Effects of a flaxseed-derived lignan supplement on C-reactive protein, IL-6
and retinol-binding protein 4 in type 2 diabetic patients
An Pan1, Wendy Demark-Wahnefried2, Xingwang Ye1, Zhijie Yu1, Huaixing Li1, Qibin Qi1, Jianqin Sun3,
Yanqiu Chen3, Xiafei Chen3, Yong Liu1and Xu Lin1*
1Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences and Graduate School of the Chinese Academy of Sciences, 294 Tai-Yuan Road, Shanghai 200031,
2Department of Behavioral Science, The University of Texas-MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston,
TX 77030, USA
3Huadong Hospital, Fudan University, 221 West Yan-An Road, 200040, Shanghai, China
(Received 25 April 2008 – Revised 7 July 2008 – Accepted 31 July 2008 – First published online 8 September 2008)
Elevated C-reactive protein (CRP), IL-6 and retinol-binding protein 4 (RBP4) levels are associated with insulin resistance and diabetes mellitus. Phy-
toestrogens (including lignans and isoflavones) may enhance the management of diabetes and are hypothesized to act through inflammation pathways.
The present study explored the effects of flaxseed-derived lignan on inflammatory factors and RBP4 concentrations in type 2 diabetics, who have higher
levels of these biomarkers. Seventy community-dwelling diabetic patients (twenty-six men and forty-four post-menopausal women) with mild hyperch-
olesterolaemia completed a randomized, double-blind, placebo-controlled, cross-over trial of supplementation with flaxseed-derived lignan capsules
(360mg/d) or placebo for 12 weeks, separated by an 8-week wash-out period. The participants maintained their habitual diets and levels of physical
activity. Baseline to follow-up concentrations of CRP increased significantly within the placebo group (1·42 (SEM 0·19) v. 1·96 (SEM 0·22) mg/l,
P,0·001), but werecomparatively unchanged in the lignan-supplemented group (1·67 (SEM 0·19) v. 1·90 (SEM 0·26) mg/l, P¼0·94); a significantdiffer-
ence was observed between treatments (20·45 (95% CI 20·76, 20·08) mg/l, P¼0·021). This effect was confined to women (P¼0·016), but not
observed in men (P¼0·49). No between-treatment differences were found with regard to IL-6 or RBP4; though IL-6 concentrations increased signifi-
cantly from baseline to follow-up in both groups (P¼0·004 and P,0·001 following lignan and placebo treatments, respectively). The study suggests
that lignan might modulate CRP levels in type 2 diabetics. These results need to be confirmed by further large clinical trials of longer duration.
Lignan: C-reactive protein: IL-6: Retinol-binding protein 4: Flaxseed: Type 2 diabetes: Inflammation
Several studies suggest that chronic inflammation, as indicated
by elevated levels of inflammatory factors such as C-reactive
protein (CRP) and IL-6, plays an important role in the patho-
genesis of diabetes mellitus(1–3). Recently, retinol-binding
protein 4 (RBP4), a protein product of hepatocytes and adipo-
cytes, has been associated with insulin resistance, diabetes(4,5)
A growing body of evidence suggests that certain chronic
disorders, including diabetes, dyslipidaemia and CVD, are
responsive to dietary phytoestrogens (isoflavones and lig-
nans)(7,8), which may modulate disease risk through inflam-
matory pathways(9). Flaxseed is the richest food source of
the plant lignan, secoisolariciresinol diglucoside(10). In a
women, Hallund et al.(11)observed significantly lower CRP
concentrations in participants receiving a lignan complex
(500mg secoisolariciresinol diglucoside/d) for 6 weeks
compared to those on placebo. Hall et al.(12)also detected
beneficial effects on CRP with 8-week supplementation of
isolated isoflavones (50mg/d) in 170 healthy post-menopausal
women. However, other studies using isoflavones have
produced null findings(13–16).
We have previously reported that a flaxseed-derived lignan
supplement moderately decreased glycated Hb levels in type 2
diabetics(17). The present study is a secondary analysis, and is
aimed at exploring the impact of lignan supplementation on
CRP, IL-6 and RBP4 levels in an effort to better understand
the effects of lignan on diabetes.
*Corresponding author: Dr Xu Lin, fax þ86 21 54920249, email email@example.com
Abbreviations: CRP, C-reactive protein; RBP4, retinol-binding protein 4.
British Journal of Nutrition (2009), 101, 1145–1149
q The Authors 2008
British Journal of Nutrition
Study design and participants
The methods of the parent study were detailed elsewhere(17).
