positive control on a wide and comprehensive array of relevant biomarkers in subjects with known risk factors for CVD. Taken together, our data show that the garlic powder has no beneficial effects on a wide variety of important biomarkers for atherosclerosis in man. This makes it unlikely that garlic exerts a beneficial effect on CVD unless some other hitherto unknown biological effect is the basis of its purported epidemiological effects. However some other associated lifestyle effect is more likely to be the cause than a biological effect of garlic.
The garlic dose administered in this study was chosen using information from the pertaining literature. Previous studies that focused on the effects of garlic on lipid metabolism, blood pressure, and heart rate in humans used garlic powder doses that varied from 0.3 g.day-1 to 1 g.day-1 (approximately equivalent to 5.1 mg alliin.day-1 to 17 mg alliin.day-1). None of these studies reported beneficial effects of garlic on any of these parameters.8-10,12-15 However, one study investigated the effects of a considerably higher dose of a garlic-derived material (7.2 g.day-1 of aged garlic extract which is mainly composed of S-allylcysteine and S-allylmercaptocysteine) and showed a significant decrease in plasma lipids, LDL-cholesterol, and blood pressure in hypercholesterolemic subjects.5 Therefore, in the present study, we choose a relatively high dose of the garlic powder (2.1 g.day -1; approximately equivalent to 107 mg alliin.day-1 or 5.2 g.day-1 of fresh garlic) to be able to detect potentially beneficial effects of garlic powder on inflammation and/or atherosclerosis related markers. Furthermore, a pharmaceutical formulation was used that complies with all requirements for optimal release and bioavailability of the supposedly active compounds of garlic.28
The compliance data suggest that the adherence to the dosing regimens was good. Obviously, measurement of garlic components and atorvastatin plasma concentrations could have corroborated this observation, but unfortunately these data are not available. The effects of atorvastation were as expected supporting the notion that compliance was good at least in the positive control group.