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In vitro evaluation of the antioxidant activities of carbohydrates
Author’s Accepted Manuscript In vitro evaluation of the antioxidant activities of carbohydrates Shuzhen Hu, Junyi Yin, Shaoping Nie, Junqiao Wang, Glyn O. Phillips, Mingyong Xie, Steve W. Cui www.elsevier.com/locate/bcdf
PII: DOI: Reference:
S2212-6198(16)30006-7 http://dx.doi.org/10.1016/j.bcdf.2016.04.001 BCDF109
To appear in: Bioactive Carbohydrates and Dietary Fibre Received date: 26 January 2016 Revised date: 7 April 2016 Accepted date: 14 April 2016 Cite this article as: Shuzhen Hu, Junyi Yin, Shaoping Nie, Junqiao Wang, Glyn O. Phillips, Mingyong Xie and Steve W. Cui, In vitro evaluation of the antioxidant activities of carbohydrates, Bioactive Carbohydrates and Dietary Fibre, http://dx.doi.org/10.1016/j.bcdf.2016.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vitro evaluation of the antioxidant activities of carbohydrates Shuzhen Hua, Junyi Yina, Shaoping Niea*, Junqiao Wanga, Glyn O. Phillipsb, Mingyong Xiea, Steve W. Cuia,c* a
State Key Laboratory of Food Science and Technology, Nanchang University, 235
Nanjing East Road, Nanchang, Jiangxi Province, 330047, China b
Phillips Hydrocolloids Research Centre, Glyndwr University, W rexham, LL11 2AW
Wales, UK c
Agriculture and Agri-Food Canada, Guelph Food Research Centre, 93 Stone Road
West, Guelph, Ontario, NIG 5C9, Canada [email protected] [email protected] *
Corresponding authors. Shao-ping Nie & Steve W. Cui. Tel.: +86-791-88304452; +1
226 217-8076. Fax: +86-791-88304452; +1 519 780-2600
Abstract
In the current study, we evaluated the antioxidant activities of highly purified monosaccharides, oligosaccharides and complex carbohydrates using six in vitro antioxidant assays, including oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), β-carotene bleaching assay, 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, hydroxyl radical scavenging 1
and superoxide radicals scavenging methods. The results suggested that monosaccharides and oligosaccharides scarcely exhibited antioxidant activities in vitro. For example in the β-carotene bleaching assay the inhibitory effects of monosaccharides at 1 mg/mL were between 0.24% to 2.25% although galacturonic acid demonstrated some inhibitory effects (8.59%). Significant lower antioxidant activities were observed for complex carbohydrates compared to ascorbic acid and BHT (e.g. ferric reducing antioxidant power by the FRAP assay, >1800 μmol Fe (Ⅱ ) /g were observed for ascorbic acid and BHT compared to only 10-60 μmol Fe (Ⅱ) /g
for complex carbohydrates, and 1.60 and 6.43 μmol Fe (Ⅱ) /g for mono and
oligosaccharides. The observed antioxidant activities for complex carbohydrates are correlated with the presence of phenolic and/or protein components. The findings from the current study indicate that it is necessary to re-evaluate the antioxidant activities claimed for complex carbohydrates in the literature as many of them did not consider the contribution of other minor components such as phenolics and proteins.
Keywords: monosaccharide, oligosaccharide, complex carbohydrate, antioxidant activity
1. Introduction
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Oxidative stress refers to the imbalance of oxidation and reduction in vivo. It leads to the production of oxidation intermediates which could cause base damage as well as strand breaks in DNA, hence become a serious threat to human health (Seifried, Anderson, Fisher, & Milner, 2007). Antioxidant components from food have been considered beneficial to human health because they can slow the effects of oxidative stress (Cook, & Samman, 1996). Antioxidants are also known to reduce the risk of Alzheimer's disease, cataracts, some types of cancer and cardiovascular diseases (Halliwell, Aeschbach, Löliger, & Aruoma, 1995; Oak, El Bedoui, & Schini-Kerth, 2005), improve human immunity and suppress inflammatory reactions (Yao, Jiang, Shi, Tomas-Barberan, Datta, Singanusong, & Chen, 2004). Thus, accurately evaluation of the activities of antioxidants of food components is important.
Many compounds from plant extracts exhibited antioxidant activities, such as pigments (fucoxanthin, anthocyanins, carotenoid e.g.) and polyphenols (phenolic acid, flavonoid, tannins e.g.) (Heo, Park, Lee, & Jeon, 2005; Soong, & Barlow, 2004; Lai, Xin, Zhao, Feng, He, Dong, ... & Wei, 2013; Van Acker, Tromp, Griffioen, Van Bennekom, Van Der Vijgh, & Bast, 1996). Numerous studies have reported that some selected polysaccharides have various biological activities, including antitumor activities, immunomodulatory effects and antiviral activities (Lv, Cheng, Zheng, Li, & Zhai, 2014; Ren, Jiao, Yang, Yuan, Guo, & Zhao, 2015; Xu, Zhang, Luo, Ma, Kou, & Huang, 2009; Zheng, Dong, Chen, Cong, & Ding, 2015). Some recent studies also 3
consider polysaccharides as sources of novel potential antioxidants (Petera et al., 2015; Chen, Ma, Liu, Liao, & Zhao, 2012; Fu, Chen, Dong, Zhang, & Zhang, 2010; Ferreira, Heleno, Reis, Stojkovic, Queiroz, Vasconcelos, & Sokovic, 2014; Ramarathnam, Osawa, Ochi, & Kawakishi, 1995). However, most of the studies on antioxidant activities of polysaccharides usually indicated they were mixed with some minor components, such as proteins and some low molecular weight substances (Chen, Zhang, Qu, & Xie, 2008; Wang et al., 2001; Wang, Zhao, Andrae-Marobela, Okatch, & Xiao, 2013). For example, Siu, Chen, & Wu (2014) suggested that the antioxidant activities of crude polysaccharide from mushrooms were mainly attributed to the phenolic and protein components, while Wang, Zhao, Andrae-Marobela, Okatch, & Xiao (2013) proved that tea polyphenols were the major antioxidants in the crude tea polysaccharides. There were also reports demonstrated that the antioxidant activities of polysaccharides could be significantly affected by modifications, such as sulfation and/or acetylation
(Delattre et al., 2015; Chen, Zhang, Wang, Nie, Li, &
Xie, 2014; Jin, Wang, Huang, Lu, & Wang, 2014; Qi, Zhang, Zhao, Chen, Zhang, Niu, & Li, 2005; Wang, Zhang, Zhang, & Li, 2008; Wang, Guo, Zhang, Wang, Zhao, Yao, & Wang, 2010). On the contrary, the antioxidant activities of purified polysaccharides and their fractions have been reported to be much weaker (Wang, & Luo, 2007). These variations on antioxidant activities of polysaccharide raised our curiosity. Does carbohydrate have antioxidant activities in vitro? Until present, little
4
information could be found in the literature on the antioxidant activity of carbohydrates.
Carbohydrate is known as polyhydroxy aldehydes and ketones, or substances that can be hydrolyzed to yield polyhydroxy aldehydes and ketones. It is covalently linked by glycosidic linkages, which are formed by condensation reaction between the glycosyl moiety of hemiacetal or hemiketal, together with the hydroxyl group of another sugar unit (Cui, 2005). Hence, there are few aldehyde or ketone group in carbohydrate solution; based on these structural characteristics we could hypothesis that pure carbohydrates do not have strong antioxidant activities. To anwser and confirm the question: the objectives of current study were to systematically evaluate the antioxidant activity of monosaccharide, oligosaccharide and some complex carbohydrate. Physical-chemical and compositions of complex carbohydrates studied were also determined to elucidate if other components contributed to the antioxidant activities of complex carbohydrates.
2. Materials and methods
2.1. Materials and chemicals
D - (+) - glucose, sucrose, lipopolysaccharide, polygalacturonic acid, pectin, xylan, arabinogalactan and D - (-) galacturonic acid were purchased from Sigma Chemical
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Co. (St. Louis, MO USA). D - (+) - galactose, D - mannose, xylose, D - lactose, raffinose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, pullulan and D - glucuronic acid were from J&K Co. (Shanghai, China). D-(-) arabinose was the product of Solarbio Co. (Shanghai, China). Galactomannan, arabinoxylan and glucomannan were obtained from Megazyme Co. (Wicklow, Ireland). Trolox, 2, 2’-Azobis (2-methylpropionamidine) dihydrochloride (AAPH), fluorescein sodium salt, 2, 4, 6-Tris (2-pyridyl)-s-triazine (TPTZ), 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) and Coomassie Brilliant Blue G-250 were obtained from Sigma Chemical Co. (St. Louis, MO USA). Linoleic acid, β-carotene and pyrogallic acid were from Aladdin Co. (Shanghai, China). Bovine serum albumin (BSA) was from Solarbio Co. (Beijing, China). Gallic acid was purchased from J&K Co. (Shanghai, China). All other chemicals were of analytical reagent grade. All aqueous solutions were prepared using freshly double distilled water.
