Industrial Crops and Products 101 (2017) 104–114

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Industrial Crops and Products

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Antioxidant/antihyperglycemic activity of phenolics from sugarcane

(Saccharum officinarum L.) bagasse and identification by
UHPLC-HR-TOFMS
Rui Zheng a,1 , Shan Su b,c,1 , Huifang Zhou a , Huaifeng Yan a , Junhua Ye a ,
Zhengang Zhao a,d,∗ , Lijun You a , Xiong Fu a,∗
a
School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China
b
Department of Oncology, Guangzhou Chest Hospital, Guangzhou 510095, China
c
Southern Medical University, Guangzhou 510515, China
d
Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, South China University of Technology, Guangzhou
510640, China

a r t i c l e i n f o a b s t r a c t

Article history: Sugarcane bagasse, one of the most abundant agro-food by-products, is a very promising raw material
Received 29 September 2016 available at low cost for recovering bioactive substances. In this study, the total phenolic content was mea-
Received in revised form 7 January 2017 sured by Folin-Ciocalteu method and the antioxidant activity of the extracts was determined by DPPH• ,
Accepted 10 March 2017
ABTS• + and ROO• (ORAC) methods. The results indicated that 30% hydroalcoholic fraction (30%E) had the
highest total phenolic content (170.68 ± 3.25 mg GAE/g DW) among the hydroalcoholic extracts and was
Keywords:
the most active fraction in antioxidant activity. Besides, intestinal ␣-glucosidase inhibitory activity was
Sugarcane bagasse
conducted to assess the antihyperglycemic ability of the extracts and 30%E showed a significant effect at
Phenolics
Antioxidant
10 mg/mL (82.64% inhibition of sucrase and 74.26% inhibition of maltase, respectively). Glucose uptake
Antidiabetes assay based on HepG2 cells was used to evaluate the glucose uptake effect of 30%E. Five phenolic com-
UHPLC-HR-TOFMS pounds (tricin 4-O-guaiacylglyceryl ether-7-O-glucopyranoside, genistin, p-coumaric acid, quercetin and
genistein) were identified in 30%E using UHPLC-HR-TOFMS. Taken together, the data indicated that sug-
arcane bagasse can be used as a potential source of bio-active phenolics and may play a role in functional
agents exploitation and agro-industrial revenue increase.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction Zhang et al., 2012). T2D is featured by postprandial hyperglycemia

Diabetes mellitus (DM) is a multifactorial endocrine disorder
involving disturbance of carbohydrate, protein and fat metabolism
(De Souza Schmidt Gonçalves et al., 2010). This disease has reached
epidemic level globally and approximately 346 million people
worldwide suffer from it and this figure is estimated to double
by the year 2030 (Lavelli et al., 2016). All of diagnosed cases of
diabetes, type 2 diabetes (T2D) occupied about 90–95%, affecting
over 170 million people throughout the world (Shen et al., 2012;
Abbreviations: ROS, reactive oxygen species; 2-NBDG, 2-(N-(7-Nitrobenz-2-oxa-
1,3-diazol-4-yl) amino)-2-deoxyglucose; AKT/PKB, protein kinase B; AMPK, 5 -AMP-
activated protein kinase; GLUT-2, glucose transporter-2.
∗ Corresponding author at: No. 381 Wushan Road, South China University of
Technology, Guangzhou, China.
E-mail addresses: fezzg@scut.edu.cn (Z. Zhao), lfxfu@scut.edu.cn (X. Fu).
1
These authors contributed equally to this work and share first authorship.
due to resistance of cells (hepatocyte and myocyte) to the func-
tion of insulin. Long-term exposure to hyperglycemic condition
is estimated to generate reactive oxygen species (ROS) continu-
ously (Mohan et al., 2013). That could alter the enzymatic activities
and decrease in vivo antioxidant levels, reportedly resulting in dia-
betes (Kang et al., 2012). Thus, plant-based products which are rich
in antioxidants including a multitude of bioactive molecules with
diverse structures may play a key role in the treatment of diabetes.
Sugarcane (Saccharum officinarum L.) is widely cultivated
around the world and has been considered as one of the most sig-
nificant economic plants (Del Río et al., 2015). Sugarcane is well
abundant in phytochemicals such as phenolics, triterpenoids, phy-
tosterols (Feng et al., 2014) and lignins (Pinheiro et al., 2017).
Previous work has assessed the antioxidant (Duarte-Almeida et al.,
2011), the antibiotic (Zhao et al., 2015), the antiproliferative
(Duarte-Almeida et al., 2007), the DNA-damage-protecting (Abbas
et al., 2014), the anti-mutation Wang et al. (2011a) activity of

