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Structural characterization and antioxidant activity of oligosaccharides from Panax ginseng C. A. Meyer
Journal Pre-proof Structural characterization and antioxidant oligosaccharides from Panax ginseng C. A. Meyer
activity
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Bin Zhao, Xinying Wang, Hao Liu, Chongning Lv, Jincai Lu PII:
S0141-8130(19)37822-5
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.02.016
Reference:
BIOMAC 14632
To appear in:
International Journal of Biological Macromolecules
Received date:
26 September 2019
Revised date:
1 February 2020
Accepted date:
3 February 2020
Please cite this article as: B. Zhao, X. Wang, H. Liu, et al., Structural characterization and antioxidant activity of oligosaccharides from Panax ginseng C. A. Meyer, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/ j.ijbiomac.2020.02.016
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© 2020 Published by Elsevier.
Journal Pre-proof Structural characterization and antioxidant activity of Oligosaccharides from Panax ginseng C. A. Meyer
Bin Zhao, Xinying Wang, Hao Liu, Chongning Lv, Jincai Lu* School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
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Shenyang 110016, PR China.
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*Corresponding author: Professor Jincai Lu, School of Traditional Chinese Materia
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Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China. E-mail:
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[email protected]. Tel (Fax): +024-23986500.
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Abstract
The purpose of present work was to investigate the antioxidant activity of
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oligosaccharides from mountain-cultivated ginseng (MCG) and cultivated ginseng
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(CG). The antioxidant activity of total oligosaccharides from MCG and CG were
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compared preliminary. And then, the total oligosaccharides of MCG, which displayed stronger activity than that of CG, were separated by Carbon–Celite column and eluted
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with water and ethanol of different concentrations (30%, 50%, 70%, 95%, v/v). Five fractions, MCGOS-H2O, MCGOS-30, MCGOS-50, MCGOS-70, MCGOS-95, were
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obtained. Seven oligosaccharides were purified from MCGOS-30~MCGOS-95. The structure features of oligosaccharides (MCGO-1~MCGO-7) were characterized using high
performance
liquid
chromatography
(HPLC),
methylation
and
gas
chromatography-mass (GC–MS), as well as nuclear magnetic resonance spectroscopy. ABTS radical scavenging assay, DPPH radical scavenging assay as well as ferric reducing antioxidant power assay were adopted for antioxidant activity of all the different oligosaccharides sub-fraction. The result showed that the fractions of MCGOS-70 and MCGOS-95 exhibited significant radical scavenging activity with DPPH and ABTS. In conclusion, the oligosaccharides from MCG possessed the significant antioxidant activity. Therefore, we propose that the oligosaccharides from Panax ginseng can be developed as natural antioxidants in food and pharmaceutical fields.
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1. Introduction
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Keywords: Panax ginseng; oligosaccharides; antioxidant activity
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Panax ginseng C.A. Meyer, which is the origin of ginseng, has been widely used as traditional herbal medicine and functional food in orient countries (eg., China,
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Japan and Korea). P. ginseng is famous for its abundant active components, such as ginsenosides, polysaccharides, oligosaccharides, volatile oil, amino acids and peptides [1, 2]. Previous researches have shown that oligosaccharides obtained from P. ginseng possessed immunoregulatory effect [3, 4], antitumor activity [1]. However, the present studies on oligosaccharides from P. ginseng are limited. The quality of ginseng products varies greatly due to growth environments. Based on the growth environment and the cultivation method, ginseng is classified into three types: mountain-wild ginseng (MWG), cultivated ginseng (CG) and mountaincultivated ginseng (MCG) [5]. The Radix et Rhizome of cultivated ginseng and mountain cultivated ginseng were showed in Fig.1. CG is the artificial planting ginseng, which has a short growth period. MWG grows in the mountains without
Journal Pre-proof manual management and only under natural conditions throughout the entire growing period [5]. MCG is planted directly in forest and mountains environment without transplantation, scarification, irrigation, or fertilization during the entire periods of growth, thus suffering much severe environmental stresses as the wild ginseng [6]. Most CG is harvested after 5~6 year of cultivation. Meanwhile, MCG is collected at ages of 10~20 years or longer, while the age of MWG is generally much older [5; 6]. The age of P. ginseng can be determined by counting the number of stem scars (Rhizome Nodes) off the rhizome. Each year of ginseng growth adds a stem scar to
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the rhizome after every stem dies back in the fall. MCG can be considered as a
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substitute of wild ginseng, which is of better quality than CG. Studies have shown that MCG has stronger anticancer activity than CG [7; 8]. However, the difference in
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chemical ingredient between CG and MCG have not been studied systematically.
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Therefore, it is important to find unique chemical components between CG and MCG.
between CG and MCG.
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This will be helpful understanding the difference of pharmacological activities
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Overproduction of free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been confirmed to cause damage to the cellular
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biomolecules, resulting degenerative diseases [9]. Hyper-physiological burden of free radicals leads to an imbalance in homeostatic phenomena between oxidants and antioxidants in the body. This imbalance leads to oxidative stress which is being suggested as the root cause of aging and various human diseases such as cancer, diabetes, atherosclerosis, stroke and neurodegenerative diseases [10]. Antioxidants play an important role in inhibiting and scavenging free radicals. Especially, the natural antioxidants from food and medicinal plants extracts exist excellent activity. For example, natural antioxidants protect the living system from oxidative stress and other chronic diseases, so they can play an important role in health care system [11]. In view of the importance of antioxidant activity, we studied the antioxidant activity of total oligosaccharides from cultivated ginseng and mountain cultivated ginseng by DPPH, ABTS and FRAP assays. Therefore, in order to better understand
Journal Pre-proof the relationship between activity and structure of ginseng oligosaccharides, we preliminary separated ginseng oligosaccharide by Carbon–Celite column and studied the antioxidant activity and monosaccharide composition of next level fraction. 2. Material and methods 2.1. Materials The roots of cultivated ginseng (5 years old) were collected from Changbai
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Mountain, Jilin Province, China in February 2019. Mountain cultivated ginseng
Huanren, Liaoning province. (DPPH),
2,4,6-tripyridyl-s-triazine
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1,1-Diphenyl-2-picryl-hydrazyl
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(MCG) roots were 15 years old, and the plants were grown at Changbai mountain,
(TPTZ),
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trifluoroacetic acid (TFA), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), ferric chloride, linoleic acid, l-ascorbic acid (VC), all of the
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monosaccharide standards and other chemical reagents were analytical grade.
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2.2. Preparation for the oligosaccharide
The dried roots of mountain-cultivated ginseng (1 kg) were cut into small pieces
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and decocted with distilled water (10 L) three times. All aqueous solutions were combined, concentrated under reduced pressure and precipitated by adding of 95% ethanol (4 volumes) at 4℃ for 24 h. After centrifugation, the supernatant was concentrated under reduced pressure to a smaller volume. The concentrated solution was loaded onto a D101 macroporous resin column and eluted with water and 60% ethanol to remove the saponins. The aqueous elution from macroporous resin was evaporated into a small volume and then loaded onto a Carbon–Celite (Charcoal and Celite; 1:1) column (4.5 × 50 cm). The Carbon–Celite column was successively eluted with water and ethanol of different concentrations (30%, 50%, 70%, 95%, v/v). All fractions were collected, concentrated and lyophilized, dry sample named MCGOS-H2O, MCGOS-30, MCGOS-50, MCGOS-70, MCGOS-95, respectively. Then, they were purified by gel filtration chromatography of Sephadex G-25 (2×90
Journal Pre-proof cm). The column was eluted with distilled water at 1.0 mL/min. The eluate was collected at 5 mL per tube and assayed for total sugar contents by the phenol–sulfuric acid method. The appropriate fractions were combined and lyophilized to get seven fractions, MCGO-1~MCGO-7 (Fig.2). As shown in Fig.3, both fractions were in a single and symmetrical peak by HPGPC, suggesting that they were homogeneous oligosaccharides.
After
separation
of
homogeneous
oligosaccharides
from
MCGOS-70 and MCGOS-95, the remaining oligosaccharides were called MCGOS-70(Ⅱ) and MCGOS-95(Ⅱ), respectively. Two nucleosides were obtained by
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Sephadex G-25 from MCGOS-30 and MCGOS-95. The total oligosaccharides of
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Mountain Cultivated ginseng and Cultivated ginseng were named MCGTOS and CGTOS, respectively. Total oligosaccharides content was determined by phenol–
-p
sulfuric acid method using glucose as standard. The contents of uronic acid were
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determined by meta-hydroxydiphenyl method, using sulfamate to eliminate the
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interference from neutral sugars, galacturonic acid was used as reference [12]. Total phenolics content was assessed by the Folin–Ciocalteu method using gallic acid as the
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standard [13].