Briefly, seventy-three type 2 diabetic patients (twenty-eight
men and forty-five post-menopausal women) aged 50–79
years were consented and randomized in a crossover design
to a double-blind, placebo-controlled study with 12-week
supplementation of lignan or placebo capsules separated by
an 8-week washout period. Randomization was performed
using stratification factors of gender and tertiled LDL-choles-
terol concentrations (the primary end-point of the parent
The lignan capsules (LinumLifee Extra; Frutarom Nether-
lands BV, Veenendaal, The Netherlands) provided a daily
dose of 360mg flaxseed-derived secoisolariciresinol digluco-
side. The three capsules provided 15·5kJ and were comprised
of 20% secoisolariciresinol diglucoside, 30% carbohydrate,
15·6% fat, 3·2% protein and 2·6% fibre. The placebo was
an identical capsule of rice flour devoid of soluble fibre. The
participants took three capsules per day (1·8g) which contrib-
uted minimally to their daily energy and nutrient intake.
Adherence was assessed by pill counts and urinary concen-
trations of lignan metabolites(17). While on study, participants
were required to take their regularly prescribed medications,
and maintain their habitual diets and physical activity levels.
Fasting venous blood samples were collected at the beginning
and completion of each intervention period. Serum CRP was
measured via a high-sensitive immunoturbidimetric assay
on a Hitachi 7080 automatic analyser using commercial
kits (Roche Diagnostics, Mannheim, Germany). IL-6 was
measured by a high-sensitive ELISA (R&D Systems, Minnea-
polis, USA). Plasma RBP4 was measured by an in-house-
developed sandwich ELISA, detailed elsewhere(5). Fasting
morning urine samples (50ml) were collected at identical
time-points using plastic jugs containing 50mg ascorbic
acid. Urinary lignan metabolites were analysed using HPLC
as reported previously(17).
The power calculations of our previous study were based on
serum lipids and were described previously(17). In the present
exploratory secondary analysis, a total of sixty participants
provide 80% power with an a level of 0·05 (two-sided) to
detect between-treatment differences of 0·30mg/l, 0·30pg/ml
and 14·6mg/ml on CRP, IL-6 and RBP4, respectively.
Data were analysed in Stata 9.2 (Statae; Texas, USA); the
two-sided P value #0·05 was considered statistically signifi-
cant. For all biomarkers, the data were natural-logarithmically
transformed prior to analysis. Paired Student’s t tests were
used to compare differences between baseline and end of treat-
ment. Differences between values after the 12-week interven-
tion were analysed using a mixed model analysis of
covariance with treatment and period as fixed factors,
participants as random factors and baseline values as covari-
ates. Further fixed terms corresponding to treatment–period
and treatment–baseline value interactions were included.
Given the weight-dependent nature of the study end-points,
baseline weight and weight changes during treatment also
were incorporated as covariates. The analyses were further
stratified by gender to explore differential effects in males
Participants with extremely high levels of CRP ($10mg/l)
or IL-6($10pg/ml) were
inflammation and were excluded from the final mixed
models. Sixty-four participants remained for CRP analyses
and sixty-seven remained for IL-6 analyses.
considered to haveacute
Of the seventy-three participants consented, three participants
dropped out for reasons described previously(17). Seventy
(twenty-six men and forty-four women) completed the study
and were available for analyses. The mean age of the
sample was 62·9 (SD 7·5) years.
Significant increases in CRP concentrations were found from
baseline to follow-up within the placebo group (P,0·001),
while those in the lignan treatment group did not experience
increases of similar magnitude (P¼0·94). Thus, compared to
placebo, increases in CRP with the lignan supplement were sig-
nificantly lower (P¼0·021). These differences were primarily
observed in women (P¼0·016), and not in men (P¼0·49)
In contrast, IL-6 concentrations significantly increased from
baseline to follow-up in both groups, with no between-treat-
ment difference observed. Likewise, no between-treatment
difference was detected in levels of RBP4, which remained
at fairly stable levels throughout the study period. Addition-
ally, urinary excretion of lignan metabolites increased signifi-
cantly after lignan treatment. As reported previously, no
significant differences were observed for energy and nutrient
intake, and physical activity between treatment phases
The CRP concentrations in the placebo group increased sig-
nificantly over the study period. This is an unexpected finding,
and the exact reasons are unclear. Elevated levels of CRP,
however, have been documented among diabetic patients
and correlated with both glycaemic control and compli-
cations(1). Speculation exists that increased CRP concen-
trations may reflect aggravated diabetes management within
these individuals. Furthermore, some participants had medi-
cation changes during the study period, which also might
have influenced their inflammatory status. Although CRP
levels increased slightly in the lignan-treated group, this
increase was not statistically significant. The present findings
are similar to a recent study by Hallund et al.(11)which
also employed a crossover design delivering placebo v. a
lignan supplement (500mg secoisolariciresinol diglucoside/
d) among twenty-two healthy post-menopausal women. They
also found greater increases in CRP during the placebo
period (from 0·80 to 1·10mg/l) than during lignan treatment
(from 0·88 to 0·92mg/l) (P¼0·028). Similarly, Teede
A. Pan et al.1146
British Journal of Nutrition
Table 1. Baseline and 12-week follow-up levels of C-reactive protein (CRP), IL-6 and retinol-binding protein 4 (RBP4) in placebo v. lignan-supplemented study groups†
(Mean values with their standard errors)
Lignan treatmentPlacebo treatment
P for between-treatment
Baseline 12 weeksBaseline 12 weeks
Mean 95% CI
All (n 64)
Male (n 25)
Female (n 39)
All (n 67)
Male (n 25)
Female (n 42)
All (n 70)
Male (n 26)
Female (n 44)
Urine lignans (mg/ml)
All (n 70)
Male (n 26)
Female (n 44)
Mean values were significantly different from those of the baseline: *P,0·05, **P,0·01, ***P,0·001.