2.2. Antioxidant activities of monosaccharide
The antioxidant activities of different monosaccharides were evaluated by oxygen radical absorbance capacity, ferric reducing antioxidant power, β-carotene bleaching
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assay, DPPH radical scavenging, hydroxyl radical scavenging and superoxide radicals scavenging assessments. Each test was performed in triplicates.
2.2.1. Oxygen radical absorbance capacity (ORAC)
The oxygen radical absorbance capacity was measured using a fluorescence microplate reader (Thermo Fisher Scientific, Thermo Electron Co., Waltham, MA, USA) described by previous report (Folch-Cano, Jullian, Speisky, & Olea-Azar, 2010), with slight modifications. The ORAC method is based on trolox as an antioxidant standard and 2, 2’-Azobis (2-methylpropionamidine) dihydrochloride (AAPH) as a peroxyl generator. The peroxyl radical scavenging was measured using fluorescein as fluorescent probe. The degree of change in fluorescence intensity reflects the extent of free radical damage. Antioxidant can inhibit the fluorescence decay caused by free radicals, reflecting its ability to inhibit free radicals (Barahona, Encinas, Imarai, Mansilla, Matsuhiro, Torres, & Valenzuela, 2014). The reaction was carried out in 75 mM sodium phosphate buffer (pH 7.4). Sample, standard and phosphate buffer (60 μL, 75mM, pH 7.4) were placed in the wells of the microplate. Then fluorescein solution (40 μL, 1.75 μM) was added. The mixture was pre-incubated for 15 min at 37 °C, before adding the AAPH solution (100 μL, 10 mM). The microplate was shaken automatically for 30 seconds and then was immediately placed in the reader. The fluorescence (excitation wave length of 460 nm, emission wave length of 515 nm) was recorded every 2 min for 120 min.
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The ORAC values were calculated as the area under the curve (AUC) and the inhibition capacity was expressed as Trolox equivalents (μmol Trolox/g).
2.2.2. Ferric reducing antioxidant power (FRAP)
The FRAP assay was performed as described previously (Xu, & Chang, 2007) with some modifications. Antioxidant donates an electron to TPTZ-Fe (Ⅲ) complex, which could be reduced to TPTZ-Fe (Ⅱ) form. TPTZ-Fe (Ⅱ) has an intensive blue color and can be monitored at 593nm (Benzie, & Strain, 1996). Briefly, The working FRAP reagent was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ and 20 mM FeCl3 in a volume ratio of 10:1:1. Sample solution (200 μL) was mixed with 1.8 mL working FRAP reagent, and incubated at 37 °C for 30 min. The absorbance was measured at 593 nm. FRAP value was calculated using the concentration of FeSO4·7H2O equivalent. Ascorbic acid and BHT were used as positive controls. The reducing capacity was expressed as μmol Fe (Ⅱ)/g. 2.2.3. β-carotene bleaching assay Antioxidant activity was carried out according to the β-carotene bleaching assay (Chen, Zhang, Wang, Nie, Li, & Xie, 2014). Linoleic acid becomes a free radical with a hydrogen atom abstracted. The radical formed then attacks β-carotene molecule and the compound loses its characteristic orange color. Antioxidant can neutralize the linoleate free radical and thus inhibit the oxidation of linoleic acid (Deba, Xuan,
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Yasuda, & Tawata, 2008). β-carotene (0.5 mg) was dissolved in 1 mL of chloroform. Twenty five μL linoleic acid and 200 mL Tween 20 were added. The chloroform was evaporated under nitrogen at 45 °C, then 100 mL distilled water was added and shook vigorously. Three hundred and fift μL of sample and 2.5 mL of the β-carotene-linoleic acid emulsion were mixed into test tube. The absorbance was immediately measured at 490 nm. The reaction mixture was incubated at 50 °C for 2 h and the absorbance was measured again. The ascorbic acid and butyl hydroxyl toluene (BHT) were used as a positive control.
2.2.4. DPPH radical scavenging activity
Relatively stable DPPH radical has been widely used to evaluate the free radical scavenging capacities of antioxidants. Antioxidant transfer hydrogen atoms to DPPH ·, leading to non-radical form DPPH-H (Li, Li, & Zhou, 2007). The antioxidant activity of a substance can be expressed as its ability in scavenging the DPPH radical. The scavenging effect of the sample on DPPH radical was measured according to the method as previously described (Wang, Liu, Huo, Zhao, Ren, & Wei, 2013). Sample solutions (1.0 mL) with variable concentrations were added with 2.0 mL DPPH 0.2mM in ethanol). The mixture was shaken and kept at room temperature in the dark. The absorbance was measured at 517 nm 30 min later. The control was done with ethanol instead of DPPH, while ethanol was used as the blank. The DPPH radical scavenging activity (%) was calculated according to the following equation:
9
Scavenging activity (%) = [1-(As-Ac)/A] × 100
Where As was the absorbance of sample and Ac was the absorbance of the ethanol control solution, and A was the absorbance of solution including 2 mL of DPPH and 1mL of ethanol. Ascorbic acid and BHT were used as standards.
2.2.5. Hydroxyl radical scavenging activity
The hydroxyl radical is known to be generated through the Fenton reaction (Erel, 2004). The scavenging of hydroxyl radical is very important due to the harmful role of free radicals in biological systems (Ke, Sun, Qiao, Wang, & Zeng, 2011). The hydroxyl radical scavenging activity of sample was evaluated according to the method described by Huang, Tu, Jiang, Xiao, Zhang, & Wang, (2012). Different concentrations of the samples (1 mL) were incubated with FeSO4 (1 mL, 4 mM), salicylic acid-ethanol (1 mL, 6 mM), and H2O2 (1 mL, 2 mM) at 37 °C for 60 min. Then the absorbance of the mixture was recorded at 510 nm. Ascorbic acid was used as positive control. The scavenging activity was calculated using the following equation:
Scavenging activity (%) = [1-(A1-A2)/A0] × 100
where A0 was the absorbance of the control (without extract), A1 was the absorbance of the sample and A2 was the absorbance without H2O2.
2.2.6. Superoxide radicals scavenging activity 10
Superoxide radicals are a precursor to active free radicals that have potential of reacting with biological macromolecules functioning in living cells, which could induce tissue damage (Gülçin, 2006). Therefore, it is extremely important to scavenge the superoxide radicals for anti-oxidation to work. The superoxide radicals were generated by autoxidation of pyrogallol in an alkalescent condition (Wang, Wang, & Quan, 2014). In the presence of antioxidant, these antioxidants could combine with the superoxide radicals and form a stable radical to terminate the radical chain reaction. The assay was carried out according to the method of Marklund, & Marklund (1974) with some modifications. Tris-HCl buffer (pH 8.2, 5 mL) was mixed with sample solution (350 μL) and incubated at 25 °C for 20 min. Then 1, 2, 3-phentriol (0.2 mL, 45 mM) was added and the mixture was shaken rapidly. The absorbance of the mixture at 325 nm was recorded every 10 s for 1 min and a slope was calculated as absorbance / min. The ability of scavenge superoxide radical was calculated as follows:
Scavenging effect (%) = (1-slope of sample / slope of control) × 100
2.3. Antioxidant activities of oligosaccharide
The antioxidant activities of oligosaccharides were investigated and evaluated by the tests described above (Section of 2.2). Oligosaccharides evaluated include sucrose,
11
lactose, raffinose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose.
2.4. Antioxidant activities and physical-chemical characteristics of complex carbohydrates
2.4.1. Antioxidant activities of complex carbohydrates
The antioxidant activities of complex carbohydrates were referred to the six assays, which was the same as assays for evaluating monosaccharide (Section of 2.2). The complex carbohydrates include lipopolysaccharide (carbohydrate compound), pectin, polygalacturonic acid, xylan, arabinogalactan, pullulan, galactomannan, arabinoxylan and glucomannan.
2.4.2. Physical-chemical properties
2.4.2.1. Protein and phenolic content
Protein content was measured according to Bradford’s method with bovine serum albumin (BSA) as a standard (Bradford, 1976). The level of total phenolic was determined with Folin-Ciocalteu’s reagent (FCR) according to Khokhar’s method (Khokhar, & Magnusdottir, 2002). All measurements were repeated thrice.
2.4.2.2. Monosaccharide compositions 12
The sample (5 mg) was placed in 1 mL of 12 M H2SO4 in a test tube, then rapidly stirring in ice-bath for 30 min. Dilution (2 M H2SO4) was carried out using ultra-pure water followed by hydrolysis at 100 °C for 2 h. The hydrolysis product was obtained by centrifugation at 4800 × g for 10 min after being neutralized by BaCO3 (Xie, Shen, Nie, Liu, Zhang, & Xie, 2013). A mixture of monosaccharide standards was used to determine the monosaccharide composition.
