Accepted Manuscript

Title: Preparation, deproteinization, characterisation, and

antioxidant activity of polysaccharide from cucumber
(Cucumis saticus L.)

Authors: Ling Chen, Gangliang Huang, Jinchuan Hu

PII: S0141-8130(17)34070-9
DOI: https://doi.org/10.1016/j.ijbiomac.2017.12.034
Reference: BIOMAC 8695

To appear in: International Journal of Biological Macromolecules

Received date: 30-10-2017

Revised date: 18-11-2017
Accepted date: 5-12-2017

Please cite this article as: Ling Chen, Gangliang Huang, Jinchuan Hu, Preparation,
deproteinization, characterisation, and antioxidant activity of polysaccharide
from cucumber (Cucumis saticus L.), International Journal of Biological
Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.12.034

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Preparation, deproteinization, characterisation, and antioxidant

activity of polysaccharide from cucumber (Cucumis saticus L.)

Ling Chen, Gangliang Huang*, Jinchuan Hu

Active Carbohydrate Research Center, College of Chemistry, Chongqing Normal University,

Chongqing, 401331, China

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E-mail: huangdoctor226@163.com

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Highlights

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 The optimal deproteinization method of cucumber polysaccharide was discussed.

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The cucumber polysaccharide was β-glycosidic linkage.
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 The cucumber polysaccharide consists of 8 monosaccharides.
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 The cucumber polysaccharide had high scavenging ability to superoxide anions.
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Abstract: Preparation, deproteinization and antioxidant activity of polysaccharide from cucumber

(Cucumis saticus L.) were investigated. The crude cucumber polysaccharide was extracted by
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hydrothermal method. It showed that the trichloroacetic acid (TCA) method had the higher

deproteinization percentage, but a little higher polysaccharide loss percentage than the CaCl2
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method. The cucumber polysaccharide is linked by the β-glycosidic linkage. It consisted of

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D-glucose, D-mannose, D-galactose, L-rhamnose, D-xylose, L-arabinose, D-glucuronic acid, and

D-galacturonic acid. Their mole ratio was 6.00:4.03:8.31:2.82:2.75:6.60:1.05:5.79. Moreover, it

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proved that the cucumber polysaccharide had high scavenging ability to superoxide anions.

Keywords: polysaccharide from cucumber (Cucumis saticus L.), preparation, deproteinization,

characterisation, antioxidant activity

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1. Introduction

Polysaccharides exist in almost all organisms, and they have many biological

functions, for example, energy storage, structure support, antigenic determinant, etc

[1]. Because the separation, purification, composition analysis, and structure

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determination of polysaccharides have achieved rapid development, moreover, their

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biological functions are further discovered and clarified, the research on

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polysaccharides has drawn great attention [2]. Polysaccharides and their derivatives

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also have some special biological functions, such as anti-inflammation,

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anti-radialization, lowering blood lipid, acting as the anticoagulant, reactive oxygen
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species scavengers [3], biological response modifiers, and so on. So, they have
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become one of the hot spots for natural medicine research in recent years.
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Cucumber (Cucumis saticus L.) is the botanic family of Cucurbitaceous. The

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carbohydrate, protein, amino acids, vitamins, and other substances are its main

ingredients [4]. The cucumber shows a strong scavenging effect on free radicals. It
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indicated that the carbohydrates in cucumber had many physiological functions [5].
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For example, they could clear heat and water, relieve poison, reduce swell, and
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regenerate body fluid for quenching thirst. The cucumber carbohydrates can treat

many illnesses, such as sore throat, common eye diseases, parenchymal jaundice,
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difficulty of urination, body heat, polydipsia, and so on. As cucumber carbohydrates

have the above-mentioned various biological functions, the corresponding preparation

and scavenging effect on superoxide anions were investigated herein. The Sevage

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method is a commonly used deproteinization method [2], but the poisonous

chloroform is used, and it is easy to pollute the environment. So, the trichloroacetic

acid (TCA) method and CaCl2 method were used in turn to investigate the

deproteinization for crude cucumber polysaccharide. Moreover, The purity, molecular

weight, sugar content, and monosaccharide composition of cucumber polysaccharide

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were analyzed, respectively.

