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Effect of garlic powder on C-reactive protein and plasma lipids in

overweight and smoking subjects


13
Martijn BA van Doorn, Sonia M Espirito Santo, Piet Meijer, Ingrid M Kamerling, Rik C Schoemaker, Verena Dirsch,
Angelika Vollmar, Thomas Haffner, Rolf Gebhardt, Adam F Cohen, Hans M Princen, and Jacobus Burggraaf
ABSTRACT
Background: Epidemiologic studies suggest that garlic may have
beneficial effects on risk factors associated with cardiovascular dis-
ease (CVD). However, these findings are not unambiguously sup-
ported by randomized placebo-controlled clinical trials.
Objective: We sought to investigate the effects of a chemically
well-characterized garlic preparation on biomarkers for inflamma-
tion, endothelial function, and lipid metabolismin subjects with risk
factors for CVD.
Design: This was a double-blind, randomized, placebo-controlled
trial in 90 overweight [body mass index (in kg/m
2
) 24.5] subjects
aged 4075 y who smoked 10 cigarettes/d. The subjects were
randomly assigned to 3 parallel treatment groups: garlic powder
(2.1 g/d), atorvastatin (40 mg/d), or placebo. Duplicate measure-
ments were performed at baseline and after 1 and 3 mo of treatment.
Treatments were comparedwithanalysis of covariance withbaseline as
the covariate, and differences between the treatments were reported as
mean percentage difference and corresponding 97.5% CI.
Results: None of the variables showed significant differences be-
tween the garlic-treated and the placebo groups. In contrast, com-
pared with the placebo group, atorvastatin treatment resulted in
significantly lower plasma concentrations of C-reactive protein
(20.2%; 1.7%, 35.3%), total cholesterol (37.2%; 33.1%, 41.1%), LDL
cholesterol (52.7%; 47.9%, 57.1%), triacylglycerols (31.9%; 20.8%,
41.5%), and tumor necrosis factor (TNF-; 41.9%; 19.0%, 58.3%)
and increased the ratio of ex vivo whole blood lipopolysaccharide-
stimulated to nonstimulated TNF- concentrations (109.7%; 37.9%,
218.9%).
Conclusion: We conclude that a chemically well-characterized gar-
lic preparationhas nosignificant effect oninflammatorybiomarkers,
endothelial function, or lipidprofile innormolipidemic subjects with
risk factors for CVD. Am J Clin Nutr 2006;84:13249.
KEY WORDS Garlic, C-reactive protein, CRP, lipids, endo-
thelial function, humans
INTRODUCTION
Beneficial effects of garlic onhumanhealthhave beenclaimed
for decades. One claim entails the beneficial effects of garlic on
riskfactors associatedwithatherosclerosis and, consequently, on
the occurrence of cardiovascular events (1, 2). To support this,
several studies found a lowering of plasma lipid concentrations,
systolic blood pressure, arterial stiffness, and enhanced fibrino-
lytic activity to be associated with garlic use (38). Through
these actions, garlic is believed to prevent progressive athero-
sclerotic changes and consequently to reduce the incidence of
cardiovascular events. However, other studies do not support
these observations and reported no significant effects on these
variables (913). These conflicting findings may be ascribed to
the use of different study designs (eg, lack of placebo group),
different patient inclusion criteria, and different garlic-derived
material (ie, raw, powder, or oil), all of which vary in production
conditions and chemical composition (14).
However, another explanation may be that garlic influences
other than the classical risk factors for atherosclerosis (15). Ev-
idence is growing to indicate that inflammation plays an impor-
tant role in the pathophysiology of atherosclerosis. It has been
shown that C-reactive protein (CRP) is one of the strongest pre-
dictors for the riskof atherosclerosis andcardiovascular events in
subjects with and without cardiovascular disease (CVD) (16
18). To our knowledge, no studies have reported the effects of
garlic on CRP or other markers of inflammation in humans. In
vitro studies show that high concentrations of garlic decrease
cytokine production in endothelial cells, suggesting antiinflam-
matory properties (1921). Hence, investigating the effects of
garlic on markers of inflammation associated with CVD may be
important and provide an explanation for the alleged benefits of
garlic on human health.
