Characterization of a Novel Alkali-Soluble Heteropolysaccharide from

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Characterization of a Novel Alkali-Soluble Heteropolysaccharide from Tetraploid Gynostemma pentaphyllum Makino and Its Potential Anti-inflammatory and Antioxidant Properties Yuge Niu,*,†,∥ Pingping Shang,†,∥ Lei Chen,† Hua Zhang,*,‡ Lu Gong,† Xiaowei Zhang,† Wenjuan Yu,§ Yuhong Xu,# Qin Wang,⊥ and Liangli (Lucy) Yu†,⊥ †

Institute of Food and Nutraceutical Science, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China ‡ School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China § Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai 200240, China # School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China ⊥ Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742, United States S Supporting Information *

ABSTRACT: A novel polysaccharide (GPP-S), with a molecular mass of 1.2 × 106 Da, was isolated from the tetraploid Gynostemma pentaphyllum Makino by alkali extraction followed by purifications using DEAE and Sephacryl S-400 column chromatographies. The monosaccharide composition of GPP-S was determined as rhamnose, arabinose, glucose, and galactose with a molar ratio of 1.00:3.72:19.49:7.82. The structural analysis suggested that the backbone of GPP-S is (1→4)-linked-glucose and (1→6)-linked-galactose with a (1→4,6)-linked-glucose branch every six monosaccharide residues. The terminals were 1-)α-arabinose, glucuronic acid, and other monosaccharides. GPP-S exhibited scavenging capacities against hydroxyl, peroxyl, and DPPH• radicals in vitro. GPP-S also had inhibitory activities on IL-1β, IL-6, and COX-2 gene expressions in RAW 264.7 mouse macrophage cells. These results suggested that GPP-S could be developed as a bioactive ingredient for functional foods and dietary supplements. KEYWORDS: Gynostemma pentaphyllum Makino polysaccharide, structure analysis, alkali extraction, antioxidant, pro-inflammatory cytokines

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believed to up-regulate and modulate the inflammatory process.12 Antioxidants are molecules that could react with ROS and terminate their intermediate hazardous effects including inflammation and are in high demand for reducing the risk of human chronic diseases.13 Polysaccharides have also been proved to be able to reduce inflammation in a mouse model in a manner independent from their antioxidant properties.14 In this study, a novel polysaccharide (GPP-S) was isolated from tetraploid G. pentaphyllum by alkali extraction followed by DEAE and Sephacryl S-400 column chromatography purifications for the first time. Its monosaccharide composition, chemical structure, and functionalities were analyzed. The results implied that GPP-S was an important functional component in G. pentaphyllum, and it could be developed as a bioactive ingredient for functional foods and dietary supplements for its potential antioxidant and anti-inflammation effects.

INTRODUCTION Gynostemma pentaphyllum Makino is a kind of Cucubitaceae herb that grows widely in China, Japan, and many other Asian countries.1 It has been used in food and tea products for thousands of years for its high saponins, which are also known as gypenosides content. Recently, there is growing evidence indicating that G. pentaphyllum has a wide variety of bioactivities, including antigastric ulcer,2 cardioprotection,3 hepatoprotection,4 chronic bronchitis mitigation treatments,5 and other health beneficial properties. Besides saponins, polysaccharides are another group of important components attributed with beneficial functions and minimal potential toxicity.6 Furthermore, polysaccharides are water-soluble or partially water-soluble and may be easily soaked out into an aqueous phase such as tea. Nowadays, natural water-soluble polysaccharides have attracted a lot of attention because of their possible antitumor,7 antioxidant,8 immune-enhancing,9 and antiexercise fatigue acivities.10 Reactive oxygen species (ROS) may attack and damage DNA, proteins, carbohydrates, and membrane lipids in an organism and cause inflammation and some inflammation-associated diseases including cancer, cardiovascular diseases, and Parkinson’s disease.11 ROS could trigger inflammation by inducing the release of proinflammatory cytokines. On the other hand, ROS, as well as cytokines and lipid mediators, have also been reported to participate in the regulation of heat shock protein (HSP) synthesis. HSPs are © 2014 American Chemical Society

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MATERIALS AND METHODS

Materials. Tetraploid whole-plant G. pentaphyllum was obtained from Asian Citrus Holdings Limited (Hong Kong). The botanical Received: Revised: Accepted: Published: 3783

