SUPPLEMENTARY MATERIAL
Protective effects of grape seed extract fractions with different
degrees of polymerisation on blood glucose, lipids, and hepatic
oxidative stress in diabetic rats
Zhaoxia WUa*, Siyao SHENa, Jiaqian JIANGb, Dehong TANa, Donghua
JIANGa, Bing BAIa, Xiyun SUNa and Shichen FUa
a
College of Food Science, Shenyang Agricultural University, Shenyang, Liaoning
Province 110866, China
b College of Animal Science & Veterinary Medicine, Shenyang Agricultural
University, Shenyang, Liaoning Province 110866, China
The purpose of this study was to evaluate the effect of grape seed procyanidin (GSPE)
fractions with different degrees of polymerisation (DP) on blood glucose, lipids, and hepatic
oxidative stress in diabetic rats. Diabetic rats received a daily oral supplement of GSPE with
different DPs for six weeks. During this period, blood glucose, body weight, and food intake
were assessed weekly. At the end of the experiment, serum lipid and hepatic oxidative stress
were assessed compared to rats that did not receive GSPE. GSPE significantly decreased
blood glucose, serum lipids, and hepatic oxidative stress. Moreover, these effects were
significantly better in the groups administered the oligomeric rather than the polymeric forms.
These results demonstrate that GSPE has a positive effect on diabetes in rats, and the
oligomeric form of GSPE may be more protective than other forms.
Keywords: procyanidins; antioxidation; diabetes; nitric oxide
*
Corresponding author. E-mail: wuzxsau@163.com
1. Experimental
1.1 Animal treatment
Sixty adult male and female Sprague-Dawley rats were obtained from Liaoning Changsheng
Biotechnology Co. LTD (Benxi, Liaoning Province, China), each weighing 180-220 g. The
animals were housed individually in stainless steel cages with wire mesh floors, and the
temperature was maintained at 22-24°C with 60-70% relative humidity on a 12-h light/dark
cycle. The rats had ad libitum access to food and tap water. For at least three days prior to the
experiment, all of the rats were fed the same basal diet, which consisted of common forage.
The rats were divided into two groups: a nondiabetic normal group (n = 10) fed common
forage and a high-energy group (n = 50) fed high-energy forage, which was prepared by
adding 20% sucrose (w/w), 18% lard (w/w), and 3% yolk to the basal diet. The rats were
grouped according to their body weight (BW) to ensure an average BW ranging from 202.6 g
to 204.8 g in each group. All high-energy diet rats were fed for four weeks and then
intraperitoneally injected with STZ (30 mg/kg BW) that was dissolved in sodium citrate
buffer (0.1 mol·L-1, pH 4.5). Of these animals, 31 with fasting blood glucose levels >11.1
mmol/L 72 h after the STZ injection were considered to be successfully modelled and
included in the experiment. They were divided into five groups: the diabetic model group
(DM, seven rats), metformin hydrochloride group (MH, six rats), oligomeric GSPE group
(DO, six rats; DP = 3.2), polymeric GSPE 1 group (PG1, six rats; DP = 6.5), and polymeric
GSPE 2 group (PG2, six rats; DP = 11.8). The grouping was again conducted in a manner to
ensure an average BW ranging from 336.2 g to 351.9 g in each group. For six weeks, the rats
in the MH group received a daily oral supplement of 200 mg·kg-1 metformin, while the rats in
the DO, PG1, and PG2 groups received 150 mg·kg-1 GSPE with DP = 3.2, 6.5, and 11.8,
respectively. The DM rats were considered the control group and orally received 150 mg·kg-1
tap water. All of the animals were fed common forage. During this six-week period, blood
glucose, body weight, and 24-h food intake were monitored. Blood glucose was measured by
the glucose oxidase method. At the end of the experiments, serum lipid and hepatic oxidative
stress were assessed.
1.2 Materials
The grape seed extract was provided by JF-NATURAL Company (Tianjin, China). The
fractions with different DP were obtained according to the protocol outlined in Figure S4,
Protocol for the fractionation of grape seed extract according to the degree of polymerisation.
The average DP of GSPE has been identified previously by spectrophotography (Yuelin et
al. 2009). The DP values of fraction (F)0 to F5 were 3.0, 3.2, 6.5, 9.4, 11.8, and 15.1,
respectively. According to our experimental design, as well as in consideration of the
production ratio of each fraction and the appropriate gradient of DP for each treatment group,
F1, F2, and F4 fractions were used for this experiment.
1.3 Blood glucose assay
In fasted animals, 400 mL·L-1 glucose solution (2 g·kg-1·BW) was administered by gavage,
and blood was sampled from the tail vein 0, 30, 60, and 120 min after gavage. The blood
glucose content was measured using a test kit (Nanjing JianCheng Bioengineering Institute,
Nanjing, China; Accu-chek Active Blood Glucose Monitoring Meter, Roche Diagnostic
Gmbh, Mannheim, Germany) based on the glucose oxidase method.
1.4 Serum lipid assay
The concentrations of TG, TC, LDL, and HDL were measured using an automatic
biochemical analyser (GS300, Shenzhen Genius Electronics Co., LTD, Shenzhen, China).
1.5 Hepatic Oxidative stress assay
Following the 6-week intervention, all of the rats received an intraperitoneal injection of 10%
chloral hydrate solution (0.3 mL·100g-1) 16 h after commencing to fast. Rats were then
sacrificed, and hepatic SOD, CAT, MDA, NO, GSH, GSH S-transferase (GSH-ST), and GSH
peroxidase (GSH-Px) levels were measured using a homologous kit (Nanjing JianCheng
Bioengineering Institute, China).
1.6 Statistical analysis
All data are expressed as mean ± SD. The differences between groups were analysed using a
one-way ANOVA and Tukey post hoc tests. P values < 0.05 were considered significant for
multiple comparison.
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Figures
Figure S1. Changes in fasting blood glucose over a 6-week period, compared between
control rats and diabetic model rats with varying levels of treatment, including grape seed
extract supplementation of differing degrees of polymerisation
Note: ND, nondiabetic normal group; DM, diabetic model group; DO, oligomeric
proanthocyanidin group (degree of polymerisation [DP] = 3.2); PG1, polymeric
proanthocyanidin group 1, (DP = 6.5); PG2, polymeric proanthocyanidin group 2 (DP =
11.8); MH, metformin hydrochloride group.
Figure S2. Changes in body weight over a 6-week period, compared between control rats
and diabetic model rats with varying levels of treatment, including grape seed extract
supplementation of differing degrees of polymerisation
Note: ND, nondiabetic normal group; DM, diabetic model group; DO, oligomeric
proanthocyanidin group (degree of polymerisation [DP] = 3.2); PG1, polymeric
proanthocyanidin group 1 (DP = 6.5); PG2, polymeric proanthocyanidin group 2 (DP = 11.8);
MH, metformin hydrochloride group.
Figure S3. Changes in 24-h food intake over a 6-week period, compared between control
rats and diabetic model rats with varying levels of treatment, including grape seed extract
supplementation of differing degrees of polymerisation
Note: ND, nondiabetic normal group; DM, diabetic model group; DO, oligomeric
proanthocyanidin group (degree of polymerisation [DP] = 3.2); PG1, polymeric
proanthocyanidin group 1 (DP = 6.5); PG2, polymeric proanthocyanidin group 2 (DP = 11.8);
MH, metformin hydrochloride group.
Figure S4: Protocol for the fractionation of grape seed extract according to the degree of
polymerisation.