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Characterization and antioxidant activity of polysaccharides

obtained from ginger pomace using two different extraction
processes

Gui-tang Chen, Biao Yuan, Hai-xiang Wang, Guo-hong Qi, Shu-

jie Cheng

PII: S0141-8130(19)34946-3
DOI: https://doi.org/10.1016/j.ijbiomac.2019.08.048
Reference: BIOMAC 13025

To appear in: International Journal of Biological Macromolecules

Received date: 1 July 2019

Revised date: 29 July 2019
Accepted date: 6 August 2019

Please cite this article as: G.-t. Chen, B. Yuan, H.-x. Wang, et al., Characterization and
antioxidant activity of polysaccharides obtained from ginger pomace using two different
extraction processes, International Journal of Biological Macromolecules(2019),
https://doi.org/10.1016/j.ijbiomac.2019.08.048

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© 2019 Published by Elsevier.

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Characterization and antioxidant activity of polysaccharides obtained

from ginger pomace using two different extraction processes

Gui-tang Chen*, Biao Yuan, Hai-xiang Wang, Guo-hong Qi, Shu-jie Cheng

Department of Food Quality and Safety, National R&D Center for Chinese Herbal

Medicine Processing, China Pharmaceutical University, 639 Longmian Avenue, Nanjing

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210009, China

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*
Corresponding author. Tel: +86 15951854251, Fax: +86 25 86185754
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Email address: cpucgt@cpu.edu.cn
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ABSTRACT
In this study, two different processes of hot water (HW) and ultrasonic–assisted (UA)

for the extraction of polysaccharide from ginger pomace (GPPs) were employed under their

respective best parameters, and the characterization and antioxidant activity of the purified

polysaccharide (HW-GPP1, HW-GPP2, HW-GPP3, and UA-GPP1, UA-GPP2, UA-GPP3,

respectively) were analyzed. The data implied that the yield of the polysaccharide obtained

by UA was higher than that of HW. Meanwhile, two kinds of GPPs possessed the different

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preliminary structural characteristics including molecular weight distributions, total sugar

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and protein content, uronic acid content, while similar monosaccharide compositions and
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sulfuric radical contents. In vitro antioxidant activity assays indicated that UA-GPP3
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showed the strongest scavenging abilities on DPPH radicals, while UA-GPP2 possessed the

strongest scavenging abilities on hydroxyl and superoxide radicals. Moreover, the

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antioxidant activity of each fractions of GPPs extracted by UA was better than that of the
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corresponding fractions of GPPs extracted by HW. These results showed that UA was more

beneficial to enhance the extraction yields of the polysaccharides, and also resulted in GPPs
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with higher bioactivity. Therefore, it indicated that UA-GPPs could be used as a potential
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natural antioxidant. Accordingly, the ginger pomace could be used as a potential source for

natural antioxidant.

KEY WORDS: Ginger pomace；Polysaccharide；Separation and purification;

Characterization; Antioxidant activity

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1. Introduction

Ginger (Zingiber officinale Roscoe) is one of the most commonly used spices around

the world and a traditional medicinal ingredient that has been widely used in China,

Ayurvedic and Unani Tibb herbal medicines for several thousand years [1]. The ginger

rhizome contains 60-70% carbohydrates, 3-8% crude fiber, 9% protein, 8% ash, 3-6% fatty

oil and 2-3% volatile oil [2]. Due to the presence of various active ingredients such as

polysaccharides, polyphenols, terpenoids, gingerol and its derivatives, ginger is effective

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against several pharmacological actions like inflammatory, high cholesterol, ulcers, tumors,

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atherosclerosis, indigestion, diabetes and cardiovascular diseases [3-8].
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consumption has increased rapidly in recent years. Increasing production of ginger makes it
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available to be processed to many other foods, such as condiment, syrup, candy, dried

powder etc. [9-11]. At the same time, there will be more and more the by-product i.e.
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ginger pomace bring out. The ginger pomace also has abundant bioactive ingredients,
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especially polysaccharides. Although several studies have focused on extraction,

purification and structural characterization of ginger polysaccharides [12-14], there is a lack

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of attention devoted to the detailed characterization and bioactive activity of

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polysaccharides from ginger pomace.

Over the past few decades, many extraction techniques are applied in the practice of

polysaccharides extraction, such as hot water extraction (HW), ultrasonic-assisted

extraction (UA), microwave-assisted extraction (MA), enzyme-assisted extraction (EA),

and so on. Previous studies have shown that different extraction techniques can lead to

differences in the extraction yields, physicochemical properties and bioactivities of

polysaccharides [15-18]. Moreover, when considering the cost, operation convenience,

environmental factors and extraction efficiency, each method has both advantages and

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disadvantages [19, 20]. For examples, HW is an easy-to-operate and environmentally

friendly extraction technology, but HW generally has the disadvantages of long operation

times, high extraction temperatures and low extraction yields. UA can enhance the

extraction yield of polysaccharides through the generation of cavitation effects, however, it

requires higher equipment cost and operator skill.