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samples were ground and treated with ethanol to remove oligosaccharides. DEAE was purchased from Whatman International Ltd. (Kent, UK). Sephacryl S-400 was purchased from GE Healthcare (Uppsala, Sweden). Dextrans with different molecular weights and standard monosaccharides (arabinose, glucose, mannose, galactose, rhamnose, fucose, and xylose) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Dihydrochloride (AAPH), 2,2′-azobis(2-amidinopropane), and myo-inositol were purchased from J&K Scientific (Beijing, China). Iron(III) chloride, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), fluorescein (FL), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). RAW 264.7 mouse macrophage was purchased from the Chinese Academy of Sciences (Shanghai, China). TRIzol reagent was bought from Invitrogen (Life Technologies, Carlsbad, CA, USA), whereas an IScript Advanced cDNA Synthesis kit was purchased from Bio-Rad (Bio-Rad Laboratories, Hercules, CA, USA). Lipopolysaccharide (LPS) from Escherichia coli 0111: B4 was obtained from Millipore (Millipore, MA, USA). All other reagents and chemicals were of the highest commercial grade and used without further purification. Extraction, Isolation, and Purification of GPP-S. Ground G. pentaphyllum was extracted by distilled water and then decocted with 2% NaOH at 80 °C three times for 2 h for each time. The extracts were neutralized and combined after filtering and concentrated in a hot water bath. Hydrogen peroxide was added to remove the pigment at 60 °C for 6 h. The supernatant was then collected after centrifugation at 4500 rpm for 10 min and precipitated by adding 4 times volumes of ethanol. The crude polysaccharide of G. pentaphyllum was collected by centrifugation, followed by washing with ethanol and acetone, and finally dried with nitrogen. The crude polysaccharide (500 mg) was dissolved in 6 mL of distilled water. After centrifugation, the supernatants were applied to a DEAE column (2.5 cm × 30 cm) and eluted with 0.6 M NaCl at a flow rate of 0.6 mL/min. Fractions were collected using an automatic fraction collector and examined with the phenol−sulfuric acid method. Each eluting peak was collected, concentrated, and loaded onto a column of Sephacryl S-400. The elutes were lyophilized to obtain the pure polysaccharide, named GPP-S. Purity and Molecular Weight Determination. The purity and molecular weight of GPP-S were determined using an HPLC-ELSD (Shimadzu Technologies, Japan) with a gel filtration column (Shodex SUGAR KS-805, 8 mm i.d. × 300 mm, Showa Denko, Japan). The mobile phase was deionized water eluting at a flow rate of 1.0 mL/min for 30 min. The calibration curve was prepared with standard dextrans with different molecular weights (1800, 2500, 4600, 7100 and 2,000,000 Da). The mass of GPP-S was calculated by comparing the retention time of the standards. Monosaccharide Composition Analysis. According to the method described previously,15 GPP-S (5 mg) was incubated at 100 °C for 8 h after being dissolved in 1 mL of 2 M trifluoroacetic acid (TFA). The excess acid was removed by codistillation with 0.1% HCl−methanol. The residue was dissolved in 1 mL of distilled water ready for the following experiments. Monosaccharide composition was analyzed according to a previously described procedure with minor modifications.16 Each of the seven monosaccharide standards and the hydrolyzed polysaccharide were reacted with NaBH4 at 65 °C for 1 h. The mixture was acetylated with pyridine and acetic anhydride at 100 °C for 2 h. Chloroform was used to extract the residues. The analysis was performed using an Agilent 7890A GC with a G4513 FID injector (Agilent Technologies, Palo Alto, CA, USA) equipped with an HP-5 capillary column (30 m × 0.32 mm, 0.25 μm). Injection volume was 1 μL. Oven temperature was first from 110 to 220 °C at 5 °C/min, held for 2 min, increased by 2 °C/min to 240 °C, held for 2 min, and then increased to a final temperature of 280 °C by 10 °C/min. PMP derivatization of monosaccharides was performed according to a protocol described previously.17 Briefly, 50 μL of seven standard monosaccharide mixtures or the hydrolyzed polysaccharide was mixed with 50 μL of NaOH (0.6 M), and then 50 μL of PMP methanol