†For details of subjects and procedures, see Methods.
‡P values are shown for the treatment effect analysed using a mixed model analysis of covariance.
Effects of lignan on inflammatory markers
British Journal of Nutrition
et al.(18)also found increased CRP levels over 3-month treat-
ments of soya (from 1·91 to 2·33mg/l) and placebo (from 1·39
to 1·87mg/l) in fifty healthy post-menopausal women, how-
ever no between-treatment difference was observed.
The present findings of moderate protective effects on CRP
are consistent with the results of five previous trials that either
tested isolated lignans(11)or isoflavones(12), flaxseed flour(19)
or soya protein(20,21). However, the present findings differ
from studies which tested isolated isoflavones(13–16)
whole flaxseed(22,23), which found no differences with respect
to CRP. It must be borne in mind that the difference that we
observed between placebo and lignan-treated groups in the
present study was primarily due to increased levels of CRP
following the placebo period, rather than a direct reduction
by lignan treatment. Therefore, given the exploratory nature
of the present study, it remains premature to draw the con-
clusion that lignan can actually lower CRP levels.
Furthermore, stratified analysis found that the significant
between-treatment difference of CRP concentrations was con-
fined to females. One potential explanation is the relatively
small sample of males. For detecting a between-treatment
difference of 0·20mg/l, 126 participants would be required
to have a power of 80%. The second possibility may be
that although not statistically significant, post-menopausal
women had moderately higher CRP levels than men, there-
fore, they were more likely to be responsive to phytoestrogens.
Previous studies suggest that the metabolism and excretion of
phytoestrogens differ between men and women(24); however,
it is currently unknown whether gender variation in the phy-
toestrogen bioavailability plays a role in CRP response.
The present findings of no effect on IL-6 are consistent with
data from another study in which forty-two post-menopausal
women with metabolic syndrome consumed soya protein or
soy nuts (30g/d containing 84–102mg isoflavones) for 8
weeks(21). In contrast, Jenkins et al.(25)found that IL-6
levels increased significantly after 1 month of a high soya
diet (containing 73mg isoflavones/d) compared to control
women. In the present study, the results of CRP and IL-6
did not follow the same pattern, which is also found in pre-
vious reports using lignan(11)or flaxseed(23). A possible expla-
nation for the present finding is that although IL-6 is the major
inducer of CRP in the liver, other pro-inflammatory factors
could also induce CRP production in other cell types(26).
RBP4 is a newly recognized adipokine associated with
obesity, insulin resistance and diabetes(4,5). A previous study
showed that RBP4 levels decreased in parallel to weight loss
and increased LDL-cholesterol catabolism after a 16-week
intervention of a hypoenergetic low-fat diet in men with meta-
bolic syndrome(27). In our previous study, we found no effects
of lignan supplementation on weight, lipid profiles and insulin
resistance(17). Therefore, it is possible that RBP4 levels were
not affected. Since whole or defatted flaxseed has consistently
shown to be effective in improving insulin resistance, and
lowering total and LDL-cholesterol(7,8), its potential impact
on RBP4 levels needs to be elucidated in future.
compared to placebo. However, further studies are needed to
validate the present results and explore the efficacy of phytoes-
trogens on inflammatory factors before consensus is achieved.
This study was funded by the Knowledge Innovation Program
Project of the Chinese Academy of Sciences (KSCX1-YW-02,
KSCX2-YW-R-116), the Science and Technology Commis-
sion of Shanghai Municipality (04DZ14007) and the Ministry
of Science and Technology of China (973 Program, Grant
2006CB503902). We thank Dr Marian Verbruggen of
Frutarom Netherlands BV and Mrs Guo Peilin of Jarrow For-
mulas Inc. for their kind donation of study capsules. A. P., W.
D.-W., Z. Y., H. L., J. S., Y. C., X. C., Y. L. and X. L. con-
tributed to the conception and design of the study. A. P., X.
Y., Z. Y. and Y. C. contributed to the data collection and man-
agement of the study subjects. A. P., X. Y., H. L. and Q. Q.
conducted the biomarkers measurement. A. P., X. Y. and
Z. Y. carried out the statistical analyses. A. P. wrote the first
draft of the manuscript with help from W. D.-W., X. Y.,
H. L., Z. Y. and X. L. All authors participated in the writing
of the final draft of the manuscript and the final interpretation
of the data. None of the authors had any conflicts of interest.
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