Monosaccharide compositions were analyzed by HPAEC. HPAEC was performed on a Dionex system using a CarboPacTM PA20 analytical column (3 mm × 150 mm) and CarboPacTM PA20 guard column (3 mm × 30 mm). Detection was pulsed amperometry (Xie, Shen, Nie, Liu, Zhang, & Xie, 2013).
2.4.2.3. Molecular weight of complex carbohydrate
The molecular weight of samples was characterized by high performance size exclusion chromatography with multi-angle laser light scattering (HPSEC-MALLS) (Yin, Nie, Guo, Wang, Cui, & Xie, 2015). The sample detection was performed by a multi-angle laser light scattering detector, a refractive index detector and a viscometer (Wyatt Technology Co., USA). A Wyatt D05SFD01 pump (Wyatt Technology Co., Goleta, CA, USA) with three columns in series were used: a SB-G guard column (8 mm×50 mm), a SB-806 HQ (8 mm×300 mm) and a SB-804 HQ (8 mm×300 mm) (Shodex Ohpak, Showa Denko K.K., Tokyo, Japan). The columns were maintained at
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35 ºC. The mobile phase was 0.1 M NaNO3 containing 0.02% NaN3 at a flow rate of 0.6 mL/min. Data was collected and analyzed using ASTRA 6.1 software.
2.5. Statistical analysis
All measurements were done in triplicates and results were expressed as mean ± standard deviation (SD). SPSS Statistics 19 was used for the data analysis. The P values were set at P < 0.05 to assess the statistically significant.
3. Results and discussions
3.1. Antioxidant activities of monosaccharide
Oxygen radical absorbance capacity (ORAC) values of monosaccharide are presented in Table 1. All the neutral monosaccharides showed very low ORAC values, ranging from 1.13 to 4.80 μmol Trolox/g. Galacturonic acid showed the highest ORAC value (7.47) while glucuronic acid showed the lowest value (1.13 μmol Trolox/g).
The results of antioxidant activities of monosaccharides were compared with ascorbic acid and BHT (Fig. 1A). All monosaccharides scarcely showed any reducing power: the FRAP values of different monosaccharides were between 1.60 and 6.43 μmol Fe (Ⅱ) /g compared to over 2000 μmol Fe (Ⅱ) /g for ascorbic acid and BHT (Fig.1A).
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It was interesting to observe that galacturonic acid had the highest FRAP value among the onosaccharides examined (~20 μmol Fe (Ⅱ) /g, Fig. 1A). The antioxidant activities of monosaccharides in the β-carotene bleaching assay are shown in Fig. 1B. The inhibitory effects of glucose, mannose, galactose, arabinose, xylose, glucuronic acid and galacturonic acid at 1 mg/mL were 1.31%, 0.37%, 1.82%, 2.25%, 0.24%, 1.78% and 8.59%, respectively, indicating that all monosaccharides scarcely inhibited the bleaching effect of β-carotene except for galacturonic acid, which demonstrated some inhibitory effects.
The scavenging activities of monosaccharides on the DPPH radical are shown in Fig. 1C. The scavenging effects of glucose, mannose, galactose, arabinose, xylose, glucuronic acid and galacturonic acid were 7.76%, 5.71%, 4.21%, 7.15%, 3.54%, 8.15% and 8.85% at 1 mg/mL for the DPPH radical, respectively, as shown in Fig.1C, indicating that all monosaccharides exhibited some scavenging activities against DPPH radical. However, compared to ascorbic acid and BHT, the scavenging activities of these monosaccharides were ignorable and hardly changed with increasing concentration (Fig.1C).
The scavenging effects against hydroxyl radical were 1.89%, 5.61%, 8.30%, 2.60%, 4.75%, 11.96% and 14.84% for glucose, mannose, galactose, arabinose, xylose, glucuronic acid and galacturonic acid at the concentration of 2 mg/mL, respectively, as shown in Fig.1D. Again, all monosacchardies tested exhibited no effect except the 15
two uronic acids; galactose also exhibited some small effect. Fig. 1E depicted the inhibitory effect of monosaccharides on superoxide radicals. The inhibitory effects were only between 0.22%, and 5.89% for all the monosaccharides examined at the concentration of
1 mg/mL compared to 87% exhibited by ascorbic acid. Based
on this observation, all monosaccharides barely inhibited the autoxidation of pyrogallol. In nature, aldopentose, aldohexose and ketohexose mainly exist in the form of closed ring, only few of them have the open-chain form in solution (Toukach, & Ananikov, 2013). Hence, there are few aldehyde or ketone groups in carbohydrate solution. Moreover, the hydrogen is inactive on the hydroxyl and carbon chain, it cannot provide proton or electron to be an effective reducing agent.
Based on the six assays, we could conclude that monosaccharides do not have antioxidant activities, except for uronic acids, which exhibited weak antioxidant activities. Among the two uronic acids tested, galacturonic acid was always slightly stronger than that of glucuronic acid. This observation is consistent with the report by Rao, & Muralikrishna (2006), possible due to the different orientation of the –OH group in galacturonic acid, hence, exhibits stronger antioxidant activity than glucuronic acid. Therefore, galacturonic acid content in acidic polysaccharides should be an important factor concerning their antioxidant activities (Asker, Manal, & Ghada, 2007; Wang, Chang, Inbaraj, & Chen, 2010). However, the detailed differences of the antioxidant mechanism between galacturonic acid and glucuronic acid are still not clear. Further studies are required to elucidate the mechanisms. 16
3.2. Antioxidant activities of oligosaccharide
Oligosaccharides contain between two and twenty monosaccharide residues linked through glycosidic linkages. The antioxidant activities of oligosaccharides as evaluated by ORAC, FRAP, β-carotene bleaching, DPPH, OH·and O2· assays are shown in Table 1 and Fig. 2. From these data we could conclude that, similar to monosaccharides, all oligosaccharides showed poor ORAC values (only ranging from 0.73 to 2.69 μmol Trolox/g) and exhibited very poor reducing power when comparing with ascorbic acid and BHT. For example, FRAP values of oligosaccharides were between 1.65 and 3.77 μmol Fe (Ⅱ) /g compared to 2144.71 and 1832.04 μmol Fe ( Ⅱ)/g for ascorbic acid and BHT up, respectively. These results are consistent with the notion that oligosaccharides cannot provide proton or electron to be effective reducing agents (Toukach, & Ananikov, 2013). The tested oligosaccharides exhibited negligible scavenging abilities against DPPH, OH· and O2· radicals. In addition, oligosaccharides barely inhibited the bleaching of β-carotene in the β-carotene-linoleic acid system. We could conclude that all the oligosaccharides evaluated scarcely showed any antioxidant activities.
3.3. Antioxidant activities of complex carbohydrates
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We have selected some polysaccharides for testing their antioxidant activities in vitro, the results are shown in Table 1 and Fig. 3. The ORAC value for pectin was 20.30 μmol Trolox/g compared to polygalacturonic acid, which was only 2.78 μmol Trolox/g (Table 1). However, polygalacturonic acid and pectin exhibited 20.4 and 23.1 μmol Fe (Ⅱ) /g for FRAP, respectively. The radical scavenging activities of pectin and polygalacturonic acid on DPPH, OH· and O2· are showed in Fig. 3C, 3D and 3E respectively. The DPPH scavenging percentages of pectin and polygalacturonic acid (at 2 mg/mL) were 37.23% and 4.11%, respectively, and it was also observed that polygalacturonic acid on DPPH scavenging activities was scarcely changed with increasing concentration. Compared with pectin, polygalacturonic acid exhibited a higher scavenging effects against OH·and O2, and a higher percentages of β-carotene bleaching at 2 mg/mL. This is in agreement with the observation by Wu, Min, Li, Li, Lai, Tang, & Yang (2013) that WP3, a pectin with higher galacturonic acid content exhibited higher antioxidant activities than WP, which had lower galacturonic acid content. Nevertheless, it is still not clear why the different antioxidant results were observed for polygalacturonic acid and pectin in ORAC, DPPH radical scavenging and β-carotene bleaching assays. We suspect that different acting mechanisms are involved with each assay, as well as the solubility of the material in the testing system would play important roles in their antioxidant effects. Further research is needed to elucidate the possible mechanisms involved.
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Weak antioxidant activities were also observed for lipopolysaccharide, xylan, arabinogalactan and arabinoxylan, as showed in Table 1 and Fig. 3. The ORAC values of lipopolysaccharide, xylan, arabinogalactan, and arabinoxylan were 19.16, 21.82, 14.59 and 15.76 μmol Trolox/g, respectively. Besides, they showed 17.3, 63.1, 33.6 and 5.6 μmol Fe (Ⅱ) /g for FRAP, same trends were observed for scavenging effects on OH· (Fig. 3D). The four polysaccharides exhibited similar antioxidant activities ·
when assessed by DPPH and O2 , and the activities hardly changed with increasing of concentration (Fig. 3C and 3E). However, lipopolysaccharide and xylan (2 mg/mL) had relatively higher inhibited percentages of β-carotene (12.36% and 24.94%, respectively) than arabinogalactan and arabinoxylan (3.44% and 3.22%, respectively), likely due to the higher solubility of the lipopolysaccharides in the lipid phase of the emulsion. Three other neutral polysaccharides, i.e. pullulan, galactomannan and glucomannan exhibited low overall antioxidant activities (Fig. 3B, 3D and 3E; Table 1).