http://dx.doi.org/10.1016/j.indcrop.2017.03.012
0926-6690/© 2017 Elsevier B.V. All rights reserved.
 R. Zheng et al. / Industrial Crops and Products 101 (2017) 104–114
sugarcane or its derived products, all of which indicated positive
results. Over 1.9 × 109 t of sugarcane are harvested annually and
0.28 × 109 t of bagasse are produced after the crushing and extrac-
tion of the juice from the sugarcane (Pinheiro et al., 2017). In most
cases bagasse is often used as primary fuel source to power the
sugar mill (Sun et al., 2004). Several attempts have been conducted
using bagasse as raw material including electricity generation, pulp
and paper production and fermented products (Pandey et al., 2000).
However, as a kind of natural products, the health-care values
of bagasse should not be ignored. Thus, exploiting the sugarcane
bagasse resources used for the promotion of human wellness is
needed. In our current work, the phenolic compounds in sugar-
cane bagasse were identified by UHPLC-HR-TOFMS method and the
antioxidant/antihyperglycemic activity of phenolic extracts from
sugarcane bagasse was also evaluated.
2. Material and methods
2.1. Chemicals and plant material
1,1-Diphenyl-2-picrylhydrazylradical (DPPH), 2,2 -azinobis-
3-ethylbenzothiazoline-6-sulfonic acid (ABTS), Folin-Ciocalteu
regent, standards of genistin, p-coumaric acid, quercetin, genistein,
Trolox, fluorescein sodium salt, 2,2-azobis(2-amidinopropane)
dihydrochloride (AAPH), rat intestinal acetone powder, 2-(N-
(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxy-D-glucose
(2-NBDG) and insulin were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Glucose C-II Test Wako kit was purchased from
Wako Pure Chemical Industries (Osaka, Japan). Acarbose and
rosiglitazone were purchased from Aladdin (Shanghai, China).
Methanol and acetic acid used for UHPLC analysis were purchased
from CNW Technologies (Dusseldorf, Germany). High glucose
Dulbecco’s modified Eagle’s medium (H-DMEM) was purchased
from Hyclone (Logan, USA) and fetal bovine serum (FBS) was pur-
chased from Sijiqing Biological Engineering Materials (Hangzhou,
China). Dimethyl sulfoxide (DMSO) was purchased from Kanglong
Biotechnology Co. (Guangzhou, China). Hank’s balanced salt solu-
tion (HBSS) and glutaraldehyde were purchased from Gibco U.S.
Biotechnology Co. (Carlsbad, CA, USA). All the other reagents used
were of analytical grade.
Sugarcane bagasse was obtained from a sugar mill belonging
to the Donta Group (Dongguan, China). Before use, bagasse was
washed with distilled water to remove foreign particles from the
surface. The sugarcane bagasse was dried at 40 ◦ C for 2 days, milled
and filtered through a 40-mesh (pore size is 0.42 mm) sieve and
stored in an air-tight container at – 20 ◦ C until analysis.
2.2. Phytochemical extraction and isolation
In brief, 10 g dried sugarcane bagasse powders were extracted
with 150 mL 60% ethanol/water (v/v) at 60 ◦ C with the assistance of
ultrasonic (40 KHz) in an ultrasonator (Kunshan Ultrasonic Instru-
ment Co., Jiangsu, China) for 90 min. The process of extraction was
repeated 3 times and the solutions pooled. After vacuum filtration,
the filtrate was combined, dried under vacuum at 42 ◦ C and was
stored at – 40 ◦ C. Before the use, the primary ethanolic extract (EF)
was suspended in distilled water. The resulting solution was parti-
tioned with various solvents (3:1, v/v) for 2 h and yielded petroleum
ether fraction (PEF), ethyl acetate fraction (EAF), n-butanol frac-
tion (BF) and aqueous fraction (AqF). All of the processes were
repeated three times. The extracts were concentrated to dryness
under reduced pressure.
EAF was further applied to a polyamide resin column for the
next purification and yield three sub-fractions (30%E, 50%E, 70%E).
The column was loaded with polyamide resin (100–150 mesh). The
105
eluant was collected and combined after thin layer chromatogra-
phy (TLC) analysis. The concentration of extracts (mg/mL) applied
in this work was obtained by completely dissolving the dry extracts
(mg) into the solvents or buffers (mL).
2.3. Determination of total phenolic content
The total phenolic content of each sample was determined using
the Folin-Ciocalteu method (Wang et al., 2017). Gallic acid (GA)
was used as standard. Briefly, samples or GA was diluted appro-
priately in test tubes with the addition of Folin-Ciocalteu regent
(100 ␮L). After mixed, the mixtures were allowed to stand for 6 min
and then reacted with 1 mL of 7% sodium carbonate (w/w) for
90 min in the dark. The absorbance was measured at 760 nm using
DU 730 Nucleic Acid/Protein Analyzer (Beckman, CA, USA). Results
were expressed as mg GA equivalents (GAE)/g (DW of bagasse or
extracts). The total phenolic content of the original extracted sugar-
cane bagasse was calculated by the addition with the free phenolic
content (EF) and the bound phenolic content. All determinations
were performed in triplicate.
2.4. Ultra-high performance liquid
chromatography/high-resolution time of flight mass spectrometry
(UHPLC-HR-TOFMS) analysis
Briefly, 30%E was dissolved in methanol (1 mg/mL), after cen-
trifugation at 12,000g, the supernatant was filtered through a
0.45 ␮m syringe filter (PVDF-D) and 0.5 ␮L was injected into a
UHPLC-HR-TOFMS system. The Agilent 1290 Infinity UHPLC sys-
tem (Agilent Technologies, Palo Alto, CA, USA) was equipped with
an Infinity binary pump (G4220A), a ZORBAX RRHD SB-C18 column
(2.1 × 50 mm, 1.8 ␮m of particle size), an Infinity 1290 autosampler
(G4226A), and an Infinity DAD (UV diode-array detector, G4212A).
The phenolic compounds were eluded using a mobile phase with
0.05% v/v aqueous acetic acid Milli-Q water solution (phase A) and
methanol (phase B). The gradient in ratios (v/v) of solvents A and B
were as follows: 0–1 min, 30% B; 1–12 min, 30% – 70% B; 12–13 min,
70–100% B; 13–15 min, 100% B. The column temperature was kept
at 30 ◦ C, with a flow rate of 0.2 mL/min. The UV absorbance was set
at 280 nm. The UHPLC system was connected to a high resolution-
time of flight (HR-TOF) mass spectrometer (MaXis Impact, Bruker,
Germany) fitted with an electrospray interface (ESI) source per-
formed in positive ion mode. The detection was carried out with
ion scanning from 50 to 1500 m/z, temperature at 180 ◦ C, ioniza-
tion voltage at 2.0 kV, nebulizer pressure at 1.0 bar, nitrogen as the
dry gas with flow rate at 4.0 L/min. The compounds presented in
30%E were identified by comparing retention time with authentic
standards as well as their molecular ions (m/z) with data from the
literature.
The peak area of individual compounds and the calibration
curves of the corresponding standards were used for identifying
and calculating the content of compounds in 30%E. The detailed
information of standards was shown in Table 2A.
2.5. Evaluation of antioxidant activity using the DPPH• method
The DPPH radical-scavenging activity of each fraction was deter-
mined using the method reported by Wang et al. (2011b) with
slight modifications. Briefly, DPPH was dissolved in 100 mL of 100%
ethanol to make the DPPH stock solution with the final concentra-
tion of 2 mM. Then, 0.1 mL of ethanol-diluted extract was placed
into a test tube (10 mL) with 3 mL of 0.2 mM DPPH radical solu-
tion. After vortex, the sample was kept in dark for 30 min and the
absorbance of the mixture was measured by a spectrophotometer
(Beckman DU 730, USA) at 517 nm. The ethanol with DPPH solution