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2. Materials and Methods

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2.1. General procedure

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Cucumber was bought at the local vegetable market. IR spectra were recorded with
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an AVATAR 360 FT-IR-Ⅱ apparatus. The sample was mixed with KBr, and was
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prepared in solid form. The wavenumbers are reported in 1/cm, and the range of data
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collection was from 4000 1/cm - 500 1/cm. The protein concentration (cp) was
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determined by ultraviolet (UV) absorption, and the relationship was as follows: cp =

1.45A280 - 0.74A260, where A280 and A260 were the absorbances at 280 and 260 nm,
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respectively. This method was corrected for any interfering absorbance because the
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nucleic acid was present in the solution [6]. The concentration of cucumber
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polysaccharide (ccp) was determined by the phenol-sulfuric acid method, and glucose

was as used the standard [7]. The total sugar content of sample (%) = (ccp × vsample
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solution)/msample × 100. The protein content of sample (%) = (cp × vsample solution)/msample ×

100. The unit of cp and ccp was mg/mL. The molecular weight of polysaccharide was

determined by viscosity method with Ubbelohde viscometer [8].

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2.2. Extraction of the crude polysaccharide from cucumber

The cucumber was washed and chopped in granular form. The granular cucumber

mashed by the triturator. The mashed mixture was defatted with the mixed solution of

diethyl ether and petroleum ether under reflux condition, and the volume of diethyl

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ether was one time that of petroleum ether. After filtration, the mash was dried. The

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200 g defatted mixture was heated with water (2 L) at 80 ℃ for 3 h, and cooled to

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room temperature. After the reaction mixture was centrifuged and the supernatant

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liquor was concentrated, the cucumber polysaccharide was precipitated with

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anhydrous ethanol. The volume of ethanol was four times that of water. The
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precipitate was washed with anhydrous ethanol and diethyl ether, respectively. The
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mixture was dried to offer 7.1 g crude product.

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2.3. Deproteinization by the TCA method

The concentrated solution of crude polysaccharide from cucumber was adjusted to

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pH 3 with 10% TCA solution, and was placed overnight. The sample was centrifuged
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at 5,000 rpm for 10 min, and the supernatant was collected to provide the
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deproteinized solution. The deproteinized polysaccharide was offered according to the

above-mentioned process. This procedure was repeated 2 - 3 times.

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2.4. Deproteinization by the CaCl2 method

The concentrated solution of crude polysaccharide from cucumber was adjusted to

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pH 8 - 9 with 2 % NaOH solution, and was heated to 85 ℃. The CaCl2 solid was

added up to the concentration of 5 % (w/v), then mixed round and boiled for 30 min.

After the mixture was cooled to room temperature and filtrated, the filtrate was

adjusted to pH 7 with 2 mol/L dilute HCl. The latter process was similar to the

above-mentioned method.

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2.5. Separation of the deproteinized polysaccharide

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The deproteinized polysaccharide was separated by column chromatography on

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Sephadex G200, and washed with distilled water. The polysaccharide solution was

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collected, which was identified to contain a dominating composition by the
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phenol-sulfuric acid method. The pure polysaccharide was provided according to the
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above-mentioned process.
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2.6. Determination of the cucumber polysaccharide content

Polysaccharide standard curve was made with glucose as the standard. The
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polysaccharide content was determined by the phenol-sulfuric acid method, and the
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absorbance value A was measured at 490 nm with a spectrophotometer. The

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regression equation was provided. When the polysaccharide content of sample was

determined, 10.00 mg cucumber polysaccharide was accurately weighed , and set to

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100 mL. After the 1.00 mL cucumber polysaccharide solution was accurately

absorbed, phenol-sulfuric acid reagent was added, and the distilled water was used as

a blank. The mixture was heated in boiling water bath for 10 min, absorbance at 490

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nm was measured, the process was repeated for 3 times, and the sugar content was

calculated.