According to the above mentioned research progress, we could find that the work on

the characterization and antioxidant activity study of polysaccharides from ginger pomace

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(GPPs) using different extraction methods has not been reported. Therefore, the aim of this

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study was to evaluate the effects of two different methods (HW and UA) on the
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physicochemical characteristics and antioxidant activities of GPPs. It is of interest to

prepare GPPs with better antioxidant activities and characterize their physicochemical
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characteristics to provide a promising application for functional foods or drugs using these
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GPPs as ingredient.
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2. Materials and methods

2.1 Materials and regents

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Freshly harvested ginger (Zingiber officinale) was purchased from Zhongcai

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Agricultural Products Wholesale Market, Nanjing city (China). The pomace used in this

study was obtained by squashing peeled ginger in a YZ-V11 fruit juicer (Joyoung Co., Ltd.,

Jinan, China). Then the mixture was filtered and the pomace was collected and dried at

60 °C for 24 h, smashed in a DFY-500 blender (Wenling Linda Machinery Co., Ltd., China),

and then sieved at 80 mesh.

1,1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma Chemical Co. (St.

Louis, MO, USA). DEAE-52 and Sephadex G-100 were purchased from Whatman Co.

(Maidstone, Kent, UK). A series of different molecular weight Pullulan and standard

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monosaccharides of chromatographic grade were purchased from Sigma-Aldrich Co.

(MilWaukee, WI). All other chemicals and solvents used in this investigation were of

analytical grade and purchased from Nanjing Chemicals Co. (Nanjing, China).

2.2 Extraction of ginger pomace polysaccharides (GPPs)

Two different processes of HW and UA for the extraction of polysaccharide from

ginger pomace were employed under their respective best parameters obtained by our

previous study. Briefly, the ginger pomace powder was extracted 3× with 85% EtOH at

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75 °C for 2 h, under reflux, to remove pigments and lipids, and the supernatant was

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removed. The residue was mixed with distilled water (40 mL/g) and extracted by using HW
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and UA techniques, and the corresponding polysaccharides obtained were named as

HW-GPPs and UA-GPPs, respectively. Each method was performed in triplicate. The
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extraction of HW-GPPs was performed in a water-bath at 70 °C for 120 min. The extraction
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of UA-GPPs was performed in an ultrasonic cell disruptor (DCTZ-2000, Beijing

Hongxianglong Biotechnology Development Co. Ltd, Beijing, China) with a power of 400
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W at 74 °C for 17 min.
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After extraction, the extracting solution of GPPs was centrifuged at 5000 × g for 15 min
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to remove the solid residue. Then the supernatant was concentrated to one-fourth of its

initial volumes using a rotary vacuum evaporator (RE-52, Yarong technology and Science

Inc., Shanghai, China) at 60 °C. Four times volume of 95% ethanol was mixed with the

concentrated extraction solution to precipitate GPPs (4 °C, 12 h). Subsequently, the

precipitates were obtained by centrifugalization at 5000 × g for 15 min, and washed with

anhydrous ethanol, acetone and ether in turn. The extracts were dissolved with distilled

water and got rid of protein by Savage reagent (1-butanol: chloroform = 1:4, v/v) method.

Lastly, the two crude GPPs were obtained by freeze drying.

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The GPPs yields was calculated by using the following equation (1):

GPPs yield (%) = (WGPPs/Wsample) × 100 （1）

where WGPPs and Wsample were the weights of GPPs and ginger pomace powder used for

extracting GPPs, respectively.

2.3 Purification of GPPs

Before determining the physicochemical characteristics and antioxidant activities of

GPPs, the GPPs were purified using an anion-exchange DEAE-52 cellulose

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chromatography and a size-exclusion Sephadex G-100 chromatography. Briefly, the GPPs

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(100 mg) was dissolved in 10 mL distilled water and filtered through 4.5 µm filters. Then
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the solution was applied to the DEAE52-cellulose column (2.6 cm × 60 cm) and eluted with
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distilled water and different concentrations of stepwise NaCl solution (0.1, 0.3, 0.5 and 0.7

M NaCl) at a flow rate of 1.0 mL/min, and with collection of 10 mL for each tube.
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Phenol-sulfuric acid method [21] was used to monitor and combine the fractions. The
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appropriate fractions were concentrated and then were further isolated by the Sephadex

G-100 column (2.6cm×80cm) with distilled water. Each fraction of 4mL was collected and
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eluted at a flow rate of 0.25mL/min. The major polysaccharide fractions were collected,
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concentrated, dialyzed and lyophilized for further study, respectively.

2.4 Characterization of GPPs fractions

2.4.1 Chemical composition analysis of GPPs fractions

Phenol-sulfuric acid method [21] was used to determine the contents of total

polysaccharides, Bradford’s method [22] was used to determine the contents of protein,

Folin-Ciocalteu assay reagent [23] was used to determine the contents of total phenolic in

GPPs fractions. Glucose, bovine serum albumin and 6-Gingerol were used as the standards

for estimation of polysaccharides, protein and phenolic contents, respectively. The content

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of uronic acid in GPPs was determined using m-hydroxybiphenyl method [24]. The content

of sulfate radicals was determined based on the report of Tang et al. [25]

2.4.2 Molecular weights analysis of GPPs fractions

High-performance gel filtration chromatography (HPGFC) was used to determine the

molecular weights of GPPs fractions. It was evaluated according to the method of

Cozzolino et al. [26] with some modification. Briefly, the purified GPPs fraction was

applied to a gel-filtration chromatography column of TSK-gel G4000 PWXl column

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(300mm × 7.8 mm, Tosoh Corp., Japan), and analyzed with HPLC (Agilent 1200, Agilent

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Technologies Co. Ltd., USA) with an evaporative light scattering detector. The column was
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maintained at a temperature of 35°C, eluted with water at a flow rate of 0.7mL/min.

