SUPPLEMENTARY MATERIAL
Decreased glycation and structural protection properties of γglutamyl-S-allyl-cysteine peptide isolated from fresh garlic scales
(Allium sativum L.)
Dehong Tana, Yao Zhangb, Lulu Chena, Ling Liua, Xuan Zhanga, Zhaoxia
Wua, Bing Baia* and Shujuan Jia
a
College of Food Science, Shenyang Agricultural University, Shenyang, Liaoning
Province 110866, P.R.China
b
Shenyang Normal University, Shenyang, Liaoning Province 110034, P.R.China
Contact details of authors:
Dehong Tan: tandehongsy@126.com
Yao Zhang: sudongbaihe@163.com
Lulu Chen: 412246090@qq.com
Ling Liu: liuling4568@sina.com
Xuan Zhang: zhxllm@163.com
Zhaoxia Wu: wuzxsau@163.com
Bing Bai: baibing@hotmail.com
Shujuan Ji: jsjsyau@sina.com
This work was supported by the [National Natural Science Foundation of China]
under Grant [31101220]; [Key Projects in the National Science and Technology Pillar
Program of China] under Grant [2012BAD38B07].
Running headers of author names: D. Tan, Y. Zhang, L. Chen, L. Liu, X. Zhang, Z.
Wu, B. Bai, S. Ji
*Corresponding author. Email: baibing@hotmail.com
Decreased glycation and structural protection properties of γglutamyl-S-allyl-cysteine peptide isolated from fresh garlic scales
(Allium sativum L.)
The antiglycative effect of γ-glutamyl-S-allyl-cysteine (GSAC) peptide
isolated from fresh garlic scales was investigated in the bovine serum
albumin (BSA)/glucose system. GSAC inhibited the increase of fluorescence
intensity at about 440 nm in a concentration-dependent manner and reduced
reacted free lysine side chains by 10.9%, 24.7% and 37.7%, as the GSAC
concentrations increased from 0.1 to 2.5 mg ml-1. Glycation specific decline
in BSA  -helix content (from 61.3% to 55.6%) and increase in β-sheet (from
2.1% to 5.4%) were prevented by GSAC (2.5 mg ml-1) in vitro, implying its
stabilization effect. GSAC treatment (2.5 mg ml-1) suppressed protein
crosslinking to form polymers. Additionally, GSAC (10, 40, and 160 g ml-1)
showed radical-scavenging and metal-chelating capacities. In conclusion,
GSAC has an antiglycative effect, which may involve its radical-scavenging
and metal chelating capacities.
Keywords: Maillard reaction; advanced glycation end products; chelating;
radical-scavenging; organosulfur peptide
1. Experimental
1.1. Plant Material
The sample of Allium sativum L. was identified by Prof Baodong Wei of the College
of Food Science, Shenyang Agricultural University. Fresh garlic bulbs harvested in
May 2013 were obtained from a local market of Xinmin, Shenyang, Liaoning
Province, China. They were freeze dried and stored at –70°C until used for analysis.
A voucher specimen (GAR 5109) was deposited at the Key Laboratory of Fruits and
Vegetables of the Shenyang Agricultural University.