3.4. Compositions of complex carbohydrates
The above results suggested that pectin had relatively higher antioxidant activities as expressed by ORAC values, reducing power and DPPH radical scavenging. Composition analysis showed that pectin contained 0.60% of phenolic and 1.42% of protein, which was slightly, but significantly higher than that of polygalacturonic acid (0.19% of phenolic, and 1.11% of protein, respectively) (Table 2). These differences
19
in composition could be responsible for the antioxidant of pectin and polygalacturonic acids, which was also reported in the literature (Siu, Chen, & Wu, 2014; Wang, Zhao, Andrae-Marobela, Okatch, & Xiao, 2013; Liu, Ooi, & Chang, 1997).
Similarly, a
study by Hromadkova et al. pointed out that phenolic compounds played a significant role on the radical scavenging effects of xylans (Hromádková, Paulsen, Polovka, Košťálová, & Ebringerová, 2013). lipopolysaccharide and xylan also showed relatively higher inhibited percentages of the bleaching of β-carotene, somewhat higher scavenging effect against hydroxyl radical than arabinogalactan and arabinoxylan, which are consistent with their relatively higher contents of proteins and phenolics (Table 2). The results indicated that both protein and phenolic compounds contributed to their antioxidant activities which is in agreement with literature reports (Moraes, da Silva Marineli, Lenquiste, Steel, de Menezes, Queiroz, & Júnior, 2015; Dlamini, Taylor, & Rooney, 2007).
Consistent with above analysis,
pullulan, galactomannan and glucomannan
exhibited lower inhibition on the bleaching of β-carotene and little scavenging activities against OH· and O2·. These results might be ascribed to the lack of phenolics or other minor components for pullulan, galactomannan and glucomannan (Table 2) (Siu, Chen, & Wu, 2014; Wu, Wang, Wang, Shen, He, Gu, & Wu, 2014). The total carbohydrate contents of these three polysacchardies were all beyond 95% (Table 2). The findings in the current study indicated that the phenolic and protein
20
constituents in complex carbohydrates may play a significant role in their antioxidant activities.
4. Conclusions
In this study, it was confirmed that monosaccharides and oligosaccharides hardly exhibited antioxidant activities as evaluated by six in vitro methods. Lipopolysaccharide, pectin and xylan exhibited weak reducing power, low ORAC values and part of hydroxyl radical scavenging effect most likely due to the relatively higher phenolic and protein contents,. Additionally, lipopolysaccharide and xylan showed relatively higher inhibited percentages of β-carotene bleaching. On the contrary, pullulan, galactomannan and glucomannan had almost no phenolic contents, showed significantly lower inhibition on the bleaching of β-carotene and little scavenging activities against OH· and O2·. The antioxidant activities of complex carbohydrates results in the literature were mainly attributed to the phenolic and protein components, rather than the carbohydrate moieties. Based on the current study and review of literature reports, we could conclude that carbohydrates do not have antioxidant activities in vitro. This finding also suggested there is a need to reevaluate the antioxidant activities claimed for complex carbohydrates in the literature.
Acknowledgements
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This study was supported by National Natural Science Foundation of China for Excellent Young Scholars (31422042), the Program for New Century Excellent Talents in University (NCET-12-0749), the Key Project of International Cooperation of Jiangxi Provincial Department of Science and Technology (20141BDH80009), the Project of Science and Technology of Jiangxi Provincial Education Department (KJLD13004)，This work was also supported by Cooperation Initiative with Guelph Food Research Centre, Agriculture and Agri-Food Canada.
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Figure 1. Antioxidant activities assessment of different monosaccharides with various methods: (A) Ferric reducing antioxidant power (FRAP) assay; (B) β-carotene bleaching assay; (C) DPPH radical scavenging activity assay; (D) Hydroxyl radical scavenging activity assay; (E) Superoxide radicals scavenging activity assay. Each test was performed thrice.
Figure 2. Antioxidant activities assessment of different oligosaccharides with various methods: (A) Ferric reducing antioxidant power (FRAP) assay; (B) β-carotene bleaching assay; (C) DPPH radical scavenging activity assay; (D) Hydroxyl radical scavenging activity assay; (E) Superoxide radicals scavenging activity assay. Each test was performed thrice.
Figure 3. Antioxidant activities assessment of different complex carbohydrates with various methods: (A) Ferric reducing antioxidant power (FRAP) assay; (B) 33
β-carotene bleaching assay; (C) DPPH radical scavenging activity assay; (D) Hydroxyl radical scavenging activity assay; (E) Superoxide radicals scavenging activity assay. Each test was performed thrice.
Table 1 Oxygen radical absorbance capacity values of carbohydrates
ORAC(μmol Trolox/g) Glucose
2.00a ± 1.10
Mannose
4.41a ± 0.72
Galactose
4.34a ± 0.38
Arabinose
4.80a ± 1.47
Xylose
4.09a ± 0.59
Glucuronic acid
1.13a ± 0.18
Galacturonic acid
7.47b ± 0.17
Sucrose
2.69a ± 0.17
Lactose
1.96b ± 0.26
Monosaccharide
Oligosaccharide
34
Carbohydrate
Complex Carbohydrate
Raffinose
1.07c ± 0.28
Maltotriose
1.25c ± 0.18
Maltotetraose
1.20c ± 0.40
Maltopentaose
0.96c ± 0.41
Maltohexaose
0.73c ± 0.49
Maltoheptaose
1.06c ± 0.50
Lipopolysaccharide
19.61a ± 0.12
Pectin
20.30a ± 1.03
Polygalacturonic acid
2.78b ± 0.07
Xylan
21.82a ± 0.82
Arabinogalactan
14.59a ± 0.34
Pullulan
1.85b ± 0.31
Galactomannan
1.52b ± 0.10
Arabinoxylan
15.76a ± 1.02
Glucomannan
10.67a ± 1.46
35
Means with different letters within monosaccharides are significantly different (P < 0.05).
Means with different letters within oligosaccharide are significantly different (P < 0.05).
Means with different letters within complex carbohydrate are significantly different (P < 0.05).
Table 2 The source, purity, molecular weight, chemical components and monosaccharide compositions of complex carbohydrates
Complex Carbohy drates
Sour ce
Lipopoly Esch saccharid erichi e a coli
Pectin
Apple
Monosaccharide composition Mol Total Total ecul Pu phen prote ar Galac rit olic in Ma Xy weig y cont cont Ara Gala Glu nno los turoni ht c % ent ent bino ctos cos se e (KD se % e % e % Acid % % % % a) % U kn A
U kn A
166 0.2
0.60a 2.08a ±0.0 ±0.0 2 5
n.d.
3.06
n.d.
n.d.
n.d .
n.d.
30-1 00B
0.60a 1.42a ±0.0 ±0.0 1 4
n.d.
n.d.
n.d.
n.d.
n.d .
77.71
36
Polygala cturonic acid
ukna
95
25-5 0B
0.19b 1.11b ±0.0 ±0.1 2 2
Xylan
Beec hwoo d
≥9 0
222. 5
0.43c 0.45b ±0.0 ±0.1 4 0
n.d.
Larc h wood
U kn
49.0
0.13b 0.65b ±0.0 ±0.2 2 2
4.54
68.9 1
n.d.
n.d.
n.d .
n.d.
454. 8
0.02d 0.28c ±0.0 ±0.0 1 5
n.d.
n.d.
95. 84
n.d.
n.d .
n.d.
107C
0.12b 0.61b ±0.0 ±0.1 1 4
n.d.
22.6 8D
n.d.
73.3 n.d 2D .
n.d.
D
n.d.
n.d.
n.d.
58. 9D
n.d.
n.d.
n.d.
39. 2D
58.8 n.d D .
n.d.
Arabinog alactan
A
Pullulan
ukna
95 .8 4
Galacto mannan
Caro b
＞ 94
Arabinox ylan
Whe at Flour
Glucoma nnan
Konj ac
95
＞ 98
56.7 C
321. 3
0.16b 1.16b ±0.0 ±0.0 0 5 0.26c 0.97b ±0.0 ±0.0 3 6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d .
＞ 90
95B
n.d.
B
36.1
n.d : Not detected. A : Unknown. B : Obtained from Sigma. C : Obtained from Megazyme by MAALS. D : Obtained from Megazyme. Means with different letters within total phenolic content column are significantly different (P < 0.05). Means with different letters within total protein content column are significantly different (P < 0.05). 37
Graphical abstract
38
Figures
Figure 1
A
B
39
C
D 40
E
41
Figure 2
A
42
B
C
43
D
44
E
45
Figure 3
A
B
46
C
47
D
E
48
Highlights
The antioxidant activities in vitro of different carbohydrates were studied by different manners.
