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Preparation and sulfation of an α-glucan from Actinidia chinensis roots and their potential activities
International Journal of Biological Macromolecules 92 (2016) 981–987
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Preparation and sulfation of an ␣-glucan from Actinidia chinensis roots and their potential activities Hong Niu, Dan Song, Yuelin Sun, Wuxia Zhang, Haibo Mu, Jinyou Duan ∗ Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of science, Northwest A&F University, Yangling 712100, Shaanxi, China
a r t i c l e
i n f o
Article history: Received 25 June 2016 Received in revised form 23 July 2016 Accepted 27 July 2016 Available online 28 July 2016 Keywords: ␣-Glucan Sulfation Moisture-preserving property Immunological activity
a b s t r a c t In this study, one homopolysaccharide (ACPA1) with a molecular weight (Mw) of 5.5 × 103 g/mol was prepared from the roots of Actinidia chinensis. It was characterized as an ␣-d-glucan consisting of predominant 4-linked ␣-d-Glcp residues branched at O-6. Three sulfated derivatives of ACPA1 (SA1, SA2 and SA3) with different degrees of sulfation (DS) were obtained by SO3 -pyridine procedure. Moisture-preserving tests demonstrated that the sulfated derivatives with the highest DS values, SA1 and SA2, exhibited better moisture-preserving abilities than ACPA1 and SA3. All sulfated derivatives exerted stimulatory effects on phagocytosis activity and nitric oxide production by RAW 264.7 macrophages while ACPA1 did not. These findings suggested that the sulfated derivatives of ACPA1 might be used as moisture-preserving or immunopotentiating reagents. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Polysaccharides are a class of natural molecules widely distributed in living organisms. Some of them exhibit various biological activities or have special functions. They contain reactive groups, such as hydroxyl, amino and carboxylic acid groups, and can be used as starting materials for different derivatives which often possess even better physicochemical properties than their raw materials [1]. Chemical modification of polysaccharides provides an opportunity to obtain new pharmacological agents with possible therapeutic uses [1]. In the last decades, much attention has focused on the biological properties of polysaccharides and their chemical derivatives, especially sulfated derivatives [2–4]. The sulfation of polysaccharides could not only enhance their water solubility but also change the chain conformation, thus resulting in the alteration of their biological activities [5]. The fruit tree Actinidia chinensis, belonging to the genus Actinidiaceae, is a medicinal plant. Its origin is supposed to be the northern Yangtse river valley, and now Actinidia chinensis is widely dispersed in the southeast of China [6]. Up to now, a great diversity of low-molecular-weight constituents including phenolic compounds, flavanoids, anthraquinones, and triterpenoids have been isolated from the roots of Actinidia chinensis [7–10]. In contrast, few
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (J. Duan). http://dx.doi.org/10.1016/j.ijbiomac.2016.07.091 0141-8130/© 2016 Elsevier B.V. All rights reserved.
reports have been concerned with the polysaccharide constituents from the roots of this species. In the present study, we isolated a homosaccharide ␣-d-glucan from A. chinensis roots and characterized its structure using chemical and spectroscopic techniques. Its sulfated derivatives were also prepared and their moisture-preserving properties and immunological activities were further investigated. 2. Materials and methods 2.1. Materials and chemicals The roots of Actinidia chinensis collected from Mei County, Shannxi province, China were same as the original material used in [11]. Sodium hyaluronate (∼2 × 106 g/mol), pyridine–sulfur trioxide complex, and neutral red were obtained from Aladdin Chemical Reagent Company, China. DEAE-52 cellulose and Sephacryl S-200 columns were purchased from Whatman Corp. The RWA 264.7 cell line was a gift from Professor Xuebo Liu of Northwest A&F University. All other reagents were of analytical grade. 2.2. Isolation and purification of ACPA1 The isolation and purification procedures were performed as described previously with some modifications [11,12]. Briefly, the dried and defatted Actinidia chinensis (AC) root powders (500 g) were extracted with hot water three times. Then the non-water soluble pellets obtained after centrifugation were solubilized with
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0.5 M NaOH at 4 ◦ C for 12 h without agitation. After the supernatant was neutralized with 2 M HCl, three volumes of cold 95% (v/v) ethanol were added to get the precipitated polysaccharide ACP. ACP was deproteinized by the method of Sevag [13]. Since the crude water or alkli-soluble polysaccharides are always contaminated with colorful polyphenols, ACP was further depigmented by 5% (v/v) H2 O2 [14] and dialyzed (Spectra/Por RC dialysis membrane, MW cutoff 500–1000 g/mol). A half volume of 95% (v/v) ethanol was added, and the supernatant was collected. Then the supernatant was loaded onto a DEAE-52 cellulose column (5 × 50 cm, Cl− form) which was eluted with distilled water. The eluate was collected by a fraction collector, and the most concentrated fractions were pooled according to the total carbohydrate content quantified by the phenol–sulfuric acid method. The collected eluate was named as ACPA and a Sephacryl S-200 column (2.5 × 100 cm) was used to remove other polysaccharides from ACPA eluate. The purified polysaccharide was designated as ACPA1, dialyzed, lyophilized, and stored in a vacuum desiccator. 2.3. Characterization of ACPA1 2.3.1. Analysis of homogeneity and molecular weight Using previously reported methods [15], the molecular weight of ACPA1 was determined by high performance gel permeation chromatography (HPGPC) on a Waters 515 GPC system at 25 ◦ C, using a Waters 2410 RI (refractive index) as the detector. Three columns (Waters Ultrahydrogel 250, 1000 and 2000; 30 cm × 7.8 mm; 6 m particles) in series were calibrated with T-series dextrans (5.2, 10, 48.6, 668, 2000 × 103 g/mol). Sodium acetate (3 mM) was used as eluant and the flow rate was kept at 0.5 mL/min. For each run, a 100 L aliquot of ACPA1 in the eluant was injected. The calibration curve of log (Mw) vs. elution volume (V) is: Log (Mw) = −0.2342 V + 10.45. 