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Chemical modification of an acidic polysaccharide (TAPA1) from Tremella aurantialba and potential biological activities
Food Chemistry 143 (2014) 336–340
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Chemical modification of an acidic polysaccharide (TAPA1) from Tremella aurantialba and potential biological activities XiuJu Du a, JingSong Zhang b,⇑, ZhiWei Lv a, LiBin Ye c, Yan Yang b, QingJiu Tang b a
College of Life Science, Liaocheng University, Liaocheng, Shandong 252059, PR China Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201106, PR China c College of Food Science and Biotechnology, Zhejiang Gongshang University, Food Safety Key Lab of Zhejiang Province, Hangzhou 310035, PR China b
a r t i c l e
i n f o
Article history: Received 25 March 2013 Received in revised form 21 June 2013 Accepted 30 July 2013 Available online 7 August 2013 Keywords: Tremella aurantialba polysaccharide Acetylation Deacetylation Immunostimulating activity Nitric oxide
a b s t r a c t TAPA1 was previously isolated from Tremella aurantialba fruiting bodies. In this paper, an acetylated derivative (TAPA1-ac) and a deactylated derivative (TAPA1-deac) of TAPA1 were prepared, and their characterization and immunostimulating activities were reported. Acetylation and deacetylation were found to occur actually by FT-IR and NMR spectra, together with calculational results. The degree of substitution (DS) of acetyl groups in TAPA1-ac was 0.23 and the content was 5.82%, which was higher than those of TAPA1 (0.03% and 0.70%, respectively) and TAPA1-deac (all were zero). Compared with TAPA1, TAPA1-ac showed significant immunostimulation effects on mouse spleen lymphocytes (MSLs) proliferation and nitric oxide (NO) production by macrophages RAW264.7, whereas TAPA1-deac showed markedly lower effects. These findings seemed to suggest that immunostimulating activities, including MSLs stimulation activity and NO production potency, might relate to the DS and content of acetyl groups, indicating that acetylation of TAPA1 was an effective way of enhancing immuno-stimulating activities. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Chemical modification of polysaccharides usually provides an opportunity to obtain new pharmacological agents with possible therapeutic uses, and it has been an important means to study the structure–activity relationships of the polysaccharide (Zhao & Wang, 2000). Acetyl group in polysaccharides were reported to potentially play a very important role in the enhancement of biological activities (Eflerbroek et al., 2004; Ma, Chen, Zhang, Zhang, & Fu, 2012). Moreover, it could change the spatial structure of the sugar chain, resulting in the exposure of a hydroxyl group, and thereby might increase its water solubility and enhance its biological activities (LilloL & Matsuhiro, 1997). In our previous work, an acidic polysaccharide fraction with a mean molecular weight of 1.35 106 Da and a carbohydrate content of 98.7%, termed as TAPA1, was isolated and purified from the fruiting bodies of Tremella aurantialba for the first time. The structure of TAPA1 had since been investigated in our laboratory using 2D-NMR spectra, monosaccharide compositional analysis and methylation data, and the results were published (shown in Fig. 1) (Du, 2009; Du et al., 2009). In TAPA1, the DS of acetyl groups was 0.03 and the acetyl content was 0.7%. The immunostimulating activity of TAPA1, determined by using the MSLs proliferation as-
⇑ Corresponding author. Tel./fax: +86 21 62201337. E-mail address: [email protected] (J. Zhang). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.07.137
say, was clear. However, compared with the clinical application of immunostimulating drugs, theeffect of the polysaccharide TAPA1 should be further enhanced. Currently there is little knowledge of acetylation and deacetylation in polysaccharides of T. aurantialba. In order to obtain new more active compounds, and further investigate structure–activity relationships of TAPA1, an acetylated derivative TAPA1-ac and a deacetylated fraction TAPA1-deac were prepared by chemical modification. Furthermore, in vitro immunostimulating activity was determined using the mouse spleen lymphocytes (MSLs) proliferation assay and nitric oxide (NO) production assay. The immunostimulating mechanism was also preliminarily investigated in the paper.
2. Materials and methods 2.1. Materials Fruiting bodies of T. aurantialba were presented by Kunming Edible Fungi Institute of General National Supply and Marketing Cooperative in People’s Republic of China. TAPA1 was purified and proved to be homogeneous in our previous work (Du et al., 2009), and stored in dryer with silica-gel desiccant at room temperature. LPS were from Sigma–Aldrich (USA). Phytohemagglutinim (PHA), Penicillin and Streptomycin were purchased from Amersco (USA). RPMI-1640 medium, DMEM medium and fetal calf serum (FCS) were bought from Gibco (USA). Alamar Blue™ reagent
X. Du et al. / Food Chemistry 143 (2014) 336–340
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was bought from Biosoure International (USA). The standard monosaccharide and dextrans were from Sigma–Aldrich (USA). All other reagents were AR grade from Chinese sources. 2.2. General methods FT-IR spectroscopy of samples mixed with dry KBr was performed in the 4000–400 cm1 region (Nexus Euro FTIR instrument). 1H NMR and 13C NMR spectra were recorded in D2O at 500 MHz (1H NMR) or 125 MHz (13C NMR) using a Bruker Avance 500 spectrometer (Germany). 13C chemical shifts were acquired in relation to DSS (d 0.00 ppm) calibrated externally, and HDO (d 4.32) was used as the internal reference signal for 1H at 70 °C (343 K). Fig. 1. The structure of polysaccharide TAPA1 from fruiting bodies of T. aurantialba.