The antioxidant activities of monosaccharide and oligosaccharide had not been found.
The antioxidant activities of complex carbohydrate were mainly attributed to the phenolic and protein components, rather than carbohydrate moiety.
49
PII: DOI: Reference:
S2212-6198(16)30006-7 http://dx.doi.org/10.1016/j.bcdf.2016.04.001 BCDF109
To appear in: Bioactive Carbohydrates and Dietary Fibre Received date: 26 January 2016 Revised date: 7 April 2016 Accepted date: 14 April 2016 Cite this article as: Shuzhen Hu, Junyi Yin, Shaoping Nie, Junqiao Wang, Glyn O. Phillips, Mingyong Xie and Steve W. Cui, In vitro evaluation of the antioxidant activities of carbohydrates, Bioactive Carbohydrates and Dietary Fibre, http://dx.doi.org/10.1016/j.bcdf.2016.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vitro evaluation of the antioxidant activities of carbohydrates Shuzhen Hua, Junyi Yina, Shaoping Niea*, Junqiao Wanga, Glyn O. Phillipsb, Mingyong Xiea, Steve W. Cuia,c* a
State Key Laboratory of Food Science and Technology, Nanchang University, 235
Nanjing East Road, Nanchang, Jiangxi Province, 330047, China b
Phillips Hydrocolloids Research Centre, Glyndwr University, W rexham, LL11 2AW
Wales, UK c
Agriculture and Agri-Food Canada, Guelph Food Research Centre, 93 Stone Road
West, Guelph, Ontario, NIG 5C9, Canada [email protected] [email protected] *
Corresponding authors. Shao-ping Nie & Steve W. Cui. Tel.: +86-791-88304452; +1
226 217-8076. Fax: +86-791-88304452; +1 519 780-2600
Abstract
In the current study, we evaluated the antioxidant activities of highly purified monosaccharides, oligosaccharides and complex carbohydrates using six in vitro antioxidant assays, including oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), β-carotene bleaching assay, 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, hydroxyl radical scavenging 1
and superoxide radicals scavenging methods. The results suggested that monosaccharides and oligosaccharides scarcely exhibited antioxidant activities in vitro. For example in the β-carotene bleaching assay the inhibitory effects of monosaccharides at 1 mg/mL were between 0.24% to 2.25% although galacturonic acid demonstrated some inhibitory effects (8.59%). Significant lower antioxidant activities were observed for complex carbohydrates compared to ascorbic acid and BHT (e.g. ferric reducing antioxidant power by the FRAP assay, >1800 μmol Fe (Ⅱ ) /g were observed for ascorbic acid and BHT compared to only 10-60 μmol Fe (Ⅱ) /g
for complex carbohydrates, and 1.60 and 6.43 μmol Fe (Ⅱ) /g for mono and
oligosaccharides. The observed antioxidant activities for complex carbohydrates are correlated with the presence of phenolic and/or protein components. The findings from the current study indicate that it is necessary to re-evaluate the antioxidant activities claimed for complex carbohydrates in the literature as many of them did not consider the contribution of other minor components such as phenolics and proteins.
Keywords: monosaccharide, oligosaccharide, complex carbohydrate, antioxidant activity
1. Introduction
2
Oxidative stress refers to the imbalance of oxidation and reduction in vivo. It leads to the production of oxidation intermediates which could cause base damage as well as strand breaks in DNA, hence become a serious threat to human health (Seifried, Anderson, Fisher, & Milner, 2007). Antioxidant components from food have been considered beneficial to human health because they can slow the effects of oxidative stress (Cook, & Samman, 1996). Antioxidants are also known to reduce the risk of Alzheimer's disease, cataracts, some types of cancer and cardiovascular diseases (Halliwell, Aeschbach, Löliger, & Aruoma, 1995; Oak, El Bedoui, & Schini-Kerth, 2005), improve human immunity and suppress inflammatory reactions (Yao, Jiang, Shi, Tomas-Barberan, Datta, Singanusong, & Chen, 2004). Thus, accurately evaluation of the activities of antioxidants of food components is important.
Many compounds from plant extracts exhibited antioxidant activities, such as pigments (fucoxanthin, anthocyanins, carotenoid e.g.) and polyphenols (phenolic acid, flavonoid, tannins e.g.) (Heo, Park, Lee, & Jeon, 2005; Soong, & Barlow, 2004; Lai, Xin, Zhao, Feng, He, Dong, ... & Wei, 2013; Van Acker, Tromp, Griffioen, Van Bennekom, Van Der Vijgh, & Bast, 1996). Numerous studies have reported that some selected polysaccharides have various biological activities, including antitumor activities, immunomodulatory effects and antiviral activities (Lv, Cheng, Zheng, Li, & Zhai, 2014; Ren, Jiao, Yang, Yuan, Guo, & Zhao, 2015; Xu, Zhang, Luo, Ma, Kou, & Huang, 2009; Zheng, Dong, Chen, Cong, & Ding, 2015). Some recent studies also 3
consider polysaccharides as sources of novel potential antioxidants (Petera et al., 2015; Chen, Ma, Liu, Liao, & Zhao, 2012; Fu, Chen, Dong, Zhang, & Zhang, 2010; Ferreira, Heleno, Reis, Stojkovic, Queiroz, Vasconcelos, & Sokovic, 2014; Ramarathnam, Osawa, Ochi, & Kawakishi, 1995). However, most of the studies on antioxidant activities of polysaccharides usually indicated they were mixed with some minor components, such as proteins and some low molecular weight substances (Chen, Zhang, Qu, & Xie, 2008; Wang et al., 2001; Wang, Zhao, Andrae-Marobela, Okatch, & Xiao, 2013). For example, Siu, Chen, & Wu (2014) suggested that the antioxidant activities of crude polysaccharide from mushrooms were mainly attributed to the phenolic and protein components, while Wang, Zhao, Andrae-Marobela, Okatch, & Xiao (2013) proved that tea polyphenols were the major antioxidants in the crude tea polysaccharides. There were also reports demonstrated that the antioxidant activities of polysaccharides could be significantly affected by modifications, such as sulfation and/or acetylation
(Delattre et al., 2015; Chen, Zhang, Wang, Nie, Li, &
Xie, 2014; Jin, Wang, Huang, Lu, & Wang, 2014; Qi, Zhang, Zhao, Chen, Zhang, Niu, & Li, 2005; Wang, Zhang, Zhang, & Li, 2008; Wang, Guo, Zhang, Wang, Zhao, Yao, & Wang, 2010). On the contrary, the antioxidant activities of purified polysaccharides and their fractions have been reported to be much weaker (Wang, & Luo, 2007). These variations on antioxidant activities of polysaccharide raised our curiosity. Does carbohydrate have antioxidant activities in vitro? Until present, little
4
information could be found in the literature on the antioxidant activity of carbohydrates.
Carbohydrate is known as polyhydroxy aldehydes and ketones, or substances that can be hydrolyzed to yield polyhydroxy aldehydes and ketones. It is covalently linked by glycosidic linkages, which are formed by condensation reaction between the glycosyl moiety of hemiacetal or hemiketal, together with the hydroxyl group of another sugar unit (Cui, 2005). Hence, there are few aldehyde or ketone group in carbohydrate solution; based on these structural characteristics we could hypothesis that pure carbohydrates do not have strong antioxidant activities. To anwser and confirm the question: the objectives of current study were to systematically evaluate the antioxidant activity of monosaccharide, oligosaccharide and some complex carbohydrate. Physical-chemical and compositions of complex carbohydrates studied were also determined to elucidate if other components contributed to the antioxidant activities of complex carbohydrates.
2. Materials and methods
2.1. Materials and chemicals
D - (+) - glucose, sucrose, lipopolysaccharide, polygalacturonic acid, pectin, xylan, arabinogalactan and D - (-) galacturonic acid were purchased from Sigma Chemical
5
Co. (St. Louis, MO USA). D - (+) - galactose, D - mannose, xylose, D - lactose, raffinose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, pullulan and D - glucuronic acid were from J&K Co. (Shanghai, China). D-(-) arabinose was the product of Solarbio Co. (Shanghai, China). Galactomannan, arabinoxylan and glucomannan were obtained from Megazyme Co. (Wicklow, Ireland). Trolox, 2, 2’-Azobis (2-methylpropionamidine) dihydrochloride (AAPH), fluorescein sodium salt, 2, 4, 6-Tris (2-pyridyl)-s-triazine (TPTZ), 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) and Coomassie Brilliant Blue G-250 were obtained from Sigma Chemical Co. (St. Louis, MO USA). Linoleic acid, β-carotene and pyrogallic acid were from Aladdin Co. (Shanghai, China). Bovine serum albumin (BSA) was from Solarbio Co. (Beijing, China). Gallic acid was purchased from J&K Co. (Shanghai, China). All other chemicals were of analytical reagent grade. All aqueous solutions were prepared using freshly double distilled water.