Preliminary calibration of the column was conducted using the Pullulan with diverse
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molecular weight (19626, 43714, 101237, 202898, 371868 and 745558).

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2.4.3 Monosaccharide composition analysis of GPPs fractions

The monosaccharide composition of GPPs fractions was analyzed using HPLC using
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the method reported by Liu et al. [27] with some modifications. Briefly, the GPPs sample
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(0.2 mg) was hydrolyzed with 0.2 mL trifluoroacetic acid (TFA, 4M) in a sealed flask
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fulfilled with N2 at 120 °C for 2 h. After hydrolysis, the excess TFA in the system was

removed by repeated coevaporation with methanol using a rotary evaporator. Subsequently,

dry hydrolysate samples of GPPs or monosaccharide standard were mixed with 0.1 ml

aqueous solution of NaOH (0.3M) and 0.5 mL methanol solution of PMP (0.5M) for

derivatization at 70°C for 100 min. After cooling, the mixture solution was neutralized with

0.1 mL HCl (0.3M), then the reaction mixture was extracted with chloroform for three

times to remove the excess PMP. The aqueous layer was filtered through a 0.45μm syringe

filter and analyzed by a HPLC system (Agilent 1200, Agilent Technologies Co. Ltd., USA)

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[28].

2.4.4 Ultraviolet spectra

The GPPs fractions were dissolved and diluted to 0.5 mg/mL respectively, and the

solutions were scanned from 200 to 400 nm with a UV-2045 ultraviolet spectrophotometer

(Shanghai Precision and Scientific Instrument Co. Ltd., Shanghai, China).

2.4.5 Infrared spectrum analysis

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The polysaccharide fractions were analyzed by FT-IR spectroscopy on a Bruker Fourier

transform infrared spectrometer (TENSOR27, Bruker Corporation, Karlsruhe, Germany).

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Dried samples were used for the FT-IR measurements and ground with potassium bromide
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power before being pressed into pellets for analysis at frequencies in the range of 4000-500
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cm−1.
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2.5 In vitro antioxidant activities of GPPs fractions

2.5.1 Measurement of scavenging effect on DPPH radical

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The effect of the polysaccharide fractions on scavenging DPPH radical was measured

by the method of Chen et al. [29] with some modifications. Sample of 1.0 ml (at different
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concentrations) was added to 1.0 ml of phosphate buffer (0.02M, pH 6.0) and 1.0 ml of 0.2
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mM DPPH in 95% ethanol. The mixture was shaken and left for 30 min at 30oC in the dark,

and the absorbance of the resulting solution was measured at 517nm by a

spectrophotometer. Ascorbic acid was used as positive control. The scavenging percentage

was calculated according to the following equation （2）:

Scavenging percentage (%)=[1-(A1－A2)/ A0]×100 （2）

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where A1 was the absorbance of sample or ascorbic acid with DPPH solution, A2 was

the absorbance of sample with deionised water, and A0 was the absorbance of DPPH

solution.

2.5.2 Measurement of hydroxyl radical scavenging activity

The hydroxyl radical scavenging activity was investigated by the method of Xie et al.

[30] with a minor modification. Briefly, sample of 1.0 ml (at different concentrations) was

mixed with 2.0 ml 0.2M phosphate buffer (pH 7.4), 1.0 ml 0.75 mM 1,10-phenanthroline,

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1.0 ml 0.75 mM FeSO4 and 1.0 ml H2 O2 (0.01%, v/v) were added into a tube. After

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incubation for 30min at 37◦C, the absorbance of the mixture was measured at 536 nm, with
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ascorbic acid as a positive control. The scavenging activity of hydroxyl radicals was

calculated by the following equation （3）:

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Scavenging activity (%) = [(A2 − A1)/(A0 − A1)] × 100 （3）

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where A1 is the absorbance of control, A2 is the absorbance of polysaccharide sample

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and A0 is the absorbance of the solution without polysaccharide sample and H2O2.
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2.5.3 Measurement of superoxide radical scavenging activity

The superoxide radical scavenging abilities of the polysaccharide fractions were

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performed according to the method of Liu et al. [31] with some modifications. In this

experiment, superoxide anion radicals were generated in 3.0 ml of sodium phosphate buffer

(0.1 M, pH 7.4) containing 156 μM nitroblue tetrazolium (NBT), 468 μM β-nicotinamide

adenine dinucleotide (NADH), and 60 μM phenazine methosulfate (PMS). After addition of

1.0 ml sample (at different concentrations), the mixture was incubated at 25℃ for 5 min,

and the absorbance of the mixture was measured at 560 nm. Deionized water and ascorbic

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acid were used as the blank control and positive control, respectively. The scavenging

activity of superoxide radicals was calculated using the following equation （4）:

Scavenging percentage (%)=[1-A2]/ A1]×100 （4）

where A1 is the absorbance of the control (deionized water, instead of sample), and A2 was

the absorbance of the test sample or ascorbic acid mixed with reaction solution.