2.3. Determination of the molecular weight The molecular weight of prepared samples was estimated according to the method of Du et al. (2009). Briefly, the sample was applied to a gel-permeation chromatography on a high-resolution Sephacryl S-500 column (XK16 100 cm) calibrated using dextrans T-150, 270, 410, 670 and 2000. Sample (150 ll) was applied to the column and the flow rate of the mobile phase (0.2 M NaNO3) was 0.5 ml/min. The column and refractive index detector (RID) temperature were maintained at 30 °C. 2.4. Preparation of derivatives of the homogeneous polysaccharide TAPA1 2.4.1. Preparation of acetylated derivative TAPA1-ac TAPA1-ac was prepared according to the method described by Ueno, Okamoto, Yamauchi, and Kato (1982), with dimethyl sulfoxide (DMSO) as the solvent in pyridine acetic anhydride system. Briefly, 50 mg TAPA1 was put into DMSO (4 ml), acetylated by the mixture of acetic anhydride (0.5 ml) and pyridine (0.6 ml) at 30 °C for 4 h, discontinued with 5 ml distilled water, dialyzed, and then lyophilized. Accordingly, an acetylated sample TAPA1ac (28 mg) was obtained. The content and the DS of the group OAc were measured according to the method reported previously by Ye (2008). 2.4.2. Preparation of deacetylated derivative TAPA1-deac TAPA1-deac was prepared according to the method of Vinogradov, Petersen, Duus, and Wasser (2004). Briefly, 50 mg TAPA1 was dissolved in a solution of 5% NH3 (20 ml) in water, kept at 50 °C for 16 h, dialyzed for 48 h, centrifuged (1776g, 5 min), and then lyophilized to yield deacetylated sample TAPA1-deac (37.5 mg). The content and the DS of the group O-Ac were measured according to Ye (2008). 2.5. In vitro spleen lymphocyte proliferation assay The MSLs proliferation assay was carried out according to the method described previously by Du et al. (2010), lymphocytes from mouse spleens were prepared. Briefly, C57 BL/6 male mice (ca. 18 g) were killed by cervical dislocation. The spleens were removed and cut into several pieces, washed thrice with PBS buffer, and then pressed through a stainless steel mesh (100 meshes) so as to obtain a single spleen cell suspension. After centrifugation (400g, 6 min), red cells in the spleen cell suspension were lysed with a Tris–HCl–NH4Cl solution (pH 7.2) (Zhang, Tang, Zimmerman-Kordmann, Reutter, & Fan, 2002). The cell suspension was further diluted with a fivefold excess of medium. After mixing and centrifugation, the cell pellets were finally re-suspended in RPMI1640 medium and adjusted to a concentration of 2 106 cells/ ml. Secondly, MSLs proliferation rate was determined by the Ala-
mar Blue Assay. Briefly, to each well of a 96-well microplate 180 ll of the cell suspension and 20 ll of different test agents were added. PBS and PHA (6 lg/ml) served as the negative and positive controls, respectively. After incubation at 37 °C in 5% CO2 atmosphere for 72 h, 20 ll Alamar Blue reagent (Biosource, Nivelles, Belgium) was added to each well and the incubation continued for another 6 h. Absorption values at 570 nm (A570) and 600 nm (A600) were measured using a micro enzyme-linked immunosorbent assay (ELISA) autoreader. The proliferation rate [absorption values at 570 nm (A570) and 600 nm (A600)] were measured using a micro enzyme-linked immunosorbent assay (ELISA) autoreader. The proliferation rate (r) was calculated according to the following formula: r(%) = [117216 A570(sample) 80856 A600(sample)]/ [117216 A570(control) 80856 A600 (control)] 100%.
2.6. NO production by macrophages RAW264.7 in vitro 2.6.1. Cell line RAW264.7, a mouse macrophage cell line, was bought from the ATCC (American Type Culture Collection) and cultured in RPMI 1640 medium containing 10% FCS (fetal calf serum), 100 U/ml streptomycin and 100 lg/ml penicillin at 37°Cin a 5% CO2 atmosphere.
2.6.2. Nitrite quantitation Nitrite accumulation was used as an indicator of NO production in the medium as previously described (Ji et al., 2007). Briefly, cells (1 106 cells/ml) were added to 96-well plates and stimulated with samples (5, 25 and 50 lg/ml) for 24 h. In this work, LPS (Lipopolysaccharide, 1 lg/ml) was served as a positive reference and PBS was as a negative control. Supernatants (100 ll) were then collected and mixed with 0.5 vol (50 ll) Griess reagent [1% (w/v) sulfanilamide, 0.1% (w/v) naphthylethylenediamine dihydrochloride, 2.5% (v/v) phosphoric acid]and incubated at room temperature for 10 min. Nitrite production was determined by comparing the absorbance at 543 nm against a standard curve generated using NaNO2.
2.7. Statistical analysis The data obtained were expressed as mean ± SD of three determinations and analyzed statistically by ANOVA method. Data were analyzed using STST2 statistical software. Significance of any differences between groups was evaluated using the Student’s t-test.