2.7. The monosaccharide composition of polysaccharide

After thorough hydrolysis of the polysaccharide with hydrochloric acid, all

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monosaccharides were derivatized to acetylated aldononitriles. Gas chromatographic

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conditions: 2m × 4mm stainless steel column, vaporization temperature of 250 ℃;

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detector temperature of 230 ℃; nitrogen flow rate of 30mL/min; column temperature

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of 200 ℃. The D-mannose, L-rhamnose, D-glucuronic acid, D-galacturonic acid,

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D-glucose, D-xylose, D-galactose, and L-arabinose were used as controls.
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2.8. The scavenging ability toward superoxide anions

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The superoxide anions were generated to use a chemiluminescence system of

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Me2SO-NaOH-O2-luminol [9]. The reaction mixture contained 0.1 mol/L NaOH (10

mL), 1×10-2 mol/L luminol (10 mL), and 70 mL phosphate buffer with pH 8.6. The
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TCA-deproteinizing cucumber polysaccharide with different concentrations (0-28

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mg/mL, 10 mL) was injected into the mixture, respectively. The final volume was the
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same (1 mL) for all assays. The sample cells were immediately placed in the

Ultra-Weak Luminescence Analyzer (Beijing, China). The chemiluminescence

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intensity was simultaneously recorded every 2 s by the processor. The

CaCl2-deproteinizing cucumber polysaccharide also was assayed according to the

above-mentioned process. The area of chemiluminescence (S) denoted the amount of

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superoxide anions. The distilled water, which didn’t contain the deproteinized

cucumber polysaccharide, was also recorded for a control comparison. The

scavenging ratio (%) = (Sdistilled water - Spolysaccharide) /Sdistilled water × 100.

3. Results and Discussion

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3.1. IR spectra analysis of the cucumber polysaccharide deproteinized by two

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methods

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The crude polysaccharide from cucumber was prepared by the hot-water extraction

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method. Starting with hot water for the initial extraction, this would possibly denature
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some proteins and remove them by centrifugation. The crude polysaccharide was
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deproteinized by TCA method and CaCl2 method, respectively. The deproteinized

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polysaccharide was separated by column chromatography on Sephadex G200 to give

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the water-soluble cucumber polysaccharide with high purity. The IR spectra of

cucumber polysaccharide were showed in Fig. 1, which were obtained from the crude
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product and above-mentioned two deproteinized methods, respectively. In every

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spectrum, it contained absorption band arising from the ν(COC) stretching vibration at
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1050 cm-1 or so, band at 893 cm-1 or so assigned to the corresponding β-glycosidic

bond (C1-H) deformation mode, and the ν(OH) band at lower frequency (3380 cm-1 or
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so), which had the highest intensity. The presence of carbonyl group band at 1650

cm-1 or so proved to have the residual protein in every sample of cucumber

polysaccharide.

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3.2. Comparison of the two methods for deproteinization

The TCA and CaCl2 were used for the deproteinization to crude polysaccharide from

cucumber, respectively. The principle of TCA method is that the protein cation can

bind TCA to form an insoluble salt at pH < pI (namely isoelectric point). The CaCl2

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method is used to deproteinize because the protein can be salted out. The results were

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shown in Table 1. It showed that the protein content of sample by CaCl2 method was

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higher than the content by TCA method. Moreover, it was confirmed that the TCA

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method had the higher percentage of deproteinization, but a little higher percentage of

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polysaccharide loss than the CaCl2 method, which was due to TCA’s ability to
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hydrolyze polysaccharides to some extent.
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3.3. The purity, molecular weight, linkage mode, and monosaccharide composition of
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cucumber polysaccharide
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The purity, molecular weight, linkage mode, and monosaccharide composition of

cucumber polysaccharide were shown in Table 2. In IR spectra, band at 893 cm-1 or so

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was assigned to the corresponding β-glycosidic bond (C1-H) deformation mode.

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According to the gas chromatography analysis, the cucumber polysaccharide

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consisted of D-glucose, D-mannose, D-galactose, L-rhamnose, D-xylose, L-arabinose,

D-glucuronic acid, and D-galacturonic acid. Their mole ratio was

6.00:4.03:8.31:2.82:2.75:6.60:1.05:5.79.

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3.4. Scavenging ability to superoxide anions

Using the chemiluminescence system of Me2SO-NaOH-O2-luminol, the superoxide

anions were measured at pH 8.6. The scavenging ratios of TCA-deproteinizing

cucumber polysaccharide and CaCl2-deproteinizing cucumber polysaccharide to

superoxide anions with different concentrations were shown in Table 3, respectively.

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The scavenging ratio of TCA-deproteinizing cucumber polysaccharide was much

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higher than that of CaCl2-deproteinizing cucumber polysaccharide at different

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concentration. It indicated that a marked increase of scavenging ratio was observed

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when the concentration of TCA-deproteinizing cucumber polysaccharide was added

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from 1 mg/mL to 15 mg/mL, but the scavenging ratio was increased a little from 15
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mg/mL to 28 mg/mL. The scavenging ratio was about 89 % when the concentration of
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TCA-deproteinizing polysaccharide was 15 mg/mL. This was proved that the

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deproteinized cucumber polysaccharide could be acted as an antioxidant.

