International Journal of Biological Macromolecules 72 (2015) 975–983
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Purification, antioxidant and immunological activities of polysaccharides from Actinidia Chinensis roots Lin Zhang a,b , Wuxia Zhang a , Qingjie Wang a , Dongdong Wang a , Dongqi Dong a , Haibo Mu a , Xin-Shan Ye b,∗∗ , Jinyou Duan a,∗ a b
College of Science, Northwest A&F University, Yangling 712100, Shaanxi, China State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xue Yuan Road No. 38, Beijing 100191, China
a r t i c l e
i n f o
Article history: Received 29 July 2014 Received in revised form 8 September 2014 Accepted 26 September 2014 Available online 12 October 2014 Keywords: Actinidia Chinensis Polysaccharides Purification Antioxidant activity Immunomodulation
a b s t r a c t Two water-soluble polysaccharides (ACPS1 and ACPS2) were isolated from the roots of Actinidia Chinensis by DEAE-52 cellulose and Sephacryl S300 chromatography. Preliminary structural characterization was conducted by physicochemical property, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) analysis. ACPS1, with an average molecular weight of 5.58 × 105 Da, was mainly composed of rhamnose, arabinose, xylose, mannose and galactose in an approximate molar ratio of 1.48:4.28:4.30:1.00:17.83. ACPS2, with a high average molecular weight of 1.23 × 106 Da, mainly contained rhamnose, arabinose and galactose in a molar ratio of approximately 1.00:2.33:6.61. Both ACPS1 and ACPS2 exhibited the remarkable antioxidant activity to scavenge the DPPH radical and significant protective effects on H2 O2 -induced HEK 293 cells death in a concentration-dependent manner. Meanwhile, in vitro immunomodulatory activities of the two polysaccharides were evaluated. The results showed that treatment with 50–300 g/mL of the samples could increase NO production and phagocytic activity of macrophages in a dose-dependent manner. The present results suggested that the two polysaccharides from Actinidia Chinensis may be potential antioxidant and immunomodulatory agents for preparing functional foods and nutraceuticals applied in food and pharmaceutical industries. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Polysaccharide is a class of biological macromolecules substance that recognizes a broad spectrum of biological response modifiers existing widely in organism. They exist widely in plants, microorganisms (fungi and bacteria), algae and animals. It is well known that polysaccharides in living organisms do not only act as energy resources but also play key biological roles in many life processes. There is much evidence that a good number of polysaccharides possess a variety of biological activities, such as antioxidant activity [1,2], antitumor activity [3,4] antimicrobial activity [5], and immunologic activity [6] among others. Because most polysaccharides derived from higher plants are relatively nontoxic and do not cause significant side effects compared with immunomodulatory bacterial polysaccharides and synthetic compounds, they are ideal candidates for modern food and medical industries [7,8].
The genus Actinidia (Actinidiaceae) consists of over 58 species and widely distributed in the Asian continent. Most species are native to temperate regions of south-western China. Actinidia Chinensis Planch is a liana plant that commonly grows in temperate climate zones. Its roots have been used for gastric carcinoma, nasopharyngeal carcinoma, breast carcinoma and hepatitis in traditional Chinese medicine [9]. Until now, there have been few reports regarding the isolation, purification, characterization, antioxidant and immunological activities of the polysaccharides from Actinidia Chinensis roots. In the present study, two polysaccharides were purified from the crude polysaccharide and preliminarily characterized. Moreover, the antioxidative effects of the purified polysaccharides on DPPH radical scavenging activity, as well as the protective effects on H2 O2 -induced HEK 293 cells death and immunomodulatory effects on RAW 264.7 macrophages were investigated. 2. Materials and methods
∗ Corresponding author. Tel.: +86 29 87092226; fax: +86 29 87092226. ∗∗ Corresponding author. Tel.: +86 10 82805736. E-mail addresses: [email protected] (X.-S. Ye), [email protected], [email protected] (J. Duan). http://dx.doi.org/10.1016/j.ijbiomac.2014.09.056 0141-8130/© 2014 Elsevier B.V. All rights reserved.
2.1. Materials and chemicals The roots of Actinidia chinensis were collected in Mei County, Shannxi province, China and identified by Professor Langran Xu
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at College of Forestry, Northwest A&F University. A voucher specimen (No.20130822) has been deposited at the Laboratory of Functional Polysaccharides, Northwest A&F University. 2, 2-diphenyl-1-picryl-hydrazyl (DPPH), neutral red and standard monosaccharide (including arabinose, rhamnose, xylose, galactose, glucose, mannose and galacturonic acid) were obtained from Aladdin Reagent Int. Bovine serum albumin (BSA), 3-(4, 5-dimethyltiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT), lipolysaccharide (LPS) and vitamin C were obtained from Sigma Chemical Co. Ltd. DEAE-52 cellulose and Sephacryl S300 were purchased from Whatman Co. Ltd. The RAW264.7 and HEK 293 (human embryonic kidney 293) cell lines were kindly gifts from Professor Xuebo Liu in Northwest A&F University. Trifluoroacetic acid (TFA), dimethylsulfoxide (DMSO) and other reagents were all of analytical grade. 2.2. Extraction and purification of crude polysaccharide The fresh Actinidia chinensis (AC) roots were collected, cleaned and dried at 40◦ C and then pulverized into powder by a disintegrator. The dried AC powder (800.0 g) was pre-extracted with 95% ethanol (8 L, 5 d, 40 ◦ C, 4 times) to remove lipids. The residue powder was extracted with distilled water at 80 ◦ C for 2 h, at the ratio of 1:10 (W/V). The mixture was centrifuged at 3000 rpm for 30 min. The precipitate was re-extracted (2 times) as described above, and the supernatants were pooled. The collected supernatants were concentrated by a rotary evaporator under reduced pressure. Then, the supernatant was mixed with 95% ethanol to final ethanol concentration of 80% and incubated for 24 h at 4 ◦ C. The precipitate was collected and dissolved in distilled water and then deproteinated by the method of Sevag [10], before being decolored by 5% H2 O2 . Finally, the resulting solution was dialyzed against distilled water (cut-off Mw: 8000 Da) and lyophilized to afford crude Actinidia chinensis polysaccharide (ACPS). The crude polysaccharide ACPS was subjected to further processing. The crude ACPS (4.0 g) was purified sequentially by chromatography using DEAE-52 cellulose and Sephacryl S300 columns. The crude ACPS solution (20 mg/mL) was applied to a DEAE-52 cellulose column (5 × 50 cm, OH− form), which was washed with deionized water, and NaCl solutions of increasing ionic strength (0.1, 0.2, 0.3, 0.5, 1.0 M) at a flow rate of 2 mL/min. The eluate (8 mL/tube) was collected automatically, and the carbohydrate content was determined using the phenol-sulfuric acid method. Five mainly fractions were obtained. Two fractions (eluates of 0.1 M and 0.2 M NaCl) were further fractionated on DEAE-52 cellulose column (5 × 50 cm, Cl− form), eluted with NaCl solutions (0.1, 0.2, 0.3 M) sequentially or Sephacryl S300 column (2.5 × 100 cm), eluted with deionized water, at a flow rate of 0.5 mL/min to obtain ACPS1 and ACPS2 solutions respectively. The relevant fractions were then collected, concentrated, dialyzed (cut-off Mw: 8000 Da) and lyophilized to yield white, purified polysaccharides ACPS1 and ACPS2 for further study. The isolation and purification steps are shown in Fig. 1. 2.3. Molecular weight determination The molecular weights of ACPS1 and ACPS2 were determined on high performance gel permeation chromatography (HPGPC), as described previously [11–13]. The operation conditions were as follows: a Waters 600 HPLC System (Waters corporation, USA); Three Waters Ultrahydrogel columns in series (250, 1000 and 2000; 30 cm × 7.8 mm, 6 m particles); A 2414 Differential Refractive Index Detector; mobile phase: 3 mM sodium acetate; flow rate: 0.5 mL/min; injection volume: 50 L. The molecular weights of the polysaccharides were estimated by referencing a calibration curve, which was created using a set of dextran standards (5.2, 10, 48.6,
668 and 2000 KDa) .The calibration curve of Log (Mw) vs. elution time (T) is: Log(Mw) = −0.1316T + 10.94
(1)
2.4. Chemical compositions analysis Neutral carbohydrate content was determined by the phenol–sulphuric acid method [14], with d-glucose as standard. Uronic acid content was measured according to hydroxybiphenyl– sulphuric acid method [15] and galacturonic acid was used as the standard. Protein content was evaluated by Bradford’s method [16] with bovine serum albumin (BSA) as standard. Polysaccharides were dissolved in distilled water for ultraviolet analysis and the solutions of the polysaccharides were scanned from 200 to 800 nm with a 722 spectrophotometer (Shanghai Precision and Scientific Instrument Co., Ltd.)[17].
2.5. Infrared spectra analysis The IR spectra of polysaccharides were determined using a Fourier transform infrared spectrophotometer (BRUKER TEMSOR 27, BRUCK, Germany).The purified polysaccharides were grounded with KBr powder and pressed into a 1 mm pellet for FTIR measurement between 400 cm−1 and 4000 cm−1 [18].