solution (0.5 M) was added to each sample. The mixture was allowed to react for 30 min at 70 °C, then cooled to room temperature, and neutralized with 100 μL of 0.3 M HCl. One milliliter of water was added to the solution, which was then extracted by chloroform (1.0 mL) three times. The aqueous layer was combined and centrifuged at 10000 rpm for 5 min. HPLC system equipment with a Zorbax Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm, Agilent Technologies) was used to detect the PMP-labeled monosaccharide monitored by UV detector at 250 nm. The mobile phase was prepared by ammonium acetate solution (pH 5.5) and acetonitrile with a ratio of 78:22. Infrared Spectral Analysis. The FT-IR spectrum was determined using a Fourier transform infrared spectrophotometer (Nicolet iN10 MX, Thermo Nicolet, USA) in the range of 4000−400 cm−1. The sample was ground with spectroscopic grade potassium bromide (KBr) powder and pressed into 1 mm pellets for FT-IR measurements. Methylation Analysis. Methylation analysis was performed according to the method of Needs and Selvendran with some modification.18 Ten milligrams of GPP-S and NaOH (100 mg) were dissolved in 5 mL of DMSO. The mixture was methylated with 2 mL of methanol iodide with stirring. The completion of the methylation of GPP-S was confirmed by the disappearance of the OH band (3200− 3700 cm−1) in the IR spectrum. The methylated product was depolymerized in 3 mL of formic acid and further hydrolyzed by 2 M TFA (3 mL) at 100 °C for 3 h. After being reduced with sodium borohydride at 60 °C for 1 h, the methylated and hydrolyzed products of the GPP-S were acetylated with acetic anhydride and pyridine (1:1 molar ratio) for 1 h at 100 °C. The resulting product was washed with distilled water and dissolved in chloroform to be subjected to GC-MS analysis and identification according to the relative retention times and fragmentation patterns. The molar ratios of each residue were calibrated from peak areas and the response factor in GC. NMR Spectroscopy. Polysaccharide was dissolved in D2O. 1D and 2D spectra were recorded on a Bruker AV-500 MHz NMR spectrometer. Oxygen Radical Absorbing Capacity (ORAC). The ORAC value was detected with fluorescein (FL) as the fluorescent probe using a Synergy 2 Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA) according to a previously reported laboratory protocol.19 Trolox was used as the standard. The initial reaction mixture contained 225 μL of 8.16 × 10−8 M FL and 30 μL of sample solution. After preheating at 37 °C for 20 min, 25 μL of 0.36 M AAPH was added into each reaction mixture. The reaction was measured every minute for 2 h. Excitation and emission wavelengths were 485 and 535 nm, respectively. Results were expressed as micromoles of trolox equivalents (TE) per gram of GPP-S. DPPH• Radical Scavenging Capacity (RDSC). A laboratory protocol was used to determine the DPPH• radical scavenging capacity of the polysaccharide using a Synergy 2 Multi-Mode Microplate Reader (BioTek).20 Briefly, each reaction mixture contained 100 μL of GPP-S, blank, or trolox standard and 100 μL of DPPH• solution. The absorbance of the reaction mixture was measured at 515 nm every minute for 1.5 h of reaction, and each sample was tested in triplicate. The RDSC was calculated using the area under the curve and reported as micromoles of TE per gram of GPP-S. Hydroxyl Radical Scavenging Capacity (HOSC). The HOSC assay was conducted using FL as the fluorescent probe on a Synergy 2 Multi-Mode Microplate Reader (BioTek) according to a laboratory protocol reported previously.21 FL solution was prepared in 75 mM sodium phosphate buffer (pH 7.4). The reaction mixture contained 170 μL of 9.28 × 10−8 M fluorescein, 30 μL of GPP-S or blank or standard, 40 μL of 0.199 M H2O2, and 60 μL of 3.43 M FeCl3. The reaction was measured every minute for 6 h at ambient temperature, with the excitation wavelength at 485 nm and the emission wavelength at 528 nm. The results were expressed as micromoles of TE per gram of GPP-S. Inhibition of IL-1β, IL-6, and COX-2 Gene Expressions in RAW 264.7 Mouse Macrophage Cells. GPP-S was dissolved in a known amount of DMSO and used for cell treatments. RAW 264.7 mouse macrophages were cultured in six-well plates overnight and reached 80% confluence. The cells were first treated with cell media 3784

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Figure 1. GC chromatograms of standard monosaccharides (A) and monosaccharide composition of GPP-S (B). Peaks: 1, rhamnose; 2, arabinose; 3, inositol; 4, mannose; 5, glucose; 6, galactose.