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3. Results and discussion 3.1. Characterization of the deacetylated derivative TAPA1-deac and acetylated derivative TAPA1-ac Recent research on polysaccharides from Inonotus obliquus, indicates that acetylationed polysaccharide (Ac-IOPS) resulted in higher antioxidant abilities on ferric-reducing power and lipid peroxidation inhibition activity compared with the native polysaccharide IOPS (Ma et al., 2012). Eflerbroek et al. (2004) assessed the contribution of acetylation and deacetylation of the polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans, to the interference with neutrophil migration. Their findings showed that the acetyl group in GXM was of necessary for its ability to interfere with neutrophil chemokinesis (Eflerbroek et al., 2004). In this work, TAPA1 was chemically acetylated and deacetylated so as to further enhance the immunomodulatory activities of TAPA1 and investigate the correlation between the acetyl content and its activity. Accordingly, a deacetylated derivative (TAPA1-deac) and an acetylated derivative (TAPA1-ac) were obtained. The average molecular weights of two derivatives were all determined to be 1.35 106 Da, which was almost identical to that of their native fraction TAPA1, indicating that no degradation had occurred in the deacetylated and acetylated reactions. The yields of TAPA1deac and TAPA1-ac were 75.0% and 56.0%, respectively. TAPA1-ac had the group O-Ac DS of 0.23 and an acetyl content of 5.82%, however, the DS and acetyl content in TAPA1-deac were determined to be zero, indicating the acetyl group in TAPA1-deac had been removed completely from its native polysaccharide TAPA1. Both derivatives TAPA1-deac and TAPA1-ac were all white, flocculent and soluble in water, which was almost similar to TAPA1. However, all fractions, TAPA1 and its derivatives TAPA1-deac and TAPA1-ac, were all insoluble in DMSO, which was due to containing some carboxyl group (–COOH) in them, including glucuronic acid and/or OAc group (Li & Xu, 2004). 3.2. Spectral characterization of TAPA1-deac and TAPA1-ac As shown in Fig. 2, two characteristic absorption bands appeared in the FT-IR spectra of TAPA1, one at near 1733.7 cm1 describing a C@O stretching vibration associated with an ester group, another at near 1249.7 cm1, representing a C–O stretching vibration of ester group (Zhou, Guo, Zheng, & Li, 2006). By comparison with TAPA1, two characteristic absorption bands (near 1733.7 and 1249.7 cm1) remarkably enhanced in the FT-IR
Fig. 3. 13C NMR (125 MHz) spectra of TAPA1 and its derivatives. 13C NMR (125 MHz) spectra of TAPA1 and its derivatives. (A) TAPA1-deac; (B) TAPA1 and (C) TAPA1-ac.
spectra of the acetylated derivative TAPA1-ac, indicating TAPA1ac was successfully acetylated (Zhou et al., 2006). However, the two bands mentioned above were disappeared or decreased in the FT-IR spectra of TAPA1-deac, indicating TAPA1-deac was actually deacetylated. The fact that acetylation and deacetylation of TAPA1 occurred successfully could also be further proven by 1H NMR (500 MHz, Fig. not shown) and 13C NMR (125 MHz, Fig. 3) spectra data. Signals of TAPA1 and TAPA1-ac at d 2.02 corresponded to the 1H signal for the CH3 moiety of an acetyl group (Serrato et al., 2008), and signals of TAPA1 and TAPA1-ac at d 23.79 upfield revealed a CH3 moiety of an acetyl group (Serrato et al., 2008), which indicated that TAPA1 and TAPA1-ac contained acetyl groups. Furthermore, two signals at d 2.02 and d 23.79 in TAPA1-ac were synchronously higher than that in TAPA1, indicating acetyled reaction of TAPA1 occurred successfully. By comparison with TAPA1, signals at d 2.02 and d 23.79 in TAPA1-deac all synchronously disappeared, showing that no acetyl groups was in TAPA1-deac, that is to say, the deacetylated reaction of TAPA1 also occurred successfully.
Fig. 2. FI-IR spectra of TAPA1 and its derivatives. FI-IR spectra of TAPA1 and its derivatives. (A) TAPA1-deac; (B) TAPA1 and (C) TAPA1-ac.
X. Du et al. / Food Chemistry 143 (2014) 336–340 Table 1 Effect of TAPA1 and its derivatives on mouse spleen lymphocyte proliferation in vitro.a Samples
TAPA1-deac TAPA1 TAPA1-ac
Proliferation rate (%) 50 (lg/ml)
200 (lg/ml)
500 (lg/ml)
102.8 ± 8.5ib 298.2 ± 13.9 f 312.5 ± 14.1e
243.0 ± 11 h 407.3 ± 6.3d 418.7 ± 6.5c
271.4 ± 2.5 g 458.7 ± 11.5b 496.5 ± 11.1a
a In this work, PHA (6 lg/ml) was used as a positive reference (467.7 ± 9.7%) and control (plus PBS) was used as negative reference (100.0 ± 4.7%). b Values within a row followed by different letters (a–i) are significantly different at p < 0.05 byt-test.
3.3. Effect of TAPA1 and its derivatives on mouse spleen lymphocyte proliferation in vitro Immunoregulation was one of the basic functions associated with polysaccharides (Su, Dai, & Yang, 2006). The mechanism of immunoregulation mainly was to stimulate macrophage, lymphocyte, T cell, B cell, NK-cell, LAK-cell and red blood cell from various levels of immunological organ, immunocyte and immunological molecules in order to increase the number of these cells and potentiate their bioactivity (He & Zhang, 2004). Therefore, in vitro cell proliferation experiments represent a suitable method for the rapid screening of polysaccharide with immunobiological activity. So the immunoregulation activities of TAPA1 and its derivatives are evaluated by determining the effect on mouse spleen lymphocyte proliferation assay. The results were shown in Table 1. It was found that TAPA1 stimulated the proliferation of MSLs in vitro in a dose-dependent manner. At tested concentrations, TAPA1-ac showed higher MSLs (p < 0.05) stimulation potency than TAPA1 did, dose-dependently, whereas TAPA1-deac showed markedly lower MSLs (p < 0.05) stimulation potency than TAPA1 did. At 500 lg/ml, the proliferation rate of the TAPA1-deac, TAPA1 and TAPA1-ac were (271.4 ± 2.5)%, (458.7 ± 11.5)% and (496.5 ± 11.1)%, respectively, which showed a significant difference (p < 0.05) among them. These findings seemed to suggest that polysaccharides with higher content and DS of the O-Ac group, showed stronger MSLs potency in immuno-enhancing activity. 3.4. Effect of TAPA1 and its derivatives on nitric oxide production by macrophages RAW264.7 in vitro Macrophage was one of the important components of the immune system, and it played an important role in immunoregulation (Albrecht, 1979). NO, produced by macrophages, was a kind of important inorganic small molecule discovered in recent years, and played an important role in inflammation, injury, defense, and so on. It had been reported that the polysaccharides could stimulate NO production by macrophages, accordingly improved the immune function.