2.6. Statistical analysis

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The data were expressed as mean ± SD of three replicates. Statistical significance was

calculated by single factor analysis of variance and Duncan’s multiple range test, and the

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differences were regarded as significant when the P-values were less than 0.05.
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3 Results
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3.1 Purification of GPPs
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The crude ginger pomace polysaccharides were extracted by HW and UA techniques

and then purified by anion exchange chromatography on a DEAE-52 cellulose column, and
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six fractions were obtained: HW-F1 to HW-F3, and UA-F1 to UA-F3, respectively (Fig.
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1a). The six fractions were collected, concentrated and purified by gel filtration

chromatography with the Sephadex G-100. Then six single and symmetrically sharp elution
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peaks (HW-GPP1, HW-GPP2, HW-GPP3, and UA-GPP1, UA-GPP2, UA-GPP3,

respectively) were obtained, indicating the six polysaccharide fractions were homogenous

polysaccharides (Fig. 1b to d).

3.2 Characterization of GPPs fractions

3.2.1 Chemical composition of GPPs fractions

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The yields and chemical composition of GPPs extracted with two different methods

were listed in Table 1. The yield of UA-GPPs (16.62%) was significantly higher than

that of HW-GPPs (12.13%) (P < 0.05), which suggested that GPPs yields were

significantly affected by extraction processes, and the ultrasonic assistance extraction

technology could notably increase the extraction yield of GPPs. The total sugar content

of UA-GPPs was significantly higher than that of HW-GPPs, but the protein content was

just the opposite. No total polyphenol was detected in all GPPs and their fractions.

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Different extraction methods had no significant effects on the sulfuric radical contents of

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GPPs (P > 0.05). However, the uronic acid content in UA-GPPs (6.09%) was significant
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higher than that in HW-GPPs (4.42%), indicated that the uronic acid contents of GPPs

were significantly affected by the extraction techniques (P< 0.05).

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As shown in Table 1, after purification, the total sugar contents of all the GPPs
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fractions were more than 95%, and there was no statistical difference between them. No

protein was detected in HW-GPP2, HW-GPP3, UA-GPP2, and UA-GPP3, but a small
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amount of it was determined in HW-GPP1 and UA-GPP1 (0.35% and 0.43%,

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respectively, P>0.05). The sulfate group contents of HW-GPP1, HW-GPP2, HW-GPP3,

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UA-GPP1, UA-GPP2 and UA-GPP3 were 0.79%, 1.31%, 1.37%, 1.03%, 1.62% and

1.83%, respectively (P< 0.05). The uronic acid contents of the six GPPs were different

followed the order of UA-GPP2 (9.29%) > UA-GPP3 (8.86%) > HW-GPP2 (7.03%)>

HW-GPP3 (6.88%) > UA-GPP1 (6.57%)> HW-GPP1 (6.24%). Which suggested that the

extraction processes had significant effect on the chemical composition of GPPs

fractions.

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Table 1 Chemical compositions of GPPs obtained by different extraction processes

Samples HW-GPPs UA-GPPs HW-GPP1 HW-GPP2 HW-GPP3 UA-GPP1 UA-GPP2 UA-GPP3

Polysaccharides yield (%) 12.13±1.15b 16.62±1.82a
Total sugar content (%) 76.42±2.32c 82.53±2.86b 95.83±2.35a 97.21±2.11a 96.14±1.93a 96.42±1.09a 98.33±2.14 a 96.47±1.54 a
Protein content (%) 8.33±0.85a 5.65±0.64b 0.35±0.03 c ND ND 0.43±0.07c ND ND
e e e c c d b
Sulfate group (%) 0.93±0.06 0.86±0.04 0.79±0.07 1.31±0.08 1.37±0.05 1.03±0.04 1.62±0.09 1.83±0.12 a
Uronic acid (%) 4.42±0.35e 6.09±0.17d 6.24±0.21c 7.03±0.16b 6.88±0.22b 6.57±0.27c 9.29±0.36 a 8.86±0.21 a
Values are expressed as the mean ± standard deviation (n = 3). ND: not detectable.

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Different letters of each value within a row indicate significant difference at P < 0.05.

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3.2.2. Molecular weight distribution of GPPs fractions
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Then, HPGFC was applied to analyze the molecular weight of GPPs fractions.

According to the calibration curve of Pullulan standards, as shown in Table 2, the average
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molecular weight of HW-GPP1, HW-GPP2, HW-GPP3 and UA-GPP1, UA-GPP1,
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UA-GPP3 were calculated as 89.2, 939.8, 1007.9 and 40.6, 868.1, 892.7 kDa, respectively.

It showed that the molecular weights of HW-GPPs were higher than that of UA-GPPs,
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which suggested that the polysaccharides molecules were flock together under the
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extraction condition of long time at higher temperature and ultrasound could decompose the

GPPs to forms the smaller ones.

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3.2.3. Monosaccharide composition of GPPs fractions

The six GPPs fractions were completely hydrolyzed by TFA, and then subjected to

HPLC analysis, the obtained results were presented in Fig. 2 and Table 2. As shown in Fig.