2.3.2. Chemical composition analysis and starch iodine test The uronic acid content of ACPA1 was measured according to the m-hydroxydiphenyl-sulphuric acid method, and galacturonic acid was used as the standard [16]. The neutral monosaccharide composition of ACPA1 was analyzed by gas chromatography (equipped with an HP-5 capillary column and flame-ionization detector) after conversion of the hydrolysate into alditol acetates as reported by [17]. The absorption curve of sample-iodine complex was measured spectrophotometrically as described in reference [18]. 2.3.3. Methylation analysis ACPA1 (10 mg) was methylated three times according to the method of Needs and Selvendran [19] using NaOH powder and methyl iodide in dimethyl sulfoxide. The fully methylated product was hydrolyzed, reduced, and acetylated. The partially methylated alditol acetates were analyzed using a gas chromatography–mass spectrometry (GC–MS) system equipped with a trace mass spectrometer and a DB-5 capillary column (0.25 mm × 30 m × 0.25 m, Thermo Finnigan Co., Santa Clara, CA). The temperature program was isothermal at 140 ◦ C for 3 min followed by a 3 ◦ C/min gradient up to 250 ◦ C [20]. The molar ratios were estimated from the peak area on GC and response factor [21,22]. 2.3.4. FT-IR and NMR analysis ACPA1 (2 mg) was ground with KBr powder and pressed into pellets for FT-IR measurement (Bruker tensor 27, Bruker, Germany) in the range of 4000 ∼ 500 cm−1 [23,24]. Dried ACPA1 was exchanged with deuterium by lyophilizing with D2 O for three times. The 1 H (500 MHz) and 13 C (125 MHz), DEPT, HSQC, HMBC and 1 H-1 H COSY NMR spectra of ACPA1 in D2 O were recorded at room temperature using a Bruker AM 500 spectrometer with a dual probe in the FT mode [25,26]. Chemical shifts are referred to the residual signal of
HOD at ı 4.74 ppm for 1 H NMR spectrum and the external standard with Me4Si for 13 C NMR spectrum. 2.4. Preparation and characterization of sulfated derivatives of ACPA1 Polysaccharides with various degrees of sulfation (DS) were prepared under mild conditions with SO3 -pyridine complex in aprotic solvents [27]. Briefly, dried polysaccharide (80 mg) was suspended in 12.8 mL of anhydrous N, N-dimethyl formamide (DMF) and then stirred for 14 h at room temperature under N2 . A required excess (15 mol/equiv of available hydroxyl group in amylose) of SO3 pyridine complex was added to the sample suspensions and then stirred for 5, 10, or 12 h (6 h, twice) at 40 ◦ C under N2 . Water was added to interrupt the reaction and the raw products were precipitated by 3 vols of cold ethanol with saturated anhydrous sodium acetate. After centrifugation, the sulfated polysaccharides were dialyzed to completely remove excess salts and lyophilized. The FTIR spectra of the samples were recorded as described previously. The DS of samples were determined by the barium chloride turbidimetric method according to the literature [28]. 2.5. Moisture-absorption and -retention tests The moisture-absorption (Ra) and −retention (Rh) abilities of the samples were tested as described in [29], and hyaluronic acid was used as the control. For the moisture absorption test, each sample (30 mg) was freeze-dried for 48 h and was kept in a silica gel drier overnight. Then, samples were put into desiccators saturated with (NH4 )2 SO4 (81%, relative humidity, RH) or Na2 CO3 (43% RH) for different periods at room temperature. The moisture absorption ability (Ra) was evaluated by the percentage of increase in weight of dry samples, based on Equation (1). Ra (%) = 100 × (Wn − Wo) /Wo
(1)
(Wo and Wn were the weights of samples before and after putting it into the desiccators, respectively. The Ra values of each sample in triplicate were averaged.) For the moisture retention test, each freeze-dried sample (30 mg) was put into a silica gel drier overnight as above. 10% (w/w) water was added to each sample and then the samples were put into desiccators saturated with (NH4 )2 SO4 or silica gel for different periods at room temperature. The moisture retention ability (Rh) was evaluated by the percentage of residual water of wet samples, according to Equation (2).
Rh (%) = 100 × Hn/Ho
(2)
(Hn and Ho were the weights of water in the samples before and after putting it into the desiccators, respectively. The Rh values of each sample in triplicate were averaged.) 2.6. Assay of immunological activities RAW 264.7 cells (1 × 106 /well) were dispensed into 96-well plates and incubated with PBS or different concentrations of polysaccharides in PBS at 37 ◦ C for 24 h. Nitric oxide production was determined, according to nitrite contents in culture supernatant which was analyzed by Griess assay [30]. The phagocytic ability of macrophages was measured by neutral red uptake assay [31]. Cells (1 × 105 /well) were incubated with different concentrations of samples for 24 h as described previously. Culture media was removed and the cells were incubated with 100 uL of 0.075% (w/v) neutral red solution for 2 h. The supernatant was discarded and the cells were washed with PBS three times. Cell lysis buffer [1% (v/v) glacial acetic acid: ethanol = 1:1, 200 L/well] was
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Fig. 1. HPGPC profile and 1D NMR spectra of ACPA1. (A) HPGPC chromatogram on ultrahydrogel columns of ACPA1 (B) 1 H NMR spectrum (C) DEPT spectrum (A: linear → 4)␣-Glcp-(1 → 4)-/Residue A, B: terminal-␣-Glcp-(1 → 4)-/Residue B, C: branch → 4)-␣-Glcp-(1 → 6)-/Residue C).
added to lyse cells. After incubation overnight at 4 ◦ C, the Abs at 540 nm was measured by an ELISA reader.
2.7. Statistical analysis Data were expressed as means ± standard deviation (SD). The scientific statistic software GraphPad Prism 5.01 was used to evaluate the significance of differences between groups. P < 0.05 was regarded as significant.
3. Results and discussion 3.1. Purification and characterization of ˛-glucan from Actinidia chinensis roots 3.1.1. Basic properties of ACPA1 The alkali-soluble crude polysaccharide, designated ACP was extracted from the dry roots of Actinidia chinensis with a yield of 2% (w/w) of root mass. After being deproteinated and depigmented, ACP was repeatedly subjected to DEAE-cellulose chromatography and Sephacryl S-200 gel-permeation chromatography, which resulted in the isolation of a polysaccharide fraction ACPA1 with a yield of 9% (w/w) of ACP mass. ACPA1 was eluted as a symmetrical narrow peak on highperformance gel-permeation chromatography (HPGPC) (Fig. 1A), and its weight-average molecular weight was estimated to be 5.5 × 103 g/mol, in reference to standard dextrans. The polydispersity index (Mw/Mn) of ACPA1 equaled 1.31, which indicated it was a relatively monodisperse polymer. The sugar composition of ACPA1 was free of uronic acid as determined by the m-hydroxydiphenylsulphuric acid method, and only glucose was detected by gas
Fig. 2. Infrared spectra of ACPA1 and SA1-SA3.
chromatography (data not shown), indicating that ACPA1 was a glucan.