2.2. Antioxidant activities of monosaccharide
The antioxidant activities of different monosaccharides were evaluated by oxygen radical absorbance capacity, ferric reducing antioxidant power, β-carotene bleaching
6
assay, DPPH radical scavenging, hydroxyl radical scavenging and superoxide radicals scavenging assessments. Each test was performed in triplicates.
2.2.1. Oxygen radical absorbance capacity (ORAC)
The oxygen radical absorbance capacity was measured using a fluorescence microplate reader (Thermo Fisher Scientific, Thermo Electron Co., Waltham, MA, USA) described by previous report (Folch-Cano, Jullian, Speisky, & Olea-Azar, 2010), with slight modifications. The ORAC method is based on trolox as an antioxidant standard and 2, 2’-Azobis (2-methylpropionamidine) dihydrochloride (AAPH) as a peroxyl generator. The peroxyl radical scavenging was measured using fluorescein as fluorescent probe. The degree of change in fluorescence intensity reflects the extent of free radical damage. Antioxidant can inhibit the fluorescence decay caused by free radicals, reflecting its ability to inhibit free radicals (Barahona, Encinas, Imarai, Mansilla, Matsuhiro, Torres, & Valenzuela, 2014). The reaction was carried out in 75 mM sodium phosphate buffer (pH 7.4). Sample, standard and phosphate buffer (60 μL, 75mM, pH 7.4) were placed in the wells of the microplate. Then fluorescein solution (40 μL, 1.75 μM) was added. The mixture was pre-incubated for 15 min at 37 °C, before adding the AAPH solution (100 μL, 10 mM). The microplate was shaken automatically for 30 seconds and then was immediately placed in the reader. The fluorescence (excitation wave length of 460 nm, emission wave length of 515 nm) was recorded every 2 min for 120 min.
7
The ORAC values were calculated as the area under the curve (AUC) and the inhibition capacity was expressed as Trolox equivalents (μmol Trolox/g).
2.2.2. Ferric reducing antioxidant power (FRAP)
The FRAP assay was performed as described previously (Xu, & Chang, 2007) with some modifications. Antioxidant donates an electron to TPTZ-Fe (Ⅲ) complex, which could be reduced to TPTZ-Fe (Ⅱ) form. TPTZ-Fe (Ⅱ) has an intensive blue color and can be monitored at 593nm (Benzie, & Strain, 1996). Briefly, The working FRAP reagent was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ and 20 mM FeCl3 in a volume ratio of 10:1:1. Sample solution (200 μL) was mixed with 1.8 mL working FRAP reagent, and incubated at 37 °C for 30 min. The absorbance was measured at 593 nm. FRAP value was calculated using the concentration of FeSO4·7H2O equivalent. Ascorbic acid and BHT were used as positive controls. The reducing capacity was expressed as μmol Fe (Ⅱ)/g. 2.2.3. β-carotene bleaching assay Antioxidant activity was carried out according to the β-carotene bleaching assay (Chen, Zhang, Wang, Nie, Li, & Xie, 2014). Linoleic acid becomes a free radical with a hydrogen atom abstracted. The radical formed then attacks β-carotene molecule and the compound loses its characteristic orange color. Antioxidant can neutralize the linoleate free radical and thus inhibit the oxidation of linoleic acid (Deba, Xuan,
8
Yasuda, & Tawata, 2008). β-carotene (0.5 mg) was dissolved in 1 mL of chloroform. Twenty five μL linoleic acid and 200 mL Tween 20 were added. The chloroform was evaporated under nitrogen at 45 °C, then 100 mL distilled water was added and shook vigorously. Three hundred and fift μL of sample and 2.5 mL of the β-carotene-linoleic acid emulsion were mixed into test tube. The absorbance was immediately measured at 490 nm. The reaction mixture was incubated at 50 °C for 2 h and the absorbance was measured again. The ascorbic acid and butyl hydroxyl toluene (BHT) were used as a positive control.
2.2.4. DPPH radical scavenging activity
Relatively stable DPPH radical has been widely used to evaluate the free radical scavenging capacities of antioxidants. Antioxidant transfer hydrogen atoms to DPPH ·, leading to non-radical form DPPH-H (Li, Li, & Zhou, 2007). The antioxidant activity of a substance can be expressed as its ability in scavenging the DPPH radical. The scavenging effect of the sample on DPPH radical was measured according to the method as previously described (Wang, Liu, Huo, Zhao, Ren, & Wei, 2013). Sample solutions (1.0 mL) with variable concentrations were added with 2.0 mL DPPH 0.2mM in ethanol). The mixture was shaken and kept at room temperature in the dark. The absorbance was measured at 517 nm 30 min later. The control was done with ethanol instead of DPPH, while ethanol was used as the blank. The DPPH radical scavenging activity (%) was calculated according to the following equation:
9
Scavenging activity (%) = [1-(As-Ac)/A] × 100
Where As was the absorbance of sample and Ac was the absorbance of the ethanol control solution, and A was the absorbance of solution including 2 mL of DPPH and 1mL of ethanol. Ascorbic acid and BHT were used as standards.
2.2.5. Hydroxyl radical scavenging activity
The hydroxyl radical is known to be generated through the Fenton reaction (Erel, 2004). The scavenging of hydroxyl radical is very important due to the harmful role of free radicals in biological systems (Ke, Sun, Qiao, Wang, & Zeng, 2011). The hydroxyl radical scavenging activity of sample was evaluated according to the method described by Huang, Tu, Jiang, Xiao, Zhang, & Wang, (2012). Different concentrations of the samples (1 mL) were incubated with FeSO4 (1 mL, 4 mM), salicylic acid-ethanol (1 mL, 6 mM), and H2O2 (1 mL, 2 mM) at 37 °C for 60 min. Then the absorbance of the mixture was recorded at 510 nm. Ascorbic acid was used as positive control. The scavenging activity was calculated using the following equation:
Scavenging activity (%) = [1-(A1-A2)/A0] × 100
where A0 was the absorbance of the control (without extract), A1 was the absorbance of the sample and A2 was the absorbance without H2O2.
2.2.6. Superoxide radicals scavenging activity 10
Superoxide radicals are a precursor to active free radicals that have potential of reacting with biological macromolecules functioning in living cells, which could induce tissue damage (Gülçin, 2006). Therefore, it is extremely important to scavenge the superoxide radicals for anti-oxidation to work. The superoxide radicals were generated by autoxidation of pyrogallol in an alkalescent condition (Wang, Wang, & Quan, 2014). In the presence of antioxidant, these antioxidants could combine with the superoxide radicals and form a stable radical to terminate the radical chain reaction. The assay was carried out according to the method of Marklund, & Marklund (1974) with some modifications. Tris-HCl buffer (pH 8.2, 5 mL) was mixed with sample solution (350 μL) and incubated at 25 °C for 20 min. Then 1, 2, 3-phentriol (0.2 mL, 45 mM) was added and the mixture was shaken rapidly. The absorbance of the mixture at 325 nm was recorded every 10 s for 1 min and a slope was calculated as absorbance / min. The ability of scavenge superoxide radical was calculated as follows:
Scavenging effect (%) = (1-slope of sample / slope of control) × 100
2.3. Antioxidant activities of oligosaccharide
The antioxidant activities of oligosaccharides were investigated and evaluated by the tests described above (Section of 2.2). Oligosaccharides evaluated include sucrose,
11
lactose, raffinose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose.
2.4. Antioxidant activities and physical-chemical characteristics of complex carbohydrates
2.4.1. Antioxidant activities of complex carbohydrates
The antioxidant activities of complex carbohydrates were referred to the six assays, which was the same as assays for evaluating monosaccharide (Section of 2.2). The complex carbohydrates include lipopolysaccharide (carbohydrate compound), pectin, polygalacturonic acid, xylan, arabinogalactan, pullulan, galactomannan, arabinoxylan and glucomannan.
2.4.2. Physical-chemical properties
2.4.2.1. Protein and phenolic content
Protein content was measured according to Bradford’s method with bovine serum albumin (BSA) as a standard (Bradford, 1976). The level of total phenolic was determined with Folin-Ciocalteu’s reagent (FCR) according to Khokhar’s method (Khokhar, & Magnusdottir, 2002). All measurements were repeated thrice.
2.4.2.2. Monosaccharide compositions 12
The sample (5 mg) was placed in 1 mL of 12 M H2SO4 in a test tube, then rapidly stirring in ice-bath for 30 min. Dilution (2 M H2SO4) was carried out using ultra-pure water followed by hydrolysis at 100 °C for 2 h. The hydrolysis product was obtained by centrifugation at 4800 × g for 10 min after being neutralized by BaCO3 (Xie, Shen, Nie, Liu, Zhang, & Xie, 2013). A mixture of monosaccharide standards was used to determine the monosaccharide composition.
Monosaccharide compositions were analyzed by HPAEC. HPAEC was performed on a Dionex system using a CarboPacTM PA20 analytical column (3 mm × 150 mm) and CarboPacTM PA20 guard column (3 mm × 30 mm). Detection was pulsed amperometry (Xie, Shen, Nie, Liu, Zhang, & Xie, 2013).