Table 2 Effect of TAPA1 and its derivatives on nitrite production by macrophages RAW264.7 in vitro.a Samples
TAPA1-deac TAPA1 TAPA1-ac
Nitrite (lmol/106cells) 5 (lg/ml)
25 (lg/ml)
50 (lg/ml)
39.8 ± 4.5db 39.3 ± 6.7d 46.0 ± 11.2d
108.2 ± 11.6c 140.5 ± 3.9b 135.3 ± 7.8b
148.4 ± 4.3b 195.3 ± 9.6a 204.6 ± 7.1a
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In cells, the NO was very spry and immediately changed into ni trite radical ðNO 2 Þ and nitrogen ðNO3 Þ, therefore the method of Griess assay (Green et al., 1982; Lin, Juan, Shen, Hsu, & Chen, 2003) was used to determine NO production, and nitrite accumulation was used as an indicator of NO production in the medium. In our work, RAW264.7 cells exhibited altered morphology and increased in size after exposure to three samples (TAPA1, TAPA1deac and TAPA1-ac). All samples also could stimulate the mouse macrophage RAW264.7 to produce NO. As shown in the Table 2, exposure of RAW264.7 cells to increasing concentrations (5– 50 lg/ml) of samples for 24 h, resulted in significant increases in NO production (p < 0.05) in comparison with the negative control [(25.0 ± 1.7) lmol/106cells]. At 50 lg/ml, cells treated with TAPA1 and TAPA1-ac, produced [(195.3 ± 9.6) lmol/106cells] and [204.6 ± 7.1 lmol/106cells] of NO, respectively, which was almost the similar to the positive control LPS [208.9 ± 8.7 lmol/106cells] (p < 0.05). At tested concentrations (5, 25 and 50 lg/ml), cells treated with TAPA1 (with the group O-Ac DS of 0.03) and TAPA1-ac (with the group O-Ac DS of 0.23), produced no significantly different NO (p < 0.05), however, TAPA1-deac produced obviously lower NO (p < 0.05) in comparison with TAPA1 and TAPA1-ac. The results indicated that effect of samples on NO production by macrophages RAW264.7 might mainly related to having the acetyl group or not, whereas, that seemed not relate to the content and the DS of acetyl group. 4. Conclusions Two derivatives of TAPA1, TAPA1-ac and TAPA1-deac, were prepared successfully, which had been proven by FT-IR spectra, NMR spectra and calculated analysis data. By comparison with TAPA1 (with DS of 0.03), TAPA1-ac (with DS of 0.23) showed markedly higher MSLs (p < 0.05) stimulation potency and produced a similar NO level at tested concentration, however, TAPA1-deac (without acetyl groups) showed markedly lower MSLs stimulation activity (p < 0.05) and markedly lower NO production potency (p < 0.05). Both MSLs proliferation assay and NO production potency demonstrated that TAPA1-ac was a very promising and immunostimulatory compound, and should be worth further researching and developing. In conclusion, polysaccharides with different DS and contents of acetyl group showed different immunostimulatory potency, which might relate to not only different DS and content of acetyl group, but also having the acetyl group or not. The structural properties of TAPA1-ac, especially the position(s) of acetylated substitution and the connection between the DS and immunostimulating activities, would be the subject of continuing research, which would promote the research on structure–activity relationships in polysaccharide field and development of carbohydrate chemistry. Acknowledgments Research work was supported by the Natural Science Foundation of Shandong Province of China (No. ZR2010CL008) and Doctoral Research Startup Foundation of Liaocheng University (no. 31805). The authors are deeply grateful to Dr. Yanfang Liu and Master Jinxia Hu for their help in activities determination, and Mrs. Liping Shi for recording the NMR spectra. References
a
In this work, LPS (1 lg/ml) was used as a positive reference (208.9 ± 8.7 lmol/ 106cells) and control (plus PBS) was used as negative reference (25.0 ± 1.7 lmol/ 6 10 cells). b Values within a row followed by different letters (a–d) are significantly different at p < 0.05 by t-test.