2.6. Scanning electron microscopy (SEM) analysis SEM was used to reveal the microstructure of the materials [19,20]. Two samples were fixed on the silicon wafer. The shape and surface characteristics were observed and recorded using a field emission scanning electron microscope (FESEM, S-4800, Hitachi, Japan). They were examined at a10 KV acceleration voltage under a high vacuum condition, as well as image magnification of 100×, 500× and 800×.
2.7. Neutral monosaccharide composition The monosaccharide composition of the sample was analyzed by GC after transesterification [20]. Briefly, approximately 5 mg of freeze-dried sample was dissolved in an ampoule containing 6 mL of 2 M trifluoroacetic acid (TFA). The sample was hydrolyzed at 121 ◦ C for 2 h. After hydrolysis, the resulting solution was concentrated in a vacuum, and the excess acid was removed by repeated co-distillations with methanol. Neutral monosaccharide was reduced to alditol using 25 mg of sodium borohydride with 3 mL of distilled water at room temperature for 2 h. Acetic acid was added to the solution to decompose excess sodium borohydride until bubble formation stopped. A stream of nitrogen gas was used to dry the solution and 3 mL of methanol was added to remove the borohydrate. The procedure was repeated four times. Acetic anhydride (5 mL) was then added to the mixture and incubated for additional 60 min at 100 ◦ C. The hydrolysate was then converted into the corresponding alditolacetates and analyzed with GC using a Shimadzu 2010 instrument equipped with a HP-5MS column (0.25 mm × 30 m × 0.25 m) and a flame-ionization detector. The initial column temperature was held at 130 ◦ C for 5 min, increased to 240 ◦ C at 4 ◦ C/min and held at 240 ◦ C for 5 min. Nitrogen gas (N2 ) was used as the carrier gas with a flow rate of 1.0 mL/min. Alditol acetates were used as standards (glucose, mannose, rhamnose, galactose, xylose, and arabinose) and they were processed using the same approach as described above for the sample.
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Fig. 1. Isolation and purification steps of ACPS1 and ACPS2.
2.8. Determination of antioxidant activities in vitro 2.8.1. DPPH radical scavenging activity The radical scavenging effects of samples on DPPH radical were estimated as described [21]. Briefly, sample solution (1.0 mL) at different concentrations was added to DPPH (0.1 mM, 3.0 mL) in 95% ethanol. The reaction solution was shaken vigorously and incubated at room temperature for 30 min, and the absorbance at 517 nm was measured. Ascorbic acid was used as a positive control. The DPPH scavenging rate (R) was calculated as follows:
R(%) = 1 −
Abs(sample) − Abs(control) × 100 Abs(blank)
the formazan. After 10 min, the absorbance was measured at 570 nm in a microplate reader (Perlong DNM-9062, China). For protective assay, when the cells reached sub-confluence, they were pretreated for 36 h with culture medium containing different concentrations of samples (10, 50, 100, 200 g/mL) that were tested in the experiments. Next, the culture supernatant was moved and cells were washed three times with phosphate-buffered saline (PBS). Subsequently, the cells were exposed to H2 O2 (1000 M) diluted in culture medium for 10 h in a humidified atmosphere of 5% CO2 at 37 ◦ C until further assay and other procedures were same as above. Assays were performed in sextuple wells for each sample.
(2)
Where the control solution contains distilled water instead of the DPPH solution, while distilled water instead of sample was used for the blank. All tests were performed in triplicate and the mean of Abs was used in the equation above. 2.8.2. Protective effects on H2 O2 -induced HEK 293 cells death These were evaluated in vitro using the MTT assay [22]. HEK 293 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 g/mL streptomycin and 100 IU/mL penicillin. To check the cell cytotoxicity, cell suspensions were seeded in 96-well plates (1 × 105 cells/well), and incubated at 37 ◦ C for 4 h, and the samples were added. After 36 h, 20 L of the MTT solution (5 mg/mL) was added into each well and the plate was further incubated for 4 h. Finally, the medium was removed and DMSO (180 L) was added to each well to dissolve
2.9. Determination of immunoregulation effects of polysaccharides 2.9.1. Effects of ACPS1 and ACPS2 on the macrophages proliferation The measurement of macrophages proliferation was determined according to the MTT-based colorimetric method. Macrophage cells were dispensed into 96-well plates and incubated at 37 ◦ C in a 5% CO2 atmosphere for 4 h. The RPMI1640 medium was discarded, and the cells were treated with various concentrations of ACPS1 and ACPS2 (10, 50, 100, 150 g/mL). The RPMI1640 medium was used as control. After incubated for 24 h, media were discarded, and MTT solution (20 L/well) was added to each well and further incubated for 4 h. Then, media were discarded, and 180 L DMSO was added. After shaking to dissolve the formazan, the optical density of each well at
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Fig. 2. HPGPC profiles of ACPS1 (A) and ACPS2 (B).
570 nm was measured by a microplate reader (Perlong DNM-9062, China).
positive control, respectively. Phagocytosis index was calculated by the following equation: Phagocytosis index =
2.9.2. Assay for nitric oxide (NO) production Nitrite accumulation was measured by Griess reagent [23–25]. RAW 264.7 cells (1 × 106 cells/well) were dispensed into 96-well plates. And then cells were stimulated with medium (for the control group), LPS (2 g/mL) and various concentrations of samples (10, 50, 100, 150 g/ml) for 24 h. After incubation, 100 L of culture supernatants were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride, and 2.5% phosphoric acid) in 96-well plates and incubated at 25 ◦ C for 10 min. The absorbance at 540 nm was measured on a microplate reader (Perlong DNM-9062, China). Nitrite concentrations in culture supernatants were measured to assess NO production in RAW 264.7 cells. NaNO2 was used as standard to calculate nitrite concentrations.
2.9.3. Assay of macrophages phagocytosis The phagocytic ability of macrophages was measured using neutral red uptake [26,27]. Briefly, cells (1 × 106 cells/well) were pipetted into 96-well plates and incubated for 4 h. Culture medium was discarded, and the cells were treated with various concentrations of samples (50, 100, 200, 300 g/mL) for 24 h. Then, 0.075% neutral red solution (100 L/well) was added and incubated for 1 h. Medium was discarded and cells in 96-well plates were washed twice with PBS to remove the neutral red that was not phagocytosed by RAW 264.7 cells. Then, cell lysis buffer (1% glacial acetic acid: ethanol = 1:1, 100 L/well) was added to lyse cells. After cells were incubated at room temperature for 15 h, the optical density of each well was measured at 540 nm using a microplate reader (Perlong DNM-9062, China). The RMPI1640 medium and LPS (10 g/mL) were used as the blank and
Abs(sample) Abs(blank control)
(3)
2.10. Statistical analysis Data were expressed as means ± standard deviation (SD). The scientific statistic software GraphPad Prism 5.0 was used to evaluate the significance of differences between groups. p < 0.05 was regarded as significant. 3. Results and discussion 3.1. Extraction, isolation and purification of polysaccharide The crude polysaccharide ACPS (38.6 g from 800.0 g) was isolated from the hot water extract of roots of Actinidia Chinensis with a yield of 4.8%. After fractionation on DEAE-52 cellulose column (OH− form), five fractions were obtained. The DEAE-52 cellulose (Cl− form) and Sephacyl S300 columns were used for further purification of the 0.1 M NaCl and 0.2 M NaCl eluent, respectively. Two polysaccharides ACPS1 (99.0 mg) and ACPS2 (104.0 mg), with the yield about 2.47% and 2.60% from ACPS, were obtained. 3.2. Molecular weight determination Molecular weight is an important index for quality control of polysaccharide products, as being closely related to their physicochemical properties and biological activities. Up to now, high performance gel permeation chromatography (HPGPC) with the advantage of rapid, high resolution and good reproducibility has been widely used in polysaccharide molecular weight determination [28–31]. As shown in Fig. 2, profiles of the ACPS1 and ACPS2 on HPGPC reveal that the fractions were represented by a
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Fig. 3. Infrared spectra of ACPS1and ACPS2.
Table 1 Neutral carbohydrate, uronic acid and protein contents of ACPS1 and ACPS2. Sample
Neutral carbohydrate (%)
Uronic acid (%)
Protein (%)
ACPS1 ACPS2
74.05 ± 1.15 68.59 ± 1.02
18.32 ± 0.78 25.36 ± 2.10
n.d.a n.d.a
a
n.d.: not detected.
single and symmetrical peak which indicated the molecular weight homogeneity and the high purity of the generated two polysaccharides. Their molecular weights were calculated to be 5.58 × 105 Da (T: 39.458) and 1.23 × 106 Da (T: 36.792), respectively. 3.3. Chemical compositions analysis The chemical compositions of ACPS1 and ACPS2 were determined and listed in Table 1. The two polysaccharides mainly contained neutral carbohydrate and uronic acid. The two polysaccharides at a concentration of 5 mg/mL had no obvious absorption at 280 nm and 260 nm in the UV spectra, indicating the absence of protein and nucleic acid. Negative responses to the Bradford test (the detection limit of BSA standard was 15 g/mL) further indicated that the two polysaccharides did not contain protein. The negative results of Fehling’s and iodine-potassium iodide tests
showed ACPS1 and ACPS2 did not contain reducing sugar and did not belong to starch-type polysaccharide [32].