To further investigate the effects of garlic on various human
health variables, the collaborative European Union program
Garlic and Health was started. This program aimed to identify
garlic species containing the highest content of the sulfur-
containing compounds believed responsible for health benefits,
to performpreclinical experiments with this garlic preparation to
define garlics role on pharmacologic targets, and to investigate
1
From the TNO Prevention and Health, Gaubius Laboratory, Leiden,
Netherlands (SMES, PM, and HMP); the Department of General Internal
Medicine, Leiden University Medical Center, Leiden, Netherlands (SMES);
the Centre for Human Drug Research, Leiden, Netherlands (MBAvD, IMK,
RCS, AFC, and JB); the Department of Pharmacy, University of Munich,
Munich, Germany (VD and AV); Lichtwer AG, Berlin, Germany (TH); and
the Institute of Biochemistry, Medical Faculty, Leipzig, Germany (RG).
2
Supportedbythe EuropeanUnionresearchproject QLK1-CT-1999-498.
3
Reprints not available. Address correspondence to MBA van Doorn,
Centre for Human Drug Research, Zernikedreef 10, 2333 CL Leiden, The
Netherlands. E-mail: mdoorn@chdr.nl.
Received September 2, 2005.
Accepted for publication July 17, 2006.
1324 Am J Clin Nutr 2006;84:13249. Printed in USA. 2006 American Society for Nutrition

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the effects of a pharmaceutical garlic preparationina clinical trial
of which the results are described in this article.
Therefore, we investigated the effects of a chemically well-
characterized and production-controlled garlic powder on
plasma CRP concentrations (primary endpoint) in subjects with
known risk factors for CVD, during 12 wk of treatment. In ad-
dition, the effects of garlic powder on plasma lipid concentra-
tions, plasma markers of inflammation, and markers of endothe-
lial function were investigated. The study was carried out with
the use of atorvastatin as a positive control because atorvastatin
was shown to decrease CRP and cholesterol concentrations in
normolipidemic subjects (22, 23).
SUBJECTS AND METHODS
Subjects
The study was carried out by using a double-blind, random-
ized, placebo-controlled design with 3 parallel groups. 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 sex in general good
health with known risk factors for atherosclerosis [ie, age 4075 y,
smoking10cigarettes/d, andbodymass index(inkg/m
2
) 24.5].
Subjects were excluded if they used chronic (ie, hormone re-
placement therapy or antihypertensives) or any other medication
(ie, aspirin, nonsteroidal antiinflammatory drugs, or lipid-
lowering drugs) that interferes with the measures of the study.
Eligibility was assessed with a general health questionnaire and
measurement of body weight, height, blood pressure, and routine
laboratory variables, including a urine pregnancy test for the
female subjects. Atotal of 150 subjects were informed about the
study; 142 signed the informed consent. Ninety subjects were
eligible and entered the study.
Study design
Eligible subjects started with a 2-wk placebo run-in period
after which blood samples for determination of liver enzymes,
creatine phosphokinase (CPK), hemoglobin, viral serology(hep-
atitis BandC, HIV), anda urine sample for pregnancytesting(for
the women) were collected. If all variables were still within the
inclusion range, subjects were randomly assigned to 1 of the 3
treatment arms. The subjects received either the garlic prepara-
tion (daily dose of 2.1 g; three 300-mg garlic tablets in the morn-
ing and four 300-mg garlic tablets in the evening plus 1
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 1 atorvastatin-matching placebo tablet in the
evening). The total treatment period was 12 wk with follow-up
visits scheduled after 4, 5, 11, and 12 wk. On these visits the
subjects reported to the research unit after an overnight fast
where their medical history was taken; adverse events were re-
corded. Also, the study medication was counted, vital signs were
measured, and blood samples were taken.