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Du, X. J., Zhang, J. S., Yang, Y., Ye, L. B., Tang, Q. J., Jia, W., et al. (2009). Structural elucidation and immuno-stimulating property of an acidic heteropolysaccharide (TAPA1) from Tremella aurantialba. Carbohydrate Research, 344(5), 672–678. Du, X. J., Zhang, J. S., Yang, Y., Tang, Q. J., Jia, W., & Pan, Y. J. (2010). Purification, chemical modification and immunostimulating activity of polysaccharide from Tremella aurantialba fruiting bodies. Journal of Zhejiang University, 11(16), 437–442. Eflerbroek, P. M., Lefeber, D. J., van Veghel, R., Scharringa, J., Brouwer, E., Gerwig, G. J., et al. (2004). O-acetylation of cryptococcal capsular glucuronoxylomannan is essential for interference with neutrophil migration. Journal of Immunology, 173(12), 7513–7520. Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishnok, J. S., & Tannenbaum, S. R. (1982). Analysis of nitrate, nitrite and [15N] nitrate in biological fluids. Analytical Biochemistry, 126, 131–138. He, Q., & Zhang, S. (2004). Advances in the studies on the mechanism of immunopotentiation effect of polysaccharides from edible-medicinal fungi. Acta Edulis Fungi, 11(2), 52–58. Ji, Z., Tang, Q. J., Zhang, J. S., Yang, Y., Jia, W., & Pan, Y. J. (2007). Immunomodulation of RAW264.7 macrophages by GLIS, a proteopolysaccharide from Ganoderma lucidum. Journal of Ethnopharmacology, 112(3), 445–450. Li, B., & Xu, S. Y. (2004). Methylation of polysaccharide undissolved in dimethyl sulfoxide. Natural Product Research and Development, 16(3), 207–209. LilloL, E., & Matsuhiro, B. (1997). Chemical modification of carboxylated chitosan. Carbohydrate Polymers, 34(4), 397–401. Lin, H. Y., Juan, S. H., Shen, S. C., Hsu, F. L., & Chen, Y. C. (2003). Inhibition of lipopolysaccharide-induced nitric oxide production by flavonoids in RAW264.7 macrophages involves heme oxygenase-1. Biochemcial Pharmacology, 66, 1821–1832.
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Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Chemical modification of an acidic polysaccharide (TAPA1) from Tremella aurantialba and potential biological activities XiuJu Du a, JingSong Zhang b,⇑, ZhiWei Lv a, LiBin Ye c, Yan Yang b, QingJiu Tang b a
College of Life Science, Liaocheng University, Liaocheng, Shandong 252059, PR China Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201106, PR China c College of Food Science and Biotechnology, Zhejiang Gongshang University, Food Safety Key Lab of Zhejiang Province, Hangzhou 310035, PR China b
a r t i c l e
i n f o
Article history: Received 25 March 2013 Received in revised form 21 June 2013 Accepted 30 July 2013 Available online 7 August 2013 Keywords: Tremella aurantialba polysaccharide Acetylation Deacetylation Immunostimulating activity Nitric oxide
a b s t r a c t TAPA1 was previously isolated from Tremella aurantialba fruiting bodies. In this paper, an acetylated derivative (TAPA1-ac) and a deactylated derivative (TAPA1-deac) of TAPA1 were prepared, and their characterization and immunostimulating activities were reported. Acetylation and deacetylation were found to occur actually by FT-IR and NMR spectra, together with calculational results. The degree of substitution (DS) of acetyl groups in TAPA1-ac was 0.23 and the content was 5.82%, which was higher than those of TAPA1 (0.03% and 0.70%, respectively) and TAPA1-deac (all were zero). Compared with TAPA1, TAPA1-ac showed significant immunostimulation effects on mouse spleen lymphocytes (MSLs) proliferation and nitric oxide (NO) production by macrophages RAW264.7, whereas TAPA1-deac showed markedly lower effects. These findings seemed to suggest that immunostimulating activities, including MSLs stimulation activity and NO production potency, might relate to the DS and content of acetyl groups, indicating that acetylation of TAPA1 was an effective way of enhancing immuno-stimulating activities. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Chemical modification of polysaccharides usually provides an opportunity to obtain new pharmacological agents with possible therapeutic uses, and it has been an important means to study the structure–activity relationships of the polysaccharide (Zhao & Wang, 2000). Acetyl group in polysaccharides were reported to potentially play a very important role in the enhancement of biological activities (Eflerbroek et al., 2004; Ma, Chen, Zhang, Zhang, & Fu, 2012). Moreover, it could change the spatial structure of the sugar chain, resulting in the exposure of a hydroxyl group, and thereby might increase its water solubility and enhance its biological activities (LilloL & Matsuhiro, 1997). In our previous work, an acidic polysaccharide fraction with a mean molecular weight of 1.35 106 Da and a carbohydrate content of 98.7%, termed as TAPA1, was isolated and purified from the fruiting bodies of Tremella aurantialba for the first time. The structure of TAPA1 had since been investigated in our laboratory using 2D-NMR spectra, monosaccharide compositional analysis and methylation data, and the results were published (shown in Fig. 1) (Du, 2009; Du et al., 2009). In TAPA1, the DS of acetyl groups was 0.03 and the acetyl content was 0.7%. The immunostimulating activity of TAPA1, determined by using the MSLs proliferation as-
⇑ Corresponding author. Tel./fax: +86 21 62201337. E-mail address: [email protected] (J. Zhang). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.07.137
say, was clear. However, compared with the clinical application of immunostimulating drugs, theeffect of the polysaccharide TAPA1 should be further enhanced. Currently there is little knowledge of acetylation and deacetylation in polysaccharides of T. aurantialba. In order to obtain new more active compounds, and further investigate structure–activity relationships of TAPA1, an acetylated derivative TAPA1-ac and a deacetylated fraction TAPA1-deac were prepared by chemical modification. Furthermore, in vitro immunostimulating activity was determined using the mouse spleen lymphocytes (MSLs) proliferation assay and nitric oxide (NO) production assay. The immunostimulating mechanism was also preliminarily investigated in the paper.