3.1.2. FT-IR analysis The FTIR spectrum of the glucan is shown in Fig. 2. The bands at 3382.91 cm−1 and 2924.87 cm−1 were due to O H and C H stretching vibration, respectively. The absorption at 918.88 cm−1 was typical for d-glucan in the pyranose form [32]. Additionally, the ␣-configuration was confirmed by the absorption at 846.61 cm−1 ; there was no absorption at 890 cm−1 indicative of the -configuration [33,34]. The negative reaction between ACPA1
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Fig. 3. 2D NMR spectra of ACPA1. (A) HSQC spectrum (B) 1 H-1 H COSY spectrum (C) HMBC spectrum (A: linear → 4)-␣-Glcp-(1 → 4)-/Residue A, B: terminal −␣-Glcp-(1 → 4)/Residue B, C: branch → 4)-␣-Glcp-(1 → 6)-/Residue C). Table 1 GC–MS test result for the methylated sugar moieties of ACPA1. Methylated sugars (as alditol acetates)
Type of linkage
Molar (%)
2,3,4,6-Me4 -Glc 2,3,6-Me3 -Glc 2,3-Me2 -Glc
Terminal Glcp 1,4-Linked Glcp 1,4,6-Linked Glcp
14.5 71.2 14.3
and iodine–potassium iodine reagent (data not shown) indicated that this ␣-glucan was a non-starch polysaccharide. 3.1.3. Methylation and NMR analysis As shown in Table 1, methylation analysis revealed the existence of three types of sugar residues in ACPA1, i.e., linear → 4)-␣-Glcp(1 → 4)- (Residue A, 71.2%), terminal-␣-Glcp-(1 → 4)- (Residue B, 14.5%), and branch → 4)-␣-Glcp-(1 → 6)- (Residue C, 14.3%). ACPA1 yields (1–4,6)-linked glucopyranosyl, which is characteristic of branch points with glucose units in (1 → 6) linkage. The 1 H NMR spectrum of ACPA1 (Fig. 1B) was similar to that of a glucan from Pseudallescheria boydii [35]. In the 1 H NMR spectrum, the signals appeared in the anomeric region at ı 5.39, 5.35 and 4.99 ppm, and were deemed Residue A, Residue B, and Residue C, respectively. The remaining upfield signals between ı 3.9 and 3.4 ppm were attributed to the proton resonances of H–2s to H–6s
compared with the previous reports [36]. In the 13 CNMR spectrum (data not shown), the anomeric carbon region for Residue A was assigned at ı 99.8 ppm, ı 99.57 ppm was assigned to Residue B, and ı 98.6 ppm was assigned to Residue C. The DEPT-135 spectrum (Fig. 1C) confirmed the assignment of the resonance at ı 60.5 ppm to C-6, as this methylene carbon signal was inverted with respect to the methine ring carbon signals [37]. A small, broad 13 C resonance at about ı 68 ppm was also inverted and may be assigned to C-6 of 6-linked glucose, although no other signals from the branch point, 4,6-linked residues could be identified. A signal observed at ı 77.1 ppm was assigned to the resonance of C-4 of 4-linked-␣Glcp, which was shifted about 5 ppm downfield compared with the resonance of standard methyl glycoside [38]. The 1 H and 13 C NMR resonances (Table 2) were assigned on the basis of the correlation of the HSQC and 1 H-1 HCOSY NMR experiments (Fig. 3A and B) which were in agreement with previous reports [36,39]. To deduce the sequence of glucopyranosyl residues of ACPA1 and to confirm the assignments made from the HSQC and 1 H1 HCOSY spectra, heteronuclear multiplebond coherence (HMBC) experiments were performed. In the HMBC spectrum (Fig. 3C), cross peaks of the H1–C1 and H2–C1 (linkages of two Residues A or B) and the cross peaks of the H1–C4 (linkages of two Residues A) were detected. Additionally, there were another two cross peaks of the
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Table 2 Summary of 1 H and 13 C NMR chemical shifts of residue A, B and C for ACPA1. (Residue A = linear → 4)-␣-Glcp-(1 → 4)-, residue B = terminal −␣-Glcp-(1 → 4)-, and residue C = branch → 4)-␣-Glcp-(1 → 6)-.). Sugar residues
A B C
Chemical shifts (ppm) H-1/C-1
H-2/C-2
H-3/C-3
H-4/C-4
H-5/C-5
H-6/C-6
5.39/99.80 5.35/99.57 4.99/98.60
3.66/71.67 3.65/71.86 3.65/71.30
3.99/73.34 3.77/73.10 3.98/ND
3.67/77.17 3.44/69.41 3.65/78.05
3.88/71.20 3.79/72.83 3.91/ND
3.86/60.5 3.86/60.5 ND/ND
Fig. 4. Moisture-absorption and −retention abilities of ACPA1 and SA1-SA3. (A) Ra at RH = 81% (B) Ra at RH = 43% (C) Rh under dry condition (D) Rh at RH = 43% (n = 3). Data are representative of three independent experiments.
H1–C4 (linkages of Residue B and Residue A; linkages of Residue B and Residue C); these results indicate that Residue B could directly link to Residue A and Residue C. Moreover, there was no cross peak found between H1 of residue C and C6 of residues A, which suggests that residue C can not directly link to residue A. These results were consistent with the results of methylation analysis. The assignments of signals within other glucosyl residues were confirmed by the cross peaks between ı 5.39 and 3.44 ppm. Taken all together, these data indicated that ACPA1 is a glucan consisting of linear 4linked ␣-d-Glcp residues substituted at position 6 with −␣-d-Glcp branches. 3.2. Characterization of sulfated derivatives of ACPA1 Three sulfated derivatives of ACPA1, named as SA1, SA2 and SA3, were obtained by the SO3 -pyridine procedure. Compared with native ACPA1, two new absorption bands at 1231.74 and 815.41 cm−1 in the FTIR spectra of SA1–SA3 appeared, corresponding to an asymmetrical S O stretching vibration and a symmetrical C O S vibration respectively (Fig. 2) [40]. The DSs of SA1, SA2 and SA3 were found to be 2.23, 2.10 and 1.78 respectively. 3.3. Moisture-preserving activities of ACPA1 and SA1–SA3 The in vitro moisture absorption and retention properties of ACPA1 and SA1–SA3 were examined gravimetrically and compared with those of hyaluronic acid, which are frequently used as
hygroscopic and humectant agents [41]. The moisture-preserving properties of ACPA1, SA1, SA2 and SA3 were explored, in comparison with those of hyaluronic acid (HA). As shown in Fig. 4A and B, for all the samples, the weight of moisture absorbed increased rapidly in the first 24 h, slowed down after that, and then became constant at both 81% and 43% RH. It appeared that the moistureabsorption ability increased significantly after the sulfation of ACPA1. Compared with that of SA3, SA1 and SA2 which had higher DS elicited a stronger moisture-absorption capacity. In the results of the moisture-retention test (Fig. 4C and D), the residual moisture in all samples decreased quickly and became constant after 20 h under the dry condition. The weights of residual moisture in the wet samples increased with time when the relative humidity was 43%. It should be noted that none of three ACPA1 sulfated derivatives were as hygroscopic or as good as water retention as hyaluronic acid, though some were pretty close. Hyaluronic acid, an important natural ingredient in cosmetics, is unique because of its excellent moisture retention ability, but the total amount is limited, and the cost of HA is high [42]. In accordance with the moisture absorption test above, SA1 and SA2 exhibited a better moisture-absorption ability than that of original ACPA1 and SA3. This observation was consistent with the concept that the sulfated group was a main active site for the moisture-absorption and moisture-retention abilities of polysaccharides extracted from algae [43]. Furthermore, it seemed that an optimal sulfation degree was indispensible for maintaining the higher moisture-preserving abilities of sulfated polysaccharides.