was used as a control while ethanol was regarded as a blank. The
106 R. Zheng et al. / Industrial Crops and Products 101 (2017) 104–114
scavenging activity of DPPH radical of each fraction was calculated
according to the following equation:
Free radical scavenging activity of DPPH• (%)
= [(A0b − A1b )/A0b ] × 100%
where A0b is the absorbance of control minus absorbance of blank
and A1b is the absorbance of sample solution minus absorbance
of blank. All determinations were performed in triplicate. Half
inhibitory concentration was defined as IC50 and the 1/IC50 values
of samples were applied to Pearson correlation test.
2.6. Evaluation of antioxidant activity using the ABTS•+ method
The ABTS radical cation (ABTS•+ ) scavenging activity of
extracted fractions was determined by a previous method reported
by Re et al. (1999). Briefly, all the samples were diluted with ethanol
and ABTS was dissolved in distilled water at a final concentration
of 7 mM. ABTS•+ was generated by adding ABTS stock solution to
2.45 mM potassium persulfate and the mixture was placed in the
dark at 25 ◦ C for 16 h. In order to study infusion, the final solu-
tion containing ABTS•+ was diluted with ethanol to an absorbance
of 0.7 (±0.02) at 734 nm, followed by the taking of reagent blank
reading. After addition of 3.0 mL of ABTS•+ dilution to 20 ␮L of sam-
ple, the reacting solution was allowed to stand in the dark at 25 ◦ C
for 30 min. The scavenging activity of ABTS•+ of each sample was
calculated according to the following equation:
Free radical scavenging activity of ABTS•+ (%)
= [(Acb − Asb )/Acb ] × 100%
where Acb is the absorbance of control minus absorbance of blank
and Asb is the absorbance in presence of sample solution minus
absorbance of blank. All determinations were performed in trip-
licate. The 1/IC50 values of samples were applied to Pearson
correlation test.
2.7. Evaluation of antioxidant activity using the oxygen radical
(ROO• ) absorbance capacity (ORAC) method
ORAC was determined by the method previously reported (Cao
et al., 1993) with some modification. Briefly, the samples were dis-
solved in ethanol and then diluted with KH2 PO4 –K2 HPO4 (75 mM;
1: 4, v/v) buffer (PBS, pH 7.4). Fluorescein sodium salt and AAPH
were well-prepared before use. To build the Trolox standard decay
curve, Trolox was weighed and dissolved in PBS, giving a concen-
tration of 10 mM. The final solution was kept at −80 ◦ C. Before use,
the final Trolox solution was diluted with PBS to a series of con-
centrations (6.25, 12.5, 25, 50, 100 ␮M). Extract dilution (20 ␮L)
was blended with 200 ␮L of fluorescein sodium salt (0.96 ␮M) in a
black 96-well microplate (Corning Scientific, USA) and shaken for
10 s. The Trolox decay and blank decay curves were established
by using 20 ␮L of Trolox or ethanol instead of sample. Afterwards,
the mixture was incubated at 37 ◦ C for 20 min followed by the
addition of 20 ␮L of AAPH (119 mM) to each well. The microplate
was immediately placed into a multimode microplate reader (Filter
Max F5, Molecular Devices, USA) with an excitation wavelength of
485 nm and an emission wavelength of 535 nm. The fluorescence
was measured every 4.5 min for 35 cycles. All the ORAC values
were calculated using a regression equation between the Trolox
concentration and the net area under the fluorescence decay curve
(AUC). The net AUC corresponding to each sample was calculated
by subtracting the AUC value from that of the blank. The results
were expressed as the scavenging activity against ROO• , micromo-
lar Trolox equivalents per gram sample (ORAC value, ␮mol TE/g,
DW of extract). All determinations were performed in triplicate.
2.8. Measurement of intestinal ˛-glucosidase inhibitory activity
The inhibitory activity against ␣-glucosidase was determined
by using the glucose oxidase method (Mutarotase-GOD method)
(1) by Glucose C-II Test Wako kit. The enzyme solution was prepared
by blending 1 g of rat intestinal acetone powder with saline solution
(0.9% NaCl) and extracted with ultrasound for 1 min, 3 times and
then centrifuged at 2095g for 30 min. The supernatant was diluted
to one-fourth of the original concentration with distilled water, and
the dilution was stored as the sucrase or maltase solution. All the
samples were diluted with 100 mM PBS (pH 6.9) to a series of con-
centrations. Maltose and sucrose were used as substrates of maltase
and sucrase, respectively. Sucrose or maltose was diluted with
100 mM PBS and the final concentration was 56 mM and 3.5 mM,
respectively. Fifty microliter of each extract (in DMSO) was mixed
with 0.4 mL sucrose solution or 0.2 mL maltose solution in a 10 mL
colorimetric tube and the mixture was incubated at 37 ◦ C for 5 min.
After the preincubation, the sucrase or maltase solution was added
and the reaction mixture was incubated for another 20 min at the
temperature of 37 ◦ C. The reaction was terminated with enzyme
inactivation by placing the mixture in boiling water for 5 min. After
cooling, the released D-glucose was measured colorimetrically by
using the Mutarotase-GOD method. The absorbance of each sam-
ple was measured at 505 nm. Values were expressed as a percent
of enzyme inhibition (%). The percentage inhibition of sucrase or
maltase by each sample was calculated according to the following
equation:
Inhibitory activities against sucrase or maltase (%)
(2) = [1 − (A1 − A0 )/(A3 − A2 )] × 100% (3)
where A0 is the absorbance of blank control, A1 is the absorbance of
blank, A2 is the absorbance of sample control, A3 is the absorbance
of sample test. All determinations were performed in triplicate. The
1/IC50 values of samples were applied to Pearson correlation test.
2.9. Glucose uptake assay on HepG2 cells
2.9.1. Cell cultures
Human hepatoma HepG2 cells were generously provided by
Jinan Biomedicine Research and Development Center (Guangzhou,
China). HepG2 cells were maintained in H-DMEM plus 10% heat-
inactivated FBS in a humidified atmosphere with 5% CO2 at 37 ◦ C.
2.9.2. Cell viability assay
The cell viability test was determined using the method reported
by Wen et al. (2015) with some modifications. Briefly, 30%E was dis-
solved in DMSO followed by dilution to various concentrations with
H-DMEM and the final concentration of DMSO in growth medium
was 0.5% (v/v). HepG2 cells were seeded at a density of 4.0 × 104
cells per well on a 96-well microplate in H-DMEM growth medium.
After incubation at 37 ◦ C for 24 h, all the medium was removed
and the cells were washed with 100 ␮L of PBS (pH 7.4), followed
by treatment with 100 ␮L of growth medium containing the sam-
ple for another 24 h. Growth medium containing 0.5%v/v DMSO
without samples served as control. Then the growth medium was
removed and all the cells were washed with 100 ␮L of PBS. Then
50 ␮L of methylene blue (MB) staining solution (98% HBSS with
1.25% glutaraldehyde and 0.6% MB) was added immediately. After
incubation for 1 h at 37 ◦ C, MB was removed and the microplate was
washed three times using deionized water. After washing, 100 ␮L of
elution buffer (49% PBS, 50% ethanol, and 1% acetic acid) was added
and the microplate was placed on a benchtop shaker for 15–20 min.
The absorbance was monitored at 570 nm by a microplate reader
(SpectraMax 190; Molecular Devices, USA). The cell viability of
 R. Zheng et al. / Industrial Crops and Products 101 (2017) 104–114 107