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2.3 Molecular weight determination The homogeneity and molecular weight distribution of ginseng oligosaccharides were determined by using high performance gel-permeation chromatography (HPGPC) with a TSK-gel G-3000 PWXL column (7.8 × 300 mm, TOSOH, Japan) connected to a Shimadzu HPLC system. 20 μL of sample (2 mg/mL) was injected, eluted with 0.7% Na2SO4 at a flow rate of 0.5 mL/min and monitored using a RID-20A detector (Shimadzu, Tokyo, Japan). D-glucose and Dextran standard set (National Institute of Metrology, China) was used as analytical standards. A calibration curve generated using the Log MW of the standard versus their retention time (RT) was obtained (Log MW= -0.3624 RT + 9.5749, R2 = 0.9933) [14]. 2.4 Hydrolysis procedures for oligosaccharides Hydrolysis of the oligosaccharides were performed according to the method with
Journal Pre-proof proper modification [15]. The oligosaccharide samples (2 mg) were dissolved in 2 mL of 2 M trifluoroacetic acid (TFA) in a screw-capped glass tube. The glass tube was screwed and kept at 120℃ for 1 h. Subsequently, the hydrolysate was evaporated to dryness under reduced pressure at 50℃, and then a small amount of methanol was added several times to remove the excess TFA. The residue was dissolved in 1 mL of distilled water. 2.5. Derivatization of hydrolysates with PMP
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The resulting hydrolysates were derivatized with 1-phenyl-3-methyl-5-pyrazolone
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(PMP) according to the method with some modifications [16]. 500 μL of the above sample solution was mixed with 500 μL of 0.3 M aqueous NaOH and 500 μL of 0.5 M
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methanol solution of PMP in a tube with lid. Then, the mixture was mixed thoroughly
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by a vortex mixer, and kept at 70℃ water-bath for 30 min. After cooling, the mixture was neutralized with 500 μL of 0.3 M HCl. The resulting solution was extracted with
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CHCl3 (1 mL) and the process was repeated three times, then the aqueous layer was filtered through a 0.22 μm membrane for HPLC analysis. A standard solution,
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containing 8 kinds of monosaccharide (Rha, Xyl, Ara, Glu, Gal, GlcA, GalA and Man
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about 1 mg/mL for each), was treated as mentioned above. 2.6. HPLC analysis
The analysis of PMP derivatives prepared was performed on Dionex Ultimate 3000 series HPLC system, equipped with a Diamonsil® C18 column (250 × 4.6 mm, 5 μm, Dikma, Japan), detected by a UV–vis DAD detector. 20 μL of the PMP derivatives was injected, eluted with 82.0% phosphate-buffered saline (PBS, 0.1 M, pH 7.0) and 18.0% acetonitrile (v/v) at a flow rate of 1.0 mL/min at 30℃. The wavelength for UV detection was 245 nm [17]. 2.7. Methylation and GC–MS analysis The methylation analysis of MCGO-1~MCGO-7 were carried out according to the method of Feng [18] with some modifications. The dried samples (3.0 mg) were
Journal Pre-proof dissolved in 1.0 mL anhydrous DMSO and then dried NaOH powder (20 mg) was added, and the mixture was stirred at room temperature for 3 h. Methyl iodide (1.0 mL) was then added slowly at ice bath. The solution was further stirred for 2 h. After reaction termination with water, CHCl3 was added to extract the resulting per-methylated products and washed with distilled water for three times. The extracts were passed through an anhydrous Na2SO4 column (0.8 cm × 1.0 cm) to remove water and evaporated by a stream of nitrogen.
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The dried methylated samples were analyzed by FT-IR spectroscopy to ensure that the peak of OH in the region 3500–3100 cm-1 almost disappeared. Then the
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per-methylated samples were hydrolyzed in 2.0 mL of 2.0 M TFA in a screw-capped
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glass tube at 120℃ for 1 h. Subsequently, the hydrolysate was evaporated to dryness under reduced pressure at 50℃, and then a small amount of methanol was added
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several times to remove the excess TFA. The obtained hydrolysates were reduced with
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sodium borohydride (5 mg) at room temperature for 12 h, and then acetylated with acetic anhydride (1.0 mL) at 120℃ for 2 h. Borate was removed by repeated additions
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and evaporations first of methanol-acetic acid (9:1) then methanol alone. The resultant partially methylated alditol acetates (PMAA) were re-dissolved in CHCl3 (2.0 mL),
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and the obtained aliquots were separated on a HP-5MS (30 m × 0.25 mm I.D., 0.2 μm film thickness, Agilent, USA), which were further analyzed by GC–MS system (Shimadzu, GCMS-TQ 8040, Japan) for linkage pattern. The injector was set at 250℃, and a 1 μL volume of the obtained aliquots was introduced in a split mode. The oven temperature was initially at 60℃, maintained for 2 minutes, raised to 180℃ at a rate of 10℃/min, held for 3 minutes, and then increased to 250℃ at a rate of 5℃/min and finally held at 250℃ for 3 minutes. 2.8. FT-IR spectroscopic analysis The FT-IR spectrum of the oligosaccharides was determined using a FT-IR spectrophotometer. Oligosaccharide samples were ground with potassium bromide powder, and then pressed into pellets for FT-IR determination in the frequency range
Journal Pre-proof of 4000-400 cm-1. 2.9 NMR spectroscopic analysis MCGO-1 and MCGO-6 were exchanged with deuterium by freeze-drying D2O (99.9 atom%) three times. The samples were finally dissolved in D2O. Both 1H, 13C and heteronuclear single quantum coherence (HSQC) spectra were recorded on a Brucker Avance III HD 600 spectrometer (Germany).
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2.10. Antioxidant activity in vitro
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2.10.1 DPPH radical scavenging assay
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The DPPH radical scavenging activity of ginseng oligosaccharides was investigated by the previous method with some modifications [19]. In brief, 100 μL of variable
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concentrations of ginseng oligosaccharides samples (0.025-2 mg/mL) was added to
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100 μL of a DPPH solution (0.1 mM in methanol). The mixture was shaken and incubated in the dark for 30 min, and the absorbance was measured at 517 nm against
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methanol as a blank. Vc was used as the positive control. The ability of the test sample to scavenge the DPPH radical was calculated using the following equation:
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DPPH scavenging rate (%) = [1-(Ai-Aj)/A0] × 100% where A0 was the absorbance of the control (methanol instead of sample), Ai was the absorbance of the sample plus DPPH-methanol solution. Aj was the absorbance of the sample solution only (methanol instead of DPPH-methanol solution). 2.10.2 ABTS radical scavenging assay The ABTS radical scavenging ability of ginseng oligosaccharides was measured by a reported method [20] with some modifications. Briefly, ABTS radical cation solution was prepared through the reaction of 7 mM ABTS solution and 2.45 mM potassium persulfate (1:1, v/v) at room temperature for 12~16 h in the dark. In the moment of use, the ABTS solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at 734 nm. All ginseng oligosaccharide samples (30 μL) with various concentrations (0.025–2
Journal Pre-proof mg/mL) was added to 170 μL of ABTS solution. After reaction at room temperature for 6 min, the absorbance was measured at 734 nm was recorded as Ai, A control sample containing the same amount of methanol and ABTS radical was measured as A0. The absorbance of the sample solution and methanol with the same amount of ABTS was recorded as Aj. Using Vc as the positive control. The ABTS radical scavenging activity was calculated as follows:
2.10.3. Ferric reducing antioxidant power assay
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ABTS scavenging rate (%) = [1-(Ai-Aj)/A0] × 100%
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The Ferric reducing antioxidant power assay (FRAP) was determined according to
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the method of Benzie and Strain with some modifications [21]. The FRAP working solution was prepared by mixing acetate buffer (0.3 mol/L, pH 3.6), TPTZ (10 mmol/L
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in 40 mM HCl) and FeCl3 solution (20 mmol/L) in the ratio 10:1:1 (v/v/v). Then, 1.00
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mL FRAP reagent and 100 μL ginseng oligosaccharide samples solution was mixed with reaction in the dark at 37℃ for 30 minutes. The absorbance was read at 593 nm
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and the antioxidant activity was measured using the prepared standard curve of FeSO4. The calibration curve was prepared using standard solutions of ferrous sulphate at
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concentrations of 0–4 mmol/L. All the measurements were conducted in triplicate and the results were expressed in mmol Fe2+ per liter sample. Vc was used as the positive control.
3. Results and discussion
3.1 Characteristic of the oligosaccharides According to the result of phenol-sulfuric method, the content of sugar of CGTOS and MCGTOS was 91.87% and 89.5%, respectively. The contents of uronic acid of CGTOS, MCGTOS, MCGOS-70(Ⅱ) and MCGOS-95(Ⅱ) were 4.02%, 4.19%, 6.14% and 5.32%, respectively. The total phenolics contents of MCGTOS, CGTOS, MCGOS-70(Ⅱ) and MCGOS-95(Ⅱ) were 2.10 μg/mg, 1.91 μg/mg, 3.51 μg/mg and 2.93 μg/mg, respectively. The UV-Vis spectrum was weakly absorbed in the range of
Journal Pre-proof 200-400 nm, indicating that CGTOS and MCGTOS contain trace amounts of nucleic acids or proteins [22]. In addition, trace nucleosides were isolated from MCGOS-30 and MCGOS-95, which verified the result of UV–vis spectral analysis. The nucleosides isolated from MCGOS-30 and MCGOS-95 were confirmed to have no antioxidant capacity by DPPH scavenging free radical experiments. The monosaccharide compositions of all the samples were determined by HPLC. The monosaccharide composition results of CGTOS, MCGTOS, MCGOS-H2O,
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MCGOS-30, MCGOS-50, MCGOS-70 and MCGOS-95 were summarized in Table 1. MCGO-1~MCGO-7 were mainly composed of glucose with minor galactose and
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mannose. The monosaccharide residues and ratios of MCGO-1~MCGO-7 were shown
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in Table 2.