2.4.2.3. Molecular weight of complex carbohydrate
The molecular weight of samples was characterized by high performance size exclusion chromatography with multi-angle laser light scattering (HPSEC-MALLS) (Yin, Nie, Guo, Wang, Cui, & Xie, 2015). The sample detection was performed by a multi-angle laser light scattering detector, a refractive index detector and a viscometer (Wyatt Technology Co., USA). A Wyatt D05SFD01 pump (Wyatt Technology Co., Goleta, CA, USA) with three columns in series were used: a SB-G guard column (8 mm×50 mm), a SB-806 HQ (8 mm×300 mm) and a SB-804 HQ (8 mm×300 mm) (Shodex Ohpak, Showa Denko K.K., Tokyo, Japan). The columns were maintained at
13
35 ºC. The mobile phase was 0.1 M NaNO3 containing 0.02% NaN3 at a flow rate of 0.6 mL/min. Data was collected and analyzed using ASTRA 6.1 software.
2.5. Statistical analysis
All measurements were done in triplicates and results were expressed as mean ± standard deviation (SD). SPSS Statistics 19 was used for the data analysis. The P values were set at P < 0.05 to assess the statistically significant.
3. Results and discussions
3.1. Antioxidant activities of monosaccharide
Oxygen radical absorbance capacity (ORAC) values of monosaccharide are presented in Table 1. All the neutral monosaccharides showed very low ORAC values, ranging from 1.13 to 4.80 μmol Trolox/g. Galacturonic acid showed the highest ORAC value (7.47) while glucuronic acid showed the lowest value (1.13 μmol Trolox/g).
The results of antioxidant activities of monosaccharides were compared with ascorbic acid and BHT (Fig. 1A). All monosaccharides scarcely showed any reducing power: the FRAP values of different monosaccharides were between 1.60 and 6.43 μmol Fe (Ⅱ) /g compared to over 2000 μmol Fe (Ⅱ) /g for ascorbic acid and BHT (Fig.1A).
14
It was interesting to observe that galacturonic acid had the highest FRAP value among the onosaccharides examined (~20 μmol Fe (Ⅱ) /g, Fig. 1A). The antioxidant activities of monosaccharides in the β-carotene bleaching assay are shown in Fig. 1B. The inhibitory effects of glucose, mannose, galactose, arabinose, xylose, glucuronic acid and galacturonic acid at 1 mg/mL were 1.31%, 0.37%, 1.82%, 2.25%, 0.24%, 1.78% and 8.59%, respectively, indicating that all monosaccharides scarcely inhibited the bleaching effect of β-carotene except for galacturonic acid, which demonstrated some inhibitory effects.
The scavenging activities of monosaccharides on the DPPH radical are shown in Fig. 1C. The scavenging effects of glucose, mannose, galactose, arabinose, xylose, glucuronic acid and galacturonic acid were 7.76%, 5.71%, 4.21%, 7.15%, 3.54%, 8.15% and 8.85% at 1 mg/mL for the DPPH radical, respectively, as shown in Fig.1C, indicating that all monosaccharides exhibited some scavenging activities against DPPH radical. However, compared to ascorbic acid and BHT, the scavenging activities of these monosaccharides were ignorable and hardly changed with increasing concentration (Fig.1C).
The scavenging effects against hydroxyl radical were 1.89%, 5.61%, 8.30%, 2.60%, 4.75%, 11.96% and 14.84% for glucose, mannose, galactose, arabinose, xylose, glucuronic acid and galacturonic acid at the concentration of 2 mg/mL, respectively, as shown in Fig.1D. Again, all monosacchardies tested exhibited no effect except the 15
two uronic acids; galactose also exhibited some small effect. Fig. 1E depicted the inhibitory effect of monosaccharides on superoxide radicals. The inhibitory effects were only between 0.22%, and 5.89% for all the monosaccharides examined at the concentration of
1 mg/mL compared to 87% exhibited by ascorbic acid. Based
on this observation, all monosaccharides barely inhibited the autoxidation of pyrogallol. In nature, aldopentose, aldohexose and ketohexose mainly exist in the form of closed ring, only few of them have the open-chain form in solution (Toukach, & Ananikov, 2013). Hence, there are few aldehyde or ketone groups in carbohydrate solution. Moreover, the hydrogen is inactive on the hydroxyl and carbon chain, it cannot provide proton or electron to be an effective reducing agent.
Based on the six assays, we could conclude that monosaccharides do not have antioxidant activities, except for uronic acids, which exhibited weak antioxidant activities. Among the two uronic acids tested, galacturonic acid was always slightly stronger than that of glucuronic acid. This observation is consistent with the report by Rao, & Muralikrishna (2006), possible due to the different orientation of the –OH group in galacturonic acid, hence, exhibits stronger antioxidant activity than glucuronic acid. Therefore, galacturonic acid content in acidic polysaccharides should be an important factor concerning their antioxidant activities (Asker, Manal, & Ghada, 2007; Wang, Chang, Inbaraj, & Chen, 2010). However, the detailed differences of the antioxidant mechanism between galacturonic acid and glucuronic acid are still not clear. Further studies are required to elucidate the mechanisms. 16
3.2. Antioxidant activities of oligosaccharide
Oligosaccharides contain between two and twenty monosaccharide residues linked through glycosidic linkages. The antioxidant activities of oligosaccharides as evaluated by ORAC, FRAP, β-carotene bleaching, DPPH, OH·and O2· assays are shown in Table 1 and Fig. 2. From these data we could conclude that, similar to monosaccharides, all oligosaccharides showed poor ORAC values (only ranging from 0.73 to 2.69 μmol Trolox/g) and exhibited very poor reducing power when comparing with ascorbic acid and BHT. For example, FRAP values of oligosaccharides were between 1.65 and 3.77 μmol Fe (Ⅱ) /g compared to 2144.71 and 1832.04 μmol Fe ( Ⅱ)/g for ascorbic acid and BHT up, respectively. These results are consistent with the notion that oligosaccharides cannot provide proton or electron to be effective reducing agents (Toukach, & Ananikov, 2013). The tested oligosaccharides exhibited negligible scavenging abilities against DPPH, OH· and O2· radicals. In addition, oligosaccharides barely inhibited the bleaching of β-carotene in the β-carotene-linoleic acid system. We could conclude that all the oligosaccharides evaluated scarcely showed any antioxidant activities.
3.3. Antioxidant activities of complex carbohydrates
17
We have selected some polysaccharides for testing their antioxidant activities in vitro, the results are shown in Table 1 and Fig. 3. The ORAC value for pectin was 20.30 μmol Trolox/g compared to polygalacturonic acid, which was only 2.78 μmol Trolox/g (Table 1). However, polygalacturonic acid and pectin exhibited 20.4 and 23.1 μmol Fe (Ⅱ) /g for FRAP, respectively. The radical scavenging activities of pectin and polygalacturonic acid on DPPH, OH· and O2· are showed in Fig. 3C, 3D and 3E respectively. The DPPH scavenging percentages of pectin and polygalacturonic acid (at 2 mg/mL) were 37.23% and 4.11%, respectively, and it was also observed that polygalacturonic acid on DPPH scavenging activities was scarcely changed with increasing concentration. Compared with pectin, polygalacturonic acid exhibited a higher scavenging effects against OH·and O2, and a higher percentages of β-carotene bleaching at 2 mg/mL. This is in agreement with the observation by Wu, Min, Li, Li, Lai, Tang, & Yang (2013) that WP3, a pectin with higher galacturonic acid content exhibited higher antioxidant activities than WP, which had lower galacturonic acid content. Nevertheless, it is still not clear why the different antioxidant results were observed for polygalacturonic acid and pectin in ORAC, DPPH radical scavenging and β-carotene bleaching assays. We suspect that different acting mechanisms are involved with each assay, as well as the solubility of the material in the testing system would play important roles in their antioxidant effects. Further research is needed to elucidate the possible mechanisms involved.
18
Weak antioxidant activities were also observed for lipopolysaccharide, xylan, arabinogalactan and arabinoxylan, as showed in Table 1 and Fig. 3. The ORAC values of lipopolysaccharide, xylan, arabinogalactan, and arabinoxylan were 19.16, 21.82, 14.59 and 15.76 μmol Trolox/g, respectively. Besides, they showed 17.3, 63.1, 33.6 and 5.6 μmol Fe (Ⅱ) /g for FRAP, same trends were observed for scavenging effects on OH· (Fig. 3D). The four polysaccharides exhibited similar antioxidant activities ·
when assessed by DPPH and O2 , and the activities hardly changed with increasing of concentration (Fig. 3C and 3E). However, lipopolysaccharide and xylan (2 mg/mL) had relatively higher inhibited percentages of β-carotene (12.36% and 24.94%, respectively) than arabinogalactan and arabinoxylan (3.44% and 3.22%, respectively), likely due to the higher solubility of the lipopolysaccharides in the lipid phase of the emulsion. Three other neutral polysaccharides, i.e. pullulan, galactomannan and glucomannan exhibited low overall antioxidant activities (Fig. 3B, 3D and 3E; Table 1).