30%E on HepG2 cells was calculated according to the following

equation:

Cell viability (%) = (Asb /Acb ) × 100% (4)

where Asb is the absorbance of the well with presence of the tested
sample minus the blank and Acb is the absorbance of control wells
minus the blank. All determinations were performed in triplicate.

2.9.3. Glucose uptake assay

Glucose uptake in HepG2 cells was conducted as previously
described with some modifications (Manaharan et al., 2013; Zhang
et al., 2012). Briefly, HepG2 cells were plated into a black 96-well
microplate at a density of 1.5 × 104 cells per well in H-DMEM
growth medium plus 10% heat-inactivated FBS. After 24 h (cells
reached 80%–90% confluence), the medium was replaced by H-
DMEM growth medium (serum free) containing 1 ␮M insulin and
50 ␮M of the fluorescent glucose analogue 2-NBDG with various
concentrations of 30%E (0.01, 0.05, 0.1 mg/mL), or rosiglitazone
(10 ␮M, used as a positive reference) or acarbose (10 ␮M, used as a
␣-glucosidase positive reference). The cells treated with H-DMEM
growth medium (serum free) with 1 ␮M of insulin and 50 ␮M of
2-NBDG served as a control. The final concentration of DMSO in
H-DMEM with all treatments was 0.5% (v/v). After 24 h, the treat-
ment was stopped by removing the growth medium and washing
the wells three times with cold PBS (4 ◦ C). Fluorescence of the cells
was measured at an excitation wavelength of 485 nm and an emis-
sion wavelength of 535 nm by a microplate reader (Filter Max F5
multi-mode microplate reader; Molecular Devices, USA).

2.10. Statistical analysis

All the data were expressed as the mean ± standard deviation

(SD). Statistical analysis was conducted by using Sigma Stat (ver-
sion 3.5, Systat software Inc, USA). Two-tailed t-test and Duncan’s
multiple range test were performed to determine the differences of
means for two groups and multiple groups at the level of p < 0.05, Fig. 1. Scheme for the preparation and isolation of phenolics from sugarcane
respectively. Pearson correlation test was performed by using SPSS bagasse and identification of 30% hydroalcoholic fraction (30%E).
version 21 software (SPSS Inc., Chicago, IL, USA) to determine the
correlation among variables and p < 0.05 (2-tailed) was considered Table 1
as statistically significant. Total phenolic contents of sugarcane bagasse extracts (expressed as mg gallic acid
equivalent/g dry sugarcane bagasse or extracts).

3. Results and discussion Fractions Total phenolic

content (mg
GAE/g, DW)
The scheme for the preparation and purification of sugarcane
bagasse is shown in Fig. 1. The extract conditions were optimized Sugarcane bagasse 7.83 ± 0.24* ,f
Free phenolics (primary ethanolic fraction) 7.35 ± 0.25* ,f
by response surface methodology (RSM) and involved data are
Bound phenolics 0.48 ± 0.01* ,h
shown in the supplementary material. Because EAF showed higher Petroleum ether fraction 7.95 ± 0.65# ,f
total phenolic content (241.42 mg GAE/g) and stronger antioxidant Ethyl acetate fraction 241.42 ± 1.91# ,a
activity (10860 ␮mol TE/g in ORAC assay), EAF was further applied n-Butanol fraction 16.77 ± 0.51# ,e
to a polyamide resin column and yielded three sub-fractions (30%E, Aqueous fraction 3.65 ± 0.11# ,g
30% Hydroalcoholic extract 170.68 ± 3.25# ,b
50%E, 70%E). 50% Hydroalcoholic extract 94.06 ± 1.66# ,c
70% Hydroalcoholic extract 73.60 ± 0.67# ,d
3.1. Total phenolic content The total phenolic content of the original extracted sugarcane bagasse was calcu-
lated by the addition with free phenolic content (EF) and the bound phenolic content.
The total phenolic content of each extract is shown in Table 1. Values with different letters have a significant difference at p < 0.05.
*
As can be seen, the total phenolic content of sugarcane bagasse Value was calculated based on dry weight of the original extracted sample.
#
Values were calculated based on dry weight of extracts.
was 7.83 mg GAE/g DW of bagasse, including the free phenolic con-
tent (EF, 7.35 mg GAE/g) and the bound phenolic content (0.48 mg
GAE/g). The highest phenolic content was found in EAF (241.42 mg also protect plants from pathogens, parasites, and predators (Wang
GAE/g), followed by BF (16.77 mg GAE/g), PEF (7.95 mg GAE/g) et al., 2017). Phenolic compounds indicate various biological activi-
and AqF (3.65 mg GAE/g). The phenolic content of hydroalcoholic ties, such as anticancer (Wen et al., 2015), antidiabetes (Zhang et al.,
extracts followed the order: 70%E <50%E <30%E, ranging from 73.60 2012) and photoprotection (Chaiprasongsuk et al., 2016), which are
to 170.68 mg GAE/g. closely associated with human wellness. The total phenolic con-
Phenolics are the secondary metabolites of plants which are not tent of sugarcane bagasse was 7.83 mg GAE/g, much higher than
only responsible for the coloring shades in fruits and vegetables but that reported previously (Zhao et al., 2015). In our work, the RSM
108 R. Zheng et al. / Industrial Crops and Products 101 (2017) 104–114

Table 2A
The information about standards used in UHPLC-HR-TOFMS.