2. Materials and methods 2.1. Materials Fruiting bodies of T. aurantialba were presented by Kunming Edible Fungi Institute of General National Supply and Marketing Cooperative in People’s Republic of China. TAPA1 was purified and proved to be homogeneous in our previous work (Du et al., 2009), and stored in dryer with silica-gel desiccant at room temperature. LPS were from Sigma–Aldrich (USA). Phytohemagglutinim (PHA), Penicillin and Streptomycin were purchased from Amersco (USA). RPMI-1640 medium, DMEM medium and fetal calf serum (FCS) were bought from Gibco (USA). Alamar Blue™ reagent
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was bought from Biosoure International (USA). The standard monosaccharide and dextrans were from Sigma–Aldrich (USA). All other reagents were AR grade from Chinese sources. 2.2. General methods FT-IR spectroscopy of samples mixed with dry KBr was performed in the 4000–400 cm1 region (Nexus Euro FTIR instrument). 1H NMR and 13C NMR spectra were recorded in D2O at 500 MHz (1H NMR) or 125 MHz (13C NMR) using a Bruker Avance 500 spectrometer (Germany). 13C chemical shifts were acquired in relation to DSS (d 0.00 ppm) calibrated externally, and HDO (d 4.32) was used as the internal reference signal for 1H at 70 °C (343 K). Fig. 1. The structure of polysaccharide TAPA1 from fruiting bodies of T. aurantialba.
2.3. Determination of the molecular weight The molecular weight of prepared samples was estimated according to the method of Du et al. (2009). Briefly, the sample was applied to a gel-permeation chromatography on a high-resolution Sephacryl S-500 column (XK16 100 cm) calibrated using dextrans T-150, 270, 410, 670 and 2000. Sample (150 ll) was applied to the column and the flow rate of the mobile phase (0.2 M NaNO3) was 0.5 ml/min. The column and refractive index detector (RID) temperature were maintained at 30 °C. 2.4. Preparation of derivatives of the homogeneous polysaccharide TAPA1 2.4.1. Preparation of acetylated derivative TAPA1-ac TAPA1-ac was prepared according to the method described by Ueno, Okamoto, Yamauchi, and Kato (1982), with dimethyl sulfoxide (DMSO) as the solvent in pyridine acetic anhydride system. Briefly, 50 mg TAPA1 was put into DMSO (4 ml), acetylated by the mixture of acetic anhydride (0.5 ml) and pyridine (0.6 ml) at 30 °C for 4 h, discontinued with 5 ml distilled water, dialyzed, and then lyophilized. Accordingly, an acetylated sample TAPA1ac (28 mg) was obtained. The content and the DS of the group OAc were measured according to the method reported previously by Ye (2008). 2.4.2. Preparation of deacetylated derivative TAPA1-deac TAPA1-deac was prepared according to the method of Vinogradov, Petersen, Duus, and Wasser (2004). Briefly, 50 mg TAPA1 was dissolved in a solution of 5% NH3 (20 ml) in water, kept at 50 °C for 16 h, dialyzed for 48 h, centrifuged (1776g, 5 min), and then lyophilized to yield deacetylated sample TAPA1-deac (37.5 mg). The content and the DS of the group O-Ac were measured according to Ye (2008). 2.5. In vitro spleen lymphocyte proliferation assay The MSLs proliferation assay was carried out according to the method described previously by Du et al. (2010), lymphocytes from mouse spleens were prepared. Briefly, C57 BL/6 male mice (ca. 18 g) were killed by cervical dislocation. The spleens were removed and cut into several pieces, washed thrice with PBS buffer, and then pressed through a stainless steel mesh (100 meshes) so as to obtain a single spleen cell suspension. After centrifugation (400g, 6 min), red cells in the spleen cell suspension were lysed with a Tris–HCl–NH4Cl solution (pH 7.2) (Zhang, Tang, Zimmerman-Kordmann, Reutter, & Fan, 2002). The cell suspension was further diluted with a fivefold excess of medium. After mixing and centrifugation, the cell pellets were finally re-suspended in RPMI1640 medium and adjusted to a concentration of 2 106 cells/ ml. Secondly, MSLs proliferation rate was determined by the Ala-
mar Blue Assay. Briefly, to each well of a 96-well microplate 180 ll of the cell suspension and 20 ll of different test agents were added. PBS and PHA (6 lg/ml) served as the negative and positive controls, respectively. After incubation at 37 °C in 5% CO2 atmosphere for 72 h, 20 ll Alamar Blue reagent (Biosource, Nivelles, Belgium) was added to each well and the incubation continued for another 6 h. Absorption values at 570 nm (A570) and 600 nm (A600) were measured using a micro enzyme-linked immunosorbent assay (ELISA) autoreader. The proliferation rate [absorption values at 570 nm (A570) and 600 nm (A600)] were measured using a micro enzyme-linked immunosorbent assay (ELISA) autoreader. The proliferation rate (r) was calculated according to the following formula: r(%) = [117216 A570(sample) 80856 A600(sample)]/ [117216 A570(control) 80856 A600 (control)] 100%.
2.6. NO production by macrophages RAW264.7 in vitro 2.6.1. Cell line RAW264.7, a mouse macrophage cell line, was bought from the ATCC (American Type Culture Collection) and cultured in RPMI 1640 medium containing 10% FCS (fetal calf serum), 100 U/ml streptomycin and 100 lg/ml penicillin at 37°Cin a 5% CO2 atmosphere.
2.6.2. Nitrite quantitation Nitrite accumulation was used as an indicator of NO production in the medium as previously described (Ji et al., 2007). Briefly, cells (1 106 cells/ml) were added to 96-well plates and stimulated with samples (5, 25 and 50 lg/ml) for 24 h. In this work, LPS (Lipopolysaccharide, 1 lg/ml) was served as a positive reference and PBS was as a negative control. Supernatants (100 ll) were then collected and mixed with 0.5 vol (50 ll) Griess reagent [1% (w/v) sulfanilamide, 0.1% (w/v) naphthylethylenediamine dihydrochloride, 2.5% (v/v) phosphoric acid]and incubated at room temperature for 10 min. Nitrite production was determined by comparing the absorbance at 543 nm against a standard curve generated using NaNO2.