3.4. Infrared spectra analysis The IR spectra of ACPS1 and ACPS2 were recorded at the range of 400–4000 cm−1 (Fig. 3). Both of them displayed a broad and intense peak nearby 3431 cm−1 and 3420 cm−1 , which were due to the hydroxyl groups stretching vibration. The bands in the region of 2926 cm−1 and 2934 cm−1 were the characteristic absorption of C H stretching vibration. The absorptions were the characteristic of antisymmetrical stretching vibration. The bands around 1627 cm−1 and 1620 cm−1 represented the carboxylate (COO ) stretching band [33], indicating that there were esterified and free carboxyl groups present in the polysaccharides from Actinidia Chinensis. It also validated the presence of uronic acids. The absorption band from 800 cm−1 to 1300 cm−1 , called “finger print” region, was related to conformation and surface structure of molecule. Although these bands are hard to explain [33], the peaks at 950–1200 cm−1 suggested the presence of C O C and C OH link bonds [34]. From the spectra, we proposed that the absorbance between 1000 cm−1 and 1200 cm−1 was due to the pyranose ring [35].
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Fig. 4. Scanning election microscope (SEM) photographs of ACPS1 (A, 100×; B, 500×; C, 800×) and ACPS2 (D, 100×; E, 500×; F, 800×).
3.5. Scanning electron microscopy (SEM) analysis The SEM photographs of the two polysaccharides were showed in Fig. 4. The surfaces of ACPS1 and ACPS2 were little different.
Images A, B and C show that ACPS1 had a rough surface like clouds with characteristic large wrinkles. In contrast, images D, E and F show that ACPS2 had a thin slice shape and the surface was very smooth.
Fig. 5. GC spectra of sample references (A), ACPS1 (B) and ACPS2 (C). Peaks in the spectra representing the follows: (1) rhamnose; (2) arabinose (3) xylose; (4) mannose; (5) galactose; (6) glucose.
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Fig. 6. Scavenging effects on DPPH radical of ACPS1 and ACPS2.
3.6. Analysis of neutral monosaccharide composition GC analysis with derivatization is an acknowledged method for the quantification of neutral sugars. By comparing the retention time with standards, the monosaccharide composition was identified. Their Gas chromatograms profiles were showed in Fig. 5. ACPS1 mainly contained neutral monosaccharide composition of rhamnose, arabinose, xylose, mannose and galactose with the molar ratio of 1.48:4.28:4.30:1.00:17.83. However, ACPS2 was mainly composed of rhamnose, arabinose, and galactose, with molar ratio of 1.00:2.33:6.61. 3.7. Determination of antioxidant activities 3.7.1. Measurement of scavenging effects on DPPH radical The model of scavenging the DPPH radical is a widely used method for evaluating the free radical-scavenging ability of
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polysaccharide [36,37].The antioxidant mechanism of DPPH radical scavenging is based on the acceptance of hydrogen by the DPPH radical, thereby converting DPPH into the non-radical form (DPPH-H). Thus the antioxidant activity is due to the hydrogen donating ability of the polysaccharide [18]. As shown in Fig. 6, the scavenging abilities of ACPS1 and ACPS2 were concentration related in all of the evaluated concentrations. An obvious growth of the scavenging ability was found at the concentration range (0.5–3 mg/mL) of the samples. At high concentrations (>1.5 mg/mL), comparing with ACPS2, ACPS1 elicited a stronger scavenging activity obviously. At the concentration of 3.0 mg/mL, the DPPH radical scavenging activity of ACPS1 and ACPS2 were 56.9% and 51.8%, respectively. The results mentioned above suggested that the polysaccharides ACPS1 and ACPS2 might act as hydrogen donator and could react with DPPH radicals to convert them to more stable products, so that they can scavenge DPPH free radical. 3.7.2. Protective effects on H2 O2 -induced HEK 293 cells death H2 O2 is able to penetrate biological membranes, and plays a radical forming role as an intermediate in the production of more reactive ROS molecules including formation of hydroxyl radical via oxidation of transition metals, and hypochlorous acid by the action of myeloperoxidase, an enzyme present in the phagosomes of neutrophils [38], H2 O2 has been used in many studies to trigger cell apoptosis. Antioxidants could prevent cell death through the suppression of H2 O2 -induced ROS formation the regulation of the endogenous oxidant–antioxidant balance [39]. HEK 293 cells are cell line originally derived from human embryonic kidney cells and widely used as a model cell line to assess the protective effects of an antioxidant on H2 O2 -induced cells death.
Fig. 7. A. Effects of ACPS1 and ACPS2 on HEK 293 cells proliferation. Cells were cultured withACPS1 and ACPS2 for 36 h. OD value at 570 nm was used to represent the proliferation of cells. n = 6. B. Viability losses in HEK 293 cells induced by various concentrations of H2 O2. **p < 0.01 compared with control. n = 6. C. Protective effects of ACPS1 and ACPS2 on viability losses in HEK 293 cells induced by H2 O2 (1000 M, 10 h). n = 6. *p < 0.05, **p < 0.01 compared with the H2 O2 alone group.
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showed that 100–1000 M of H2 O2 could result in the HEK 293 cells death dramatically (Fig. 7B) after 10 h. For instance, as showed in (Fig. 7 C) the incubation of HEK 293 cells with 1000 M H2 O2 for 10 h resulted in a cell viability rate of 38.9% compared to the control. However, when pretreating the cells with four different concentrations (10, 50, 100, 200 g/mL) of ACPS1 and ACPS2, the cell viabilities were significantly increased to 68.6% and 72.3%, respectively. 3.8. Determination of immunoregulation effects
Fig. 8. Effects of ACPS1 and ACPS2 on the proliferation of RAW 264.7 macrophages. Cells were cultured with samples for 24 h. OD value at 570 nm was used to represent the proliferation of cells. n = 6.
We first tested whether ACPS1 and ACPS2 affected the viability of HEK 293 cells, which was determined by MTT assay. Two samples, ACPS1 and ACPS2, at the dose of 10–200 g/ml had no significant toxic effects on HEK 293 cells (Fig. 7A). The MTT assay
3.8.1. Effects of ACPS1and ACPS2 on the proliferation of macrophages It was necessary to evaluate the cytotoxic or proliferation effects of the two polysaccharides on RAW 264.7 cells before further tests were carried out. MTT assay indicated that ACPS1 and ACPS2 did not show cytotoxicity or stimulation on the proliferation of RAW 264.7 cells in 24 h (Fig. 8) significantly. Based on these results, the measurement of NO production and phagocytosis can well represent the cell function without any changes in cell quantity.
Fig. 9. Effects of ACPS1 (A) and ACPS2 (B) on the NO production of RAW 264.7 cells. Cells were pretreated with ACPS1 and ACPS2 at different concentrations (50, 100, 200, 300 g/mL) or LPS (2 g/ml) for 24 h. The supernatant nitrite levels were determined using Griess reagent. *p < 0.05, **p < 0.01 compared with control. n = 6.
Fig. 10. Effects of the ACPS1 (A) and ACPS2 (B) on the phagocytosis index of RAW 264.7 macrophages by a neutral red uptake assay. After treatment with the two polysaccharide samples (50, 100, 200, 300 g/mL) or LPS (10 g/mL) for 24 h, cells were used to test phagocytic activity. *p < 0.05, **p < 0.01 compared with control. n = 6.