Compound PubChem ID Molecular formula Molecular weight (g/mol) Spectral max. (nm)

genistin 5281377 C21 H20 O10 432 258, 326a

p-coumaric acid 637542 C9 H8 O3 164 230, 310b
quercetin 5280343 C15 H10 O7 302 255, 374c
genistein 5280961 C15 H10 O5 270 205, 258a
a
Łuczkiewicz et al. (2004).
b
Mateos et al. (2001).
c
Ang et al. (2015).

was applied to determine the optimal condition of extraction and it
was found that 60% ethanol/water (v/v) solution at 60 ◦ C was more
efficient than 70% ethanol/water (v/v) solution at 4 ◦ C reported in
(Zhao et al., 2015). More importantly, the methodology with ultra-
sonic was a major contributor to the higher content of phenolics
in our work. A previous research indicated that various kinds of
phenolic acids exist in the cell walls of sugarcane bagasse, such
as ferulic and p-coumaric acid (Xu et al., 2005) and the ultrasonic
enhancement should be ascribed to the acoustic effects of cavita-
tion in the solvent, causing disruption of bagasse cell walls which
facilitated the release of phenolics into the solvent (Ma et al., 2008).
3.2. Phytochemical characterization of 30%E by
UHPLC-HR-TOFMS analysis
The 30%E fraction was injected into the UHPLC system and then
the separation of compounds was performed by the column chro-
matography (C18 column). The elution produced by UHPLC was
then applied to the HR-TOFMS system for further analysis. The
MS/MS data and content of individual compound are shown in
Table 2B. Fraction 1 showed a molecular cation at m/z = 639.1944.
Then a loss of 162 u (a hexose unit) resulted in the fragment of
m/z 477.1401. Another ion fragment (m/z = 447.1303) was observed
after the cleavage of a methoxy group (30 u) from m/z 477.1401,
which was proposed to be the aglycon. However, the literature
reporting this compound in detail was insufficient. Thus compound
1 was tentatively identified as an unknown glucoside. Fraction 2
indicated a protonated molecule at m/z 689.2106 and a major frag-
ment ion was observed at m/z 331.1555, which was attributed to
the protonated aglycone tricin ([M+H]+ , 331). In addition, the m/z
331.0828 fragment was also observed in the MS/MS spectrum. The
MS and MS/MS data in our research were identical to that reported
in previous work (Colombo et al., 2005). Hence, Fraction 2 was
tentatively identified as tricin 4-O-(threo or erythro guaiacylglyc-
eryl) ether-7-O-glucopyranoside. More data would be required to
distinguish the threo and erythro forms. Fraction 3 indicated pre-
cursor ions at 433.1512 ([M+H]+ ) as well as a fragment ion at
m/z 419.0992 ([M+Na−2H2 O]+ ). Whilst, a MS/MS fragment ion m/z
313.1107 ([M+H−120]+ ) was viewed. The spectrum data in our
work was similar to that reported in previous research (Otieno et al.,
2007). Consequently, Fraction 3 was identified as genistin. Fraction
4 showed a base peak at m/z 147.0450 ([M−H2 O+H]+ ), a quasi-
molecular ion at m/z 186.9572 ([M+Na]+ ) and a molecular ion at
m/z 163.9413 ([M]+ ); meanwhile, fragment ions with m/z 119.0492
and 91.0548 were observed in MS/MS analysis. Comparing with
data in the literature, Fraction 4 was identified as p-coumaric acid
(Chiang et al., 2003). The mass spectrum of Fraction 5 showed a base
peak at m/z 302.3072 ([M]+ ) and a quasi-molecular ion occurred at
m/z 303.3104 ([M+H]+ ). The mass data of Fraction 5 was consis-
tent with data reported by (Cremin and Zeng, 2002). Thus, Fraction
5 was determined as quercetin. The mass spectrum of Fraction 6
showed a base peak at m/z 270.2803 ([M]+ ) and a quasi-molecular
ion occurred at m/z 271.2835 ([M+H]+ ). The mass data of Frac-
tion 6 was consistent with data reported by (Cremin and Zeng,
2002). Thus, Fraction 6 was determined as genistein. The calibration
curves of the corresponding standards were used for calculating
the content of compounds. As can been seen in Table 2B, genis-
tein was one of predominant compounds in 30%E (15.22 mg/g, DW

Table 2B
Phenolic compounds detected in the most active hydroalcoholic fraction (30% hydroalcoholic extract) by UHPLC-HR-TOFMS.

Fraction Retention time Molecular and Phenolic Identity Content (mg/g

(min) product ionsa compound conformation DW of 30%E)

1 0.97 639.1944 (30.2%), unknown glucoside none –b

477.1401 (97.6%)
447.1303 (100%)
2 1.78 689.2106 (100%), T4G7G Colombo et al. (2005) –
331.1555 (59.8%)
331.0828 (100%,
MS/MS)
3 2.51 433.1512 (100%), genistin . trace
419.0992 (50.3%), standard and
313.1107 (42%, (Otieno et al., 2007).
MS/MS)
4 3.10 186.9572 (71.7%), p-coumaric acid standard and (Chiang et al., 2003) 12.62 ± 0.34
163.9413 (36.7%),
147.0450 (100%),
119.0492 (25.6%,
MS/MS),
91.0548 (100%,
MS/MS)
5 8.15 303.3104 (19%), 302.3072 (100%) quercetin standard and (Cremin and Zeng, 2002) 9.98 ± 0.40
6 8.45 271.2835 (24.3%), genistein standard and (Cremin and Zeng, 2002) 15.22 ± 1.28
270.2803 (100%)

T4G7G, tricin 4-O-(threo or erythro guaiacylglyceryl) ether-7-O-glucopyranoside.