Treatments
The garlic powder (Printanor) was produced under high
sulfur-fertilization levels during the cultivation procedures as a
part of the European Union research program Garlic and Health
carried out by a consortium of 15 independent research groups
from6 countries. This garlic powder was produced and supplied
byone of the participants (INRA, Dijon, France) andanalyzedby
standard HPLC procedure for the content of garlic sulfur-
containing compounds (ie, allicin liberation capacity) (14). The
allicin liberation capacity was 4.5 g allicin/mg garlic powder.
This means that the subjects with a daily intake of 2.1 g garlic
powder/d had the capacity to liberate 9.4 mg allicin/d. Subse-
quently, 300-mg tablets of this garlic powder and matching pla-
cebo tablets were produced under good manufacturing practice
standards (Lichtwer Pharma AG, Berlin, Germany). The tablets
were enteric coated, such that the tablets were (gastric) acid
resistant for at least 2 h (24). Atorvastatin (Lipitor 40 mg) tablets
were purchased by the LUMC hospital pharmacy, and matching
placebo tablets were manufactured (Katwijk Pharma, Katwijk,
Netherlands). All study medication was packed, labeled, and
dispensed by the LUMC pharmacy.
Measurements
Tolerability assessment consisted of adverse event assess-
ment, measurements of vital signs, 12-lead electrocardiogram
recordings (Cardiofax V equipped with ECAPS12 analysis pro-
gram; Nihon Kohden, Tokyo, Japan) and routine laboratory
safety measurements (aspartate aminotransferase, alanine ami-
notransferase, alkaline phosphatase, -glutamyl transpeptidase,
lactate dehydrogenase, CPK, total bilirubin, and conjugated bil-
irubin) on all visits.
Blood sampling and assays
All blood samples were collected at screening, at random
assignment, after 4 and 5 wk (1 mo) of treatment, and after 11 and
12 wk (3 mo) of treatment. The samples were taken after an
overnight fast and at least 10 min of supine rest. Blood samples
for hemoglobin concentration (at screening) were collected in
tubes containing EDTA and measured by the Central Clinical
Hematology Laboratory of LUMC with the use of a standard
automated assay. Blood samples for aspartate aminotransferase,
alanine aminotransferase, alkaline phosphatase, -glutamyl
transpeptidase, lactate dehydrogenase, CPK, bilirubin, choles-
terol, HDLcholesterol, LDLcholesterol, and triacylglycerols (at
screening and during study) were collected in plain serum tubes
and measured by the Central Clinical Chemistry Laboratory of
LUMC with the use of standard methods.
Bloodsamples for CRP, vonWillebrandfactor, andfibrinogen
(STA fibrinogen methods; Roche Diagnostics, Mannheim, Ger-
many) were collected on ice in tubes containing 0.105 M citrate
and processed within 60 min and measured by using previously
described methods (25, 26). Blood samples for tumor necrosis
factor (TNF-) were collected in tubes containing EDTA,
stored at 37 C, and processed within 30 min. TNF- was mea-
sured with the use of the Quantikine TNF- enzyme-linked im-
munoabsorbent assay (R&D Systems, Abington, United King-
dom) in EDTA plasma of a whole blood lipopolysaccharide
stimulation test with the use of 0 and 10 ng lipopolysaccha-
ride/mL (final concentration) and overnight incubation at 37 C
in a 5%CO
2
incubator (27). Blood samples for markers of vessel
wall activation (soluble vascular cell adhesion molecule, soluble
intercellular adhesion molecule, and soluble selectin) were col-
lected on ice in Li-Heparin tubes, processed within 60 min, and
measured by commercial kits (R&D Systems).
GARLIC AND BIOMARKERS FOR CARDIOVASCULAR DISEASE 1325

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Statistics
The a priori power of the study was calculated with the use of
data from an earlier experiment that investigated the effects of
atorvastatin on plasma CRP concentrations (26). On the basis
of that data, the present study had a power of 0.72 to detect a 25%
decrease in CRP or 0.80 to detect a 30% decrease in CRP (at a
2-sided level of 0.05) in each treatment group of 30 subjects.