2.7. Statistical analysis The data obtained were expressed as mean ± SD of three determinations and analyzed statistically by ANOVA method. Data were analyzed using STST2 statistical software. Significance of any differences between groups was evaluated using the Student’s t-test.
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3. Results and discussion 3.1. Characterization of the deacetylated derivative TAPA1-deac and acetylated derivative TAPA1-ac Recent research on polysaccharides from Inonotus obliquus, indicates that acetylationed polysaccharide (Ac-IOPS) resulted in higher antioxidant abilities on ferric-reducing power and lipid peroxidation inhibition activity compared with the native polysaccharide IOPS (Ma et al., 2012). Eflerbroek et al. (2004) assessed the contribution of acetylation and deacetylation of the polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans, to the interference with neutrophil migration. Their findings showed that the acetyl group in GXM was of necessary for its ability to interfere with neutrophil chemokinesis (Eflerbroek et al., 2004). In this work, TAPA1 was chemically acetylated and deacetylated so as to further enhance the immunomodulatory activities of TAPA1 and investigate the correlation between the acetyl content and its activity. Accordingly, a deacetylated derivative (TAPA1-deac) and an acetylated derivative (TAPA1-ac) were obtained. The average molecular weights of two derivatives were all determined to be 1.35 106 Da, which was almost identical to that of their native fraction TAPA1, indicating that no degradation had occurred in the deacetylated and acetylated reactions. The yields of TAPA1deac and TAPA1-ac were 75.0% and 56.0%, respectively. TAPA1-ac had the group O-Ac DS of 0.23 and an acetyl content of 5.82%, however, the DS and acetyl content in TAPA1-deac were determined to be zero, indicating the acetyl group in TAPA1-deac had been removed completely from its native polysaccharide TAPA1. Both derivatives TAPA1-deac and TAPA1-ac were all white, flocculent and soluble in water, which was almost similar to TAPA1. However, all fractions, TAPA1 and its derivatives TAPA1-deac and TAPA1-ac, were all insoluble in DMSO, which was due to containing some carboxyl group (–COOH) in them, including glucuronic acid and/or OAc group (Li & Xu, 2004). 3.2. Spectral characterization of TAPA1-deac and TAPA1-ac As shown in Fig. 2, two characteristic absorption bands appeared in the FT-IR spectra of TAPA1, one at near 1733.7 cm1 describing a C@O stretching vibration associated with an ester group, another at near 1249.7 cm1, representing a C–O stretching vibration of ester group (Zhou, Guo, Zheng, & Li, 2006). By comparison with TAPA1, two characteristic absorption bands (near 1733.7 and 1249.7 cm1) remarkably enhanced in the FT-IR
Fig. 3. 13C NMR (125 MHz) spectra of TAPA1 and its derivatives. 13C NMR (125 MHz) spectra of TAPA1 and its derivatives. (A) TAPA1-deac; (B) TAPA1 and (C) TAPA1-ac.
spectra of the acetylated derivative TAPA1-ac, indicating TAPA1ac was successfully acetylated (Zhou et al., 2006). However, the two bands mentioned above were disappeared or decreased in the FT-IR spectra of TAPA1-deac, indicating TAPA1-deac was actually deacetylated. The fact that acetylation and deacetylation of TAPA1 occurred successfully could also be further proven by 1H NMR (500 MHz, Fig. not shown) and 13C NMR (125 MHz, Fig. 3) spectra data. Signals of TAPA1 and TAPA1-ac at d 2.02 corresponded to the 1H signal for the CH3 moiety of an acetyl group (Serrato et al., 2008), and signals of TAPA1 and TAPA1-ac at d 23.79 upfield revealed a CH3 moiety of an acetyl group (Serrato et al., 2008), which indicated that TAPA1 and TAPA1-ac contained acetyl groups. Furthermore, two signals at d 2.02 and d 23.79 in TAPA1-ac were synchronously higher than that in TAPA1, indicating acetyled reaction of TAPA1 occurred successfully. By comparison with TAPA1, signals at d 2.02 and d 23.79 in TAPA1-deac all synchronously disappeared, showing that no acetyl groups was in TAPA1-deac, that is to say, the deacetylated reaction of TAPA1 also occurred successfully.
Fig. 2. FI-IR spectra of TAPA1 and its derivatives. FI-IR spectra of TAPA1 and its derivatives. (A) TAPA1-deac; (B) TAPA1 and (C) TAPA1-ac.
X. Du et al. / Food Chemistry 143 (2014) 336–340 Table 1 Effect of TAPA1 and its derivatives on mouse spleen lymphocyte proliferation in vitro.a Samples
TAPA1-deac TAPA1 TAPA1-ac
Proliferation rate (%) 50 (lg/ml)
200 (lg/ml)
500 (lg/ml)
102.8 ± 8.5ib 298.2 ± 13.9 f 312.5 ± 14.1e
243.0 ± 11 h 407.3 ± 6.3d 418.7 ± 6.5c
271.4 ± 2.5 g 458.7 ± 11.5b 496.5 ± 11.1a
a In this work, PHA (6 lg/ml) was used as a positive reference (467.7 ± 9.7%) and control (plus PBS) was used as negative reference (100.0 ± 4.7%). b Values within a row followed by different letters (a–i) are significantly different at p < 0.05 byt-test.