a
Data obtained in MS or MS/MS analysis.
b
Standard was not available.
 R. Zheng et al. / Industrial Crops and Products 101 (2017) 104–114
Fig. 2. Evaluation of antioxidant capacities of the primary ethanolic fraction (EF) using DPPH• (A), ABTS• + (B) and ROO• (ORAC) (C) methods. Values represent the mean ± SD
(n = 3). Values with different letters have a significant difference at p < 0.05 at the same concentration. The concentration of extracts (mg/mL) applied in this work was obtained
by completely dissolving the dry extracts (mg) into the solvents or buffers (mL).
of 30%E, R2 = 0.999) and followed by p-coumaric acid (12.62 mg/g,
DW of 30%E, R2 = 0.999) and quercetin (9.98 mg/g, DW of 30%E,
R2 = 0.9993). Phenolics, especially phenolic acids are abundant in
sugarcane. Gallic, ferulic and p-coumaric acids are the major phe-
nolic acids in sugarcane bagasse (Zhao et al., 2015). In sugarcane
juice, ten phenolic acids were identified by Zhao et al. (2008) and
swertisin, isoorientin-7, 3 -dimethyl ether and tricin-7-O-glycoside
were reportedly rich (Zhu et al., 2010). The predominant pheno-
lics in culms were caffeic, chlorogenic and coumaric acid while the
content of flavones (apigenin, tricin and luteolin derivatives) was
in lower amounts (Duarte-Almeida et al., 2011).
3.3. Antioxidant activities
For a comprehensive predication, it is generally recommended
to use more than one method to investigate the antioxidant capac-
ities of plants and their compounds (Moon and Shibamoto, 2009).
Hence, DPPH• assay, ABTS•+ assay and ORAC (the scavenging activ-
ity against ROO• ) assay were performed to evaluate the antioxidant
activities of the fractions in this research.
As shown in Fig. 2A and B, the primary ethanolic extract (EF) pos-
sessed scavenging capacities against DPPH radical and ABTS radical
cation in a dose-dependent manner; the radical scavenging activ-
ities of extracts improved with increasing concentration (from 0.1
to 2 mg/mL). Compared to the positive control Vitamin C (Vc), the
antioxidant activities of EF were much lower than those of Vc in
DPPH• and ABTS•+ method at all ranges of concentrations. Signif-
icant differences (p < 0.05) existed at all levels except at 0.1 and
0.25 mg/mL in ABTS•+ assay.
109
Fig. 3 indicates the antioxidant activities of EAF, BF, PEF and
AqF evaluated by these three antioxidant evaluation methods. As
can be seen, the antioxidant activities of all treatment improved
with increasing concentration and EAF expressed the greatest
antioxidant activities among four extracted samples. At the peak
concentration (2 mg/mL) of treatment in Fig. 3B, no statistical
significance (p > 0.05) was observed between EAF and positive con-
trol Vc. The ORAC values of the extracts were in the sequence of
EAF > BF > PEF > AqF, ranging from 902 ␮mol TE/g to 10,860 ␮mol
TE/g. EAF showed a significantly higher ORAC value (Fig. 3C) than
that of Vc (10213 ␮mol TE/g) (p < 0.05).
Fig. 4A–C demonstrates the antioxidant activities of the ethanol
elution fractions. Among the fractions, 30%E showed advantages
over other fractions in DPPH radical and ABTS radical cation scav-
enging capacities, with the scavenging activity of DPPH• (%) at
95.10% and ABTS•+ (%) at 95.24% at 2 mg/mL, respectively, both of
which were significantly higher than 50%E and 70%E. No signifi-
cant differences were observed between Vc and 30%E at 2 mg/mL
in the two methods (p > 0.05). The ORAC value of 30%E was
9830 ± 146 ␮mol TE/g higher than 50%E (6642 ± 158 ␮mol TE/g)
but lower than Vc (10213 ± 61 ␮mol TE/g) and significant differ-
ences (p < 0.05) were found among them (Fig. 4C). Except for 30%E,
50%E also possessed the scavenging activity of ABTS•+ with 87.89%
at 2 mg/mL (Fig. 4B).
The results of a single assay cannot give a precise assessment
of the antioxidant activity of tested samples due to the compli-
cated reaction systems and differences between components and
functional groups, chemical polarity and behaviors (Sacchetti et al.,
2005). Thus, multiple methods are advisably applied in research
110 R. Zheng et al. / Industrial Crops and Products 101 (2017) 104–114

Fig. 3. Evaluation of antioxidant capacities of organic solvent elution phases using DPPH• (A), ABTS• + (B) and ROO• (ORAC) (C) methods. PEF, petroleum ether fraction; EAF,
ethyl acetate fraction; BF, n-butanol fraction; AqF, aqueous fraction. Values represent the mean ± SD (n = 3). Values with different letters have a significant difference at
p < 0.05 at the same concentration. The concentration of extracts (mg/mL) applied in this work was obtained by completely dissolving the dry extracts (mg) into the solvents
or buffers (mL).

Table 3
Correlation between total phenolic contents and bioactivities of sugarcane bagasse extracts.a

TPC DPPH• ABTS• + ROO• sucrase maltase

TPC 1 0.894* 0.911* 0.891* 0.851* 0.844*

DPPH• 1 0.989* 0.956* 0.990* 0.978*
ABTS• + 1 0.971* 0.978* 0.948*
ROO• 1 0.958* 0.947*
sucrase 1 0.980*
maltase 1
a
Correlation is significant at (*) p < 0.05 level (2-tailed). TPC, total phenolic content; DPPH• , the scavenging activity against DPPH• ; ABTS• + , the scavenging activity against
ABTS• + ; ROO• , the scavenging activity against ROO• (ORAC). Except TPC and ROO• , other original values were transformed into 1/IC50 for the Pearson correlation analysis.