Data were initially analyzed by calculating change frombase-
line and including the values after baseline only, with the use of
SAS PROC MIXED with an unstructured covariance matrix,
with subject as random effect, time and treatment by time inter-
action as fixed effects, and with baseline and baseline by time as
covariates. The treatment-by-time interaction tests for overall
parallel time profiles. Subsequently, the endpoints were ana-
lyzed by mixed-model analyses of covariance (with the use of
SAS PROC MIXED with an unstructured covariance matrix)
with subject as a random effect; treatment, time, and treatment-
by-time as fixed effects; and with baseline as covariate. Data
were analyzed and log-transformed to correct for the observed
log-normal distribution of the data (which is common for con-
centration measurements). The analysis results were back-
transformed and presented as geometric mean and percentage of
difference and the corresponding 97.5% CI for the treatment
contrasts. To correct for multiple comparisons of the 2 prespeci-
fied contrasts, P values 0.025 were considered statistically
significant, and contrasts are reported with 97.5% CIs. In addi-
tion, baseline variables were compared by using analysis of vari-
ance. All calculations were performed with the use of SAS for
WINDOWS version 9.1.2 (SAS Institute Inc, Cary, NC).
RESULTS
Ten subjects (3 subjects in the atorvastatin group, 5 subjects in
the garlic group, and 2 subjects in the placebo group) did not
complete the study because of personal reasons (n 3) or ad-
verse events (n 7). Two of these adverse events (abdominal
discomfort andsevere garlic odor) occurredinsubjects randomly
assigned to receive garlic, whereas the remaining 5 events were
consideredunrelated. Five of these subjects didcontribute data to
the analysis because they provided data at the 1-mo assessment.
Two subjects (in the atorvastatin group) were replaced with
newly recruited volunteers. In addition, 3 subjects were excluded
from the statistical analysis before unblinding (all 3 in the pla-
cebo group) because of intercurrent inflammatory conditions,
which were expected to interfere with the assessment of CRP
status.
Therefore, the final study population consisted of 84 subjects;
30 subjects in the atorvastatin group, 28 subjects in the garlic
group, and 26 subjects in the placebo group. Characteristics of
the study population are listed in Table 1. No significant differ-
ences were observed in the baseline characteristics among the 3
groups except for diastolic blood pressure, which was slightly
lower in the atorvastatin group. This difference in diastolic blood
pressure was considered not physiologically important.
Adverse events
The adverse events reported in the course of the present study
were mild or moderate and considered unrelated to study drug
administration except for the severe garlic odor. Vital signs,
electrocardiogram, and routine laboratory variables were virtu-
ally unchanged apart from a mild rise in liver enzyme concen-
trations noted in the atorvastatin group.
Compliance
The compliance data showed only minor differences among
subjects in tablet counts of returned study medication on each
visit. The average daily intake of garlic was 7.1 0.1 tablets
(range: 6.97.2 tablets), and the average daily intake of atorva-
statin was 1.0 0.1 tablets (range: 0.91.2 tablets).
Inflammation markers
During 12 wk of garlic treatment, the mean CRPconcentration
increased nonsignificantly by 17.8%(97.5%CI: 5.0%, 46.0%)
compared with placebo treatment; whereas atorvastatin treat-
ment resulted in a 20.2% (97.5% CI: 1.7%, 35.3%) decrease in
mean CRP concentration compared with placebo treatment
(Table 2). After garlic treatment, plasma (nonstimulated) TNF-
concentrations and the ratio between lipopolysaccharide-
stimulated and nonstimulated TNF- concentrations remained
virtually unchanged. In contrast, after atorvastatin treatment, a
significantly lower mean plasma TNF- concentration and
higher ratio of the lipopolysaccharide-stimulated concentration
to nonstimulated mean TNF- concentration was observed
(Table 2). Plasma fibrinogen concentrations were unaffected in
both the garlic and the atorvastatin treatment groups (Table 2).