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3.8.2. Measurement of NO production In recent years, NO was found to be a novel signaling molecule and a key mediator of signal transduction in the immune system [40,41]. Bacterial products such as lipopolysaccharide (LPS), a major constituent of the outer membrane of Gram-negative bacteria, have been shown to activate macrophages to release pro-inflammatory cytokines and NO [42–44]. The effects of two samples on the NO production of RAW 264.7 cells were determined by Griess assay. As shown in Fig. 9, the positive control, LPS at a concentration of 2 g/mL had a strong potential in stimulating NO production in macrophages. The two polysaccharides could stimulate macrophages to produce higher levels of NO in a dose dependent manner at different concentrations (50, 100, 200, 300 g/mL) compared with control (p < 0.01). NO is one of the most important mediators in the regulation of immunologic functions. Our results confirmed that the two samples may act as stimulators of NO release in macrophages and have the function of activation of macrophages. 3.8.3. Assay of macrophages phagocytosis Activated macrophages not only participate in both specific immune reactions and non-specific immune reactions, but also are the “bridge cell” of these two kinds of immune reactions [45]. One of the most distinguished features of macrophages activation would be an increase in phagocytic activity. It is well known that macrophages can phagocytose some dyes such as neutral red and malachite green in vitro. Therefore, in the present study we carried out the neutral red phagocytosis assay to evaluate the effects of ACPS1 and ACPS2 on phagocytic activity of RAW 264.7 macrophages. As shown in Fig. 10, phagocytosis index of ACPS1and ACPS2 all exceeded 1.0 and increased in a dose-dependent manner at the test concentrations, indicating that all the two fractions had abilities to enhanced phagocytic activity of RAW 264.7 cells. Compared with the blank control, ACPS1 and ACPS2 could significantly enhance the phagocytosis of macrophages (≥100 g/mL, p < 0.05) as well as LPS action (10 g/mL, p < 0.01). Macrophages can phagocytose aging bacteria, damage cells and necrotic tissues invading the body. Phagocytic capacity is one of the most important indicators of the body’s non-specific immunity [41]. In this study, the results indicated that the ACPS1 and ACPS2 could significantly enhance the phagocytosis of the macrophages, which may be due to the binding of the polysaccharide with a specific receptor on the surface of macrophages [41,46]. 4. Conclusions The roots of Actinidia Chinensis have been used for the treatment of cancers in the Chinese folk medicine, and were proved to have antitumor and immunopotentiating activities [9]. In this investigation, we have succeeded in abstracting the crude polysaccharide ACPS. Two water-soluble polysaccharides (ACPS1 and ACPS2) with the molecular weights of 5.58 × 105 Da and 1.23 × 106 Da, respectively, were purified from the roots of Actinidia Chinensis by combination use of DEAE-52 cellulose and Sephacryl S300 columns. ACPS1 and ACPS2 were defined as acid heteropolysaccharides with different content of uronic acids as well as the ratio of neutral sugars. Moreover, the two polysaccharides showed significant DPPH radical scavenging activity, protection of the HEK 293 cells from H2 O2 damage, stimulatory effects on the NO production and phagocytosis activity of RAW 264.7 macrophages. These finding indicated that ACPS1 and ACPS2 have the potential to be antioxidative and immunopotentiating agents that could be further developed in the food and pharmaceutical industries.
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Acknowledgments This work was financially supported by the National Natural Science Foundation of China (NSFC) (Grant No. 31270860), Program for New Century Excellent Talents in University (NCET-13-0480), the State Key Laboratory of Natural and Biomimetic Drugs, peking University (K20120216), and ‘Interdisciplinary Cooperation Team’ Program for Science and Technology Innovation of the Chinese Academy of Science. References [1] X.-K. Zhong, X. Jin, F.-Y. Lai, Q.-S. Lin, J.-G. Jiang, Carbohyd. Polym. 82 (2010) 722–727. [2] C.-L. Ye, Q. Huang, Carbohyd. Polym. 89 (2012) 1131–1137. [3] S.-D. Park, Y.-S. Lai, C.-H. Kim, Life Sci. 75 (2004) 2621–2632. [4] X. Ding, Y. Hou, W. Hou, Carbohyd. Polym. 89 (2012) 397–402. [5] J.-H. Xie, M.-Y. Shen, M.-Y. Xie, S.-P. Nie, Y. Chen, C. Li, D.-F. Huang, Y.-X. Wang, Carbohyd. Polym. 89 (2012) 177–184. [6] R. Chen, Y. Li, H. Dong, Z. Liu, S. Li, S. Yang, X. Li, Ultrason. Sonochem. 19 (2012) 1160–1168. [7] D. Zhang, S. Li, Q. Xiong, C. Jiang, X. Lai, Carbohyd. Polym. 95 (2013) 114–122. [8] R. Wang, P. Chen, F. Jia, J. Tang, F. Ma, Int. J. Biol. Macromol. 50 (2012) 331–336. [9] J.N.M. College, in, Science and Technology Press of Shanghai Shanghai, 1977. [10] M. Sevag, D.B. Lackman, J. Smolens, J. Biol. Chem. 124 (1938) 425–436. [11] J. Duan, X. Wang, Q. Dong, J.-N. Fang, X. Li, Carbohyd. Res. 338 (2003) 1291–1297. [12] W. Zhang, H. Mu, A. Zhang, G. Cui, H. Chen, J. Duan, S. Wang, Glycoconjugate J. 30 (2013) 577–583. [13] G. Cui, W. Zhang, Q. Wang, A. Zhang, H. Mu, H. Bai, J. Duan, Carbohyd. Polym. 111 (2014) 245–255. [14] T. Masuko, A. Minami, N. Iwasaki, T. Majima, S.-I. Nishimura, Y.C. Lee, Anal. Biochem. 339 (2005) 69–72. [15] N. Blumenkrantz, G. Asboe-Hansen, Anal. Biochem. 54 (1973) 484–489. [16] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254. [17] L. Zhao, Y. Dong, G. Chen, Q. Hu, Carbohyd. Polym. 80 (2010) 783–789. [18] T. Zhao, G. Mao, W. Feng, R. Mao, X. Gu, T. Li, Q. Li, Y. Bao, L. Yang, X. Wu, Carbohyd. Polym. 105 (2014) 26–33. [19] Q. Zhao, J.F. Kennedy, X. Wang, X. Yuan, B. Zhao, Y. Peng, Y. Huang, Int. J. Biol. Macromol. 49 (2011) 181–187. [20] Y. Zhu, Q. Li, G. Mao, Y. Zou, W. Feng, D. Zheng, W. Wang, L. Zhou, T. Zhang, J. Yang, Carbohyd. Polym. 101 (2014) 606–613. [21] G. Cui, W. Zhang, A. Zhang, H. Mu, H. Bai, J. Duan, C. Wu, Int. J. Biol. Macromol. 57 (2013) 278–284. [22] S.-M. Lee, M.-Y. Yoon, H.-R. Park, Biosci. Biotechnol. Biochem. 72 (2008) 1272–1277. [23] M. Shi, Y. Yang, D. Guan, Y. Zhang, Z. Zhang, Carbohyd. Polym. 89 (2012) 1268–1276. [24] D. Guan, Z. Zhang, Y. Yang, G. Xing, J. Liu, Int. J. Biol. 3 (2011) p3. [25] A.M. Gamal-Eldeen, H. Amer, W.A. Helmy, R.M. Talaat, H. Ragab, Int. Immunopharmacol. 7 (2007) 871–878. [26] W. Chen, Z. Zhao, S.-F. Chen, Y.-Q. Li, Biores. Technol. 99 (2008) 3187–3194. [27] X. Li, L. Zhao, Q. Zhang, Q. Xiong, C. Jiang, Carbohyd. Polym. 102 (2014) 912–919. [28] J. Dawkins, Pure Appl. Chem. 54 (1982) 281–292. [29] J.-H. Xie, M.-Y. Xie, S.-P. Nie, M.-Y. Shen, Y.-X. Wang, C. Li, Food Chem. 119 (2010) 1626–1632. [30] G. Zhao, J. Kan, Z. Li, Z. Chen, Carbohyd. Polym. 61 (2005) 125–131. [31] T. Dreher, D. Hawthorne, B. Grant, J. Chromatogr. A 174 (1979) 443–446. [32] X. Yang, Y. Zhao, Y. Lv, Carbohyd. Polym. 71 (2008) 372–379. [33] R. Gnanasambandam, A. Proctor, Food Chem. 68 (2000) 327–332. [34] M. Kacurakova, P. Capek, V. Sasinkova, N. Wellner, A. Ebringerova, Carbohyd. Polym. 43 (2000) 195–203. [35] F. Lai, Q. Wen, L. Li, H. Wu, X. Li, Carbohyd. Polym. 81 (2010) 323–329. [36] Y. Chen, M.-Y. Xie, S.-P. Nie, C. Li, Y.-X. Wang, Food Chem. 107 (2008) 231–241. [37] D. Qiao, C. Ke, B. Hu, J. Luo, H. Ye, Y. Sun, X. Yan, X. Zeng, Carbohyd. Polym. 78 (2009) 199–204. [38] J. Nordberg, E.S. Arnér, Free Radical Biol. Med. 31 (2001) 1287–1312. [39] H.Y. Xue, G.Z. Gao, Q.Y. Lin, L.J. Jin, Y.P. Xu, Phytother. Res. 26 (2012) 369–374. [40] J. MacMicking, Q.-w. Xie, C. Nathan, Annu. Rev. Immunol. 15 (1997) 323–350. [41] I.A. Schepetkin, M.T. Quinn, Int. Immunopharmacol. 6 (2006) 317–333. [42] X. Zhang, Y. Li, G. Tai, G. Xu, P. Zhang, Y. Yang, F. Lao, Z. Liu, Cell Mol. Immunol. 2 (2005) 63–67. [43] G. Drozina, J. Kohoutek, T. Nishiya, B.M. Peterlin, J. Biol. Chem. 281 (2006) 39963–39970. [44] K.P. Crume, J.H. Miller, A.C. La Flamme, Exp. Biol. Med. 232 (2007) 607–613. [45] X. Chen, J. Lu, Y. Zhang, J. He, X. Guo, G. Tian, L. Jin, Int. J. Biol. Macromol. 43 (2008) 252–256. [46] J. Tai-Nin Chow, D.A. Williamson, K.M. Yates, W.J. Goux, Carbohyd. Res. 340 (2005) 1131–1142.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Purification, antioxidant and immunological activities of polysaccharides from Actinidia Chinensis roots Lin Zhang a,b , Wuxia Zhang a , Qingjie Wang a , Dongdong Wang a , Dongqi Dong a , Haibo Mu a , Xin-Shan Ye b,∗∗ , Jinyou Duan a,∗ a b
College of Science, Northwest A&F University, Yangling 712100, Shaanxi, China State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xue Yuan Road No. 38, Beijing 100191, China
a r t i c l e
i n f o
Article history: Received 29 July 2014 Received in revised form 8 September 2014 Accepted 26 September 2014 Available online 12 October 2014 Keywords: Actinidia Chinensis Polysaccharides Purification Antioxidant activity Immunomodulation
a b s t r a c t Two water-soluble polysaccharides (ACPS1 and ACPS2) were isolated from the roots of Actinidia Chinensis by DEAE-52 cellulose and Sephacryl S300 chromatography. Preliminary structural characterization was conducted by physicochemical property, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) analysis. ACPS1, with an average molecular weight of 5.58 × 105 Da, was mainly composed of rhamnose, arabinose, xylose, mannose and galactose in an approximate molar ratio of 1.48:4.28:4.30:1.00:17.83. ACPS2, with a high average molecular weight of 1.23 × 106 Da, mainly contained rhamnose, arabinose and galactose in a molar ratio of approximately 1.00:2.33:6.61. Both ACPS1 and ACPS2 exhibited the remarkable antioxidant activity to scavenge the DPPH radical and significant protective effects on H2 O2 -induced HEK 293 cells death in a concentration-dependent manner. Meanwhile, in vitro immunomodulatory activities of the two polysaccharides were evaluated. The results showed that treatment with 50–300 g/mL of the samples could increase NO production and phagocytic activity of macrophages in a dose-dependent manner. The present results suggested that the two polysaccharides from Actinidia Chinensis may be potential antioxidant and immunomodulatory agents for preparing functional foods and nutraceuticals applied in food and pharmaceutical industries. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Polysaccharide is a class of biological macromolecules substance that recognizes a broad spectrum of biological response modifiers existing widely in organism. They exist widely in plants, microorganisms (fungi and bacteria), algae and animals. It is well known that polysaccharides in living organisms do not only act as energy resources but also play key biological roles in many life processes. There is much evidence that a good number of polysaccharides possess a variety of biological activities, such as antioxidant activity [1,2], antitumor activity [3,4] antimicrobial activity [5], and immunologic activity [6] among others. Because most polysaccharides derived from higher plants are relatively nontoxic and do not cause significant side effects compared with immunomodulatory bacterial polysaccharides and synthetic compounds, they are ideal candidates for modern food and medical industries [7,8].