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Fig. 5. Immunological activities of ACPA1 and SA1–SA3. (A) Effect of samples on phagocytic activity of macrophages. After treating with ACPA1 and SA1–SA3 (1, 10, 50 g/mL) for 24 h, RAW 264.7 macrophages were used to test the phagocytosis by neutral red uptake assay. (B) Effect of samples on the NO production of macrophages. Cells were pretreated with the samples as described previously. The supernatant nitrite levels were determined using Griess reagent. The significant difference from the control group was evaluated with t-test (***p < 0.0001). Values are mean ± SD (n = 5). Data are representative of three independent experiments.
3.4. Immunological activities of ACPA1 and SA1–SA3
4. Conclusion
Macrophages play a significant role in the host defense mechanism. When activated they inhibit the growth of a wide variety of tumor cells and microorganisms [44]. One of the key ways in which macrophage activation is assayed is to measure whether phagocytic activity is increased. The phagocytic activity can be determined through the evaluation of the uptake of some dyes such as neutral red and malachite green in vitro [31]. The neutral red uptake assay is performed to evaluate the effects of ACPA1 and its sulfated derivatives on phagocytic activity of RAW 264.7 macrophages. As shown in Fig. 5A, the treatment with native ACPA1 had no effects on the uptake of neutral red by macrophages at three concentrations tested. In contrast, SA1, SA2 and SA3 could enhance phagocytic activity of RAW 264.7 macrophages at a concentration of 10 or 50 g/mL. Nitric oxide (NO) is reported to be a vital signaling molecule and a key mediator of signal transduction in the immune system [45]. Nitric oxide is involved in the cytolytic function of macrophages [46]. Stimulation of murine macrophages by LPS and IFN-␥ results in the expression of an inducible NO synthase (iNOS), which catalyzes the production of large amounts of NO from L-arginine and molecular oxygen [47]. The effects of ACPA1 and its sulfated derivatives on the NO production of RAW 264.7 macrophages was determined by Griess assay [30]. As shown in Fig. 5B, all sulfated derivatives at a concentration of 10 or 50 g/mL ostensibly stimulated NO production in macrophages. These results indicate that ACPA1sulfated derivatives may activate immune cells such as murine macrophages. Several studies had demonstrated that sulfated modification could enhance immune function of polysaccharides. For example, the adjuvanticity of the sulfated polysaccharide lentinan was greatly enhanced in comparison with that of the native lentinan [48]. Additionally, the introduction of sulfate groups to a glucan isolated from Grifola frondosa enhanced the peritoneal macrophages phagocytosis [49]. It should be noted that the activity of sulfated polysaccharides strongly depended on the degree of sulfation, with high DS being associated with higher biological activity [50]. However, the DS of sulfated polysaccharides must be within optimum scope since the cytotoxicity should also be concerned for the higher DS. In the present study, we demonstrated that the sulfated ␣-d-glucan with DS (1.78-2.23) exerted stimulatory effects on the phagocytosis activity and NO production of RAW 264.7 macrophages. As an immunopotentiating agent, sulfated derivatives with appropriate DS remained to be optimized in future research.
In this study, we purified a homogenous polysaccharide fraction designated ACPA1 from the roots of Actinidia chinensis. A combination of chemical and spectroscopic analysis indicated that ACPA1 was a non-starch ␣-d-glucan consisting of predominantly 4linked ␣-d-Glcp residues branched at O-6. The mild SO3 -pyridine procedure gave three sulfated derivatives of the glucan. The moisture-preserving and immunological activities of these sulfated derivatives and the native ␣-d-glucan were further evaluated. Results demonstrated that sulfated ACPA1derivatives had moderate moisture-absorption and moisture-retention abilities and could activate immune cells. These findings implied that ␣-d-glucan from the roots of Actinidia chinensis might be useful starting materials to develop moisture-preserving or immune-stimulatory reagents through chemical derivatization such as O-sulfation. Conflict of interest We declare that we have no conflict of interest. Acknowledgements This work was supported by Program for New Century Excellent Talents in University (NCET-13-0480) and the National Natural Science Foundation of China (NSFC) [Grant 31270860]. References [1] M. Yalpani, Polysaccharides: Syntheses, Modifications and Structure/Property Relations, Elsevier, 2013. [2] X. Mingyong, W. Zhijun, X. Jianhua, J. Chin. Inst. Food Sci. Technol. 15 (2015) 1–8. [3] H.S.D. Santa, P.R.T. Romão, V. Sovrani, F.R. Oliveira, A. Peres, M.C. Monteiro, Polysacch. Bioactivity Biotechnol. (2015) 1991–2016. [4] Y. Chen, H. Zhang, Y. Wang, S. Nie, C. Li, M. Xie, Food Chem. 186 (2015) 231–238. [5] L. Zhang, M. Zhang, Q. Zhou, J. Chen, F. Zeng, Biosci. Biotechnol. Biochem. 64 (2000) 2172–2178. [6] J. Zhou, Y. Liu, H. Huang, Am. J. Bot. 98 (2011) e100–102. [7] J. Chang, R. Case, Planta Med. 71 (2005) 955–959. [8] X.F. Zhou, P. Zhang, H.F. Pi, Y.H. Zhang, H.L. Ruan, H. Wang, J.Z. Wu, Chem. Biodivers. 6 (2009) 1202–1207. [9] W.J. Zhu, D.H. Yu, M. Zhao, M.G. Lin, Q. Lu, Q.W. Wang, Y.Y. Guan, G.X. Li, X. Luan, Y.F. Yang, X.M. Qin, C. Fang, G.H. Yang, H.Z. Chen, Anticancer Agents Med. Chem. 13 (2013) 195–198. [10] Z. Ji, X. Liang, Acta Pharm. Sin. 20 (1985) 778. [11] L. Zhang, W. Zhang, Q. Wang, D. Wang, D. Dong, H. Mu, X.-S. Ye, J. Duan, Int. J. Biol. Macromol. 72 (2015) 975–983. [12] H. Mu, A. Zhang, W. Zhang, G. Cui, S. Wang, J. Duan, Int. J. Mol. Sci. 13 (2012) 9194–9206. [13] A. Staub, Methods Carbohydr. Chem. 5 (1965) 5–6.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Preparation and sulfation of an ␣-glucan from Actinidia chinensis roots and their potential activities Hong Niu, Dan Song, Yuelin Sun, Wuxia Zhang, Haibo Mu, Jinyou Duan ∗ Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of science, Northwest A&F University, Yangling 712100, Shaanxi, China
a r t i c l e
i n f o
Article history: Received 25 June 2016 Received in revised form 23 July 2016 Accepted 27 July 2016 Available online 28 July 2016 Keywords: ␣-Glucan Sulfation Moisture-preserving property Immunological activity