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The molecular weight distribution of all oligosaccharide samples were analyzed using HPGPC. The molecular weight of CGTOS and MCGTOS were ranging from
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100 Da to 2200 Da. The molecular weight distribution of MCGOS-H2O, MCGOS-30, MCGOS-50, MCGOS-70 and MCGOS-95 were 107-930 Da, 100-1464 Da, 100-1794
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Da, 125-2185 Da and 100-2069 Da, respectively. The HPLC chromatograms of all the
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samples showed a non-single peak, therefore, the above five parts of the product are all mixtures and contain more components. 3.2. FT-IR spectra of CGTOS and MCGTOS FT-IR spectroscopy was usually used for the identification of characteristic groups in oligosaccharides. As shown in Fig.4, the FT-IR spectra of CGTOS and MCGTOS displayed a typical absorption peaks of oligosaccharides in the range of 4000– 400 cm−1. A broad and intense peak around at 3401-3408 cm−1 was assigned to the stretching vibration of hydroxyl groups [23]. The weak absorption peaks at 2927-2928 cm−1 were characteristic of C-H stretching vibration. Furthermore, a stretching peak appeared at 1633-1630 cm−1 and a weak stretching peak at 1408 cm-1 were ascribed to the deprotonated carboxylic group, suggesting the presence of uronic acid in the oligosaccharide structure [24]. The absorbance in the range of 1145–
Journal Pre-proof 1060 cm−1 indicated the pyranose unit [25, 26]. The absorption peak at 1105 and 1039 cm−1 represented uronic acid [27]. The absorption at 920 cm−1 referred to the amount of D-glucopyranosyl [25]. 3.3 Methylation analysis Methylation analysis was performed to determine the linkage pattern of MCGO-1~MCGO-7, and the linkage patterns were summarized in Table 2. The procedure
involved
derivation
of
the
monosaccharide
component
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MCGO-1~MCGO-7 to partially methylated alditol acetates, which were analyzed by
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GC-MS. There were six types of partially methylated alditol acetates, namely 2,3,4,6-Me4-Glcp, 2,3,6-Me3-Glcp, 2,3-Me2-Glcp, 3,4,6-Me3-Manp, 2,3,4-Me3-Gal
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and 2,4,6-Me3-Gal; which were assigned to 1→linked Glcp, (1→4)-linked Glcp,
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(1→4,6)-linked Glcp, (1→2)-linked Manp, (1→6)-linked Galp and (1→3)-linked
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Galp residues, respectively. 3.4 NMR analysis
H, 13C NMR and 2D NMR (HSQC) spectra were used to confirm the structure of
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1
MCGO-1 and MCGO-6. The 1H NMR spectrum (Fig. 5A) showed H-1 signals from 13
C NMR spectrum (Fig. 5B) showed C-1 signals from 90.0 to
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4.5–5.5 ppm and the
110.0 ppm. In general, chemical shifts between 4.4-4.9 ppm are typical of the anomeric protons of these β-linked residues, whereas α-anomeric protons will appear in the region of 4.9-5.5 ppm [28]. As shown in 1H NMR, MCGO-1 and MCGO-6 had anomeric proton signals at δ 4.64, in which the characteristic signals ranging from 4.4 ppm to 4.9 ppm showed the presence of β-configuration. MCGO-1 and MCGO-6 had four anomeric proton signals appeared in the anomeric region at 4.96, 5.22 and 5.35-5.39 ppm, in which the characteristic signals ranging from 4.9 ppm to 5.5 ppm showed the presence of α-configuration. The spectra of MCGO-1 were partially assigned on the basis of the HSQC experiment (Fig. 5C) and by comparison with previous studies. The signals at H-1/C-1 5.35-5.39 ppm and 99.32-99.90 ppm were assigned to →4)-α-D-Glcp-(1→ residues [29, 30]. Similarly, two signals at H-1 5.22
Journal Pre-proof and C-1 91.85 ppm were assigned to →4,6)-α-D-Glcp-(1→ residues [30]. The 1
H-NMR spectra revealed signal for anomeric protons of terminal β-D-Glcp residues
at δ 4.64 ppm, while the corresponding chemical shift in the anomeric carbon was δ 95.71 ppm [29]. Taking the results of methylation analysis into consideration, there were 1,2-Manp residue and 1,6- Galp residue in MCGO-1. The signals at δ 5.22/100.49
and
4.96/98.30
ppm
were
assigned
to
the
H-1/C-1
of
→2)-α-D-Manp-(1→ residue and →6)-α-D-Galp-(1→ residue, respectively [31, 32]. As shown in 1H and
13
C NMR, MCGO-6 and MCGO-1 had similar structural
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properties. Five sugar residues were inferred to be 1,4-Glcp, 1,4,6-Glcp, 1, 6-Galp, 1,
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2-Manp and T- β -D-Glcp, which was consistent with the results of methylation
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analysis. The branches were attached to the backbone at the O-6 position of Glcp
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residues.
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3.5 Antioxidant activity
3.5.1 DPPH radical scavenging activity
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DPPH radical is stable free radical at room temperature and is commonly used as a tool to evaluate the scavenging activity of antioxidants. Scavenging activity of all
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ginseng oligosaccharide samples and Vc on DPPH radicals was determined and the results were shown in Fig.6. All samples showed concentration-dependent DPPH radical scavenging activity. When the concentration increased to 2 mg/mL, the DPPH radical scavenging activity of MCGOS-70 and MCGOS-95 were almost no longer increased, and their DPPH radical-scavenging activity was close to that of Vc. The 50% inhibition (IC50) values of MCGOS-95 and MCGOS-70 for DPPH were 0.155 and 0.154 mg/mL, respectively. The scavenging activity of DPPH radicals by different oligosaccharide samples were in the following order at 2.0 mg/mL: MCGOS-70 > MCGOS-95 > MCGOS-30 > MCGTOS > MCGOS-50 > CGTOS > MCGOS-H2O. The results indicated that MCGOS-70 and MCGOS-95 fractions had a noticeable effect on scavenging DPPH free radicals, especially at high concentrations. The MCGTOS showed the stronger DPPH radical scavenging activity than CGTOS.
Journal Pre-proof Previous studies have showed that the antioxidant activity of polysaccharides were related to their uronic acid content, monosaccharide composition, molecular weight and the type of glycosidic linkage [
]. The antioxidant activity of
oligosaccharides was also related to the content of uronic acid, which was confirmed by the experimental results. The antioxidant activity of MCGO-1~MCGO-7, MCGOS-70(II) and MCGOS-90(II) were investigated by DPPH radical scavenging activity. The antioxidant activity of MCGO-1~MCGO-7 were lower than that of MCGOS-70(II) and MCGOS-95(II). In addition, the antioxidant activity of
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MCGOS-70(II) was higher than that of MCGOS-70, and MCGOS-95(II) was higher
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than MCGOS-95. MCGO-1~MCGO-7 were all neutral oligosaccharides with no obvious antioxidant. The total content of phenolics in MCGOS-70(II), MCGOS-95(II),
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CGTOS and MCGTOS decreased in the order MCGOS-70(II) > MCGOS-95(II) >
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MCGTOS > CGTOS. A similar trend was observed for the uronic acid concentration,
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indicating that the DPPH radical scavenging activities of MCGOS-70(II), MCGOS-95(II), CGTOS and MCGTOS could be attributed to their uronic acid and
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phenolics. However, phenolics component was extremely low, indicating that the uronic acid constituents in each fraction play a dominant role.
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3.5.2 ABTS radical scavenging activity The ABTS radical is often used to estimate the total antioxidant power of compounds. The ABTS radical scavenging abilities of all the samples compared with VC as a control standard, as shown in Fig.7. All the sample exhibited dose-dependent increases in their ABTS radical scavenging activity for concentrations in the range of 0.025–2.00 mg/mL. The ABTS scavenging ability of MCGOS-70 at the concentration of 2 mg/mL was slightly lower than that of Vc, which showed that the MCGOS-70 had good scavenging activity against ABTS free radicals. The ABTS radical scavenging rates of MCGOS-70 and MCGOS-95 were 96.17% and 90.39%, respectively, at a concentration of 2 mg/mL. When the samples concentration was 2.0 mg/mL, MCGOS-70 and MCGOS-95 showed the higher ABTS radical
Journal Pre-proof scavenging activities, whereas MCGOS-50, MCGOS-30 and MCGTOS had modest activities, and MCGOS-H2O manifested relatively low ABTS radical scavenging activity. The ABTS radical scavenging activity of MCGTOS was higher than that of CGTOS at a concentration of 0.25-2 mg/mL. The data from the methylation analysis suggested that MCGO-1~MCGO-7 was mainly composed of a Glcp backbone linked 1→4 and the branching points of the Glcp chain were at O-6 positions. The antioxidant activity of MCGO-1~MCGO-7 was studied by ABTS free radical scavenging activity, and it was found that MCGO-1~MCGO-7 did not exist
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significant antioxidant activity. Therefore, the antioxidant mechanism of MCGOS-70
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and MCGOS-95 in this study may be attributed to uronic acid contents, monosaccharide composition and glycosidic linkage types of oligosaccharides. Thus,
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a combination of several factors would affect the antioxidant activities of
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oligosaccharides [36].