The genus Actinidia (Actinidiaceae) consists of over 58 species and widely distributed in the Asian continent. Most species are native to temperate regions of south-western China. Actinidia Chinensis Planch is a liana plant that commonly grows in temperate climate zones. Its roots have been used for gastric carcinoma, nasopharyngeal carcinoma, breast carcinoma and hepatitis in traditional Chinese medicine [9]. Until now, there have been few reports regarding the isolation, purification, characterization, antioxidant and immunological activities of the polysaccharides from Actinidia Chinensis roots. In the present study, two polysaccharides were purified from the crude polysaccharide and preliminarily characterized. Moreover, the antioxidative effects of the purified polysaccharides on DPPH radical scavenging activity, as well as the protective effects on H2 O2 -induced HEK 293 cells death and immunomodulatory effects on RAW 264.7 macrophages were investigated. 2. Materials and methods
∗ Corresponding author. Tel.: +86 29 87092226; fax: +86 29 87092226. ∗∗ Corresponding author. Tel.: +86 10 82805736. E-mail addresses: [email protected] (X.-S. Ye), [email protected], [email protected] (J. Duan). http://dx.doi.org/10.1016/j.ijbiomac.2014.09.056 0141-8130/© 2014 Elsevier B.V. All rights reserved.
2.1. Materials and chemicals The roots of Actinidia chinensis were collected in Mei County, Shannxi province, China and identified by Professor Langran Xu
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at College of Forestry, Northwest A&F University. A voucher specimen (No.20130822) has been deposited at the Laboratory of Functional Polysaccharides, Northwest A&F University. 2, 2-diphenyl-1-picryl-hydrazyl (DPPH), neutral red and standard monosaccharide (including arabinose, rhamnose, xylose, galactose, glucose, mannose and galacturonic acid) were obtained from Aladdin Reagent Int. Bovine serum albumin (BSA), 3-(4, 5-dimethyltiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT), lipolysaccharide (LPS) and vitamin C were obtained from Sigma Chemical Co. Ltd. DEAE-52 cellulose and Sephacryl S300 were purchased from Whatman Co. Ltd. The RAW264.7 and HEK 293 (human embryonic kidney 293) cell lines were kindly gifts from Professor Xuebo Liu in Northwest A&F University. Trifluoroacetic acid (TFA), dimethylsulfoxide (DMSO) and other reagents were all of analytical grade. 2.2. Extraction and purification of crude polysaccharide The fresh Actinidia chinensis (AC) roots were collected, cleaned and dried at 40◦ C and then pulverized into powder by a disintegrator. The dried AC powder (800.0 g) was pre-extracted with 95% ethanol (8 L, 5 d, 40 ◦ C, 4 times) to remove lipids. The residue powder was extracted with distilled water at 80 ◦ C for 2 h, at the ratio of 1:10 (W/V). The mixture was centrifuged at 3000 rpm for 30 min. The precipitate was re-extracted (2 times) as described above, and the supernatants were pooled. The collected supernatants were concentrated by a rotary evaporator under reduced pressure. Then, the supernatant was mixed with 95% ethanol to final ethanol concentration of 80% and incubated for 24 h at 4 ◦ C. The precipitate was collected and dissolved in distilled water and then deproteinated by the method of Sevag [10], before being decolored by 5% H2 O2 . Finally, the resulting solution was dialyzed against distilled water (cut-off Mw: 8000 Da) and lyophilized to afford crude Actinidia chinensis polysaccharide (ACPS). The crude polysaccharide ACPS was subjected to further processing. The crude ACPS (4.0 g) was purified sequentially by chromatography using DEAE-52 cellulose and Sephacryl S300 columns. The crude ACPS solution (20 mg/mL) was applied to a DEAE-52 cellulose column (5 × 50 cm, OH− form), which was washed with deionized water, and NaCl solutions of increasing ionic strength (0.1, 0.2, 0.3, 0.5, 1.0 M) at a flow rate of 2 mL/min. The eluate (8 mL/tube) was collected automatically, and the carbohydrate content was determined using the phenol-sulfuric acid method. Five mainly fractions were obtained. Two fractions (eluates of 0.1 M and 0.2 M NaCl) were further fractionated on DEAE-52 cellulose column (5 × 50 cm, Cl− form), eluted with NaCl solutions (0.1, 0.2, 0.3 M) sequentially or Sephacryl S300 column (2.5 × 100 cm), eluted with deionized water, at a flow rate of 0.5 mL/min to obtain ACPS1 and ACPS2 solutions respectively. The relevant fractions were then collected, concentrated, dialyzed (cut-off Mw: 8000 Da) and lyophilized to yield white, purified polysaccharides ACPS1 and ACPS2 for further study. The isolation and purification steps are shown in Fig. 1. 2.3. Molecular weight determination The molecular weights of ACPS1 and ACPS2 were determined on high performance gel permeation chromatography (HPGPC), as described previously [11–13]. The operation conditions were as follows: a Waters 600 HPLC System (Waters corporation, USA); Three Waters Ultrahydrogel columns in series (250, 1000 and 2000; 30 cm × 7.8 mm, 6 m particles); A 2414 Differential Refractive Index Detector; mobile phase: 3 mM sodium acetate; flow rate: 0.5 mL/min; injection volume: 50 L. The molecular weights of the polysaccharides were estimated by referencing a calibration curve, which was created using a set of dextran standards (5.2, 10, 48.6,
668 and 2000 KDa) .The calibration curve of Log (Mw) vs. elution time (T) is: Log(Mw) = −0.1316T + 10.94
(1)
2.4. Chemical compositions analysis Neutral carbohydrate content was determined by the phenol–sulphuric acid method [14], with d-glucose as standard. Uronic acid content was measured according to hydroxybiphenyl– sulphuric acid method [15] and galacturonic acid was used as the standard. Protein content was evaluated by Bradford’s method [16] with bovine serum albumin (BSA) as standard. Polysaccharides were dissolved in distilled water for ultraviolet analysis and the solutions of the polysaccharides were scanned from 200 to 800 nm with a 722 spectrophotometer (Shanghai Precision and Scientific Instrument Co., Ltd.)[17].
2.5. Infrared spectra analysis The IR spectra of polysaccharides were determined using a Fourier transform infrared spectrophotometer (BRUKER TEMSOR 27, BRUCK, Germany).The purified polysaccharides were grounded with KBr powder and pressed into a 1 mm pellet for FTIR measurement between 400 cm−1 and 4000 cm−1 [18].
