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Antioxidant activity and antitumor activity (in vitro) of xyloglucan selenious ester and surfated xyloglucan
International Journal of Biological Macromolecules 45 (2009) 231–235
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Antioxidant activity and antitumor activity (in vitro) of xyloglucan selenious ester and surfated xyloglucan Yu Cao ∗ , Isao Ikeda ∗∗ Department of Applied Chemistry & Biotechnology, Graduate School of Engineering, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan
a r t i c l e
i n f o
Article history: Received 19 March 2009 Accepted 20 May 2009 Available online 27 May 2009 Keywords: Xyloglucan selenious ester Sulfated xyloglucan Antioxidant Antitumor Selenition
a b s t r a c t Two kinds of xyloglucan derivatives (xyloglucan selenious ester and sulfated xyloglucan) were prepared and evaluated on antioxidant activity and antitumor activity. Compared with xyloglucan, xyloglucan derivatives have new bioactivity against oxidative damage and tumor. Furthermore, xyloglucan selenious ester is more potent than sulfated xyloglucan at antioxidant activity and antitumor activity in vitro. The current data suggest for the first time that selenition of xyloglucan significantly increases its bioactivity and the chemical modification of polysaccharide may allow the preparation of derivatives with new properties and a variety of applications. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Polysaccharide, distributed in nature extensively, plays an important role in life. With the development of polysaccharide science and technology, polysaccharide and its derivatives have a prospective application in many fields especially in biomedicine. Recently, the research on antioxidant activity and antitumor activity of polysaccharide and its derivatives has attracted much attention [1–3]. Xyloglucan is a neutral, nontoxic polysaccharide, and a major heteropolysaccharide in the primary cell walls of higher plants. Xyloglucan is used as skin patches, oral or rectal delivery of drugs, and for intraperitoneal injections. Xyloglucan provides stronger bioavailability of the relevant drug and longer residence times than previous commercial suppositories. Importantly there was no apparent tissue damage implying that xyloglucan is a biocompatible material that can be implanted noninvasively by injection in medical applications [2]. The biological role of selenium (Se) has received considerable attention since early times. Its biological functions are at least in part associated with its antioxidant activity. As known, many biological molecules such as glutathione peroxidase, and/or thioredoxin reductasaes, as well as numerous small organic molecules show promising antioxidant activity [4,5]. However, to date, the
∗ Corresponding author. Present address: College of Chemistry, Central China Normal University, 152#, Luoyu Road, Wuhan, Hubei 430079, PR China. Tel.: +86 27 61311087; fax: +86 27 67867141. ∗∗ Corresponding author. Tel.: +81 776 278637; fax: +81 776 278747. E-mail addresses: [email protected] (Y. Cao), [email protected] (I. Ikeda). 0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2009.05.007
antioxidant activities and anticarcinogenic activities of polysaccharides containing Se have received little attention. Partly, it is due to very low Se content (<1%) in commercial selenopolysaccharide [6–8]. Based on this thinking, xyloglucan selenious ester and sulfated xyloglucan were prepared and determined on antioxidant activity and antitumor activity. 2. Materials and methods 2.1. Materials Xyloglucan (commercially available as Glyloid from Dainippon Pharmaceutical, Osaka, Japan) was purified by precipitation from its aqueous solution into 2-propanol. The supernatant was lyophilized. Sources of all chemicals/materials were analytical grade unless otherwise stated. 2.2. Preparation of sulfated xyloglucan [9] Briefly, 1 g of xyloglucan was suspended in 150 ml of N,Ndimethylformamide (DMF) and was then stirred for 4 h at room temperature under N2 . A needed excess (over 10 mol equiv of available hydroxyl group in polysaccharide) of pyridine–sulfur trioxide complex was added to the sample suspension, and the mixture was stirred at 50 ◦ C for 10 h under N2 . After the reaction was interrupted by addition of 100 ml of H2 O, the mixture was cooled and neutralized. The product was isolated by extensive dialysis against distilled water for 3 days. The dialyzate was concentrated and freeze-dried to give xyloglucan sulfate.