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containing GPP-S for 24 h at 100 and 10 μg/mL concentrations. An initial concentration of 10 ng/mL of LPS was added into each well followed by incubation at 37 °C with 5% CO2 for another 4 h. Then cells were collected for RNA isolation and real-time PCR analysis according to the previously published protocol.22,23 After incubation, the TRIzol reagent was added for total RNA isolation, and the StrataScript First Strand complementary DNA Synthesis kit was used to reverse transcribe complementary DNA. Real-time PCR was performed using an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with an AB Power SYBR Green PCR Master Mix. The following amplification parameters were used for PCR: 50 °C for 2 min, 95 °C for 10 min, and 46 cycles of amplification at 95 °C for 15 s and at 60 °C for 1 min. Statistical Analysis. Data were recorded as the mean ± SD for triplicate determinations. Tukey’s test and one-way ANOVA were used to identify differences in means. Correlation was presented by a twotailed Pearson’s correlation test. Statistics were analyzed using SPSS for Windows (version rel. 10.0.5, 1999, SPSS Inc., Chicago, IL, USA). Statistical significance was declared at P < 0.05.

RESULTS AND DISCUSSION

Extraction and Purification of GPP-S. The crude polysaccharide was isolated from the alkaline extraction of tetraploid G. pentaphyllum with a yield of 5.07 g/100 g. After purification using DEAE and Sephacryl S-400 columns, GPP-S was obtained from the crude polysaccharides eluted by NaCl solution and distilled water, respectively. GPP-S showed a single sharp peak on the high-performance gel permeation chromatograph with a purity of 99.9% (Supporting Information Figure S1). No absorbance was detected at 260 or 280 nm in the UV spectrum, indicating that GPP-S was free of protein and nucleic acid. Molecular Mass and Monosaccharide Composition of GPP-S. The average molecular mass of GPP-S was calculated as 1.2 × 106 Da on the basis of the standard curve of a series of standard dextran. According to the retention time showed in Figure 1 of alditol acetate derivatives of GPP-S hydrolysate in 3785

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Figure 2. HPLC chromatograms of PMP derivatives of (A) seven standard monosaccharides and (B) GPP-S. Peaks: 1, mannose; 2, PMP; 3, rhamnose; 4, glucuronic acid; 5, glucose; 6, galactose; 7, arabinose; 8, fucose.

identified as 2,3,6-tri-O-methylglucitol, 2,3-di-O-methylglucitol, 2,3,4-tri-O-methylgalactitol, and 2,3,4-tri-O-arabinitol with a molar ratio of 17.10:2.78:1.51:1.00. The results of methylation analysis indicated that the relative amount of (1→4,6)-linked glucose was 12.4%, suggesting that the backbone of GPP-S consisted of a (1→6)-linked branch every six residues. The 1H and 13C NMR spectra of GPP-S are shown in Figure 4A,B. Four main signals from δ 95 to 105 were detected in the anomeric region. The anomeric proton signals (δ 5.41, 5.12, 5.14, and 5.28) and the anomeric carbon signals (δ 99.3, 101.1, 107.2, and 108.9) corresponded with H-1 and C-1 of four anomeric residues, including residue A as (1→4)-α-glucopyranose, residue B as (1→4,6)-α-glucopyranose, residue C as (1→6)-αgalactopyranose, and residue D as (1→)-α-arabinopyranose. All other carbon and proton signals are presented in Table 2 on the basis of the 1D and 2D NMR spectroscopy.25−27 The signals of rhamnose residue were not detected in methylation and NMR analysis due to its low content. The HMBC spectra of GPP-S (Figure 4C) also showed the cross peak of H-1 and C-4 of residue A,