a b s t r a c t In this study, one homopolysaccharide (ACPA1) with a molecular weight (Mw) of 5.5 × 103 g/mol was prepared from the roots of Actinidia chinensis. It was characterized as an ␣-d-glucan consisting of predominant 4-linked ␣-d-Glcp residues branched at O-6. Three sulfated derivatives of ACPA1 (SA1, SA2 and SA3) with different degrees of sulfation (DS) were obtained by SO3 -pyridine procedure. Moisture-preserving tests demonstrated that the sulfated derivatives with the highest DS values, SA1 and SA2, exhibited better moisture-preserving abilities than ACPA1 and SA3. All sulfated derivatives exerted stimulatory effects on phagocytosis activity and nitric oxide production by RAW 264.7 macrophages while ACPA1 did not. These findings suggested that the sulfated derivatives of ACPA1 might be used as moisture-preserving or immunopotentiating reagents. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Polysaccharides are a class of natural molecules widely distributed in living organisms. Some of them exhibit various biological activities or have special functions. They contain reactive groups, such as hydroxyl, amino and carboxylic acid groups, and can be used as starting materials for different derivatives which often possess even better physicochemical properties than their raw materials [1]. Chemical modification of polysaccharides provides an opportunity to obtain new pharmacological agents with possible therapeutic uses [1]. In the last decades, much attention has focused on the biological properties of polysaccharides and their chemical derivatives, especially sulfated derivatives [2–4]. The sulfation of polysaccharides could not only enhance their water solubility but also change the chain conformation, thus resulting in the alteration of their biological activities [5]. The fruit tree Actinidia chinensis, belonging to the genus Actinidiaceae, is a medicinal plant. Its origin is supposed to be the northern Yangtse river valley, and now Actinidia chinensis is widely dispersed in the southeast of China [6]. Up to now, a great diversity of low-molecular-weight constituents including phenolic compounds, flavanoids, anthraquinones, and triterpenoids have been isolated from the roots of Actinidia chinensis [7–10]. In contrast, few
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (J. Duan). http://dx.doi.org/10.1016/j.ijbiomac.2016.07.091 0141-8130/© 2016 Elsevier B.V. All rights reserved.
reports have been concerned with the polysaccharide constituents from the roots of this species. In the present study, we isolated a homosaccharide ␣-d-glucan from A. chinensis roots and characterized its structure using chemical and spectroscopic techniques. Its sulfated derivatives were also prepared and their moisture-preserving properties and immunological activities were further investigated. 2. Materials and methods 2.1. Materials and chemicals The roots of Actinidia chinensis collected from Mei County, Shannxi province, China were same as the original material used in [11]. Sodium hyaluronate (∼2 × 106 g/mol), pyridine–sulfur trioxide complex, and neutral red were obtained from Aladdin Chemical Reagent Company, China. DEAE-52 cellulose and Sephacryl S-200 columns were purchased from Whatman Corp. The RWA 264.7 cell line was a gift from Professor Xuebo Liu of Northwest A&F University. All other reagents were of analytical grade. 2.2. Isolation and purification of ACPA1 The isolation and purification procedures were performed as described previously with some modifications [11,12]. Briefly, the dried and defatted Actinidia chinensis (AC) root powders (500 g) were extracted with hot water three times. Then the non-water soluble pellets obtained after centrifugation were solubilized with
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0.5 M NaOH at 4 ◦ C for 12 h without agitation. After the supernatant was neutralized with 2 M HCl, three volumes of cold 95% (v/v) ethanol were added to get the precipitated polysaccharide ACP. ACP was deproteinized by the method of Sevag [13]. Since the crude water or alkli-soluble polysaccharides are always contaminated with colorful polyphenols, ACP was further depigmented by 5% (v/v) H2 O2 [14] and dialyzed (Spectra/Por RC dialysis membrane, MW cutoff 500–1000 g/mol). A half volume of 95% (v/v) ethanol was added, and the supernatant was collected. Then the supernatant was loaded onto a DEAE-52 cellulose column (5 × 50 cm, Cl− form) which was eluted with distilled water. The eluate was collected by a fraction collector, and the most concentrated fractions were pooled according to the total carbohydrate content quantified by the phenol–sulfuric acid method. The collected eluate was named as ACPA and a Sephacryl S-200 column (2.5 × 100 cm) was used to remove other polysaccharides from ACPA eluate. The purified polysaccharide was designated as ACPA1, dialyzed, lyophilized, and stored in a vacuum desiccator. 2.3. Characterization of ACPA1 2.3.1. Analysis of homogeneity and molecular weight Using previously reported methods [15], the molecular weight of ACPA1 was determined by high performance gel permeation chromatography (HPGPC) on a Waters 515 GPC system at 25 ◦ C, using a Waters 2410 RI (refractive index) as the detector. Three columns (Waters Ultrahydrogel 250, 1000 and 2000; 30 cm × 7.8 mm; 6 m particles) in series were calibrated with T-series dextrans (5.2, 10, 48.6, 668, 2000 × 103 g/mol). Sodium acetate (3 mM) was used as eluant and the flow rate was kept at 0.5 mL/min. For each run, a 100 L aliquot of ACPA1 in the eluant was injected. The calibration curve of log (Mw) vs. elution volume (V) is: Log (Mw) = −0.2342 V + 10.45. 2.3.2. Chemical composition analysis and starch iodine test The uronic acid content of ACPA1 was measured according to the m-hydroxydiphenyl-sulphuric acid method, and galacturonic acid was used as the standard [16]. The neutral monosaccharide composition of ACPA1 was analyzed by gas chromatography (equipped with an HP-5 capillary column and flame-ionization detector) after conversion of the hydrolysate into alditol acetates as reported by [17]. The absorption curve of sample-iodine complex was measured spectrophotometrically as described in reference [18]. 