3.4. Compositions of complex carbohydrates
The above results suggested that pectin had relatively higher antioxidant activities as expressed by ORAC values, reducing power and DPPH radical scavenging. Composition analysis showed that pectin contained 0.60% of phenolic and 1.42% of protein, which was slightly, but significantly higher than that of polygalacturonic acid (0.19% of phenolic, and 1.11% of protein, respectively) (Table 2). These differences
19
in composition could be responsible for the antioxidant of pectin and polygalacturonic acids, which was also reported in the literature (Siu, Chen, & Wu, 2014; Wang, Zhao, Andrae-Marobela, Okatch, & Xiao, 2013; Liu, Ooi, & Chang, 1997).
Similarly, a
study by Hromadkova et al. pointed out that phenolic compounds played a significant role on the radical scavenging effects of xylans (Hromádková, Paulsen, Polovka, Košťálová, & Ebringerová, 2013). lipopolysaccharide and xylan also showed relatively higher inhibited percentages of the bleaching of β-carotene, somewhat higher scavenging effect against hydroxyl radical than arabinogalactan and arabinoxylan, which are consistent with their relatively higher contents of proteins and phenolics (Table 2). The results indicated that both protein and phenolic compounds contributed to their antioxidant activities which is in agreement with literature reports (Moraes, da Silva Marineli, Lenquiste, Steel, de Menezes, Queiroz, & Júnior, 2015; Dlamini, Taylor, & Rooney, 2007).
Consistent with above analysis,
pullulan, galactomannan and glucomannan
exhibited lower inhibition on the bleaching of β-carotene and little scavenging activities against OH· and O2·. These results might be ascribed to the lack of phenolics or other minor components for pullulan, galactomannan and glucomannan (Table 2) (Siu, Chen, & Wu, 2014; Wu, Wang, Wang, Shen, He, Gu, & Wu, 2014). The total carbohydrate contents of these three polysacchardies were all beyond 95% (Table 2). The findings in the current study indicated that the phenolic and protein
20
constituents in complex carbohydrates may play a significant role in their antioxidant activities.
4. Conclusions
In this study, it was confirmed that monosaccharides and oligosaccharides hardly exhibited antioxidant activities as evaluated by six in vitro methods. Lipopolysaccharide, pectin and xylan exhibited weak reducing power, low ORAC values and part of hydroxyl radical scavenging effect most likely due to the relatively higher phenolic and protein contents,. Additionally, lipopolysaccharide and xylan showed relatively higher inhibited percentages of β-carotene bleaching. On the contrary, pullulan, galactomannan and glucomannan had almost no phenolic contents, showed significantly lower inhibition on the bleaching of β-carotene and little scavenging activities against OH· and O2·. The antioxidant activities of complex carbohydrates results in the literature were mainly attributed to the phenolic and protein components, rather than the carbohydrate moieties. Based on the current study and review of literature reports, we could conclude that carbohydrates do not have antioxidant activities in vitro. This finding also suggested there is a need to reevaluate the antioxidant activities claimed for complex carbohydrates in the literature.
Acknowledgements
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This study was supported by National Natural Science Foundation of China for Excellent Young Scholars (31422042), the Program for New Century Excellent Talents in University (NCET-12-0749), the Key Project of International Cooperation of Jiangxi Provincial Department of Science and Technology (20141BDH80009), the Project of Science and Technology of Jiangxi Provincial Education Department (KJLD13004)，This work was also supported by Cooperation Initiative with Guelph Food Research Centre, Agriculture and Agri-Food Canada.
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Figure 1. Antioxidant activities assessment of different monosaccharides with various methods: (A) Ferric reducing antioxidant power (FRAP) assay; (B) β-carotene bleaching assay; (C) DPPH radical scavenging activity assay; (D) Hydroxyl radical scavenging activity assay; (E) Superoxide radicals scavenging activity assay. Each test was performed thrice.
Figure 2. Antioxidant activities assessment of different oligosaccharides with various methods: (A) Ferric reducing antioxidant power (FRAP) assay; (B) β-carotene bleaching assay; (C) DPPH radical scavenging activity assay; (D) Hydroxyl radical scavenging activity assay; (E) Superoxide radicals scavenging activity assay. Each test was performed thrice.
Figure 3. Antioxidant activities assessment of different complex carbohydrates with various methods: (A) Ferric reducing antioxidant power (FRAP) assay; (B) 33
β-carotene bleaching assay; (C) DPPH radical scavenging activity assay; (D) Hydroxyl radical scavenging activity assay; (E) Superoxide radicals scavenging activity assay. Each test was performed thrice.
Table 1 Oxygen radical absorbance capacity values of carbohydrates
ORAC(μmol Trolox/g) Glucose
2.00a ± 1.10
Mannose
4.41a ± 0.72
Galactose
4.34a ± 0.38
Arabinose
4.80a ± 1.47
Xylose
4.09a ± 0.59
Glucuronic acid
1.13a ± 0.18
Galacturonic acid
7.47b ± 0.17
Sucrose
2.69a ± 0.17
Lactose
1.96b ± 0.26
Monosaccharide
Oligosaccharide
34
Carbohydrate
Complex Carbohydrate
Raffinose
1.07c ± 0.28
Maltotriose
1.25c ± 0.18
Maltotetraose
1.20c ± 0.40
Maltopentaose
0.96c ± 0.41
Maltohexaose
0.73c ± 0.49
Maltoheptaose
1.06c ± 0.50
Lipopolysaccharide
19.61a ± 0.12
Pectin
20.30a ± 1.03
Polygalacturonic acid
2.78b ± 0.07
Xylan
21.82a ± 0.82
Arabinogalactan
14.59a ± 0.34
Pullulan
1.85b ± 0.31
Galactomannan
1.52b ± 0.10
Arabinoxylan
15.76a ± 1.02
Glucomannan
10.67a ± 1.46
35
Means with different letters within monosaccharides are significantly different (P < 0.05).
Means with different letters within oligosaccharide are significantly different (P < 0.05).
Means with different letters within complex carbohydrate are significantly different (P < 0.05).
Table 2 The source, purity, molecular weight, chemical components and monosaccharide compositions of complex carbohydrates
Complex Carbohy drates
Sour ce
Lipopoly Esch saccharid erichi e a coli
Pectin
Apple
Monosaccharide composition Mol Total Total ecul Pu phen prote ar Galac rit olic in Ma Xy weig y cont cont Ara Gala Glu nno los turoni ht c % ent ent bino ctos cos se e (KD se % e % e % Acid % % % % a) % U kn A
U kn A
166 0.2
0.60a 2.08a ±0.0 ±0.0 2 5
n.d.
3.06
n.d.
n.d.
n.d .
n.d.
30-1 00B
0.60a 1.42a ±0.0 ±0.0 1 4
n.d.
n.d.
n.d.
n.d.
n.d .
77.71
36
Polygala cturonic acid
ukna
95
25-5 0B
0.19b 1.11b ±0.0 ±0.1 2 2
Xylan
Beec hwoo d
≥9 0
222. 5
0.43c 0.45b ±0.0 ±0.1 4 0
n.d.
Larc h wood
U kn
49.0
0.13b 0.65b ±0.0 ±0.2 2 2
4.54
68.9 1
n.d.
n.d.
n.d .
n.d.
454. 8
0.02d 0.28c ±0.0 ±0.0 1 5
n.d.
n.d.
95. 84
n.d.
n.d .
n.d.
107C
0.12b 0.61b ±0.0 ±0.1 1 4
n.d.
22.6 8D
n.d.
73.3 n.d 2D .
n.d.
D
n.d.
n.d.
n.d.
58. 9D
n.d.
n.d.
n.d.
39. 2D
58.8 n.d D .
n.d.
Arabinog alactan
A
Pullulan
ukna
95 .8 4
Galacto mannan
Caro b
＞ 94
Arabinox ylan
Whe at Flour
Glucoma nnan
Konj ac
95
＞ 98
56.7 C
321. 3
0.16b 1.16b ±0.0 ±0.0 0 5 0.26c 0.97b ±0.0 ±0.0 3 6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d .
＞ 90
95B
n.d.
B
36.1
n.d : Not detected. A : Unknown. B : Obtained from Sigma. C : Obtained from Megazyme by MAALS. D : Obtained from Megazyme. Means with different letters within total phenolic content column are significantly different (P < 0.05). Means with different letters within total protein content column are significantly different (P < 0.05). 37
Graphical abstract
38
Figures
Figure 1
A
B
39
C
D 40
E
41
Figure 2
A
42
B
C
43
D
44
E
45
Figure 3
A
B
46
C
47
D
E
48
Highlights
The antioxidant activities in vitro of different carbohydrates were studied by different manners.
The antioxidant activities of monosaccharide and oligosaccharide had not been found.
The antioxidant activities of complex carbohydrate were mainly attributed to the phenolic and protein components, rather than carbohydrate moiety.
49