2, the peaks of 10 standard monosaccharides were well-proportioned and the interval

between peaks was obvious, which indicated that each standard monosaccharide could be

separated well in liquid chromatography. According to the monosaccharide standards, the

six GPPs were all contained mannose, rhamnose and glucose, and glucose was noted to be

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the predominant monosaccharide (50.01-68.33%) (Table 2). Moreover, the HW-GPP2 and

UA-GPP2 also contained xylose and arabinose. Besides xylose and arabinose, the

HW-GPP3 and UA-GPP3 also contain some Galactose. This finding suggested that the

GPPs extracted from ginger pomace were heteropolysaccharides with different molar

percentages. Particularly, the monosaccharide analysis results indicated the corresponding

fractions of HW-GPPs and UA-GPPs had a similar composition, but different extraction

techniques had significant effects on the contents of monosaccharides in GPPs (P<0.05).

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Table 2 Molecular weight and monosaccharide composition of ginger polysaccharide fractions

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HW-GPP1 HW-GPP2 HW-GPP3 UA-GPP1 UA-GPP2 UA-GPP3
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Molecular weight (kDa) 89.2 939.8 1007.9 40.6 868.1 892.7
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Mannose (%) 19.40±0.06a 11.84±0.13d 11.33±0.05e 17.56±0.11b 13.18±0.05c 8.32±0.09f
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Rhamnose (%) 12.27±0.05b 9.36±0.02c 13.90±0.03a 7.72±0.29e 9.03±0.08d 9.01±0.02d

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Glucose (%) 68.33±0.24b 58.05±0.07e 50.01±0.13f 74.72±0.27a 63.78±0.14c 59.28±0.11d

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Galactose (%) ND ND 10.96±0.13a ND ND 4.33±0.03b

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Xylose (%) ND 12.68±0.15a 4.73±0.09d ND 8.97±0.15c 12.19±0.12b

Arabinose (%) ND 8.07±0.08b 9.07±0.14a ND 5.04±0.08d 6.87±0.05c

The results were given as mean values ± standard deviation (n = 3). ND: not detectable.

Different letters of each value within a row indicate significant difference at P < 0.05.

3.2.4 UV spectrum of GPPs fractions

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The UV spectrum indicated that there was no obviously absorption peak at 260 nm

and 280 nm (Fig. 3), suggesting the GPPs fractions had little or no nucleic acid and protein,

which consistent the results of Table 1.

3.2.5 FT-IR spectra of GPPs fractions

The FT-IR spectra recorded in the region of 4000-500 cm-1 for GPPs fractions were

shown in Fig. 4. All the six fractions exhibited a broad intense peak near 3500-3100 cm-1

for the intramolecular and intermolecular O-H stretching vibration, a weak peak near

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3000-2900 cm-1 for the C-H stretching vibration and symmetrical deformation vibration,

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respectively [25]. These two absorption peaks are characteristic peaks of polysaccharides.
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The absorption peaks at 2350.68, 2349.90, 2363.85, 2331.31, 2350.68, and 2330.54 cm-1

are characteristic absorption peaks of X-H bonds (X is an element, such as diazo salts,
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phosphorus, or silicon). The absorption peaks at 1632.54, 1637.96, 1638.73, 1632.54,

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1626.34, and 1637.96 cm-1 are symmetrical vibration peaks of carboxyl groups (C=O

symmetrical stretching), meanwhile, the existence of C=O indicates polysaccharides

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contain amide bond or carboxylic acid [12]. The absorption peaks near 1237 cm-1 and 827
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cm-1 are S=O stretching vibration of sulfate group. HW-GPP2, HW-GPP3 and UA-GPP2,
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UA-GPP3 have the stronger absorption peak, indicating that the sulfate group content in

GPP2 and GPP3 are higher. While HW-GPP1 and UA-GPP1 have no obvious absorption

peak, it shows that the sulfate group content in GPP1 is very low. These results were

consistent with the determination results of sulfate group content in Table 1.

Previous studies have shown that there are two strong absorption peaks of furanose, and

three strong absorption peaks of pyranose in the middle of 1200-900 cm-1 [32]. In addition,

the region ranging from 1200 to 950 cm−1 suggests the presence of C-O-C, C-O-H link

bonds and hydroxyl of pyranose ring, and it demonstrates the presence of pyranose [33, 34]

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As shown in Fig. 4, three absorption peaks appeared in all the six GPPs fractions at 1100

-1010 cm-1, this showed that they were linked by pyran-cycloglycoside. In addition, when

pyranocyclic glycosides are linked, the absorption peaks at 890 cm -1 are generally

expressed as beta-glycoside bonds, and at 840 cm-1, the absorption peaks show that they are

alpha-glycoside bonds. From Fig. 4, it can be seen that the three polysaccharide

components extracted by UA and HW-GPP3 are alpha-pyranose, while the HW-GPP1 and

HW-GPP2 are beta-pyranose. It suggested that ultrasound could break and recombine the

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glycoside bonds of polysaccharides and change the glycoside bond configuration of

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polysaccharides.

3.3 Antioxidant activity

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3.3.1 DPPH radical-scavenging assay
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Due to the convenient and reproducibility, DPPH radical has been widely used to
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determine the free radical scavenging abilities of various natural compounds. Results in Fig.