2.3.3. Methylation analysis ACPA1 (10 mg) was methylated three times according to the method of Needs and Selvendran [19] using NaOH powder and methyl iodide in dimethyl sulfoxide. The fully methylated product was hydrolyzed, reduced, and acetylated. The partially methylated alditol acetates were analyzed using a gas chromatography–mass spectrometry (GC–MS) system equipped with a trace mass spectrometer and a DB-5 capillary column (0.25 mm × 30 m × 0.25 m, Thermo Finnigan Co., Santa Clara, CA). The temperature program was isothermal at 140 ◦ C for 3 min followed by a 3 ◦ C/min gradient up to 250 ◦ C [20]. The molar ratios were estimated from the peak area on GC and response factor [21,22]. 2.3.4. FT-IR and NMR analysis ACPA1 (2 mg) was ground with KBr powder and pressed into pellets for FT-IR measurement (Bruker tensor 27, Bruker, Germany) in the range of 4000 ∼ 500 cm−1 [23,24]. Dried ACPA1 was exchanged with deuterium by lyophilizing with D2 O for three times. The 1 H (500 MHz) and 13 C (125 MHz), DEPT, HSQC, HMBC and 1 H-1 H COSY NMR spectra of ACPA1 in D2 O were recorded at room temperature using a Bruker AM 500 spectrometer with a dual probe in the FT mode [25,26]. Chemical shifts are referred to the residual signal of
HOD at ı 4.74 ppm for 1 H NMR spectrum and the external standard with Me4Si for 13 C NMR spectrum. 2.4. Preparation and characterization of sulfated derivatives of ACPA1 Polysaccharides with various degrees of sulfation (DS) were prepared under mild conditions with SO3 -pyridine complex in aprotic solvents [27]. Briefly, dried polysaccharide (80 mg) was suspended in 12.8 mL of anhydrous N, N-dimethyl formamide (DMF) and then stirred for 14 h at room temperature under N2 . A required excess (15 mol/equiv of available hydroxyl group in amylose) of SO3 pyridine complex was added to the sample suspensions and then stirred for 5, 10, or 12 h (6 h, twice) at 40 ◦ C under N2 . Water was added to interrupt the reaction and the raw products were precipitated by 3 vols of cold ethanol with saturated anhydrous sodium acetate. After centrifugation, the sulfated polysaccharides were dialyzed to completely remove excess salts and lyophilized. The FTIR spectra of the samples were recorded as described previously. The DS of samples were determined by the barium chloride turbidimetric method according to the literature [28]. 2.5. Moisture-absorption and -retention tests The moisture-absorption (Ra) and −retention (Rh) abilities of the samples were tested as described in [29], and hyaluronic acid was used as the control. For the moisture absorption test, each sample (30 mg) was freeze-dried for 48 h and was kept in a silica gel drier overnight. Then, samples were put into desiccators saturated with (NH4 )2 SO4 (81%, relative humidity, RH) or Na2 CO3 (43% RH) for different periods at room temperature. The moisture absorption ability (Ra) was evaluated by the percentage of increase in weight of dry samples, based on Equation (1). Ra (%) = 100 × (Wn − Wo) /Wo
(1)
(Wo and Wn were the weights of samples before and after putting it into the desiccators, respectively. The Ra values of each sample in triplicate were averaged.) For the moisture retention test, each freeze-dried sample (30 mg) was put into a silica gel drier overnight as above. 10% (w/w) water was added to each sample and then the samples were put into desiccators saturated with (NH4 )2 SO4 or silica gel for different periods at room temperature. The moisture retention ability (Rh) was evaluated by the percentage of residual water of wet samples, according to Equation (2).
Rh (%) = 100 × Hn/Ho
(2)
(Hn and Ho were the weights of water in the samples before and after putting it into the desiccators, respectively. The Rh values of each sample in triplicate were averaged.) 2.6. Assay of immunological activities RAW 264.7 cells (1 × 106 /well) were dispensed into 96-well plates and incubated with PBS or different concentrations of polysaccharides in PBS at 37 ◦ C for 24 h. Nitric oxide production was determined, according to nitrite contents in culture supernatant which was analyzed by Griess assay [30]. The phagocytic ability of macrophages was measured by neutral red uptake assay [31]. Cells (1 × 105 /well) were incubated with different concentrations of samples for 24 h as described previously. Culture media was removed and the cells were incubated with 100 uL of 0.075% (w/v) neutral red solution for 2 h. The supernatant was discarded and the cells were washed with PBS three times. Cell lysis buffer [1% (v/v) glacial acetic acid: ethanol = 1:1, 200 L/well] was
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Fig. 1. HPGPC profile and 1D NMR spectra of ACPA1. (A) HPGPC chromatogram on ultrahydrogel columns of ACPA1 (B) 1 H NMR spectrum (C) DEPT spectrum (A: linear → 4)␣-Glcp-(1 → 4)-/Residue A, B: terminal-␣-Glcp-(1 → 4)-/Residue B, C: branch → 4)-␣-Glcp-(1 → 6)-/Residue C).
added to lyse cells. After incubation overnight at 4 ◦ C, the Abs at 540 nm was measured by an ELISA reader.
2.7. Statistical analysis Data were expressed as means ± standard deviation (SD). The scientific statistic software GraphPad Prism 5.01 was used to evaluate the significance of differences between groups. P < 0.05 was regarded as significant.
3. Results and discussion 3.1. Purification and characterization of ˛-glucan from Actinidia chinensis roots 3.1.1. Basic properties of ACPA1 The alkali-soluble crude polysaccharide, designated ACP was extracted from the dry roots of Actinidia chinensis with a yield of 2% (w/w) of root mass. After being deproteinated and depigmented, ACP was repeatedly subjected to DEAE-cellulose chromatography and Sephacryl S-200 gel-permeation chromatography, which resulted in the isolation of a polysaccharide fraction ACPA1 with a yield of 9% (w/w) of ACP mass. ACPA1 was eluted as a symmetrical narrow peak on highperformance gel-permeation chromatography (HPGPC) (Fig. 1A), and its weight-average molecular weight was estimated to be 5.5 × 103 g/mol, in reference to standard dextrans. The polydispersity index (Mw/Mn) of ACPA1 equaled 1.31, which indicated it was a relatively monodisperse polymer. The sugar composition of ACPA1 was free of uronic acid as determined by the m-hydroxydiphenylsulphuric acid method, and only glucose was detected by gas
Fig. 2. Infrared spectra of ACPA1 and SA1-SA3.
chromatography (data not shown), indicating that ACPA1 was a glucan.