3.3. Effect of TAPA1 and its derivatives on mouse spleen lymphocyte proliferation in vitro Immunoregulation was one of the basic functions associated with polysaccharides (Su, Dai, & Yang, 2006). The mechanism of immunoregulation mainly was to stimulate macrophage, lymphocyte, T cell, B cell, NK-cell, LAK-cell and red blood cell from various levels of immunological organ, immunocyte and immunological molecules in order to increase the number of these cells and potentiate their bioactivity (He & Zhang, 2004). Therefore, in vitro cell proliferation experiments represent a suitable method for the rapid screening of polysaccharide with immunobiological activity. So the immunoregulation activities of TAPA1 and its derivatives are evaluated by determining the effect on mouse spleen lymphocyte proliferation assay. The results were shown in Table 1. It was found that TAPA1 stimulated the proliferation of MSLs in vitro in a dose-dependent manner. At tested concentrations, TAPA1-ac showed higher MSLs (p < 0.05) stimulation potency than TAPA1 did, dose-dependently, whereas TAPA1-deac showed markedly lower MSLs (p < 0.05) stimulation potency than TAPA1 did. At 500 lg/ml, the proliferation rate of the TAPA1-deac, TAPA1 and TAPA1-ac were (271.4 ± 2.5)%, (458.7 ± 11.5)% and (496.5 ± 11.1)%, respectively, which showed a significant difference (p < 0.05) among them. These findings seemed to suggest that polysaccharides with higher content and DS of the O-Ac group, showed stronger MSLs potency in immuno-enhancing activity. 3.4. Effect of TAPA1 and its derivatives on nitric oxide production by macrophages RAW264.7 in vitro Macrophage was one of the important components of the immune system, and it played an important role in immunoregulation (Albrecht, 1979). NO, produced by macrophages, was a kind of important inorganic small molecule discovered in recent years, and played an important role in inflammation, injury, defense, and so on. It had been reported that the polysaccharides could stimulate NO production by macrophages, accordingly improved the immune function.
Table 2 Effect of TAPA1 and its derivatives on nitrite production by macrophages RAW264.7 in vitro.a Samples
TAPA1-deac TAPA1 TAPA1-ac
Nitrite (lmol/106cells) 5 (lg/ml)
25 (lg/ml)
50 (lg/ml)
39.8 ± 4.5db 39.3 ± 6.7d 46.0 ± 11.2d
108.2 ± 11.6c 140.5 ± 3.9b 135.3 ± 7.8b
148.4 ± 4.3b 195.3 ± 9.6a 204.6 ± 7.1a
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In cells, the NO was very spry and immediately changed into ni trite radical ðNO 2 Þ and nitrogen ðNO3 Þ, therefore the method of Griess assay (Green et al., 1982; Lin, Juan, Shen, Hsu, & Chen, 2003) was used to determine NO production, and nitrite accumulation was used as an indicator of NO production in the medium. In our work, RAW264.7 cells exhibited altered morphology and increased in size after exposure to three samples (TAPA1, TAPA1deac and TAPA1-ac). All samples also could stimulate the mouse macrophage RAW264.7 to produce NO. As shown in the Table 2, exposure of RAW264.7 cells to increasing concentrations (5– 50 lg/ml) of samples for 24 h, resulted in significant increases in NO production (p < 0.05) in comparison with the negative control [(25.0 ± 1.7) lmol/106cells]. At 50 lg/ml, cells treated with TAPA1 and TAPA1-ac, produced [(195.3 ± 9.6) lmol/106cells] and [204.6 ± 7.1 lmol/106cells] of NO, respectively, which was almost the similar to the positive control LPS [208.9 ± 8.7 lmol/106cells] (p < 0.05). At tested concentrations (5, 25 and 50 lg/ml), cells treated with TAPA1 (with the group O-Ac DS of 0.03) and TAPA1-ac (with the group O-Ac DS of 0.23), produced no significantly different NO (p < 0.05), however, TAPA1-deac produced obviously lower NO (p < 0.05) in comparison with TAPA1 and TAPA1-ac. The results indicated that effect of samples on NO production by macrophages RAW264.7 might mainly related to having the acetyl group or not, whereas, that seemed not relate to the content and the DS of acetyl group. 4. Conclusions Two derivatives of TAPA1, TAPA1-ac and TAPA1-deac, were prepared successfully, which had been proven by FT-IR spectra, NMR spectra and calculated analysis data. By comparison with TAPA1 (with DS of 0.03), TAPA1-ac (with DS of 0.23) showed markedly higher MSLs (p < 0.05) stimulation potency and produced a similar NO level at tested concentration, however, TAPA1-deac (without acetyl groups) showed markedly lower MSLs stimulation activity (p < 0.05) and markedly lower NO production potency (p < 0.05). Both MSLs proliferation assay and NO production potency demonstrated that TAPA1-ac was a very promising and immunostimulatory compound, and should be worth further researching and developing. In conclusion, polysaccharides with different DS and contents of acetyl group showed different immunostimulatory potency, which might relate to not only different DS and content of acetyl group, but also having the acetyl group or not. The structural properties of TAPA1-ac, especially the position(s) of acetylated substitution and the connection between the DS and immunostimulating activities, would be the subject of continuing research, which would promote the research on structure–activity relationships in polysaccharide field and development of carbohydrate chemistry. Acknowledgments Research work was supported by the Natural Science Foundation of Shandong Province of China (No. ZR2010CL008) and Doctoral Research Startup Foundation of Liaocheng University (no. 31805). The authors are deeply grateful to Dr. Yanfang Liu and Master Jinxia Hu for their help in activities determination, and Mrs. Liping Shi for recording the NMR spectra. References
a
In this work, LPS (1 lg/ml) was used as a positive reference (208.9 ± 8.7 lmol/ 106cells) and control (plus PBS) was used as negative reference (25.0 ± 1.7 lmol/ 6 10 cells). b Values within a row followed by different letters (a–d) are significantly different at p < 0.05 by t-test.
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