5a demonstrated the DPPH scavenging activities of the six GPPs fractions and ascorbic acid,
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it is easily seen that the scavenging activity is dose-dependent from 0 to 2.0 mg/mL, in
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which ascorbic acid exhibited outstanding scavenging ability. At the concentration of 2.0
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mg/mL, the order of scavenging activity of six fractions was UA-GPP3 > UA-GPP2 >

HW-GPP3 > HW-GPP2 > UA-GPP1 > HW-GPP1, and the scavenging rate was

76.4±3.4 %, 68.7±1.4%, 61.7±3.3%, 58.8±1.5%, 42.6±2.2%, 37.6±2.4 %, respectively. The

results showed the DPPH free radical scavenging activity of each fractions of ginger

pomace polysaccharide extracted by ultrasound is better than that of the corresponding

components extracted by hot water.

3.3.2 Hydroxyl radical assay

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Hydroxyl radical is the most reactive free radical which can react with almost all the

biomolecules and cause serious damage to the adjacent biomolecules. Therefore, removing

hydroxyl radical is very important for antioxidant defense in cell or food systems. In the

present study, though the scavenging ability was lower than ascorbic acid, GPPs expressed

outstanding ability to scavenge hydroxyl radical at concentrations from 0.075 mg/mL to 2.0

mg/mL, and in a concentration-dependent manner (Fig. 5B). At the concentration of 2.0

mg/mL, the order of scavenging activity of six GPPs fractions was UA-GPP2 > UA-GPP3 >

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HW-GPP2 > UA-GPP1 > HW-GPP3 > HW-GPP1, and the scavenging rate was

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71.2±1.9 %, 59.6±2.1%, 54.3±1.5%, 48.6±1.4%, 44.7±1.8%, 42.4±1.6 %, respectively.
-p
These results indicated that GPPs might act as electron or hydrogen donor to scavenge

hydroxyl radicals, and it can be possible to conclude that the UA-GPPs had a stronger
re

capability of scavenging hydroxyl radical than that of the HW-GPPs.

lP

3.3.3 Superoxide radical assay

Superoxide anions radical is considered as the initial free radicals formed from the
na

mitochondrial electron transport system. Fig. 5c illustrated the different superoxide radical
ur

scavenging abilities of the six polysaccharides in dose-dependent manners at all tested

Jo

concentrations. At the concentration of 2.0 mg/mL, the scavenging activity of the six

samples on superoxide anion followed the order: UA-GPP2 > UA-GPP3 >

UA-GPP1 >HW-GPP3 > HW-GPP1 > HW-GPP2, and the scavenging rate was 77.5±1.5 %,

64.8±1.1%, 56.2±1.5%, 50.7±2.3%, 45.1±1.0%, 39.5±1.6%, respectively. Similar to

scavenging hydroxyl radicals, UA-GPPs had stronger scavenging ability than HW-GPPs,

and the UA-GPP2 had the highest scavenging activity.

4. Discussion

16
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In this study, two different techniques of HW and UA for the extraction of

polysaccharide from ginger pomace were employed under their respective best

parameters, and the physicochemical properties and in vitro antioxidant activity of the

purified polysaccharide were evaluated. The data showed that the yield of the

polysaccharide obtained by UA was higher than that of HW. It possibly due to the UA

could produce cavitation effect through collapse of ultrasonic bubbles, and then release

tremendous energy, capable of obtaining more polysaccharides [35]. Meanwhile, the

of
HW-GPPs possessed the larger molecular weight, higher protein content, lower total

ro
sugar and uronic acid content, but their monosaccharide composition and sulfate content
-p
were similar to the UA-GPPs. This may be because ultrasound breaks down some

glycoside bonds and glycol-peptide bonds, and HW helps to dissolve some

re

protein-bound polysaccharide fraction. After purification, the total sugar content of the
lP

two kinds of polysaccharides had no significant difference, but the sulfate content and

glucuronic acid content of UA-GPPs fractions were higher than those of HW-GPPs
na

fractions. These results suggested that the different extraction processes had significant
ur

effect on the yield of polysaccharide and chemical composition of GPPs fractions.

Jo

According to the in vitro antioxidant activity analysis, the antioxidant activities of the

GPPS fractions in various reactive systems were different, among which UA-GPP3

showed strongest scavenging abilities on DPPH radicals, while UA-GPP2 possessed

strongest scavenging abilities on hydroxyl and superoxide radicals. Moreover, the

antioxidant activity of each fractions of GPPs extracted by UA was stronger than that of

the corresponding fractions of GPPs extracted by HW. These data indicated that UA

resulted in GPPs with higher antioxidant activity.