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3.5.3 Ferric reducing antioxidant power (FRAP) result FRAP is a colorimetric method based on the reduction of a ferric
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2,4,6-tripyridyl-s-triazine complex (Fe3+-TPTZ) to the ferrous form (Fe2+-TPTZ) by
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antioxidants, and Fe2+-TPTZ has a strong absorption peak at 595 nm [37]. The antioxidant activity of a sample is assessed by measuring its ability to reduce Fe3+-TPTZ.
The standard curve was plotted for FeSO4 with a concentration range of 0-4 mM, and there was a strong correlation between FeSO4 concentration and antioxidant capacity (R2 = 0.9996) and the equation was Y=1.0329x+0.0678. The standard curve displayed a linear trend between 0 and 4 mM FeSO4. As can be seen from Fig.8, ferric reducing activity of all samples were lower than the positive control Vc. When the sample concentration was 0.025-2 mg/mL, there was a dose-dependent relationship between reducibility and samples concentration. The results showed that MCGOS-95 had stronger reducing power, followed by MCGOS-70. At a concentration of 2 mg/mL, the ferric reducing activity decreased in the order MCGOS-95 >
Journal Pre-proof MCGOS-70 > MCGOS-30> MCGTOS > CGTOS > MCGOS-50 > MCGOS-H2O. The ferric reducing activity of MCGTOS was higher than that of CGTOS at a concentration of 0.25-2 mg/mL. The activities of antioxidants were ascribed to various mechanisms, such as prevention of chain initiation, decomposition of peroxides, reducing capacity and radical scavenging [38]. In addition, the antioxidant effects were assigned to their hydrogen-donating abilities, previous studies have shown that the presence of uronic acid groups in the polysaccharides could activate the hydrogen atom of the anomeric carbon [39]. Therefore, the content of uronic acid
of
in oligosaccharide had a great influence on its antioxidant activity.
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Based on the results of DPPH, ABTS and FRAP assay, MCGOS-70 and
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MCGOS-95 showed significant antioxidant, suggesting its potential as an effective natural antioxidant. Among the three antioxidant models, the antioxidant activity of
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MCGTOS was stronger than that of CGTOS. However, further studies are required to
4. Conclusion
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activities.
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investigate the relationships between the chemical structure and the antioxidant
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The present work investigated the antioxidant activities of cultivated ginseng and mountain cultivated ginseng oligosaccharides. The results showed that the antioxidant activity of MCGTOS was stronger than that of CGTOS. The total oligosaccharides of MCG were separated by Carbon–Celite column and the activity of each fraction was studied. We studied the monosaccharide composition and molecular weight of oligosaccharides.
Furthermore,
the
antioxidant
activity
of
homogeneous
oligosaccharides MCGO-1~MCGO-7 isolated from MCGTOS were investigated by DPPH radical scavenging activity and found that they didn't exist strong antioxidant properties. Two oligosaccharides fractions MCGOS-70(Ⅱ) and MCGOS-95(Ⅱ) with the higher uronic acid content (6.14% and 5.32%, respectively) obtained from MCGOS-70 and MCGOS-95 showed stronger free radical scavenging activities than MCGO-1~MCGO-7 containing no uronic acid.
Journal Pre-proof Previous studies found that tea polysaccharide conjugates were found to exhibit antioxidant activities, and there was a direct relationship between the content of uronic acid and the radical-scavenging effects of tea polysaccharide conjugates [40]. Our studies were similar to Chen’s reports that the uronic acid was very important to the antioxidant activities of oligosaccharides. The antioxidant mechanism of all samples in this study may be attributed to the synergistic action of carbohydrates, uronic acid and phenolics. Overall, the antioxidant activities of oligosaccharides are not determined by a single factor but a combination of several related factors [36].
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Future research is required to better understand the relationships between the
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antioxidant activity and the structure characteristics of the ginseng oligosaccharides. These findings provided a reference for the potential applications of the
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oligosaccharides from P. ginseng to be developed as natural antioxidants in food and
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pharmaceutical area.
None.
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Acknowledgements
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Conflicts of interest
This work was granted by the National Key R&D Program of China (2017YFC1702302), Liaoning Programs for Science and Technology Development (2017226009), Foundation of Liaoning Education Department(2017LZD02) and the fourth national survey on Chinese materia medica resources in Liaoning Province (2018018). References [1] L.L. Jiao, X.Y. Zhang, B. Li, Z. Liu, M.Z. Wang, S.Y. Liu, Anti-tumour and immunomodulatory activities of oligosaccharides isolated from Panax ginseng C. A. Meyer, Int. J. Biol. Macromol. 65 (2014) 229-233. https://doi.org/10.1016/j.ijbiomac.2014.01.039.
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Journal Pre-proof Table 1. Monosaccharide composition of CGTOS, MCGTOS, MCGOS-H2O~MCGOS-95 Samples
Sugar components (%)
Gal
GlcA
GalA
Rha
Man
Ara
CGTOS
96.67
1.10
0.18
2.78
---
0.23
0.37
---
MCGTOS
92.59
1.22
0.21
3.18
---
0.67
0.52
---
MCGOS-H2O
95.51
1.42
---
---
0.27
0.30
---
MCGOS-30
95.92
1.15
0.19
---
---
0.77
0.50
0.22
MCGOS-50
94.49
1.33
0.39
---
---
0.41
0.51
---
MCGOS-70
90.55
1.97
0.62
2.71
---
0.38
0.75
2.44
MCGOS-95
86.43
1.00
0.56
2.23
---
0.22
0.81
2.69
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Glu
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ro
---
Xyl
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Table 2. GC–MS analysis of the methylated products of MCGO-1~MCGO-7
Methylated
Type of linkage
Mass fragments (m/z)
MCGO-1~ MCGO-7 (Mol.%)a
sugars
1
2,3,4,6-Me4-Glc
β-D-Glcp-(1→
43,71,87,101,117,129,145,161,205
2,3,6-Me3-Glc
→4)-α-D-Glcp-(1→
45,71,87,99,101,113,117,129,131,161,173,
→4,6)-α-D-Glcp-(1→
3,4,6-Me3-Man
→2)-α-D-Manp-(1→
2,3,4-Me3-Gal
→6)-α-D-Galp-(1→
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3
4
5
6
7
20.69
20.86
24.44
20.22
19.05
26.92
18.60
51.88
53.49
61.36
56.23
71.55
58.09
66.51
8.46
12.49
12.13
10.53
8.98
---
43,71,87,101,129,161,173,205
5.27
---
---
---
3.61
---
---
43,71,87, 101, 117, 129,161, 173,189, 233
2.50
1.94
---
2.39
2.52
1.8
1.38
233
2,3-Me2-Glc
f o
2
l a
p e
r P
n r u
43,57,71,85,99,101,117,127,159,201,261
o J
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2,4,6-Me3-Gal
a
→3)-α-D-Galp-(1→
43,58,71,87,101,113,117,129,161,233
---
2.65
---
Relative molar ratio, calculated from the ratio of peak areas.
f o
l a
o J
n r u
r P
e
o r p
2.65
0.87
---
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Caption of figures Fig. 1. Cultivated Ginseng (A) and Mountain Cultivated Ginseng (B) Fig. 2. Elution profile of MCGOS-30 (a), MCGOS-50 (b), MCGOS-70 (c), MCGOS-95 (d) on Sephadex G-25 gel chromatography column with distilled water. Fig. 3. HPGPC elution profiles of the purified oligosaccharides.
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Fig. 4. Infrared spectrum of total oligosaccharides of MCG and CG.
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Fig. 5. A. 1H NMR spectra of MCGO-1 and MCGO-6 in D2O solution.
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C. HSQC spectrum of MCGO-1.
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B. 13C NMR spectra of MCGO-1 and MCGO-6 in D2O solution
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Fig.6. DPPH scavenging effect of oligosaccharides from panax ginseng. Fig.7. ABTS• scavenging effect of oligosaccharides from panax ginseng
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ginseng.
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Fig.8. Ferric reducing antioxidant power (FRAP) of oligosaccharides from Panax
Journal Pre-proof Authors Statement Conceptualization: Bin Zhao; methodology: Bin Zhao; investigation: Bin Zhao, Xinying Wang and Hao Liu; Writing- Original draft preparation: Bin Zhao. Supervision: Jincai Lu, Chongning Lv; Writing- Reviewing and Editing: Jincai Lu. All
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authors have read and agreed to the published version of the manuscript.
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Highlights
Investigation of the antioxidant activity of oligosaccharides from different cultivation methods Total oligosaccharides of Mountain cultivated ginseng possessed better antioxidant activity than cultivated ginseng
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70% ethanol elution and 95% ethanol elution from a Carbon–Celite column exhibited significant antioxidant activity
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Preliminarily discussed the antioxidant mechanism of Panax ginseng oligosaccharides
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
activity
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Bin Zhao, Xinying Wang, Hao Liu, Chongning Lv, Jincai Lu PII:
S0141-8130(19)37822-5
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.02.016
Reference:
BIOMAC 14632
To appear in:
International Journal of Biological Macromolecules
Received date:
26 September 2019
Revised date:
1 February 2020
Accepted date:
3 February 2020
Please cite this article as: B. Zhao, X. Wang, H. Liu, et al., Structural characterization and antioxidant activity of oligosaccharides from Panax ginseng C. A. Meyer, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/ j.ijbiomac.2020.02.016
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Journal Pre-proof Structural characterization and antioxidant activity of Oligosaccharides from Panax ginseng C. A. Meyer
Bin Zhao, Xinying Wang, Hao Liu, Chongning Lv, Jincai Lu* School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
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Shenyang 110016, PR China.