2.6. Scanning electron microscopy (SEM) analysis SEM was used to reveal the microstructure of the materials [19,20]. Two samples were fixed on the silicon wafer. The shape and surface characteristics were observed and recorded using a field emission scanning electron microscope (FESEM, S-4800, Hitachi, Japan). They were examined at a10 KV acceleration voltage under a high vacuum condition, as well as image magnification of 100×, 500× and 800×.
2.7. Neutral monosaccharide composition The monosaccharide composition of the sample was analyzed by GC after transesterification [20]. Briefly, approximately 5 mg of freeze-dried sample was dissolved in an ampoule containing 6 mL of 2 M trifluoroacetic acid (TFA). The sample was hydrolyzed at 121 ◦ C for 2 h. After hydrolysis, the resulting solution was concentrated in a vacuum, and the excess acid was removed by repeated co-distillations with methanol. Neutral monosaccharide was reduced to alditol using 25 mg of sodium borohydride with 3 mL of distilled water at room temperature for 2 h. Acetic acid was added to the solution to decompose excess sodium borohydride until bubble formation stopped. A stream of nitrogen gas was used to dry the solution and 3 mL of methanol was added to remove the borohydrate. The procedure was repeated four times. Acetic anhydride (5 mL) was then added to the mixture and incubated for additional 60 min at 100 ◦ C. The hydrolysate was then converted into the corresponding alditolacetates and analyzed with GC using a Shimadzu 2010 instrument equipped with a HP-5MS column (0.25 mm × 30 m × 0.25 m) and a flame-ionization detector. The initial column temperature was held at 130 ◦ C for 5 min, increased to 240 ◦ C at 4 ◦ C/min and held at 240 ◦ C for 5 min. Nitrogen gas (N2 ) was used as the carrier gas with a flow rate of 1.0 mL/min. Alditol acetates were used as standards (glucose, mannose, rhamnose, galactose, xylose, and arabinose) and they were processed using the same approach as described above for the sample.
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Fig. 1. Isolation and purification steps of ACPS1 and ACPS2.
2.8. Determination of antioxidant activities in vitro 2.8.1. DPPH radical scavenging activity The radical scavenging effects of samples on DPPH radical were estimated as described [21]. Briefly, sample solution (1.0 mL) at different concentrations was added to DPPH (0.1 mM, 3.0 mL) in 95% ethanol. The reaction solution was shaken vigorously and incubated at room temperature for 30 min, and the absorbance at 517 nm was measured. Ascorbic acid was used as a positive control. The DPPH scavenging rate (R) was calculated as follows:
R(%) = 1 −
Abs(sample) − Abs(control) × 100 Abs(blank)
the formazan. After 10 min, the absorbance was measured at 570 nm in a microplate reader (Perlong DNM-9062, China). For protective assay, when the cells reached sub-confluence, they were pretreated for 36 h with culture medium containing different concentrations of samples (10, 50, 100, 200 g/mL) that were tested in the experiments. Next, the culture supernatant was moved and cells were washed three times with phosphate-buffered saline (PBS). Subsequently, the cells were exposed to H2 O2 (1000 M) diluted in culture medium for 10 h in a humidified atmosphere of 5% CO2 at 37 ◦ C until further assay and other procedures were same as above. Assays were performed in sextuple wells for each sample.
(2)
Where the control solution contains distilled water instead of the DPPH solution, while distilled water instead of sample was used for the blank. All tests were performed in triplicate and the mean of Abs was used in the equation above. 2.8.2. Protective effects on H2 O2 -induced HEK 293 cells death These were evaluated in vitro using the MTT assay [22]. HEK 293 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 g/mL streptomycin and 100 IU/mL penicillin. To check the cell cytotoxicity, cell suspensions were seeded in 96-well plates (1 × 105 cells/well), and incubated at 37 ◦ C for 4 h, and the samples were added. After 36 h, 20 L of the MTT solution (5 mg/mL) was added into each well and the plate was further incubated for 4 h. Finally, the medium was removed and DMSO (180 L) was added to each well to dissolve
2.9. Determination of immunoregulation effects of polysaccharides 2.9.1. Effects of ACPS1 and ACPS2 on the macrophages proliferation The measurement of macrophages proliferation was determined according to the MTT-based colorimetric method. Macrophage cells were dispensed into 96-well plates and incubated at 37 ◦ C in a 5% CO2 atmosphere for 4 h. The RPMI1640 medium was discarded, and the cells were treated with various concentrations of ACPS1 and ACPS2 (10, 50, 100, 150 g/mL). The RPMI1640 medium was used as control. After incubated for 24 h, media were discarded, and MTT solution (20 L/well) was added to each well and further incubated for 4 h. Then, media were discarded, and 180 L DMSO was added. After shaking to dissolve the formazan, the optical density of each well at
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Fig. 2. HPGPC profiles of ACPS1 (A) and ACPS2 (B).
570 nm was measured by a microplate reader (Perlong DNM-9062, China).
positive control, respectively. Phagocytosis index was calculated by the following equation: Phagocytosis index =
2.9.2. Assay for nitric oxide (NO) production Nitrite accumulation was measured by Griess reagent [23–25]. RAW 264.7 cells (1 × 106 cells/well) were dispensed into 96-well plates. And then cells were stimulated with medium (for the control group), LPS (2 g/mL) and various concentrations of samples (10, 50, 100, 150 g/ml) for 24 h. After incubation, 100 L of culture supernatants were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride, and 2.5% phosphoric acid) in 96-well plates and incubated at 25 ◦ C for 10 min. The absorbance at 540 nm was measured on a microplate reader (Perlong DNM-9062, China). Nitrite concentrations in culture supernatants were measured to assess NO production in RAW 264.7 cells. NaNO2 was used as standard to calculate nitrite concentrations.
2.9.3. Assay of macrophages phagocytosis The phagocytic ability of macrophages was measured using neutral red uptake [26,27]. Briefly, cells (1 × 106 cells/well) were pipetted into 96-well plates and incubated for 4 h. Culture medium was discarded, and the cells were treated with various concentrations of samples (50, 100, 200, 300 g/mL) for 24 h. Then, 0.075% neutral red solution (100 L/well) was added and incubated for 1 h. Medium was discarded and cells in 96-well plates were washed twice with PBS to remove the neutral red that was not phagocytosed by RAW 264.7 cells. Then, cell lysis buffer (1% glacial acetic acid: ethanol = 1:1, 100 L/well) was added to lyse cells. After cells were incubated at room temperature for 15 h, the optical density of each well was measured at 540 nm using a microplate reader (Perlong DNM-9062, China). The RMPI1640 medium and LPS (10 g/mL) were used as the blank and
Abs(sample) Abs(blank control)
(3)
2.10. Statistical analysis Data were expressed as means ± standard deviation (SD). The scientific statistic software GraphPad Prism 5.0 was used to evaluate the significance of differences between groups. p < 0.05 was regarded as significant. 3. Results and discussion 3.1. Extraction, isolation and purification of polysaccharide The crude polysaccharide ACPS (38.6 g from 800.0 g) was isolated from the hot water extract of roots of Actinidia Chinensis with a yield of 4.8%. After fractionation on DEAE-52 cellulose column (OH− form), five fractions were obtained. The DEAE-52 cellulose (Cl− form) and Sephacyl S300 columns were used for further purification of the 0.1 M NaCl and 0.2 M NaCl eluent, respectively. Two polysaccharides ACPS1 (99.0 mg) and ACPS2 (104.0 mg), with the yield about 2.47% and 2.60% from ACPS, were obtained. 3.2. Molecular weight determination Molecular weight is an important index for quality control of polysaccharide products, as being closely related to their physicochemical properties and biological activities. Up to now, high performance gel permeation chromatography (HPGPC) with the advantage of rapid, high resolution and good reproducibility has been widely used in polysaccharide molecular weight determination [28–31]. As shown in Fig. 2, profiles of the ACPS1 and ACPS2 on HPGPC reveal that the fractions were represented by a
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Fig. 3. Infrared spectra of ACPS1and ACPS2.
Table 1 Neutral carbohydrate, uronic acid and protein contents of ACPS1 and ACPS2. Sample
Neutral carbohydrate (%)
Uronic acid (%)
Protein (%)
ACPS1 ACPS2
74.05 ± 1.15 68.59 ± 1.02
18.32 ± 0.78 25.36 ± 2.10
n.d.a n.d.a
a
n.d.: not detected.
single and symmetrical peak which indicated the molecular weight homogeneity and the high purity of the generated two polysaccharides. Their molecular weights were calculated to be 5.58 × 105 Da (T: 39.458) and 1.23 × 106 Da (T: 36.792), respectively. 3.3. Chemical compositions analysis The chemical compositions of ACPS1 and ACPS2 were determined and listed in Table 1. The two polysaccharides mainly contained neutral carbohydrate and uronic acid. The two polysaccharides at a concentration of 5 mg/mL had no obvious absorption at 280 nm and 260 nm in the UV spectra, indicating the absence of protein and nucleic acid. Negative responses to the Bradford test (the detection limit of BSA standard was 15 g/mL) further indicated that the two polysaccharides did not contain protein. The negative results of Fehling’s and iodine-potassium iodide tests
showed ACPS1 and ACPS2 did not contain reducing sugar and did not belong to starch-type polysaccharide [32].