work. However, it is noteworthy that both DPPH and ABTS assay
use non-physiological radicals and ORAC assay is a method that
takes the kinetic action of antioxidants. It can determine both
the hydrophobic and hydrophilic antioxidant activity of biological
samples using a single, physiologically relevant free radical source
(Dudonné et al., 2009; Prior et al., 2003). Although the ORAC value
of EF was low (3118 ± 81 ␮mol TE/g), parts of successive extracts
(EAF, BF, 30%E, 50%E etc.) showed good effect against peroxyl rad-
icals (ROO• ) generated by AAPH (Huang et al., 2002). In addition,
our work presented that the fractions with higher total phenolic
content showed stronger antioxidants activities. The correlation
analysis was conducted among the total phenolic contents and
the antioxidant ability and the results were shown in Table 3. The
total phenolic content exhibited significant correlations (p < 0.05)
with DPPH (r = 0.894), ABTS (r = 0.911) as well as ORAC (r = 0.891).
In addition, significant correlations (r > 0.950, p < 0.05) among the
three antioxidant methods were also found, which suggests these
assays are nearly interchangeable in characterizing the bagasse
antioxidant abilities. The results strongly indicate that the pheno-
lic compounds in sugarcane bagasse are chiefly responsible for its
antioxidant property and this conclusion is in agreement with pre-
vious reports (Sun et al., 2002; Thaipong et al., 2006; Wojdylo et al.,
2007). Thus plants with rich phenolic contents can be a valuable
source of antioxidants.
3.4. Intestinal ˛-glucosidase inhibitory activity
The inhibitory values of DMSO were calculated and the data
of samples minus the solvent value were used for analysis. As
shown in Tables 4A and 4B, all fractions of bagasse extracts indi-
cated inhibitory activities against the ␣-glucosidase and the effects
improved with the increase of concentrations. 30%E was the most
 R. Zheng et al. / Industrial Crops and Products 101 (2017) 104–114 111

Fig. 4. Evaluation of antioxidant capacities of 30, 50, 70%E using DPPH• (A), ABTS• + (B) and ROO• (ORAC) (C) methods. 30–70%E, 30–70% hydroalcoholic extract. Values
represent the mean ± SD (n = 3). Values with different letters have a significant difference at p < 0.05 at the same concentration. The concentration of extracts (mg/mL) applied
in this work was obtained by completely dissolving the dry extracts (mg) into the solvents or buffers (mL).

Table 4A
Inhibitory activities of sugarcane extracts against sucrase.

Extracts % Inhibition

0.625 mg/mL 1.25 mg/mL 2.5 mg/mL 5 mg/mL 10 mg/mL

EF 25.63 ± 2.41* 30.15 ± 2.58 34.56 ± 2.75 40.18 ± 2.95 45.35 ± 3.06
PEF 19.86 ± 2.16 30.52 ± 2.42 47.63 ± 2.1 52.37 ± 2.04 56.45 ± 1.95
EAF 30.17 ± 2.29 52.38 ± 2.13 65.42 ± 2.16 70.04 ± 1.93 72.96 ± 2.3
BF 24.36 ± 2.1 40.12 ± 2.04 56.34 ± 2.37 62.81 ± 2.15 68.35 ± 2.5
AqF 14.22 ± 1.94 23.63 ± 1.8 26.45 ± 2.33 30.21 ± 2.05 38.36 ± 2.14
30% E 35.48 ± 1.9 52.67 ± 1.94 64.41 ± 2.21 73.58 ± 2.35 82.64 ± 2.19
50% E 29.53 ± 2.17 39.39 ± 2.48 47.45 ± 3.01 58.74 ± 2.28 67.08 ± 2.22
70% E 18.99 ± 2.14 30.57 ± 3.05 40.44 ± 2.36 50.62 ± 2.21 55.31 ± 1.85

Table 4B
Inhibitory activities of sugarcane extracts against maltase.

Extracts % Inhibition

0.625 mg/mL 1.25 mg/mL 2.5 mg/mL 5 mg/mL 10 mg/mL

EF 10.83 ± 2.7* 20.34 ± 2.23 26.49 ± 2.35 35.03 ± 2.93 38.18 ± 2.16
PEF 20.17 ± 2.4 27.8 ± 2.23 40.26 ± 1.89 51.57 ± 2.36 58.32 ± 2.45
EAF 28.64 ± 2.17 49.52 ± 2.36 58.43 ± 2.26 67.58 ± 2.08 70.88 ± 2.56
BF 21.54 ± 2.88 30.87 ± 1.84 51.33 ± 2.23 60.08 ± 2.39 64.5 ± 1.94
AqF 8.64 ± 2.10 16.73 ± 2.26 20.32 ± 2.43 26.67 ± 1.96 28.25 ± 2.04
30% E 28.15 ± 2.35 44.62 ± 2.4 59.34 ± 2.18 68.91 ± 2.05 74.26 ± 1.96
50% E 21.35 ± 2.29 30.4 ± 2.38 41.65 ± 2.17 53.27 ± 2.21 58.33 ± 2.06
70% E 18.44 ± 2.38 26.92 ± 2.15 35.71 ± 2.06 40.88 ± 1.95 45.51 ± 1.88

EF, primary ethanolic fraction; PEF, petroleum ether fraction; EAF, ethyl acetate fraction; BF, n-butanol fraction; AqF, aqueous fraction; 30–70%E, 30–70% hydroalcoholic
fraction.
*
Values were expressed as a percent of enzyme inhibition (%).
112 R. Zheng et al. / Industrial Crops and Products 101 (2017) 104–114

Fig. 5. Cell viability of 30% hydroalcoholic extract (30%E) on HepG2 cells. Values are expressed relative to control group (n = 3) (A). Different concentrations of 30%E (mg/mL),
acarbose (AB) (10 ␮M) and rosiglitazone (RG) (10 ␮M) on the glucose uptake of HepG2 cells (B). Values are expressed as mean ± SD (n = 3). Values with different letters have
a significant difference at p < 0.05. The concentration of extracts (mg/mL) applied in this work was obtained by completely dissolving the dry extracts (mg) into the solvents
or buffers (mL).