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2.3. Preparation of xyloglucan selenious ester from sulfated xyloglucan Catalyzed with Ba2+ and H+ , the solution of sulfated xyloglucan was heated with the selenious acid in 45 ◦ C for 48 h. The sulfuric acid solution was dropped gradually into reaction solution on room temperature and the BaSO4 precipitated from solution in order to remove Ba2+ ion. Then reaction solution was filtrated in order to eliminate BaSO4 . The clear reaction solution was adjusted to pH 7. Ethanol was drip-fed to the supernatant and up to an ethanol concentration of 70%. The crude product was dissolved in water, and some insoluble material was removed by centrifugation at 8000 rpm. The ethanol was added to the supernatant solution, and the resulting precipitate was filtered with Whatman filter paper No. 1 and washed with acetone. The purification was re-dissolved in water and freeze-dried. 2.4. Analytical methods The sulfate content was determined according to the method of BaSO4 colorimetry [10], and the selenium content (Se%) in the selenited sample was determined by the Variamine Blue method [11]. Fourier-transform infrared (FTIR) spectra were recorded from the sample in KBr pellets on a JASCO FT/IR-620 system spectrometer (JASCO Co., Japan). UV analyses were conducted on a JASCO V-550 UV/VIS spectrophotometer (JASCO Co., Japan). 2.5. Antioxidant activity assays 2.5.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity The scavenging activity of the DPPH radical was assayed according to the method of Shimada et al. [12]. Briefly, a 0.1 mM of ethanolic DPPH solution was prepared. The initial absorbance of the DPPH in ethanol was measured at 517 nm and did not change throughout the period of assay. An aliquot (0.1 ml) of each sample (with appropriate dilution if necessary) was added to 3.0 ml of ethanolic DPPH solution. Discolorations were measured at 517 nm after incubation for 30 min at 30 ◦ C in the dark. Measurements were performed at least in triplicate. The percentage of DPPH which was scavenged was calculated by the following equation:
K(%) =
1−
Asample − Ablank Acontrol
× 100
Here ethanol (3.0 ml) plus sample solution (0.1 ml) was used as a blank and 3 ml of DPPH–ethanol solution plus ethanol (0.1 ml) was also used as a negative control. 2.5.2. Superoxide anion scavenging activity The superoxide radicals were generated in a pyrogallol system [13]. In this experiment, the Tris–HCl buffer (16 mM, pH 8.0) and samples were attempered for 20 min at 25 ◦ C. As soon as pyrogallol (0.1 mM) was added, the absorbances of the reaction mixtures were measured at 510 nm against a reagent blank every 1 min for 5 min. Superoxide anion scavenging activity (OD, %) was calculated by the following equation: OD(%) =
Ao − As × 100 Ao
where Ao was the change rate of absorbance of the pyrogallol system without sample, and As was the change rate of absorbance of samples reacted with the pyrogallol.
2.5.3. Hydroxyl radical scavenging activity The scavenging activity of hydroxyl radicals was measured with the system of o-Phenanthroline Fenton [13]. Reaction mixtures, containing different samples, were incubated with oPhenanthroline (0.75 mM), H2 O2 (2 mM), FeSO4 (0.75 mM) in potassium phosphate buffer (20 mM, pH 7.4) for 60 min at 37 ◦ C. The absorbances of the mixtures were measured at 536 nm against a reagent blank. Hydroxyl radical scavenging activity (S, %) was calculated by the following equation: S(%) =
As − Ao × 100 Ac − Ao
where Ac was the absorbance of the samples, As was the absorbance of samples reacted with H2 O2 , and Ao was the absorbance of the mixture without sample. 2.5.4. Assays of lipid peroxidation using vitellose The inhibition of lipid peroxidation was determined by quantification of MDA decomposed from the lipid peroxide, which based on the egg vitellose reacting to thiobarbituric acid. The principal procedures were a modified method published [14]. For the in vitro studies, the fresh vitellose was dissected and homogenized in icecold PSB (20 mM, pH 7.4) to produce a 10% homogenate (v/v). The homogenate was centrifuged at 4000 rpm for 20 min to remove precipitation. 1 ml aliquots of the supernatant were incubated with the test samples in the presence of 5 mM FeSO4 at 37 ◦ C for 1 h. The reaction was stopped by addition of 1.0 ml trichloroacetic acid (TCA, 20%, w/v) and 1.5 ml thiobarbituric acid (TBA, 1%, w/v) in succession, and the solution was then heated at 100 ◦ C for 15 min. After centrifugation at 4000 rpm for 20 min to remove precipitated protein, the color of the complex was detected at 532 nm. The control group was run in parallel without sample under similar conditions, except that 1.0 ml trichloroacetic acid (TCA, 20%, w/v) was added before incubation. The lipid peroxidation scavenging activity (K, %) was calculated by the following equation: K(%) =
Ac − As × 100% Ac
where Ac was the absorbance of the control, and As was the absorbance of samples. 2.6. Antitumor activity in vitro The cytotoxicity of the synthesized compounds was measured against human liver cancer cell line HepG2 with the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenytetrazolium) assay [15]. Fifty microliters of the cell suspension of 1.85 × 105 cells/ml were seeded into wells of a 24-well plate. For HepG2 cells, the experiments were performed after 24 h. Cell viability was determined after the treated cells were incubated for 44 h. In brief, 10 l MTT was added to the wells, the cells were cultured for 3 h at 37 ◦ C in an incubator with an atmosphere of 5% CO2 . Then 100 l of 10% SDS was added to the wells and the cells were cultured overnight. The formation of formazan was measured at 570 nm using microplate spectrophotometer. The inhibitory rate was calculated as follows: inhibitory rate % =
Acontrol − Asample Acontrol
× 100.