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*Corresponding author: Professor Jincai Lu, School of Traditional Chinese Materia
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Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China. E-mail:
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[email protected]. Tel (Fax): +024-23986500.
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Abstract
The purpose of present work was to investigate the antioxidant activity of
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oligosaccharides from mountain-cultivated ginseng (MCG) and cultivated ginseng
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(CG). The antioxidant activity of total oligosaccharides from MCG and CG were
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compared preliminary. And then, the total oligosaccharides of MCG, which displayed stronger activity than that of CG, were separated by Carbon–Celite column and eluted
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with water and ethanol of different concentrations (30%, 50%, 70%, 95%, v/v). Five fractions, MCGOS-H2O, MCGOS-30, MCGOS-50, MCGOS-70, MCGOS-95, were
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obtained. Seven oligosaccharides were purified from MCGOS-30~MCGOS-95. The structure features of oligosaccharides (MCGO-1~MCGO-7) were characterized using high
performance
liquid
chromatography
(HPLC),
methylation
and
gas
chromatography-mass (GC–MS), as well as nuclear magnetic resonance spectroscopy. ABTS radical scavenging assay, DPPH radical scavenging assay as well as ferric reducing antioxidant power assay were adopted for antioxidant activity of all the different oligosaccharides sub-fraction. The result showed that the fractions of MCGOS-70 and MCGOS-95 exhibited significant radical scavenging activity with DPPH and ABTS. In conclusion, the oligosaccharides from MCG possessed the significant antioxidant activity. Therefore, we propose that the oligosaccharides from Panax ginseng can be developed as natural antioxidants in food and pharmaceutical fields.
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1. Introduction
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Keywords: Panax ginseng; oligosaccharides; antioxidant activity
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Panax ginseng C.A. Meyer, which is the origin of ginseng, has been widely used as traditional herbal medicine and functional food in orient countries (eg., China,
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Japan and Korea). P. ginseng is famous for its abundant active components, such as ginsenosides, polysaccharides, oligosaccharides, volatile oil, amino acids and peptides [1, 2]. Previous researches have shown that oligosaccharides obtained from P. ginseng possessed immunoregulatory effect [3, 4], antitumor activity [1]. However, the present studies on oligosaccharides from P. ginseng are limited. The quality of ginseng products varies greatly due to growth environments. Based on the growth environment and the cultivation method, ginseng is classified into three types: mountain-wild ginseng (MWG), cultivated ginseng (CG) and mountaincultivated ginseng (MCG) [5]. The Radix et Rhizome of cultivated ginseng and mountain cultivated ginseng were showed in Fig.1. CG is the artificial planting ginseng, which has a short growth period. MWG grows in the mountains without
Journal Pre-proof manual management and only under natural conditions throughout the entire growing period [5]. MCG is planted directly in forest and mountains environment without transplantation, scarification, irrigation, or fertilization during the entire periods of growth, thus suffering much severe environmental stresses as the wild ginseng [6]. Most CG is harvested after 5~6 year of cultivation. Meanwhile, MCG is collected at ages of 10~20 years or longer, while the age of MWG is generally much older [5; 6]. The age of P. ginseng can be determined by counting the number of stem scars (Rhizome Nodes) off the rhizome. Each year of ginseng growth adds a stem scar to
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the rhizome after every stem dies back in the fall. MCG can be considered as a
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substitute of wild ginseng, which is of better quality than CG. Studies have shown that MCG has stronger anticancer activity than CG [7; 8]. However, the difference in
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chemical ingredient between CG and MCG have not been studied systematically.
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Therefore, it is important to find unique chemical components between CG and MCG.
between CG and MCG.
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This will be helpful understanding the difference of pharmacological activities
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Overproduction of free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been confirmed to cause damage to the cellular
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biomolecules, resulting degenerative diseases [9]. Hyper-physiological burden of free radicals leads to an imbalance in homeostatic phenomena between oxidants and antioxidants in the body. This imbalance leads to oxidative stress which is being suggested as the root cause of aging and various human diseases such as cancer, diabetes, atherosclerosis, stroke and neurodegenerative diseases [10]. Antioxidants play an important role in inhibiting and scavenging free radicals. Especially, the natural antioxidants from food and medicinal plants extracts exist excellent activity. For example, natural antioxidants protect the living system from oxidative stress and other chronic diseases, so they can play an important role in health care system [11]. In view of the importance of antioxidant activity, we studied the antioxidant activity of total oligosaccharides from cultivated ginseng and mountain cultivated ginseng by DPPH, ABTS and FRAP assays. Therefore, in order to better understand
Journal Pre-proof the relationship between activity and structure of ginseng oligosaccharides, we preliminary separated ginseng oligosaccharide by Carbon–Celite column and studied the antioxidant activity and monosaccharide composition of next level fraction. 2. Material and methods 2.1. Materials The roots of cultivated ginseng (5 years old) were collected from Changbai
of
Mountain, Jilin Province, China in February 2019. Mountain cultivated ginseng
Huanren, Liaoning province. (DPPH),
2,4,6-tripyridyl-s-triazine
-p
1,1-Diphenyl-2-picryl-hydrazyl
ro
(MCG) roots were 15 years old, and the plants were grown at Changbai mountain,
(TPTZ),
re
trifluoroacetic acid (TFA), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), ferric chloride, linoleic acid, l-ascorbic acid (VC), all of the
lP
monosaccharide standards and other chemical reagents were analytical grade.
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2.2. Preparation for the oligosaccharide
The dried roots of mountain-cultivated ginseng (1 kg) were cut into small pieces
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and decocted with distilled water (10 L) three times. All aqueous solutions were combined, concentrated under reduced pressure and precipitated by adding of 95% ethanol (4 volumes) at 4℃ for 24 h. After centrifugation, the supernatant was concentrated under reduced pressure to a smaller volume. The concentrated solution was loaded onto a D101 macroporous resin column and eluted with water and 60% ethanol to remove the saponins. The aqueous elution from macroporous resin was evaporated into a small volume and then loaded onto a Carbon–Celite (Charcoal and Celite; 1:1) column (4.5 × 50 cm). The Carbon–Celite column was successively eluted with water and ethanol of different concentrations (30%, 50%, 70%, 95%, v/v). All fractions were collected, concentrated and lyophilized, dry sample named MCGOS-H2O, MCGOS-30, MCGOS-50, MCGOS-70, MCGOS-95, respectively. Then, they were purified by gel filtration chromatography of Sephadex G-25 (2×90
Journal Pre-proof cm). The column was eluted with distilled water at 1.0 mL/min. The eluate was collected at 5 mL per tube and assayed for total sugar contents by the phenol–sulfuric acid method. The appropriate fractions were combined and lyophilized to get seven fractions, MCGO-1~MCGO-7 (Fig.2). As shown in Fig.3, both fractions were in a single and symmetrical peak by HPGPC, suggesting that they were homogeneous oligosaccharides.
After
separation
of
homogeneous
oligosaccharides
from
MCGOS-70 and MCGOS-95, the remaining oligosaccharides were called MCGOS-70(Ⅱ) and MCGOS-95(Ⅱ), respectively. Two nucleosides were obtained by
of
Sephadex G-25 from MCGOS-30 and MCGOS-95. The total oligosaccharides of
ro
Mountain Cultivated ginseng and Cultivated ginseng were named MCGTOS and CGTOS, respectively. Total oligosaccharides content was determined by phenol–
-p
sulfuric acid method using glucose as standard. The contents of uronic acid were
re
determined by meta-hydroxydiphenyl method, using sulfamate to eliminate the
lP
interference from neutral sugars, galacturonic acid was used as reference [12]. Total phenolics content was assessed by the Folin–Ciocalteu method using gallic acid as the
na
standard [13].