3.1.2. FT-IR analysis The FTIR spectrum of the glucan is shown in Fig. 2. The bands at 3382.91 cm−1 and 2924.87 cm−1 were due to O H and C H stretching vibration, respectively. The absorption at 918.88 cm−1 was typical for d-glucan in the pyranose form [32]. Additionally, the ␣-configuration was confirmed by the absorption at 846.61 cm−1 ; there was no absorption at 890 cm−1 indicative of the -configuration [33,34]. The negative reaction between ACPA1
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Fig. 3. 2D NMR spectra of ACPA1. (A) HSQC spectrum (B) 1 H-1 H COSY spectrum (C) HMBC spectrum (A: linear → 4)-␣-Glcp-(1 → 4)-/Residue A, B: terminal −␣-Glcp-(1 → 4)/Residue B, C: branch → 4)-␣-Glcp-(1 → 6)-/Residue C). Table 1 GC–MS test result for the methylated sugar moieties of ACPA1. Methylated sugars (as alditol acetates)
Type of linkage
Molar (%)
2,3,4,6-Me4 -Glc 2,3,6-Me3 -Glc 2,3-Me2 -Glc
Terminal Glcp 1,4-Linked Glcp 1,4,6-Linked Glcp
14.5 71.2 14.3
and iodine–potassium iodine reagent (data not shown) indicated that this ␣-glucan was a non-starch polysaccharide. 3.1.3. Methylation and NMR analysis As shown in Table 1, methylation analysis revealed the existence of three types of sugar residues in ACPA1, i.e., linear → 4)-␣-Glcp(1 → 4)- (Residue A, 71.2%), terminal-␣-Glcp-(1 → 4)- (Residue B, 14.5%), and branch → 4)-␣-Glcp-(1 → 6)- (Residue C, 14.3%). ACPA1 yields (1–4,6)-linked glucopyranosyl, which is characteristic of branch points with glucose units in (1 → 6) linkage. The 1 H NMR spectrum of ACPA1 (Fig. 1B) was similar to that of a glucan from Pseudallescheria boydii [35]. In the 1 H NMR spectrum, the signals appeared in the anomeric region at ı 5.39, 5.35 and 4.99 ppm, and were deemed Residue A, Residue B, and Residue C, respectively. The remaining upfield signals between ı 3.9 and 3.4 ppm were attributed to the proton resonances of H–2s to H–6s
compared with the previous reports [36]. In the 13 CNMR spectrum (data not shown), the anomeric carbon region for Residue A was assigned at ı 99.8 ppm, ı 99.57 ppm was assigned to Residue B, and ı 98.6 ppm was assigned to Residue C. The DEPT-135 spectrum (Fig. 1C) confirmed the assignment of the resonance at ı 60.5 ppm to C-6, as this methylene carbon signal was inverted with respect to the methine ring carbon signals [37]. A small, broad 13 C resonance at about ı 68 ppm was also inverted and may be assigned to C-6 of 6-linked glucose, although no other signals from the branch point, 4,6-linked residues could be identified. A signal observed at ı 77.1 ppm was assigned to the resonance of C-4 of 4-linked-␣Glcp, which was shifted about 5 ppm downfield compared with the resonance of standard methyl glycoside [38]. The 1 H and 13 C NMR resonances (Table 2) were assigned on the basis of the correlation of the HSQC and 1 H-1 HCOSY NMR experiments (Fig. 3A and B) which were in agreement with previous reports [36,39]. To deduce the sequence of glucopyranosyl residues of ACPA1 and to confirm the assignments made from the HSQC and 1 H1 HCOSY spectra, heteronuclear multiplebond coherence (HMBC) experiments were performed. In the HMBC spectrum (Fig. 3C), cross peaks of the H1–C1 and H2–C1 (linkages of two Residues A or B) and the cross peaks of the H1–C4 (linkages of two Residues A) were detected. Additionally, there were another two cross peaks of the
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Table 2 Summary of 1 H and 13 C NMR chemical shifts of residue A, B and C for ACPA1. (Residue A = linear → 4)-␣-Glcp-(1 → 4)-, residue B = terminal −␣-Glcp-(1 → 4)-, and residue C = branch → 4)-␣-Glcp-(1 → 6)-.). Sugar residues
A B C
Chemical shifts (ppm) H-1/C-1
H-2/C-2
H-3/C-3
H-4/C-4
H-5/C-5
H-6/C-6
5.39/99.80 5.35/99.57 4.99/98.60
3.66/71.67 3.65/71.86 3.65/71.30
3.99/73.34 3.77/73.10 3.98/ND
3.67/77.17 3.44/69.41 3.65/78.05
3.88/71.20 3.79/72.83 3.91/ND
3.86/60.5 3.86/60.5 ND/ND
Fig. 4. Moisture-absorption and −retention abilities of ACPA1 and SA1-SA3. (A) Ra at RH = 81% (B) Ra at RH = 43% (C) Rh under dry condition (D) Rh at RH = 43% (n = 3). Data are representative of three independent experiments.
H1–C4 (linkages of Residue B and Residue A; linkages of Residue B and Residue C); these results indicate that Residue B could directly link to Residue A and Residue C. Moreover, there was no cross peak found between H1 of residue C and C6 of residues A, which suggests that residue C can not directly link to residue A. These results were consistent with the results of methylation analysis. The assignments of signals within other glucosyl residues were confirmed by the cross peaks between ı 5.39 and 3.44 ppm. Taken all together, these data indicated that ACPA1 is a glucan consisting of linear 4linked ␣-d-Glcp residues substituted at position 6 with −␣-d-Glcp branches. 3.2. Characterization of sulfated derivatives of ACPA1 Three sulfated derivatives of ACPA1, named as SA1, SA2 and SA3, were obtained by the SO3 -pyridine procedure. Compared with native ACPA1, two new absorption bands at 1231.74 and 815.41 cm−1 in the FTIR spectra of SA1–SA3 appeared, corresponding to an asymmetrical S O stretching vibration and a symmetrical C O S vibration respectively (Fig. 2) [40]. The DSs of SA1, SA2 and SA3 were found to be 2.23, 2.10 and 1.78 respectively. 3.3. Moisture-preserving activities of ACPA1 and SA1–SA3 The in vitro moisture absorption and retention properties of ACPA1 and SA1–SA3 were examined gravimetrically and compared with those of hyaluronic acid, which are frequently used as
hygroscopic and humectant agents [41]. The moisture-preserving properties of ACPA1, SA1, SA2 and SA3 were explored, in comparison with those of hyaluronic acid (HA). As shown in Fig. 4A and B, for all the samples, the weight of moisture absorbed increased rapidly in the first 24 h, slowed down after that, and then became constant at both 81% and 43% RH. It appeared that the moistureabsorption ability increased significantly after the sulfation of ACPA1. Compared with that of SA3, SA1 and SA2 which had higher DS elicited a stronger moisture-absorption capacity. In the results of the moisture-retention test (Fig. 4C and D), the residual moisture in all samples decreased quickly and became constant after 20 h under the dry condition. The weights of residual moisture in the wet samples increased with time when the relative humidity was 43%. It should be noted that none of three ACPA1 sulfated derivatives were as hygroscopic or as good as water retention as hyaluronic acid, though some were pretty close. Hyaluronic acid, an important natural ingredient in cosmetics, is unique because of its excellent moisture retention ability, but the total amount is limited, and the cost of HA is high [42]. In accordance with the moisture absorption test above, SA1 and SA2 exhibited a better moisture-absorption ability than that of original ACPA1 and SA3. This observation was consistent with the concept that the sulfated group was a main active site for the moisture-absorption and moisture-retention abilities of polysaccharides extracted from algae [43]. Furthermore, it seemed that an optimal sulfation degree was indispensible for maintaining the higher moisture-preserving abilities of sulfated polysaccharides.