GC, GPP-S consisted of rhamnose, arabinose, glucose, and galactose with a molar ratio of 1.00:3.72:19.49:7.82. To further validate the GC analysis results, a PMP-HPLC analysis was carried out to determine the possible presence of uronic acid in GPP-S. As shown in Figure 2, GPP-S contained rhamnose, arabinose, glucose, galactose, and a small amount of glucuronic acid, which could prove the result of GC analysis. It needs to be pointed out that PMP derivatives were not stable, which made it difficult to accurately quantify the ratio of monosaccharides. Structure Characterization of GPP-S. As shown in Figure 3, the IR spectrum of GPP-S displayed a broad stretching peak at 3346 cm−1, which indicated the presence of hydroxyl groups. The weak peak at 2933 cm−1 was due to C−H stretching vibrations. Strong IR bands in the range of 1200−1030 cm−1 suggested the existence of C−O stretching vibrations of the pyranose rings. Methylation analysis is widely used to determine the type of glucosidic bonds of polysaccharides.24 After four methylations, the methylated polysaccharide was hydrolyzed and analyzed by GC-MS. As shown in Table 1, the four major peaks were 3786

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Figure 3. FT-IR spectrum of GPP-S.

galactose with a (1→4,6)-linked glucose branch every six residues. The predicted structure of GPP-S is shown in Figure 5. Antioxidant Activity of GPP-S. The antioxidant ability of polysaccharides should be evaluated by different types of in vitro assays to avoid the influence of testing method on the antioxidant property estimation. Radical deactivation in the oxidation process has two major mechanisms, single electron transfer (SET) and hydrogen atom transfer (HAT). RDSC is a SET assay and measures the DPPH• scavenging capacity of GPP-S. ORAC measures the oxygen radical absorbance capacity. HOSC evaluates the scavenging capacity against OH radical generated by the Fenton reaction of Fe(III) and peroxide. These two assays are based on the HAT mechanism. As shown in Figure 6, GPP-S showed the stronger scavenging capacity against O2•− radical and HO• radical with ORAC and HOSC values of 28 and 19 μmol TE/g, respectively, whereas the DPPH• radical scavenging capacity was 2 μmol TE/g. The results suggested that GPP-S may scavenge the radicals by both hydrogen atom or proton transfer mechanisms. Effects of GPP-S on IL-1β, IL-6, and COX-2 mRNA expressions. Chronic inflammation is implicated in the pathogenesis of several chronic diseases including atherosclerosis, obesity, metabolic syndrome, diabetes, neurodegenerative diseases, and cancers.28 The inhibitory effect of the pro-inflammatory

Table 1. GC-MS Results of GPP-S methylated sugar

retention time (min)

molar ratio

mass fragments (m/z)

2,3,6-Me3-Glc

13.64

17.1

2,3-Me2-Glc

15.23

2.78

2,3,4-Me3-Gal

12.3

1.51

2,3,4-Me3-Ara

26.31

1.00

43, 71, 87, 99, 101, 117, 129, 161, 173, 233 43, 85, 101, 117, 142, 159, 201, 261 45, 87, 101, 129, 161, 189, 233 59, 87, 101, 117, 161

linkage type 1,4-linked Glc 1,4,6-linked Glc 1,6-linked Gal 1-linked Ara

which suggested the linkage of two 1,4-α-Glcp residues. Both analyses of monosaccharide composition and methylation indicated that (1→4)-linked glucose was the residue at the largest amount, which formed the main structure of GPP-S. Cross peak A H1/B C4 and cross peak B H1/A C4 indicated that residue A was linked to residue B. Cross peak A H1/C C6 was observed in Figure 4C, which was possibly due to the linkage of residues A and C. No corresponding signal between residues B and C implied the branch residue linked only with 1,4-α-Glcp residues. In summary, the structural analysis suggested that the backbone of GPP-S is (1→4)-linked glucose and (1→6)-linked Table 2. 13C and 1H Chemical Shifts of GPP-S

chemical shifts, δ sugar residue

C1/H1

C2/H2

C3/H3

C4/H4

C5/H5

C6/H6

(A) →4)-α-Glcp-(1→ (B) →4,6)-α-Glcp-(1→ (C) →6)-α-Glcp-(1→ (D) α-Ara-(1→

99.3/5.41 101.1/5.12 108.9/5.28 106.9/5.20

76.6/3.67 76.0/4.15 81.0/4.25 72.9/3.59

73.8/4.05 74.5/3.75 77.4/3.98 76.3/3.97

72.9/3.73 76.6/3.87 71.2/3.66 77.2/4.18

70.9/3.86 71.1/3.67 69.5/3.70 62.6/3.78

60.2/3.85 69.1/3.44 70.7/3.86

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Figure 4. 13C (A), 1H (B), and HMBC (C) NMR spectra of GPP-S in D2O solution at 30 °C.

cytokine genes expression of GPP-S was evaluated by measuring its ability to suppress the mRNA expressions of pro-inflammatory

cytokines in RAW 264.7 mouse macrophage cells stimulated by LPS. 3788

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Figure 5. Possible repeating unit of GPP-S.