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2.3 Molecular weight determination The homogeneity and molecular weight distribution of ginseng oligosaccharides were determined by using high performance gel-permeation chromatography (HPGPC) with a TSK-gel G-3000 PWXL column (7.8 × 300 mm, TOSOH, Japan) connected to a Shimadzu HPLC system. 20 μL of sample (2 mg/mL) was injected, eluted with 0.7% Na2SO4 at a flow rate of 0.5 mL/min and monitored using a RID-20A detector (Shimadzu, Tokyo, Japan). D-glucose and Dextran standard set (National Institute of Metrology, China) was used as analytical standards. A calibration curve generated using the Log MW of the standard versus their retention time (RT) was obtained (Log MW= -0.3624 RT + 9.5749, R2 = 0.9933) [14]. 2.4 Hydrolysis procedures for oligosaccharides Hydrolysis of the oligosaccharides were performed according to the method with
Journal Pre-proof proper modification [15]. The oligosaccharide samples (2 mg) were dissolved in 2 mL of 2 M trifluoroacetic acid (TFA) in a screw-capped glass tube. The glass tube was screwed and kept at 120℃ for 1 h. Subsequently, the hydrolysate was evaporated to dryness under reduced pressure at 50℃, and then a small amount of methanol was added several times to remove the excess TFA. The residue was dissolved in 1 mL of distilled water. 2.5. Derivatization of hydrolysates with PMP
of
The resulting hydrolysates were derivatized with 1-phenyl-3-methyl-5-pyrazolone
ro
(PMP) according to the method with some modifications [16]. 500 μL of the above sample solution was mixed with 500 μL of 0.3 M aqueous NaOH and 500 μL of 0.5 M
-p
methanol solution of PMP in a tube with lid. Then, the mixture was mixed thoroughly
re
by a vortex mixer, and kept at 70℃ water-bath for 30 min. After cooling, the mixture was neutralized with 500 μL of 0.3 M HCl. The resulting solution was extracted with
lP
CHCl3 (1 mL) and the process was repeated three times, then the aqueous layer was filtered through a 0.22 μm membrane for HPLC analysis. A standard solution,
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containing 8 kinds of monosaccharide (Rha, Xyl, Ara, Glu, Gal, GlcA, GalA and Man
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about 1 mg/mL for each), was treated as mentioned above. 2.6. HPLC analysis
The analysis of PMP derivatives prepared was performed on Dionex Ultimate 3000 series HPLC system, equipped with a Diamonsil® C18 column (250 × 4.6 mm, 5 μm, Dikma, Japan), detected by a UV–vis DAD detector. 20 μL of the PMP derivatives was injected, eluted with 82.0% phosphate-buffered saline (PBS, 0.1 M, pH 7.0) and 18.0% acetonitrile (v/v) at a flow rate of 1.0 mL/min at 30℃. The wavelength for UV detection was 245 nm [17]. 2.7. Methylation and GC–MS analysis The methylation analysis of MCGO-1~MCGO-7 were carried out according to the method of Feng [18] with some modifications. The dried samples (3.0 mg) were
Journal Pre-proof dissolved in 1.0 mL anhydrous DMSO and then dried NaOH powder (20 mg) was added, and the mixture was stirred at room temperature for 3 h. Methyl iodide (1.0 mL) was then added slowly at ice bath. The solution was further stirred for 2 h. After reaction termination with water, CHCl3 was added to extract the resulting per-methylated products and washed with distilled water for three times. The extracts were passed through an anhydrous Na2SO4 column (0.8 cm × 1.0 cm) to remove water and evaporated by a stream of nitrogen.
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The dried methylated samples were analyzed by FT-IR spectroscopy to ensure that the peak of OH in the region 3500–3100 cm-1 almost disappeared. Then the
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per-methylated samples were hydrolyzed in 2.0 mL of 2.0 M TFA in a screw-capped
-p
glass tube at 120℃ for 1 h. Subsequently, the hydrolysate was evaporated to dryness under reduced pressure at 50℃, and then a small amount of methanol was added
re
several times to remove the excess TFA. The obtained hydrolysates were reduced with
lP
sodium borohydride (5 mg) at room temperature for 12 h, and then acetylated with acetic anhydride (1.0 mL) at 120℃ for 2 h. Borate was removed by repeated additions
na
and evaporations first of methanol-acetic acid (9:1) then methanol alone. The resultant partially methylated alditol acetates (PMAA) were re-dissolved in CHCl3 (2.0 mL),
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and the obtained aliquots were separated on a HP-5MS (30 m × 0.25 mm I.D., 0.2 μm film thickness, Agilent, USA), which were further analyzed by GC–MS system (Shimadzu, GCMS-TQ 8040, Japan) for linkage pattern. The injector was set at 250℃, and a 1 μL volume of the obtained aliquots was introduced in a split mode. The oven temperature was initially at 60℃, maintained for 2 minutes, raised to 180℃ at a rate of 10℃/min, held for 3 minutes, and then increased to 250℃ at a rate of 5℃/min and finally held at 250℃ for 3 minutes. 2.8. FT-IR spectroscopic analysis The FT-IR spectrum of the oligosaccharides was determined using a FT-IR spectrophotometer. Oligosaccharide samples were ground with potassium bromide powder, and then pressed into pellets for FT-IR determination in the frequency range
Journal Pre-proof of 4000-400 cm-1. 2.9 NMR spectroscopic analysis MCGO-1 and MCGO-6 were exchanged with deuterium by freeze-drying D2O (99.9 atom%) three times. The samples were finally dissolved in D2O. Both 1H, 13C and heteronuclear single quantum coherence (HSQC) spectra were recorded on a Brucker Avance III HD 600 spectrometer (Germany).
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2.10. Antioxidant activity in vitro
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2.10.1 DPPH radical scavenging assay
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The DPPH radical scavenging activity of ginseng oligosaccharides was investigated by the previous method with some modifications [19]. In brief, 100 μL of variable
re
concentrations of ginseng oligosaccharides samples (0.025-2 mg/mL) was added to
lP
100 μL of a DPPH solution (0.1 mM in methanol). The mixture was shaken and incubated in the dark for 30 min, and the absorbance was measured at 517 nm against
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methanol as a blank. Vc was used as the positive control. The ability of the test sample to scavenge the DPPH radical was calculated using the following equation:
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DPPH scavenging rate (%) = [1-(Ai-Aj)/A0] × 100% where A0 was the absorbance of the control (methanol instead of sample), Ai was the absorbance of the sample plus DPPH-methanol solution. Aj was the absorbance of the sample solution only (methanol instead of DPPH-methanol solution). 2.10.2 ABTS radical scavenging assay The ABTS radical scavenging ability of ginseng oligosaccharides was measured by a reported method [20] with some modifications. Briefly, ABTS radical cation solution was prepared through the reaction of 7 mM ABTS solution and 2.45 mM potassium persulfate (1:1, v/v) at room temperature for 12~16 h in the dark. In the moment of use, the ABTS solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at 734 nm. All ginseng oligosaccharide samples (30 μL) with various concentrations (0.025–2
Journal Pre-proof mg/mL) was added to 170 μL of ABTS solution. After reaction at room temperature for 6 min, the absorbance was measured at 734 nm was recorded as Ai, A control sample containing the same amount of methanol and ABTS radical was measured as A0. The absorbance of the sample solution and methanol with the same amount of ABTS was recorded as Aj. Using Vc as the positive control. The ABTS radical scavenging activity was calculated as follows:
2.10.3. Ferric reducing antioxidant power assay
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ABTS scavenging rate (%) = [1-(Ai-Aj)/A0] × 100%
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The Ferric reducing antioxidant power assay (FRAP) was determined according to
-p
the method of Benzie and Strain with some modifications [21]. The FRAP working solution was prepared by mixing acetate buffer (0.3 mol/L, pH 3.6), TPTZ (10 mmol/L
re
in 40 mM HCl) and FeCl3 solution (20 mmol/L) in the ratio 10:1:1 (v/v/v). Then, 1.00
lP
mL FRAP reagent and 100 μL ginseng oligosaccharide samples solution was mixed with reaction in the dark at 37℃ for 30 minutes. The absorbance was read at 593 nm
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and the antioxidant activity was measured using the prepared standard curve of FeSO4. The calibration curve was prepared using standard solutions of ferrous sulphate at
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concentrations of 0–4 mmol/L. All the measurements were conducted in triplicate and the results were expressed in mmol Fe2+ per liter sample. Vc was used as the positive control.
3. Results and discussion
3.1 Characteristic of the oligosaccharides According to the result of phenol-sulfuric method, the content of sugar of CGTOS and MCGTOS was 91.87% and 89.5%, respectively. The contents of uronic acid of CGTOS, MCGTOS, MCGOS-70(Ⅱ) and MCGOS-95(Ⅱ) were 4.02%, 4.19%, 6.14% and 5.32%, respectively. The total phenolics contents of MCGTOS, CGTOS, MCGOS-70(Ⅱ) and MCGOS-95(Ⅱ) were 2.10 μg/mg, 1.91 μg/mg, 3.51 μg/mg and 2.93 μg/mg, respectively. The UV-Vis spectrum was weakly absorbed in the range of
Journal Pre-proof 200-400 nm, indicating that CGTOS and MCGTOS contain trace amounts of nucleic acids or proteins [22]. In addition, trace nucleosides were isolated from MCGOS-30 and MCGOS-95, which verified the result of UV–vis spectral analysis. The nucleosides isolated from MCGOS-30 and MCGOS-95 were confirmed to have no antioxidant capacity by DPPH scavenging free radical experiments. The monosaccharide compositions of all the samples were determined by HPLC. The monosaccharide composition results of CGTOS, MCGTOS, MCGOS-H2O,
of
MCGOS-30, MCGOS-50, MCGOS-70 and MCGOS-95 were summarized in Table 1. MCGO-1~MCGO-7 were mainly composed of glucose with minor galactose and
ro
mannose. The monosaccharide residues and ratios of MCGO-1~MCGO-7 were shown
-p
in Table 2.