Endothelial function markers
Neither garlic nor atorvastatin treatments affected the mean
plasma concentrations of vonWillebrandfactor, soluble vascular
cell adhesion molecule, soluble intercellular adhesion molecule,
and soluble selectin compared with placebo treatment.
TABLE 1
Baseline characteristics of the study population
1
Placebo group Garlic group Atorvastatin group P at baseline
Sex
Female 13 16 17 NA
Male 13 12 13
Age (y) 48.8 6.6
2
48.5 6.9 47.1 5.6 0.5497
BMI (kg/m
2
) 29.8 3.3 29.1 3.1 29.7 4.3 0.7580
Systolic blood pressure (mm Hg) 124.7 13.7 128.4 13.5 123.0 14.3 0.3308
Diastolic blood pressure (mm Hg) 78.8 9.9 81.5 11.5 72.6 11.0 0.0080
1
NA, not applicable.
2
x SD (all such values).
1326 VAN DOORN ET AL

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.
GARLIC AND BIOMARKERS FOR CARDIOVASCULAR DISEASE 1327

b
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Lipids and lipoproteins
Garlic treatment did not influence the total cholesterol, HDL-
cholesterol, LDL-cholesterol, and triacylglycerol concentrations
when compared with placebo treatment. In contrast, atorvastatin
treatment resulted in lower concentrations of total cholesterol
(37.2%; 97.5% CI: 33.1%, 41.1%), LDL cholesterol (52.7%;
97.5% CI: 47.9%, 57.1%), and triacylglycerols (31.9%; 97.5%
CI: 20.8%, 41.5%) when compared with placebo treatment.
HDL-cholesterol concentrations were not significantly affected
by atorvastatin (P 0.34; Table 3).
DISCUSSION
The present placebo-controlled study investigated the effects
of a 12-wk treatment intervention with a chemically well-
characterized and production-controlled garlic powder in sub-
jects with known risk factors for CVD. A group receiving ator-
vastatin was used as positive control. The study aimed to identify
the effects of garlic powder on inflammatory markers (in partic-
ular CRP), endothelial function, and measures of lipid metabo-
lism. The main finding of the study is that garlic powder has no
detectable effects on CRP, TNF-, in the basal and stimulated
state or lipid concentrations, whereas these concentrations were
positively affected by atorvastatin. This makes it unlikely that
garlic exerts a beneficial effect on CVD by antiinflammatory or
lipid-lowering mechanisms.
The emphasis on the potential antiinflammatory effects of
garlic was chosen because of the observations of in vitro studies
which showed that high concentrations of garlic decreased cy-
tokine production in endothelial cells which in turn suggested
distinct antiinflammatory properties (20, 21, 28). In addition, a
growing body of evidence suggests that inflammatory processes
play an important role in the pathophysiology of atherosclerosis.
Because it has been shown that CRP is one of the strongest
predictors for the risk of atherosclerosis and cardiovascular
events in subjects with and without CVD (1618), this marker
was chosen as primary endpoint in the current study. Note that
our study had an a priori power of 7080% to detect a 2530%
decrease in plasma CRPconcentration. This percentage decrease
was considered relevant because other studies have shown sim-
ilar effects of atorvastatin on CRP (26). Because our study pop-
ulation had a relatively high mean plasma concentration of CRP
(2 mg/L) when compared with healthy volunteers (2 mg/L),
this allowedus todetect possible effects of garlic andatorvastatin
treatment on the inflammatory status of these subjects.
It can be argued that the results obtained in our population
might not be representative for patients with a higher risk of
CVD. Indeed, our population had only 2 known risk factors
(overweight and smoking) for CVD, and the participants were
normotensive and normocholestrolemic. We feel, however, that
the choice for this population is justified. First, it may be con-
sidered unethical to performa similar study in patients with overt
hypercholesterolemia because indicated medical treatments for
these conditions are available. Second, it has been shown that
elevatedCRPconcentrations as observedinour populationare an
independent risk factor for CVD. Finally, it has been shown that
effective treatments have a CRP- and lipid-lowering effect in
overweight subjects (22) and even in healthy volunteers (23).