17
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It is widely accepted that the bioactivity of polysaccharides is highly related to their

molecular weight, chemical composition, and monosaccharide composition. Among all the

influence factors mentioned above, a lot of reports suggested that the antioxidant activity

is mainly associated with molecular weight of polysaccharides. It was supposed that

polysaccharides with lower molecular weights exhibited more significant activities

compared with the higher ones, because at an equal mass basis, polysaccharides with a

lower molecular weights have more reductive hydroxyl group terminals to accept and

of
eliminate the free radicals [36]. However, opposite results were also described. For

ro
example, Cheng et al. [37] obtained polysaccharides from Epimedium acuminatum with
-p
larger molecular weight displayed better antioxidant actions, which was consistent with

the results of UA-GPP3 on DPPH radical scavenging activity, when compared with
re

UA-GPP1 and UA-GPP2. However, when compared with HW-GPP3, UA-GPP3 has
lP

lower molecular weight and stronger DPPH radical scavenging activity. Hence, it is still

uncertain about the relationship between the molecular weight and the antioxidant activity
na

of polysaccharides.
ur

Furthermore, uronic acid content is considered to be another important indicator

Jo

reflecting the antioxidant activity of the polysaccharides. Generally, polysaccharides

possessed the radical scavenging activities due to their electron- or hydrogen-donating

abilities. Uronic acid groups existing in polysaccharides could interact with the hydrogen

atom of the anomeric carbon. Therefore, the higher content of uronic acid, the stronger

antioxidant activity of polysaccharides [38]. In the present study, the UA-GPP3 and

UA-GPP2 have the highest uronic acid content and the strongest antioxidant activity,

which in agreement with the results of previous reports [15, 19, 39].

18
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In conclusion, taking into consideration the differences among the physicochemical

properties of the GPPs samples, the antioxidant activity of polysaccharides might be not a

function of a single factor but a combination of several factors. Differences in origin

materials, extraction processes, and even drying methods that influence the molecular

weight, monosaccharide composition, physiochemical properties, or conformation of

polysaccharides will lead to differences in antioxidant activity.

Conflict of interest

of
The authors declare that they have no conflict of interest.

ro
Acknowledgments -p
This work was supported by the State key research and development plan "modern food
re
processing and food storage and transportation technology and equipment"

(No.2017YFD0400203).
lP

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Legends to Figures

Fig.1. Purification of the ginger pomace polysaccharides obtained from two extraction

processes. (a) The crude polysaccharide was applied to a column of DEAE-cellulose-52

and eluted as described in Section 2.3. The fractions containing the polysaccharides were

pooled and named as HW-F1, HW-F2, HW-F3, and UA-F1, UA-F2, UA-F3,

respectively. (b) HW-F1 and UA-F1 obtained on DEAE-cellulose-52 was applied to a

Dextran G-100 and eluted as described in Section 2.3. (c) HW-F2 and UA-F2 obtained

of
on DEAE-cellulose-52 was applied to a Dextran G-100 and eluted as described in

ro
Section 2.3. (d) HW-F3 and UA-F3 obtained on DEAE-cellulose-52 was applied to a
-p
Dextran G-100 and eluted as described in Section 2.3.

Fig.2 HPLC spectra of monosaccharide composition: monosaccharide reference (a),

re

HW-GPP1 (b), HW-GPP2 (c), HW-GPP3 (d), UA-GPP1 (e), UA-GPP2 (f), and
lP

UA-GPP3 (g). (1, Mannose; 2, Ribose; 3, Rhamnose; 4, Glucuronic acid; 5,

Galacturonic acid; 6, Glucose; 7, Galactose; 8, Xylose; 9, Arabinose; 10, Fucose)

na

Fig 3 UV spectra of ginger dregs polysaccharide

ur

Fig 4 FT-IR spectra of GPPs fractions (a: HW-GPP1; b: HW-GPP2; c: HW-GPP3; d:

Jo

UA-GPP1; e: UA-GPP2; f: UA-GPP3)

Fig. 5 Antioxidant activities of ginger pomace polysaccharide fractions: DPPH radical

scavenging activity (a); Hydroxyl radical scavenging activity (b); Superoxide radical

scavenging activity (c).

25
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1.4 a UA-GPPs HW-GPPs NaCl 0.7

0.4
b
1.2 UA-F2 0.6
HW-F2 UA-GPP1

NaCl concentration/M
Absorbance at 490 nm

1.0 0.5 0.3

Absorbance at 490 nm
UA-F1
HW-F1 HW-GPP1
0.8 0.4
0.2
0.6 0.3
UA-F3

0.4 HW-F3 0.2 0.1

0.2 0.1

of
0.0
0.0 0.0 0 5 10 15 20 25 30
0 50 100 150 200 250 300
Tuber number
Tube number

ro
1.00
c
-p 0.8
d

UA-GPP2
Absorbance at 490 nm

UA-GPP3
re
0.6
0.75
Absorbance at 490 nm

HW-GPP3
lP

0.50 0.4
HW-GPP2

0.25 0.2
na

0.00 0.0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
ur

Tube number Tube number

Fig.1. Purification of the ginger pomace polysaccharides obtained from two extraction processes. (a) The crude
Jo

polysaccharide was applied to a column of DEAE-cellulose-52 and eluted as described in Section 2.3. The fractions

containing the polysaccharides were pooled and named as HW-F1, HW-F2, HW-F3, and UA-F1, UA-F2, UA-F3,

respectively. (b) HW-F1 and UA-F1 obtained on DEAE-cellulose-52 was applied to a Dextran G-100 and eluted as

described in Section 2.3. (c) HW-F2 and UA-F2 obtained on DEAE-cellulose-52 was applied to a Dextran G-100 and

eluted as described in Section 2.3. (d) HW-F3 and UA-F3 obtained on DEAE-cellulose-52 was applied to a Dextran

G-100 and eluted as described in Section 2.3.