re
The molecular weight distribution of all oligosaccharide samples were analyzed using HPGPC. The molecular weight of CGTOS and MCGTOS were ranging from
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100 Da to 2200 Da. The molecular weight distribution of MCGOS-H2O, MCGOS-30, MCGOS-50, MCGOS-70 and MCGOS-95 were 107-930 Da, 100-1464 Da, 100-1794
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Da, 125-2185 Da and 100-2069 Da, respectively. The HPLC chromatograms of all the
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samples showed a non-single peak, therefore, the above five parts of the product are all mixtures and contain more components. 3.2. FT-IR spectra of CGTOS and MCGTOS FT-IR spectroscopy was usually used for the identification of characteristic groups in oligosaccharides. As shown in Fig.4, the FT-IR spectra of CGTOS and MCGTOS displayed a typical absorption peaks of oligosaccharides in the range of 4000– 400 cm−1. A broad and intense peak around at 3401-3408 cm−1 was assigned to the stretching vibration of hydroxyl groups [23]. The weak absorption peaks at 2927-2928 cm−1 were characteristic of C-H stretching vibration. Furthermore, a stretching peak appeared at 1633-1630 cm−1 and a weak stretching peak at 1408 cm-1 were ascribed to the deprotonated carboxylic group, suggesting the presence of uronic acid in the oligosaccharide structure [24]. The absorbance in the range of 1145–
Journal Pre-proof 1060 cm−1 indicated the pyranose unit [25, 26]. The absorption peak at 1105 and 1039 cm−1 represented uronic acid [27]. The absorption at 920 cm−1 referred to the amount of D-glucopyranosyl [25]. 3.3 Methylation analysis Methylation analysis was performed to determine the linkage pattern of MCGO-1~MCGO-7, and the linkage patterns were summarized in Table 2. The procedure
involved
derivation
of
the
monosaccharide
component
of
of
MCGO-1~MCGO-7 to partially methylated alditol acetates, which were analyzed by
ro
GC-MS. There were six types of partially methylated alditol acetates, namely 2,3,4,6-Me4-Glcp, 2,3,6-Me3-Glcp, 2,3-Me2-Glcp, 3,4,6-Me3-Manp, 2,3,4-Me3-Gal
-p
and 2,4,6-Me3-Gal; which were assigned to 1→linked Glcp, (1→4)-linked Glcp,
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(1→4,6)-linked Glcp, (1→2)-linked Manp, (1→6)-linked Galp and (1→3)-linked
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Galp residues, respectively. 3.4 NMR analysis
H, 13C NMR and 2D NMR (HSQC) spectra were used to confirm the structure of
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1
MCGO-1 and MCGO-6. The 1H NMR spectrum (Fig. 5A) showed H-1 signals from 13
C NMR spectrum (Fig. 5B) showed C-1 signals from 90.0 to
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4.5–5.5 ppm and the
110.0 ppm. In general, chemical shifts between 4.4-4.9 ppm are typical of the anomeric protons of these β-linked residues, whereas α-anomeric protons will appear in the region of 4.9-5.5 ppm [28]. As shown in 1H NMR, MCGO-1 and MCGO-6 had anomeric proton signals at δ 4.64, in which the characteristic signals ranging from 4.4 ppm to 4.9 ppm showed the presence of β-configuration. MCGO-1 and MCGO-6 had four anomeric proton signals appeared in the anomeric region at 4.96, 5.22 and 5.35-5.39 ppm, in which the characteristic signals ranging from 4.9 ppm to 5.5 ppm showed the presence of α-configuration. The spectra of MCGO-1 were partially assigned on the basis of the HSQC experiment (Fig. 5C) and by comparison with previous studies. The signals at H-1/C-1 5.35-5.39 ppm and 99.32-99.90 ppm were assigned to →4)-α-D-Glcp-(1→ residues [29, 30]. Similarly, two signals at H-1 5.22
Journal Pre-proof and C-1 91.85 ppm were assigned to →4,6)-α-D-Glcp-(1→ residues [30]. The 1
H-NMR spectra revealed signal for anomeric protons of terminal β-D-Glcp residues
at δ 4.64 ppm, while the corresponding chemical shift in the anomeric carbon was δ 95.71 ppm [29]. Taking the results of methylation analysis into consideration, there were 1,2-Manp residue and 1,6- Galp residue in MCGO-1. The signals at δ 5.22/100.49
and
4.96/98.30
ppm
were
assigned
to
the
H-1/C-1
of
→2)-α-D-Manp-(1→ residue and →6)-α-D-Galp-(1→ residue, respectively [31, 32]. As shown in 1H and
13
C NMR, MCGO-6 and MCGO-1 had similar structural
of
properties. Five sugar residues were inferred to be 1,4-Glcp, 1,4,6-Glcp, 1, 6-Galp, 1,
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2-Manp and T- β -D-Glcp, which was consistent with the results of methylation
-p
analysis. The branches were attached to the backbone at the O-6 position of Glcp
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residues.
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3.5 Antioxidant activity
3.5.1 DPPH radical scavenging activity
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DPPH radical is stable free radical at room temperature and is commonly used as a tool to evaluate the scavenging activity of antioxidants. Scavenging activity of all
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ginseng oligosaccharide samples and Vc on DPPH radicals was determined and the results were shown in Fig.6. All samples showed concentration-dependent DPPH radical scavenging activity. When the concentration increased to 2 mg/mL, the DPPH radical scavenging activity of MCGOS-70 and MCGOS-95 were almost no longer increased, and their DPPH radical-scavenging activity was close to that of Vc. The 50% inhibition (IC50) values of MCGOS-95 and MCGOS-70 for DPPH were 0.155 and 0.154 mg/mL, respectively. The scavenging activity of DPPH radicals by different oligosaccharide samples were in the following order at 2.0 mg/mL: MCGOS-70 > MCGOS-95 > MCGOS-30 > MCGTOS > MCGOS-50 > CGTOS > MCGOS-H2O. The results indicated that MCGOS-70 and MCGOS-95 fractions had a noticeable effect on scavenging DPPH free radicals, especially at high concentrations. The MCGTOS showed the stronger DPPH radical scavenging activity than CGTOS.
Journal Pre-proof Previous studies have showed that the antioxidant activity of polysaccharides were related to their uronic acid content, monosaccharide composition, molecular weight and the type of glycosidic linkage [
]. The antioxidant activity of
oligosaccharides was also related to the content of uronic acid, which was confirmed by the experimental results. The antioxidant activity of MCGO-1~MCGO-7, MCGOS-70(II) and MCGOS-90(II) were investigated by DPPH radical scavenging activity. The antioxidant activity of MCGO-1~MCGO-7 were lower than that of MCGOS-70(II) and MCGOS-95(II). In addition, the antioxidant activity of
of
MCGOS-70(II) was higher than that of MCGOS-70, and MCGOS-95(II) was higher
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than MCGOS-95. MCGO-1~MCGO-7 were all neutral oligosaccharides with no obvious antioxidant. The total content of phenolics in MCGOS-70(II), MCGOS-95(II),
-p
CGTOS and MCGTOS decreased in the order MCGOS-70(II) > MCGOS-95(II) >
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MCGTOS > CGTOS. A similar trend was observed for the uronic acid concentration,
lP
indicating that the DPPH radical scavenging activities of MCGOS-70(II), MCGOS-95(II), CGTOS and MCGTOS could be attributed to their uronic acid and
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phenolics. However, phenolics component was extremely low, indicating that the uronic acid constituents in each fraction play a dominant role.
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3.5.2 ABTS radical scavenging activity The ABTS radical is often used to estimate the total antioxidant power of compounds. The ABTS radical scavenging abilities of all the samples compared with VC as a control standard, as shown in Fig.7. All the sample exhibited dose-dependent increases in their ABTS radical scavenging activity for concentrations in the range of 0.025–2.00 mg/mL. The ABTS scavenging ability of MCGOS-70 at the concentration of 2 mg/mL was slightly lower than that of Vc, which showed that the MCGOS-70 had good scavenging activity against ABTS free radicals. The ABTS radical scavenging rates of MCGOS-70 and MCGOS-95 were 96.17% and 90.39%, respectively, at a concentration of 2 mg/mL. When the samples concentration was 2.0 mg/mL, MCGOS-70 and MCGOS-95 showed the higher ABTS radical
Journal Pre-proof scavenging activities, whereas MCGOS-50, MCGOS-30 and MCGTOS had modest activities, and MCGOS-H2O manifested relatively low ABTS radical scavenging activity. The ABTS radical scavenging activity of MCGTOS was higher than that of CGTOS at a concentration of 0.25-2 mg/mL. The data from the methylation analysis suggested that MCGO-1~MCGO-7 was mainly composed of a Glcp backbone linked 1→4 and the branching points of the Glcp chain were at O-6 positions. The antioxidant activity of MCGO-1~MCGO-7 was studied by ABTS free radical scavenging activity, and it was found that MCGO-1~MCGO-7 did not exist
of
significant antioxidant activity. Therefore, the antioxidant mechanism of MCGOS-70
ro
and MCGOS-95 in this study may be attributed to uronic acid contents, monosaccharide composition and glycosidic linkage types of oligosaccharides. Thus,
-p
a combination of several factors would affect the antioxidant activities of
re
oligosaccharides [36].
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3.5.3 Ferric reducing antioxidant power (FRAP) result FRAP is a colorimetric method based on the reduction of a ferric
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2,4,6-tripyridyl-s-triazine complex (Fe3+-TPTZ) to the ferrous form (Fe2+-TPTZ) by
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antioxidants, and Fe2+-TPTZ has a strong absorption peak at 595 nm [37]. The antioxidant activity of a sample is assessed by measuring its ability to reduce Fe3+-TPTZ.