active in both sucrase (82.64%, 10 mg/mL) and maltase (74.26%, by a previous research on ginger which also showed strong correla-
10 mg/mL) inhibitory activities, followed by EAF and BF. The 50%E tion between the two bioactivities (r = 0.99) (Alu’Datt et al., 2016).
and PEF also showed good inhibitory activities in the two assays. Although several bioactivities of sugarcane extracts were studied
As shown in Table 3, the total phenolic contents indicate significant (Duarte-Almeida et al., 2007; Feng et al., 2014; Lee et al., 2012),
correlations with antihyperglycemic activities, namely inhibitory we report here the first study of the strong antidiabetic activities
activities against sucrase (r = 0.851) and inhibitory activities against of sugarcane bagasse extracts and these results may make some
maltase (r = 0.844) at p < 0.05 level. Also, the antioxidant activi- contribution to the further use of this by-product from cane-sugar
ties correlate significantly with the antihyperglycemic activities, industry.
suggesting that the fractions with strong antioxidant properties
possess strong antidiabetic capacities.
3.5. Glucose uptake effect of 30% E
Reduction of the increased postprandial blood glucose level
through inhibitory effects on the carbohydrate-hydrolyzing enzy-
T2D is characterized by insulin resistance possibly combining
matic activities is a well-accepted treatment for T2D (Kang et al.,
with the reduction of insulin secretion (Du et al., 2012). Insulin
2014). Among the carbohydrate-hydrolyzing enzymes, disaccha-
capacitates cells of organs and tissues to take up glucose from
ridases are the main target for the treatment of diabetes because
blood and transform glucose into energy or to synthetize glyco-
human clinical studies have shown that disaccharidases are highly
gen. In this study, the cell viability effect of 30%E (from 0.01 to
active in DM patients’ small intestine (Zhang et al., 2015). ␣-
0.1 mg/mL) was evaluated and indicated no toxicity on human liver
Glucosidase inhibitors, such as acarbose, voglibose and miglitol
HepG2 cells (Fig. 5A). The insulin-stimulated 2-NBDG uptake activ-
have already been clinically applied for the treatment of DM,
ity of HepG2 cells was used to evaluate the hypoglycemic capacity
however, these drugs frequently generate side effects including
of the bio-active 30%E as well as the positive reference acarbose
flatulence, diarrhea and abdominal pain (Zhang et al., 2015). Large
and rosiglitazone. As shown in Fig. 5B, all concentrations of 30%
amounts of evidence have confirmed that dietary phenolics from
E and rosiglitazone (10 ␮M) but not acarbose (10 ␮M) improved
plants (quercetin genistin and genistein etc.) based on in vitro
the 2-NBDG uptake of HepG2 cells significantly compared to con-
studies, animal models and clinical trials have anti-diabetic activ-
trol (insulin, 0.5% DMSO (v/v), p < 0.05). In addition, the dose-effect
ities (Bahadoran et al., 2013; Li et al., 2009; Wu et al., 2012).
relationship arose when the concentration of 30%E increased and
Additionally, phenolics with antioxidant capacities show beneficial
significant difference was observed between the 30%E 0.1 mg/mL
antidiabetic effects via adjusting the disturbed oxidative milieu in
group and the positive reference (rosiglitazone).
diabetic conditions and can protect ˇ-cells from oxidative dam-
High level of glucose induces a reduction in the phosphorylation
age and dysfunction (Abdelmoaty et al., 2010; Bansal et al., 2012).
level of protein kinase B (AKT/PKB) and 5 -AMP-activated protein
The results obtained in our work indicate the positive relationship
kinase (AMPK) in part, leading to the decrease of glycogen con-
between antioxidant and antidiabetic activities and are supported
tent and glucose transporter-2 (GLUT-2) levels, which suggests an
 R. Zheng et al. / Industrial Crops and Products 101 (2017) 104–114
insulin-resistant situation (Cordero-Herrera et al., 2014). For peo-
ple who have T2D, specific cells (skeletal muscle and liver cells)
have no or weak ability to take up glucose from blood, which
results in a high level of blood glucose (hyperglycemia). Improv-
ing the insulin-stimulated glucose uptake and inhibiting insulin
resistance could prevent or retard the generation of T2D. AMPK
is a key energy sensor involved in insulin metabolism and acti-
vation by phosphorlation can facilitate glucose uptake but the
phosphorylation activity was suppressed in insulin-resistant cells.
As reported previously, p-coumaric acid was able to improve 2-
NBDG uptake by activating AMPK in L6 skeletal muscle cells (Yoon
et al., 2013). A study suggested that quercetin expressed antidi-
abetic activity by influencing the glucose homeostasis of skeletal
muscle and liver cells via the mechanism associated with AMPK
activation (Eid et al., 2015). Cordero-Herrera et al. also reported
that cocoa phenolic extract can elevate total and phosphorlated
level of AMPK in HepG2 cells which could be helpful to prevent
insulin-resistance and hyperglymia (Cordero-Herrera et al., 2014).
Although various mechanisms involved in hypoglycemic activities
of phenolics should be studied at molecular level, these findings
suggested, at least, phenolics can be a potential candidate for the
treatment of DM.
4. Conclusions
The combined analysis of total phenolic contents and the antiox-
idant/antihyperglycemic activity of the extracts gave new insight
into the general understanding of the additional values of bagasse.
Several methods have been applied to determine the phenolic com-
pounds in plants, such as high-performance liquid chromatography
(HPLC), thin-layer chromatograph (TLC) and HPLC-MS (Zhao et al.,
2015; Khatoon et al., 2008; Zhao et al., 2013). However, these
methods suffer from obvious drawbacks, including low resolution
and long-time analysis with considerable amount of solvents con-
sumption. UHPLC-HR-TOFMS shows dominant advantages in rapid
detection of targeted compounds due to its much higher selectiv-
ity/sensitivity with very lower consumption of time and solvents
(Singh et al., 2016).
This research underlines the initial effort to assess the total
phenolic content and antioxidant/antihyperglycemic activities of
different fractions isolated from sugarcane bagasse. Several phe-
nolic compounds were identified for the primary elucidation for
the biological activities of sugarcane bagasse extract. The antiox-
idant activities demonstrated by the extracts prove sugarcane
bagasse is an excellent source of natural antioxidants. Thus bagasse
extracts can serve the cosmetic and pharmaceutical industries. In
addition, bagasse extracts have high inhibitory activities against
␣-glucosidase, suggesting that bagasse extracts can be useful ther-
apeutic agents to treat T2D patients. Future work should focus on
the utilization of sugarcane phenolics and the exploitation of other
phytochemicals (triterpenoids, sterols and lignins, etc.) in sugar-
cane. Overall, the obtained results demonstrate sugarcane bagasse
can be helpful in new agents discovery and agro-industrial revenue
increase.
Conflict of interests
The authors declare no conflicts of interest.
Acknowledgments
This work was supported by the National Natural Science Foun-
dation of China (31301506); Guangdong Science and Technology
Program (2014B090904063); the Fundamental Research Funds
for the Central University and the Leading Talent of Guangdong
113
Province. The Leading Talent of Guangdong Province is an inde-
pendent sponsor and we kindly ask you to change this.
We are grateful for technical support from Analytical and Test-
ing Center, South China University of Technology, Guangzhou,
China.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.indcrop.2017.03.
012.
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