26
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mAU
mAU
400
a 5 200
b 6

300 150
1
2
200 100
3
8 1
4 6 9
7
100 10 50 3

0 0

10 20 30 40 50 60 70 0 10 20 30 40 50 60 70

min min

mAU
250
c 6
mAU
100
d

of
200 6
80
150
60

ro
100
3
40
7 1 7 9
50 1 3 8
20 8

0
0
-p
0 10 20 30 40 50 60 70
0 10 20 30 40 50 60 70
min
min
re
mAU
mAU
300
300 e f 6
6
lP

250
250

200
200

150
150
na

100
100
1 1
50 3 8
50 3 9
ur

0
0

0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
min min
Jo

mAU
250 g
6
200

150

100

8
50 3
1 7 9

0 10 20 30 40 50 60 70
min

Fig.2 HPLC spectra of monosaccharide composition: monosaccharide reference (a), HW-GPP1 (b),
HW-GPP2 (c), HW-GPP3 (d), UA-GPP1 (e), UA-GPP2 (f), and UA-GPP3 (g). (1, Mannose; 2,
Ribose; 3, Rhamnose; 4, Glucuronic acid; 5, Galacturonic acid; 6, Glucose; 7, Galactose; 8, Xylose;
9, Arabinose; 10, Fucose)

27
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3.0 b
2.5 a

2.5
2.0

Absorbance
2.0
Absorbance

HW-GPP1
1.5
1.5 UA-GPP1
HW-GPP3
1.0 UA-GPP3
HW-GPP2 1.0
UA-GPP2
0.5 0.5

0.0 0.0

of
200 250 300 350 400 200 250 300 350 400
Wavelength/nm wavelength/nm

ro
Fig 3 UV spectra of ginger dregs polysaccharide
-p
re
lP
na
ur
Jo

28
 % Transmittance % Transmittance
% Transmittance

65
70
75
80
85
90
95
100
105
110
60
70
80
90
100
110
0
20
40
60
80
100

4000

4000
4000

e
a

3500
3421.01

3500
3500
3392.64 3282.63

2985.81

3000
3000

3000
2920.08 2926.27

2500
2363.85

2500

2500
2350.68 2350.68

2000
Wavenumbers/cm
-1
2000
Wavenumbers/cm

2000
Wavenumbers/cm
-1

-1
1638.73
Jo
1528.73 1632.54
1626.34 1457.46

1500
1535.70 1399.54 1355.05 1509.36 1418.72

1500

1500
1386.96 1255.05 1367.59 1315.68
1237.44 1069.34 1147.58
1140.47

1000
ur 1101.87 1076.31
1056.94 817.59 1030.60
739.31
1000

908.20 895.03 927.57

1000
888.06 661.85 823.76
778.05 829.95

500
na 500

500
lP
% Transmittance

29
% Transmittance

0
20
40
60
80

-20

50
60
70
80
90
100
110
120
130
4000
% Transmittance
d

4000
re
65
70
75
80
85
90
95
100
105

f
4000

3424.40

3500
b

3500
3346.16
-p
Journal Pre-proof

3500

3398.06

3000
2958.81
2938.67

3000
ro 2925.50
3000

2500
2331.31

d: UA-GPP1; e: UA-GPP2; f: UA-GPP3)

2500
2330.54
2500

of
2349.90
2000
Wavenumbers/cm
-1

2000
1632.54

Wavenumbers/cm
-1
2000
Wavenumbers/cm
-1

1399.35
1500

1637.96 1347.45
1527.95 1147.58 1637.96
1444.29

1500
1076.30 1534.93
1405.55 1450.48
1500

1030.60 1417.95
1230.47
1000

1346.67 1307.94
1217.30
1049.19
1049.19

1000
894.25
1000

822.98 887.28
500

816.78
757.91
661.07

500
500

Fig 4 FT-IR spectra of GPPs fractions (a: HW-GPP1; b: HW-GPP2; c: HW-GPP3;

 Journal Pre-proof

100
a 

 

80 


Scavenging rate/%
 

 
60  





  
40 

 

 
 

   

 
20 


HW-GPP1 HW-GPP2 HW-GPP3

  UA-GPP1 UA-GPP2 UA-GPP3
  Ascorbic acid



0 

0.0 0.5 1.0 1.5 2.0

Concentration (mg/mL)

of
100
b 

 

ro
Scavenging rate/%

80 




60 

-p 







 

 
40  
   
 

 
re




  HW-GPP1 HW-GPP2 HW-GPP3
20  
 UA-GPP1 UA-GPP2 UA-GPP3



 
Ascorbic acid




lP

0 

0.0 0.5 1.0 1.5 2.0

Concentration (mg/mL)
na

100
c 




80


ur Scavenging rate/%


60 
 

 


  
 
Jo

40    
 
 


 

 
 

 
20   HW-GPP1 HW-GPP2 HW-GPP3
 
 UA-GPP1 UA-GPP2 UA-GPP3

 Ascorbic acid


0 

0.0 0.5 1.0 1.5 2.0

Concentration (mg/mL)

Fig. 5 Antioxidant activities of ginger pomace polysaccharide fractions: DPPH radical

scavenging activity (a); Hydroxyl radical scavenging activity (b); Superoxide radical

scavenging activity (c).

30