Thus, by choosing this population with not readily amendable
risk factors for CVD a relevant population was studied.
The relatively high garlic dose administered in the present
study was chosen because there are some indications that higher
garlic doses are associated with greater effects. Indeed, previous
studies that showed no beneficial effects of garlic on lipid me-
tabolismand blood pressure in humans used garlic powder doses
that variedfrom0.3g/dto1g/d(913) for whichit maybe argued
that the garlic dose was too low. Therefore, in the present study,
we choose a relatively high dose of the garlic powder (2.1 g/d,
approximately equivalent to 9.4 mg allicin/d or 5.2 g fresh gar-
lic/d) to be able to detect potentially beneficial effects of garlic
powder. Furthermore, a pharmaceutical formulation was used that
complies withall requirements for optimal release andbioavailabil-
ity of the supposedly active compounds of garlic (25, 29).
The compliance data suggested that the adherence to the dos-
ing regimens was good. Obviously, measurement of garlic com-
ponents and atorvastatin plasma concentrations could have cor-
roborated this observation, but, unfortunately, these data were
not available. The effects of atorvastatin were as expected, sup-
porting the notion that compliance was good, at least in the
positive control group.
Apart from the primary endpoint, other markers of inflamma-
tion (fibrinogen and leukocyte TNF- production in response to
lipopolysaccharide), our data show that garlic powder had no
significant effects on any of these markers, whereas atorvastatin
showed antiinflammatory effects also on TNF-concentrations.
This suggests that garlic has no significant effect on the inflam-
matory processes associated with atherosclerosis.
For a long time the lipid- and lipoprotein-lowering properties
of garlic have been raised as a mechanism by which garlic could
exert a beneficial effect on atherogenesis, although this is not
unambiguously supported. Our data appear to be in keeping with
the latter findings because we did not observe any effect of garlic
on plasma lipids and lipoproteins. In contrast, atorvastatin treat-
ment significantly decreased total cholesterol, LDL-cholesterol,
and triacylglycerol concentrations. A posteriori power analysis
withthe data obtainedinthe present studyindicatedthat the study
had a power of 80% at a 2-sided level, P 0.05, to detect a
decrease of 0.95 mmol/L in total cholesterol concentration (a
17% decrease).
In conclusion, our data showed that 12 wk of treatment with a
high-dose, chemically well-characterized, production-controlled
garlic powder had no antiinflammatory or lipid-lowering effect
in a normolipidemic population with known risk factors for
CVD. As such, we conclude that evidence does not showthat the
garlic powder Printanor that was speciallyselectedandoptimally
pharmaceutically formulated has beneficial effects on athero-
genesis and, consequently, cardiovascular events in a relevant
population through antiinflammatory processes or lipid-
lowering effects. The findings of the present study lead us to
speculate that garlic powder (and probably garlic in general) has
no relevant place in the prevention or treatment of the inflam-
matory and dyslipidemic features of atherosclerosis.
We thank all the people involved in the European Union project Garlic and
Health for their collaboration.
SMES analyzed the data and wrote the manuscript; MBAvDexecuted the
study, analyzed the data, and wrote the manuscript; PM, VD, and AV per-
formedthe laboratoryassays; IMKwrote the protocol andexecutedthe study;
RCS developed the protocol, analyzed the statistics, and wrote the manu-
script; TH developed the garlic and garlic-placebo formulations; RG per-
formed the study setup; AFCdeveloped the protocol, executed the study, and
1328 VAN DOORN ET AL

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reviewed the manuscript; HMP developed the protocol, supervised the lab-
oratory assays, and reviewed the manuscript; JB developed the protocol,
executedthe study, analyzedthe data, wrote andreviewedthe manuscript and
review, and was the principal investigator. SMES and MBAvD contributed
equallytothe study. THis anemployee of Lichtwehr Pharma AG; none of the
other authors had a conflict of interest.
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