3.4. Infrared spectra analysis The IR spectra of ACPS1 and ACPS2 were recorded at the range of 400–4000 cm−1 (Fig. 3). Both of them displayed a broad and intense peak nearby 3431 cm−1 and 3420 cm−1 , which were due to the hydroxyl groups stretching vibration. The bands in the region of 2926 cm−1 and 2934 cm−1 were the characteristic absorption of C H stretching vibration. The absorptions were the characteristic of antisymmetrical stretching vibration. The bands around 1627 cm−1 and 1620 cm−1 represented the carboxylate (COO ) stretching band [33], indicating that there were esterified and free carboxyl groups present in the polysaccharides from Actinidia Chinensis. It also validated the presence of uronic acids. The absorption band from 800 cm−1 to 1300 cm−1 , called “finger print” region, was related to conformation and surface structure of molecule. Although these bands are hard to explain [33], the peaks at 950–1200 cm−1 suggested the presence of C O C and C OH link bonds [34]. From the spectra, we proposed that the absorbance between 1000 cm−1 and 1200 cm−1 was due to the pyranose ring [35].
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Fig. 4. Scanning election microscope (SEM) photographs of ACPS1 (A, 100×; B, 500×; C, 800×) and ACPS2 (D, 100×; E, 500×; F, 800×).
3.5. Scanning electron microscopy (SEM) analysis The SEM photographs of the two polysaccharides were showed in Fig. 4. The surfaces of ACPS1 and ACPS2 were little different.
Images A, B and C show that ACPS1 had a rough surface like clouds with characteristic large wrinkles. In contrast, images D, E and F show that ACPS2 had a thin slice shape and the surface was very smooth.
Fig. 5. GC spectra of sample references (A), ACPS1 (B) and ACPS2 (C). Peaks in the spectra representing the follows: (1) rhamnose; (2) arabinose (3) xylose; (4) mannose; (5) galactose; (6) glucose.
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Fig. 6. Scavenging effects on DPPH radical of ACPS1 and ACPS2.
3.6. Analysis of neutral monosaccharide composition GC analysis with derivatization is an acknowledged method for the quantification of neutral sugars. By comparing the retention time with standards, the monosaccharide composition was identified. Their Gas chromatograms profiles were showed in Fig. 5. ACPS1 mainly contained neutral monosaccharide composition of rhamnose, arabinose, xylose, mannose and galactose with the molar ratio of 1.48:4.28:4.30:1.00:17.83. However, ACPS2 was mainly composed of rhamnose, arabinose, and galactose, with molar ratio of 1.00:2.33:6.61. 3.7. Determination of antioxidant activities 3.7.1. Measurement of scavenging effects on DPPH radical The model of scavenging the DPPH radical is a widely used method for evaluating the free radical-scavenging ability of
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polysaccharide [36,37].The antioxidant mechanism of DPPH radical scavenging is based on the acceptance of hydrogen by the DPPH radical, thereby converting DPPH into the non-radical form (DPPH-H). Thus the antioxidant activity is due to the hydrogen donating ability of the polysaccharide [18]. As shown in Fig. 6, the scavenging abilities of ACPS1 and ACPS2 were concentration related in all of the evaluated concentrations. An obvious growth of the scavenging ability was found at the concentration range (0.5–3 mg/mL) of the samples. At high concentrations (>1.5 mg/mL), comparing with ACPS2, ACPS1 elicited a stronger scavenging activity obviously. At the concentration of 3.0 mg/mL, the DPPH radical scavenging activity of ACPS1 and ACPS2 were 56.9% and 51.8%, respectively. The results mentioned above suggested that the polysaccharides ACPS1 and ACPS2 might act as hydrogen donator and could react with DPPH radicals to convert them to more stable products, so that they can scavenge DPPH free radical. 3.7.2. Protective effects on H2 O2 -induced HEK 293 cells death H2 O2 is able to penetrate biological membranes, and plays a radical forming role as an intermediate in the production of more reactive ROS molecules including formation of hydroxyl radical via oxidation of transition metals, and hypochlorous acid by the action of myeloperoxidase, an enzyme present in the phagosomes of neutrophils [38], H2 O2 has been used in many studies to trigger cell apoptosis. Antioxidants could prevent cell death through the suppression of H2 O2 -induced ROS formation the regulation of the endogenous oxidant–antioxidant balance [39]. HEK 293 cells are cell line originally derived from human embryonic kidney cells and widely used as a model cell line to assess the protective effects of an antioxidant on H2 O2 -induced cells death.
Fig. 7. A. Effects of ACPS1 and ACPS2 on HEK 293 cells proliferation. Cells were cultured withACPS1 and ACPS2 for 36 h. OD value at 570 nm was used to represent the proliferation of cells. n = 6. B. Viability losses in HEK 293 cells induced by various concentrations of H2 O2. **p < 0.01 compared with control. n = 6. C. Protective effects of ACPS1 and ACPS2 on viability losses in HEK 293 cells induced by H2 O2 (1000 M, 10 h). n = 6. *p < 0.05, **p < 0.01 compared with the H2 O2 alone group.
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showed that 100–1000 M of H2 O2 could result in the HEK 293 cells death dramatically (Fig. 7B) after 10 h. For instance, as showed in (Fig. 7 C) the incubation of HEK 293 cells with 1000 M H2 O2 for 10 h resulted in a cell viability rate of 38.9% compared to the control. However, when pretreating the cells with four different concentrations (10, 50, 100, 200 g/mL) of ACPS1 and ACPS2, the cell viabilities were significantly increased to 68.6% and 72.3%, respectively. 3.8. Determination of immunoregulation effects
Fig. 8. Effects of ACPS1 and ACPS2 on the proliferation of RAW 264.7 macrophages. Cells were cultured with samples for 24 h. OD value at 570 nm was used to represent the proliferation of cells. n = 6.
We first tested whether ACPS1 and ACPS2 affected the viability of HEK 293 cells, which was determined by MTT assay. Two samples, ACPS1 and ACPS2, at the dose of 10–200 g/ml had no significant toxic effects on HEK 293 cells (Fig. 7A). The MTT assay
3.8.1. Effects of ACPS1and ACPS2 on the proliferation of macrophages It was necessary to evaluate the cytotoxic or proliferation effects of the two polysaccharides on RAW 264.7 cells before further tests were carried out. MTT assay indicated that ACPS1 and ACPS2 did not show cytotoxicity or stimulation on the proliferation of RAW 264.7 cells in 24 h (Fig. 8) significantly. Based on these results, the measurement of NO production and phagocytosis can well represent the cell function without any changes in cell quantity.
Fig. 9. Effects of ACPS1 (A) and ACPS2 (B) on the NO production of RAW 264.7 cells. Cells were pretreated with ACPS1 and ACPS2 at different concentrations (50, 100, 200, 300 g/mL) or LPS (2 g/ml) for 24 h. The supernatant nitrite levels were determined using Griess reagent. *p < 0.05, **p < 0.01 compared with control. n = 6.
Fig. 10. Effects of the ACPS1 (A) and ACPS2 (B) on the phagocytosis index of RAW 264.7 macrophages by a neutral red uptake assay. After treatment with the two polysaccharide samples (50, 100, 200, 300 g/mL) or LPS (10 g/mL) for 24 h, cells were used to test phagocytic activity. *p < 0.05, **p < 0.01 compared with control. n = 6.