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Fig. 5. Immunological activities of ACPA1 and SA1–SA3. (A) Effect of samples on phagocytic activity of macrophages. After treating with ACPA1 and SA1–SA3 (1, 10, 50 g/mL) for 24 h, RAW 264.7 macrophages were used to test the phagocytosis by neutral red uptake assay. (B) Effect of samples on the NO production of macrophages. Cells were pretreated with the samples as described previously. The supernatant nitrite levels were determined using Griess reagent. The significant difference from the control group was evaluated with t-test (***p < 0.0001). Values are mean ± SD (n = 5). Data are representative of three independent experiments.
3.4. Immunological activities of ACPA1 and SA1–SA3
4. Conclusion
Macrophages play a significant role in the host defense mechanism. When activated they inhibit the growth of a wide variety of tumor cells and microorganisms [44]. One of the key ways in which macrophage activation is assayed is to measure whether phagocytic activity is increased. The phagocytic activity can be determined through the evaluation of the uptake of some dyes such as neutral red and malachite green in vitro [31]. The neutral red uptake assay is performed to evaluate the effects of ACPA1 and its sulfated derivatives on phagocytic activity of RAW 264.7 macrophages. As shown in Fig. 5A, the treatment with native ACPA1 had no effects on the uptake of neutral red by macrophages at three concentrations tested. In contrast, SA1, SA2 and SA3 could enhance phagocytic activity of RAW 264.7 macrophages at a concentration of 10 or 50 g/mL. Nitric oxide (NO) is reported to be a vital signaling molecule and a key mediator of signal transduction in the immune system [45]. Nitric oxide is involved in the cytolytic function of macrophages [46]. Stimulation of murine macrophages by LPS and IFN-␥ results in the expression of an inducible NO synthase (iNOS), which catalyzes the production of large amounts of NO from L-arginine and molecular oxygen [47]. The effects of ACPA1 and its sulfated derivatives on the NO production of RAW 264.7 macrophages was determined by Griess assay [30]. As shown in Fig. 5B, all sulfated derivatives at a concentration of 10 or 50 g/mL ostensibly stimulated NO production in macrophages. These results indicate that ACPA1sulfated derivatives may activate immune cells such as murine macrophages. Several studies had demonstrated that sulfated modification could enhance immune function of polysaccharides. For example, the adjuvanticity of the sulfated polysaccharide lentinan was greatly enhanced in comparison with that of the native lentinan [48]. Additionally, the introduction of sulfate groups to a glucan isolated from Grifola frondosa enhanced the peritoneal macrophages phagocytosis [49]. It should be noted that the activity of sulfated polysaccharides strongly depended on the degree of sulfation, with high DS being associated with higher biological activity [50]. However, the DS of sulfated polysaccharides must be within optimum scope since the cytotoxicity should also be concerned for the higher DS. In the present study, we demonstrated that the sulfated ␣-d-glucan with DS (1.78-2.23) exerted stimulatory effects on the phagocytosis activity and NO production of RAW 264.7 macrophages. As an immunopotentiating agent, sulfated derivatives with appropriate DS remained to be optimized in future research.
In this study, we purified a homogenous polysaccharide fraction designated ACPA1 from the roots of Actinidia chinensis. A combination of chemical and spectroscopic analysis indicated that ACPA1 was a non-starch ␣-d-glucan consisting of predominantly 4linked ␣-d-Glcp residues branched at O-6. The mild SO3 -pyridine procedure gave three sulfated derivatives of the glucan. The moisture-preserving and immunological activities of these sulfated derivatives and the native ␣-d-glucan were further evaluated. Results demonstrated that sulfated ACPA1derivatives had moderate moisture-absorption and moisture-retention abilities and could activate immune cells. These findings implied that ␣-d-glucan from the roots of Actinidia chinensis might be useful starting materials to develop moisture-preserving or immune-stimulatory reagents through chemical derivatization such as O-sulfation. Conflict of interest We declare that we have no conflict of interest. Acknowledgements This work was supported by Program for New Century Excellent Talents in University (NCET-13-0480) and the National Natural Science Foundation of China (NSFC) [Grant 31270860]. References [1] M. Yalpani, Polysaccharides: Syntheses, Modifications and Structure/Property Relations, Elsevier, 2013. [2] X. Mingyong, W. Zhijun, X. Jianhua, J. Chin. Inst. Food Sci. Technol. 15 (2015) 1–8. [3] H.S.D. Santa, P.R.T. Romão, V. Sovrani, F.R. Oliveira, A. Peres, M.C. Monteiro, Polysacch. Bioactivity Biotechnol. (2015) 1991–2016. [4] Y. Chen, H. Zhang, Y. Wang, S. Nie, C. Li, M. Xie, Food Chem. 186 (2015) 231–238. [5] L. Zhang, M. Zhang, Q. Zhou, J. Chen, F. Zeng, Biosci. Biotechnol. Biochem. 64 (2000) 2172–2178. [6] J. Zhou, Y. Liu, H. Huang, Am. J. Bot. 98 (2011) e100–102. [7] J. Chang, R. Case, Planta Med. 71 (2005) 955–959. [8] X.F. Zhou, P. Zhang, H.F. Pi, Y.H. Zhang, H.L. Ruan, H. Wang, J.Z. Wu, Chem. Biodivers. 6 (2009) 1202–1207. [9] W.J. Zhu, D.H. Yu, M. Zhao, M.G. Lin, Q. Lu, Q.W. Wang, Y.Y. Guan, G.X. Li, X. Luan, Y.F. Yang, X.M. Qin, C. Fang, G.H. Yang, H.Z. Chen, Anticancer Agents Med. Chem. 13 (2013) 195–198. [10] Z. Ji, X. Liang, Acta Pharm. Sin. 20 (1985) 778. [11] L. Zhang, W. Zhang, Q. Wang, D. Wang, D. Dong, H. Mu, X.-S. Ye, J. Duan, Int. J. Biol. Macromol. 72 (2015) 975–983. [12] H. Mu, A. Zhang, W. Zhang, G. Cui, S. Wang, J. Duan, Int. J. Mol. Sci. 13 (2012) 9194–9206. [13] A. Staub, Methods Carbohydr. Chem. 5 (1965) 5–6.
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