Figure 6. Antioxidant activities of GPP-S. Data are expressed as Trolox equivalents (TE) in μmol TE/g GPP-S. Antioxidant capacity was calculated using the area under the curve (AUC) against the Trolox standard. Vertical bars represent standard deviation (SD) for triplicate measurements.

The mRNA expression of IL-1β, IL-6, and COX-2 in the cells treated by 100 μg/mL GPP-S had 20.54, 82.15, and 41.75% reductions, respectively (Figure 7). The GPP-S of low concentration (10 μg/mL) showed significant suppression on IL-6 mRNA expression by 79.86% (Figure 7B), not suggesting a possible dose dependence. In conclusion, GPP-S exhibited significant inhibitory activity against mRNA expressions of the pro-inflammatory cytokine genes. Chronic inflammation involves the lining of blood vessels and many internal systems, which might contribute to the development of cancer, cardiovascular disease, diabetes, immune system decline, and other aging-associated health problems. The inflammatory process was often associated with oxidative stress and free radical damage. The antioxidant and potential anti-inflammatory activities of GPP-S implied that G. pentaphyllum could be used for a potential functional food ingredient to reduce the risk of chronic inflammatory diseases, warranting additional animal and human studies to further investigate and confirm the anti-inflammatory effects of GPP-S. In summary, a novel alkaline-soluble polysaccharide (GPP-S), with a molecular mass of 1.2 × 106 Da, was isolated from tetraploid G. pentaphyllum Makino. It had a monosaccharide composition of rhamnose, arabinose, glucose, and galactose at a ratio of 1.00:3.72:19.49:7.82. The backbone of GPP-S is (1→4)linked glucose and (1→6)-linked galactose with an occasional (1→4,6)-linked glucose, which terminated with 1-)-α-arabinose, glucuronic acid, and other monosaccharides. In our previous research, two different kinds of polysaccharides, named GPP and GPP-TL, have been extracted by hot water from diploid and tetraploid G. pentaphyllum Makino, respectively.15,29 Their molecular weights, monosaccharide compositions, and chemical structures were different from those of GPP-S, which led to the different antioxidant activities.

Figure 7. Effects of GPP-S on (A) IL-1β, (B) IL-6, and (C) COX-2 mRNA expressions in lipopolysaccharide (LPS)-stimulated RAW 264.7 mouse macrophage cells. DMSO was the vehicle. The solid and open bars represent initial GPP-S concentrations of 100 and 10 μg/mL, respectively. The vertical bars represent the standard deviation (n = 3) of each data point. Different letters represent significant differences (P < 0.05).

GPP-S showed higher scavenging ability against peroxide radicals than GPP and GPP-TL, whereas it had lower absorbance capacities for DPPH• and HO• than GPP and GPP-TL. In addition, GPP-S had significant inhibition of IL-1β, IL-6, and COX-2 gene expressions in RAW 264.7 mouse macrophage cells, suggesting its potential application in functional food and dietary supplement products. 3789

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1. Profile of GPP-S on a high-performance gel permeation chromatograph. Figure S2. H−H COSY NMR spectra of GPP-S. Figure S3. HSQC NMR spectra of GPP-S. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(Y.N.) Phone: (86)-21-34204538. E-mail: [email protected]. *(H.Z.) Phone: (86)-21-60877220. E-mail: [email protected]. Author Contributions ∥

Y.N. and P.S. contributed equally to this work.

Funding

This research was supported by a Special Financial Grant and a General Grant from the Chinese Postdoctoral Science Foundation (Grants 2013T60450 and 2012M511098), a special fund for Agroscientific Research in the Public Interest (Grant 201203069), SJTU 985-III disciplines platform and talent fund (Grants TS0414115001 and TS0320215001), and a grant from the Wilmar (Shanghai) Biotechnology Research & Development Center Co., Ltd. Notes

The authors declare no competing financial interest.

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REFERENCES

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dx.doi.org/10.1021/jf500438s | J. Agric. Food Chem. 2014, 62, 3783−3790