The standard curve was plotted for FeSO4 with a concentration range of 0-4 mM, and there was a strong correlation between FeSO4 concentration and antioxidant capacity (R2 = 0.9996) and the equation was Y=1.0329x+0.0678. The standard curve displayed a linear trend between 0 and 4 mM FeSO4. As can be seen from Fig.8, ferric reducing activity of all samples were lower than the positive control Vc. When the sample concentration was 0.025-2 mg/mL, there was a dose-dependent relationship between reducibility and samples concentration. The results showed that MCGOS-95 had stronger reducing power, followed by MCGOS-70. At a concentration of 2 mg/mL, the ferric reducing activity decreased in the order MCGOS-95 >
Journal Pre-proof MCGOS-70 > MCGOS-30> MCGTOS > CGTOS > MCGOS-50 > MCGOS-H2O. The ferric reducing activity of MCGTOS was higher than that of CGTOS at a concentration of 0.25-2 mg/mL. The activities of antioxidants were ascribed to various mechanisms, such as prevention of chain initiation, decomposition of peroxides, reducing capacity and radical scavenging [38]. In addition, the antioxidant effects were assigned to their hydrogen-donating abilities, previous studies have shown that the presence of uronic acid groups in the polysaccharides could activate the hydrogen atom of the anomeric carbon [39]. Therefore, the content of uronic acid
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in oligosaccharide had a great influence on its antioxidant activity.
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Based on the results of DPPH, ABTS and FRAP assay, MCGOS-70 and
-p
MCGOS-95 showed significant antioxidant, suggesting its potential as an effective natural antioxidant. Among the three antioxidant models, the antioxidant activity of
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MCGTOS was stronger than that of CGTOS. However, further studies are required to
4. Conclusion
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activities.
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investigate the relationships between the chemical structure and the antioxidant
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The present work investigated the antioxidant activities of cultivated ginseng and mountain cultivated ginseng oligosaccharides. The results showed that the antioxidant activity of MCGTOS was stronger than that of CGTOS. The total oligosaccharides of MCG were separated by Carbon–Celite column and the activity of each fraction was studied. We studied the monosaccharide composition and molecular weight of oligosaccharides.
Furthermore,
the
antioxidant
activity
of
homogeneous
oligosaccharides MCGO-1~MCGO-7 isolated from MCGTOS were investigated by DPPH radical scavenging activity and found that they didn't exist strong antioxidant properties. Two oligosaccharides fractions MCGOS-70(Ⅱ) and MCGOS-95(Ⅱ) with the higher uronic acid content (6.14% and 5.32%, respectively) obtained from MCGOS-70 and MCGOS-95 showed stronger free radical scavenging activities than MCGO-1~MCGO-7 containing no uronic acid.
Journal Pre-proof Previous studies found that tea polysaccharide conjugates were found to exhibit antioxidant activities, and there was a direct relationship between the content of uronic acid and the radical-scavenging effects of tea polysaccharide conjugates [40]. Our studies were similar to Chen’s reports that the uronic acid was very important to the antioxidant activities of oligosaccharides. The antioxidant mechanism of all samples in this study may be attributed to the synergistic action of carbohydrates, uronic acid and phenolics. Overall, the antioxidant activities of oligosaccharides are not determined by a single factor but a combination of several related factors [36].
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Future research is required to better understand the relationships between the
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antioxidant activity and the structure characteristics of the ginseng oligosaccharides. These findings provided a reference for the potential applications of the
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oligosaccharides from P. ginseng to be developed as natural antioxidants in food and
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pharmaceutical area.
None.
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Acknowledgements
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Conflicts of interest
This work was granted by the National Key R&D Program of China (2017YFC1702302), Liaoning Programs for Science and Technology Development (2017226009), Foundation of Liaoning Education Department(2017LZD02) and the fourth national survey on Chinese materia medica resources in Liaoning Province (2018018). References [1] L.L. Jiao, X.Y. Zhang, B. Li, Z. Liu, M.Z. Wang, S.Y. Liu, Anti-tumour and immunomodulatory activities of oligosaccharides isolated from Panax ginseng C. A. Meyer, Int. J. Biol. Macromol. 65 (2014) 229-233. https://doi.org/10.1016/j.ijbiomac.2014.01.039.
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[2] B. Zhao, C.N. Lv, J.C. Lu, Natural occurring polysaccharides from Panax ginseng C. A. Meyer: A review of isolation, structures, and bioactivities, Int. J. Biol. Macromol. 133 (2019) 324-336. https://doi.org/10.1016/j.ijbiomac.2019.03.229.
[3] D.B. Wan, L.L. Jiao, H.M. Yang, S.Y. Liu, Structural characterization and immunological activities of the water-soluble oligosaccharides isolated from the Panax ginseng roots, Planta. 235
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[4] L. Jiao, D. Wan, X. Zhang, B. Li, H. Zhao, S. Liu, Characterization and immunostimulating
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effects on murine peritoneal macrophages of oligosaccharide isolated from Panax ginseng C.A.
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Meyer., J. Ethnopharmacol. 144 (2012) 490-496. https://doi.org/10.1016/j.jep.2012.09.004.
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[5] X.F. Xu, X.L. Cheng, Q.H. Lin, S.S. Li, et al., Identification of mountain-cultivated ginseng
na
and cultivated ginseng using UPLC/Q-TOF MSE with a multivariate statistical sample-profiling
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strategy, J. Ginseng Res. 40 (2016) 344-350. https://doi.org/10.1016/j.jgr.2015.11.001.
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Journal Pre-proof Table 1. Monosaccharide composition of CGTOS, MCGTOS, MCGOS-H2O~MCGOS-95 Samples
Sugar components (%)
Gal
GlcA
GalA
Rha
Man
Ara
CGTOS
96.67
1.10
0.18
2.78
---
0.23
0.37
---
MCGTOS
92.59
1.22
0.21
3.18
---
0.67
0.52
---
MCGOS-H2O
95.51
1.42
---
---
0.27
0.30
---
MCGOS-30
95.92
1.15
0.19
---
---
0.77
0.50
0.22
MCGOS-50
94.49
1.33
0.39
---
---
0.41
0.51
---
MCGOS-70
90.55
1.97
0.62
2.71
---
0.38
0.75
2.44
MCGOS-95
86.43
1.00
0.56
2.23
---
0.22
0.81
2.69
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Glu
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---
Xyl
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Table 2. GC–MS analysis of the methylated products of MCGO-1~MCGO-7
Methylated
Type of linkage
Mass fragments (m/z)
MCGO-1~ MCGO-7 (Mol.%)a
sugars
1
2,3,4,6-Me4-Glc
β-D-Glcp-(1→
43,71,87,101,117,129,145,161,205
2,3,6-Me3-Glc
→4)-α-D-Glcp-(1→
45,71,87,99,101,113,117,129,131,161,173,
→4,6)-α-D-Glcp-(1→
3,4,6-Me3-Man
→2)-α-D-Manp-(1→
2,3,4-Me3-Gal
→6)-α-D-Galp-(1→
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3
4
5
6
7
20.69
20.86
24.44
20.22
19.05
26.92
18.60
51.88
53.49
61.36
56.23
71.55
58.09
66.51
8.46
12.49
12.13
10.53
8.98
---
43,71,87,101,129,161,173,205
5.27
---
---
---
3.61
---
---
43,71,87, 101, 117, 129,161, 173,189, 233
2.50
1.94
---
2.39
2.52
1.8
1.38
233
2,3-Me2-Glc
f o
2
l a
p e
r P
n r u
43,57,71,85,99,101,117,127,159,201,261
o J
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2,4,6-Me3-Gal
a
→3)-α-D-Galp-(1→
43,58,71,87,101,113,117,129,161,233
---
2.65
---
Relative molar ratio, calculated from the ratio of peak areas.
f o
l a
o J
n r u
r P
e
o r p
2.65
0.87
---
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Caption of figures Fig. 1. Cultivated Ginseng (A) and Mountain Cultivated Ginseng (B) Fig. 2. Elution profile of MCGOS-30 (a), MCGOS-50 (b), MCGOS-70 (c), MCGOS-95 (d) on Sephadex G-25 gel chromatography column with distilled water. Fig. 3. HPGPC elution profiles of the purified oligosaccharides.
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Fig. 4. Infrared spectrum of total oligosaccharides of MCG and CG.
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Fig. 5. A. 1H NMR spectra of MCGO-1 and MCGO-6 in D2O solution.
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C. HSQC spectrum of MCGO-1.
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B. 13C NMR spectra of MCGO-1 and MCGO-6 in D2O solution
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Fig.6. DPPH scavenging effect of oligosaccharides from panax ginseng. Fig.7. ABTS• scavenging effect of oligosaccharides from panax ginseng
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ginseng.
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Fig.8. Ferric reducing antioxidant power (FRAP) of oligosaccharides from Panax
Journal Pre-proof Authors Statement Conceptualization: Bin Zhao; methodology: Bin Zhao; investigation: Bin Zhao, Xinying Wang and Hao Liu; Writing- Original draft preparation: Bin Zhao. Supervision: Jincai Lu, Chongning Lv; Writing- Reviewing and Editing: Jincai Lu. All
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authors have read and agreed to the published version of the manuscript.
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Highlights
Investigation of the antioxidant activity of oligosaccharides from different cultivation methods Total oligosaccharides of Mountain cultivated ginseng possessed better antioxidant activity than cultivated ginseng
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70% ethanol elution and 95% ethanol elution from a Carbon–Celite column exhibited significant antioxidant activity
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Preliminarily discussed the antioxidant mechanism of Panax ginseng oligosaccharides
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8