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3.8.2. Measurement of NO production In recent years, NO was found to be a novel signaling molecule and a key mediator of signal transduction in the immune system [40,41]. Bacterial products such as lipopolysaccharide (LPS), a major constituent of the outer membrane of Gram-negative bacteria, have been shown to activate macrophages to release pro-inflammatory cytokines and NO [42–44]. The effects of two samples on the NO production of RAW 264.7 cells were determined by Griess assay. As shown in Fig. 9, the positive control, LPS at a concentration of 2 g/mL had a strong potential in stimulating NO production in macrophages. The two polysaccharides could stimulate macrophages to produce higher levels of NO in a dose dependent manner at different concentrations (50, 100, 200, 300 g/mL) compared with control (p < 0.01). NO is one of the most important mediators in the regulation of immunologic functions. Our results confirmed that the two samples may act as stimulators of NO release in macrophages and have the function of activation of macrophages. 3.8.3. Assay of macrophages phagocytosis Activated macrophages not only participate in both specific immune reactions and non-specific immune reactions, but also are the “bridge cell” of these two kinds of immune reactions [45]. One of the most distinguished features of macrophages activation would be an increase in phagocytic activity. It is well known that macrophages can phagocytose some dyes such as neutral red and malachite green in vitro. Therefore, in the present study we carried out the neutral red phagocytosis assay to evaluate the effects of ACPS1 and ACPS2 on phagocytic activity of RAW 264.7 macrophages. As shown in Fig. 10, phagocytosis index of ACPS1and ACPS2 all exceeded 1.0 and increased in a dose-dependent manner at the test concentrations, indicating that all the two fractions had abilities to enhanced phagocytic activity of RAW 264.7 cells. Compared with the blank control, ACPS1 and ACPS2 could significantly enhance the phagocytosis of macrophages (≥100 g/mL, p < 0.05) as well as LPS action (10 g/mL, p < 0.01). Macrophages can phagocytose aging bacteria, damage cells and necrotic tissues invading the body. Phagocytic capacity is one of the most important indicators of the body’s non-specific immunity [41]. In this study, the results indicated that the ACPS1 and ACPS2 could significantly enhance the phagocytosis of the macrophages, which may be due to the binding of the polysaccharide with a specific receptor on the surface of macrophages [41,46]. 4. Conclusions The roots of Actinidia Chinensis have been used for the treatment of cancers in the Chinese folk medicine, and were proved to have antitumor and immunopotentiating activities [9]. In this investigation, we have succeeded in abstracting the crude polysaccharide ACPS. Two water-soluble polysaccharides (ACPS1 and ACPS2) with the molecular weights of 5.58 × 105 Da and 1.23 × 106 Da, respectively, were purified from the roots of Actinidia Chinensis by combination use of DEAE-52 cellulose and Sephacryl S300 columns. ACPS1 and ACPS2 were defined as acid heteropolysaccharides with different content of uronic acids as well as the ratio of neutral sugars. Moreover, the two polysaccharides showed significant DPPH radical scavenging activity, protection of the HEK 293 cells from H2 O2 damage, stimulatory effects on the NO production and phagocytosis activity of RAW 264.7 macrophages. These finding indicated that ACPS1 and ACPS2 have the potential to be antioxidative and immunopotentiating agents that could be further developed in the food and pharmaceutical industries.
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Acknowledgments This work was financially supported by the National Natural Science Foundation of China (NSFC) (Grant No. 31270860), Program for New Century Excellent Talents in University (NCET-13-0480), the State Key Laboratory of Natural and Biomimetic Drugs, peking University (K20120216), and ‘Interdisciplinary Cooperation Team’ Program for Science and Technology Innovation of the Chinese Academy of Science. References [1] X.-K. Zhong, X. Jin, F.-Y. Lai, Q.-S. Lin, J.-G. Jiang, Carbohyd. Polym. 82 (2010) 722–727. [2] C.-L. Ye, Q. Huang, Carbohyd. Polym. 89 (2012) 1131–1137. [3] S.-D. Park, Y.-S. Lai, C.-H. Kim, Life Sci. 75 (2004) 2621–2632. [4] X. Ding, Y. Hou, W. Hou, Carbohyd. Polym. 89 (2012) 397–402. [5] J.-H. Xie, M.-Y. Shen, M.-Y. Xie, S.-P. Nie, Y. Chen, C. Li, D.-F. Huang, Y.-X. Wang, Carbohyd. Polym. 89 (2012) 177–184. [6] R. Chen, Y. Li, H. Dong, Z. Liu, S. Li, S. Yang, X. Li, Ultrason. Sonochem. 19 (2012) 1160–1168. [7] D. Zhang, S. Li, Q. Xiong, C. Jiang, X. Lai, Carbohyd. Polym. 95 (2013) 114–122. [8] R. Wang, P. Chen, F. Jia, J. Tang, F. Ma, Int. J. Biol. Macromol. 50 (2012) 331–336. [9] J.N.M. College, in, Science and Technology Press of Shanghai Shanghai, 1977. [10] M. Sevag, D.B. Lackman, J. Smolens, J. Biol. Chem. 124 (1938) 425–436. [11] J. Duan, X. Wang, Q. Dong, J.-N. Fang, X. Li, Carbohyd. Res. 338 (2003) 1291–1297. [12] W. Zhang, H. Mu, A. Zhang, G. Cui, H. Chen, J. Duan, S. Wang, Glycoconjugate J. 30 (2013) 577–583. [13] G. Cui, W. Zhang, Q. Wang, A. Zhang, H. Mu, H. Bai, J. Duan, Carbohyd. Polym. 111 (2014) 245–255. [14] T. Masuko, A. Minami, N. Iwasaki, T. Majima, S.-I. Nishimura, Y.C. Lee, Anal. Biochem. 339 (2005) 69–72. [15] N. Blumenkrantz, G. Asboe-Hansen, Anal. Biochem. 54 (1973) 484–489. [16] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254. [17] L. Zhao, Y. Dong, G. Chen, Q. Hu, Carbohyd. Polym. 80 (2010) 783–789. [18] T. Zhao, G. Mao, W. Feng, R. Mao, X. Gu, T. Li, Q. Li, Y. Bao, L. Yang, X. Wu, Carbohyd. Polym. 105 (2014) 26–33. [19] Q. Zhao, J.F. Kennedy, X. Wang, X. Yuan, B. Zhao, Y. Peng, Y. Huang, Int. J. Biol. Macromol. 49 (2011) 181–187. [20] Y. Zhu, Q. Li, G. Mao, Y. Zou, W. Feng, D. Zheng, W. Wang, L. Zhou, T. Zhang, J. Yang, Carbohyd. Polym. 101 (2014) 606–613. [21] G. Cui, W. Zhang, A. Zhang, H. Mu, H. Bai, J. Duan, C. Wu, Int. J. Biol. Macromol. 57 (2013) 278–284. [22] S.-M. Lee, M.-Y. Yoon, H.-R. Park, Biosci. Biotechnol. Biochem. 72 (2008) 1272–1277. [23] M. Shi, Y. Yang, D. Guan, Y. Zhang, Z. Zhang, Carbohyd. Polym. 89 (2012) 1268–1276. [24] D. Guan, Z. Zhang, Y. Yang, G. Xing, J. Liu, Int. J. Biol. 3 (2011) p3. [25] A.M. Gamal-Eldeen, H. Amer, W.A. Helmy, R.M. Talaat, H. Ragab, Int. Immunopharmacol. 7 (2007) 871–878. [26] W. Chen, Z. Zhao, S.-F. Chen, Y.-Q. Li, Biores. Technol. 99 (2008) 3187–3194. [27] X. Li, L. Zhao, Q. Zhang, Q. Xiong, C. Jiang, Carbohyd. Polym. 102 (2014) 912–919. [28] J. Dawkins, Pure Appl. Chem. 54 (1982) 281–292. [29] J.-H. Xie, M.-Y. Xie, S.-P. Nie, M.-Y. Shen, Y.-X. Wang, C. Li, Food Chem. 119 (2010) 1626–1632. [30] G. Zhao, J. Kan, Z. Li, Z. Chen, Carbohyd. Polym. 61 (2005) 125–131. [31] T. Dreher, D. Hawthorne, B. Grant, J. Chromatogr. A 174 (1979) 443–446. [32] X. Yang, Y. Zhao, Y. Lv, Carbohyd. Polym. 71 (2008) 372–379. [33] R. Gnanasambandam, A. Proctor, Food Chem. 68 (2000) 327–332. [34] M. Kacurakova, P. Capek, V. Sasinkova, N. Wellner, A. Ebringerova, Carbohyd. Polym. 43 (2000) 195–203. [35] F. Lai, Q. Wen, L. Li, H. Wu, X. Li, Carbohyd. Polym. 81 (2010) 323–329. [36] Y. Chen, M.-Y. Xie, S.-P. Nie, C. Li, Y.-X. Wang, Food Chem. 107 (2008) 231–241. [37] D. Qiao, C. Ke, B. Hu, J. Luo, H. Ye, Y. Sun, X. Yan, X. Zeng, Carbohyd. Polym. 78 (2009) 199–204. [38] J. Nordberg, E.S. Arnér, Free Radical Biol. Med. 31 (2001) 1287–1312. [39] H.Y. Xue, G.Z. Gao, Q.Y. Lin, L.J. Jin, Y.P. Xu, Phytother. Res. 26 (2012) 369–374. [40] J. MacMicking, Q.-w. Xie, C. Nathan, Annu. Rev. Immunol. 15 (1997) 323–350. [41] I.A. Schepetkin, M.T. Quinn, Int. Immunopharmacol. 6 (2006) 317–333. [42] X. Zhang, Y. Li, G. Tai, G. Xu, P. Zhang, Y. Yang, F. Lao, Z. Liu, Cell Mol. Immunol. 2 (2005) 63–67. [43] G. Drozina, J. Kohoutek, T. Nishiya, B.M. Peterlin, J. Biol. Chem. 281 (2006) 39963–39970. [44] K.P. Crume, J.H. Miller, A.C. La Flamme, Exp. Biol. Med. 232 (2007) 607–613. [45] X. Chen, J. Lu, Y. Zhang, J. He, X. Guo, G. Tian, L. Jin, Int. J. Biol. Macromol. 43 (2008) 252–256. [46] J. Tai-Nin Chow, D.A. Williamson, K.M. Yates, W.J. Goux, Carbohyd. Res. 340 (2005) 1131–1142.