1.2. Isolation and identification of GSAC from fresh garlic scales
GSAC was isolated from fresh garlic scales according to the methods of Lawson et
al. (1991) and Kubec & Musah (2005) with slight modifications. Briefly, whole
garlic scales (1000 g) were carefully cleaned and incubated in ethyl alcohol (800 ml)
at 70 °C for 5 min. The pieces were homogenized using a blender and the slurries
obtained were filtered. The crude extracts were concentrated to about 300 ml by
vacuum evaporation (40 °C). The pH value of extracts was adjusted to 2.0 by
addition of 2 M HCl. The precipitate was filtered off and the filtrate was loaded on a
column (60 cm × 5.0 cm) of Dowex 50 × 4 - 400 cation-exchange resin (SigmaAldrich, Shanghai, China) saturated with H+. After washed the column with H2O
(1000 ml), the dipeptide fraction was eluted with 1000 ml of 1 M NH4OH. The
yellowish eluent obtained was concentrated to about 300 mL by vacuum evaporation
at 60 °C. The eluate was adjusted to pH 7.0 with glacial acetic acid and subjected to
a column (40 cm × 5.0 cm) of Dowex 2 × 8 - 200 anion-exchange resin (SigmaAldrich, Shanghai, China) saturated with OH+. The column was washed with 800 ml
of H2O and the dipeptide fraction was eluted with 10% acetic acid (800 ml). The
eluate fraction was lyophilized to a white powder which was further purified using a
preparative liquid chromatography (Hitachi Elite D-2000, Japan), equipped with a
C-18 prep column (250 mm × 9.4 mm i.d. 5 μm, Agilent, USA) and with
water/MeOH (2:3, v/v) as the mobile phase. The fraction eluting at 9.3 min was
repeatedly collected. From 1 kg of fresh garlic scales, 377 mg dry weight of GSAC
was obtained by freeze-drying as a white hygroscopic powder. The identity of
GSAC was confirmed by comparisons of HPLC-MS (1100LC/MSD Agilent, USA),
1
H NMR and 13C NMR (AVANCE III HD Spectrometer, Brucker, Germany) data
with published data (Mütsch-Eckner et al. 1992).
The powder obtained in the dipeptide-containing fraction was confirmed to
yield the expected [M-H] –of 288.8 (GASC, C11H18O5N2S, 290.0937) on HPLC-MS
(Figure S1) analysis. The 1H and 13C NMR spectra (Figure S2) of GASC was
confirmed as follows. 1H NMR (600 MHz, D2O), δ: 2.32 (1H, dd, J=7.2, 9.0 Hz,
CH2CH2CH-a), 2.43 (1H, dd, J=6.0, 8.4 Hz, CH2CH2CH-b), 2.66 (2H, s,
CH2CH2CH), 2.74~2.78 (H, m, SCH2CH-a), 2.82~2.85 (H, m, SCH2CH-b), 3.21
(2H, d, J=7.2 Hz, SCH2CH=CH2), 3.45~3.47 (1H, m, Glu-CH), 4.72~4.83 (1H, m,
Cys-CH), 5.17~5.22 (2H, m, SCH2CH=CH2), 5.82~5.89 (1H, m, SCH2CH=CH2).
13
C NMR (600 MHz; D2O),δ:27.8 (CH2CH2CH), 32.6 (CH2CH2CH), 36.4
(SCH2CH), 37.9 (SCH2CH=CH2), 57.7 (Glu-CH, Cys-CH), 120.2 (SCH2CH=CH2),
136.9 (SCH2CH=CH2), 182.5 (Glu-COOH), 183.2 (NHCOCH2), 184.4 (CysCOOH).
1.3. Preparation of the BSA/glucose reaction system
The reaction system was prepared as described previously (Peng et al. 2008). Briefly,
0.2 mg ml-1 BSA was incubated at 37 °C in a solution of 2 mg ml-1 D-glucose in 0.1 M
phosphate buffer (pH 7.4) in the absence (control) or presence of different
concentrations (0,1, 0,5, 2.5 mg ml-1) of GSAC, with added NaN3 (final concentration
0.2%). After 7 d, BSA-glucose solutions were filtered through low protein-binding
filters (0.22-μm filter unit, Merck-Millipore, Billerica, MA) and dialyzed against
autoclaved phosphate-buffered saline (PBS) at 4 °C to remove free glucose molecules.
BSA solution (0.2 mg ml-1) incubated under the same conditions was used as the
blank.
Protein concentrations were determined by the Pierce-BCA protein assay
(Thermo Scientific, China) calibrated with a BSA standard.
Optical density of glycation samples were recorded by measuring the absorbance
at wavelength of 190–600 nm in a PERSEE TU-1810 spectrophotometer.