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4. Conclusion
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The polysaccharide from cucumber was prepared by the hot-water extraction method.
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The TCA method had the better ability to deproteinization for the crude
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polysaccharide from cucumber, but its percentage of polysaccharide loss was the

higher than CaCl2 methods. At the same time, it showed that the CaCl2 method was a
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more perfect deproteinization method for cucumber polysaccharide by comparison of

the above-mentioned two methods, because the percentage of deproteinization was

about 90%, but the percentage of polysaccharide loss was about 4.3% by CaCl2

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method. The polysaccharide was linked by the β-glycosidic linkage. It consisted of

D-glucose, D-mannose, D-galactose, L-rhamnose, D-xylose, L-arabinose,

D-glucuronic acid, and D-galacturonic acid. Moreover, it was proved that the

cucumber polysaccharide had high scavenging ability to superoxide anions by

chemiluminescence method.

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Acknowledgements

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The Project Sponsored by the Scientific Research Foundation for the Returned

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Overseas Chinese Scholars, State Education Ministry. The work was also supported

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by Chongqing Key Research Project of Basic Science & Frontier Technology (No.
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cstc2017jcyjBX0012), Scientific and Technological Research Program of Chongqing
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Municipal Education Commission (Grant No. KJ1400523), Foundation Project of

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Chongqing Normal University (No. 14XYY020), Chongqing General Research

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Project of Basic Science & Frontier Technology (No. cstc2015jcyjA10054),

Chongqing Normal University Postgraduate's Research and Innovation Project (No.

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YKC17004), Open Foundation Project of University Engineering Research Center for

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Bioactive Substances in Chongqing (No. AS201614), Chongqing University Students'

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Training Project of Innovation & Undertaking (No. 201610637076), China.

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of different fruit juices used as carbon source on cucumber seedling under in-vitro cultures, Afr.

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carbohydrate status and fermentative enzyme activities in cucumber (Cucumis saticus L.)

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seedlings under hypoxia, Plant Growth Regul. 57 (2009) 259-269.
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[6] G.L. Huang, J.T. Tan, X.C. Tan, D.Q. Peng, Preparation of polysaccharides from wax gourd,
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Int. J. Food Sci. Nutr. 62 (2011) 480-483.

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[7] G.L. Huang, Q. Yang, Z.B. Wang, Extraction and deproteinization of mannan oligosaccharides,
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Z. Naturforsch. 65c (2010) 387-390.

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147-163.

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 A Figures

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Fig. 1. IR spectra (in KBr) of the cucumber polysaccharide deproteinized by (A) the crude product,
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(B) the TCA method, and (C) the CaCl2 method, respectively.
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Tables

Table 1. Comparison of two deproteinization methods a.

Method Total sugar content Protein content Percentage of Percentage of

of sample (%) of sample (%) deproteinization (%) polysaccharide loss (%)

TCA 94.6±1.2 1.3±0.5 95.3±0.7 18.9±0.5

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CaCl2 88.2±0.7 4.7±0.8 89.6±0.6 4.3±0.9

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a Values are the mean ± standard deviations of three separate determinations.

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Table 2. The purity, molecular weight, sugar content, and monosaccharide composition of

cucumber polysaccharide.
Method purity% molecular weight linkage mode
β-glycosidic bond
U monosaccharide composition
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TCA 94.6±1.2 2.37×104 D-glucose, D-mannose,

CaCl2 88.2±0.7 2.96×104 D-galactose, L-rhamnose,

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D-xylose, L-arabinose
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D-glucuronic acid,

D-galacturonic acid
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Table 3. The scavenging effect of two deproteinized cucumber polysaccharides on superoxide

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anions.
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Polysaccharide concentration Scavenging ratio (%)

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(mg/mL) TCA CaCl2

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 1 18 7

3 30 13

5 40 27

7.5 53 40

10 62 50

11 70 58

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13 80 64

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15 89 65

17.5 89.3 66

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20 89.5 68

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21 89.6 70

23 89.8 70.2

25 89.9
U 70.5
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28 90 71
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E PT
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A

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