1.4. Analysis of AGE formation
Using methods described by Wu et al. (2009), AGE-related autofluorescence of
relevant samples (0.2 mg ml-1) was analyzed after excitation at 370 nm and
monitoring the emission at 400–500 nm and at 360–380 nm using a
spectrofluorometer (Hitachi F-4600, Japan).
1.5. Determination of free lysine residues
Determination of free lysine residues was accomplished using the 2-4-6trinitrobenzene sulfonic acid (TNBSA) method (Sattarahmady et al. 2008). Briefly,
4% NaHCO3 and 0.1% TNBSA were added to the protein solution, reacted at 37°C
for 1 h, and solubilized with 10% sodium dodecyl sulfonate (SDS); 1 M HCl was
added to stop the reaction. Ultraviolet absorbance was then measured at 335 nm using
a spectrophotometer.
1.6. Congo Red assay
The Congo Red binding assay was performed according to methods described by
Miroliaei et al. (2011). In brief, 800 μl of protein solution (100 μM) was incubated
with 200 μl of Congo Red solution (100 μM Congo Red in 10% PBS-ethanol).
Absorbance was recorded at 530 nm with a spectrophotometer.
1.7. Circular dichroism (CD) spectropolarimetry
A quantitative analysis of the secondary structure elements upon glycation was
performed within far-UV CD spectra (190–240 nm) at room temperature on a
JASCO-810 spectropolarimeter (Japan) using solutions with a BSA concentration of
0.2 mg ml-1. Sample groups with the exception of native BSA were incubated in
Water-jacket constant temperature incubator at 37 °C for 7 d to glycation.
Results are expressed as molar ellipticity [Θ] (mdeg). The molar ellipticity was
determined as [Θ] = mdeg/(cl), where l, is the light path length in millimeter, c, is the
protein concentration in mM, and Θ is the measured ellipticity in degrees at the
relevant wavelength. The relative percentages of the secondary structure elements
were estimated using SELCON3 software.
1.8. Gel electrophoresis assay
Protein cross-linking was determined by SDS polyacrylamide gel electrophoresis
(SDS-PAGE, 10% polyacrylamide gels) (Ahmad et al. 2007). Briefly, protein samples
(native BSA, glycated BSA control, and glycated BSA in the 10 mg/ml GSAC
treatment group) were diluted with loading buffer (containing 0.1% 2mercaptoethanol) and boiled for 5 min. Samples (containing 10 μg protein) were
loaded into the wells and subjected to electrophoresis (Bio-Rad PowerPace Basic,
Hercules, USA). The gels were removed and stained in 2.5 g kg-1 Coomassie Blue R
solution (50% methanol and 10% acetic acid) for 0.5 h and then destained in a
solution containing 25% methanol and 7% acetic acid for more than 3 h.
1.9. Evaluation of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical–scavenging
capacity
The DPPH radical-scavenging capacity was assessed using the microplate method
(Ho et al. 2010). Briefly, different concentrations (10, 40, and 160 g ml-1) of GSAC
(20 l) were reacted with 180 l of 0.2 mM DPPH methanolic solution in a 96-well
microplate for 5 min. Absorbance at 520 nm was then measured using a microtiter
plate reader.
1.10. Evaluation of metal-chelating capacity
Metal-chelating capacity was determined according to methods described by Ho et al.
(2010). Different concentrations (10, 40, and 160 g ml-1) of GSAC were reacted with
40 M ferrous chloride and 200 M ferrozine at ambient temperature for 10 min.
Absorbance at 540 nm was then determined using a microtiter plate reader.
1.11. Statistical analysis
The experiment was performed in 3 independent replicates. Data were analyzed using
the statistical analysis system software package SPSS 16.0. Analyses of variance were
performed using ANOVA. Significant differences between means were determined
using Duncan’s multiple range tests.
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Figure S1. HPLC-MS-MS of GSAC. a: MS of GSAC, b: MS2 of GSAC with
precursor ion of 288.7. isolated from fresh garlic scales.
Figure S2. 1H NMR (a) and 13C NMR (b) spectra of GSAC.
1
2
Figure S1.
Figure S2.