2.7. Statistical analysis Data was expressed as means ± standard deviations of three replicated determinations, and followed by the Student’s t-test. Differences were considered to be statistically significant if P < 0.05.
Y. Cao, I. Ikeda / International Journal of Biological Macromolecules 45 (2009) 231–235
233
Fig. 3. Free radical scavenging capacity of sulfated xyloglucan () and xyloglucan selenious ester () on DPPH (1,1-diphenyl-2-picrylhydrazyl). Values are expressed in terms of mean ± S.D. for three observations.
Fig. 1. FTIR spectra of xyloglucan (a), sulfated xyloglucan (b) and xyloglucan selenious ester (c).
3. Results and discussion 3.1. The characterization of xyloglucan selenious ester in FTIR spectra and UV–visible spectra Compared with xyloglucan, Fig. 1 shows the FTIR spectral characterization of sulfated polysaccharide. The signal at 1253 cm−1 was attributed to the asymmetric stretching of S O and is wide and strong. The absorption at 808 cm−1 , because of sulfur groups, was assigned to C–O–S bond stretching. After the hydroxy sulfated, the bands near 3400 cm−1 became narrow and moved to a high wavenumber (3450 cm−1 ) and the bands near 1100 cm−1 decreased. It revealed a decrease in hydroxy groups. In xyloglucan selenious ester, the band at 818 cm−1 signaled the Se–O stretching vibration, while the band at 930 cm−1 was representative of the vibration of Se O groups. There was no absorbance peak in UV–vis spectra of xyloglucan from Fig. 2. Sulfated xyloglucan displayed an absorption band near 260 nm, as did other sulfated polysaccharides, while absorption of xyloglucan selenious ester was found to be around 200 nm. The sulfated content of the sulfated xyloglucan was 32.93%, while the selenium content is 28.2% in the xyloglucan selenious ester. Few kinds of polysaccharide containing selenium have been prepared and commercialized as a health food due to low selenium
Fig. 2. UV–visible spectra of xyloglucan (a), sulfated xyloglucan (b) and xyloglucan selenious ester (c).
content (<1%) until now [6]. In our study, selenium content was up to 28.2% in pure xyloglucan selenious ester, which gave a chance to find the new medicine application of polysaccharide selenious ester. 3.2. Antioxidant activity of xyloglucan derivatives The DPPH free radical is a stable free radical, which has been widely accepted as a tool for estimating the free radical scavenging activities of antioxidants in a relatively short time. In this study, the antioxidant activities of the xyloglucan selenious ester and xyloglcan sulfate were noticeable at the tested concentration determined by DPPH free radical scavenging assay, and their scavenging effects of its derivatives increased with increasing concentration. As shown in Fig. 3, the percentage inhibition of DPPH radical by 500 g/ml xyloglucan seleninic ester was near 70%, while this ratio at this concentration for sulfated xyloglucan was found as 34.3%. Superoxide anion, which is formed in viable cells during several biochemical reactions, can be magnified by its effect on cell damage because it produces other types of free radicals and oxidizing agent. Superoxide anion scavenging activity of the polysaccharide derivatives was measured using the pyrogallol system and the inhibitory effect of xyloglucan derivatives was marked and concentration related. As shown in Fig. 4, the percentage inhibition of superoxide, generated by 400 g/ml xyloglucan selenious ester, was found about 70%, while this ratio was 52.3% for the sulfated xyloglucan. Xyloglucan was found to have no superoxide radical scavenging activity. Although superoxide is a relatively weak oxidant, it decomposes to form stronger, reactive oxidative species, such as singlet oxygen and hydroxyl radicals, which initiate peroxidation of lipids.
Fig. 4. Inhibitory effect of xyloglucan (), sulfated xyloglucan () and xyloglucan selenious ester () against superoxide radicals. Values are expressed in terms of mean ± S.D. for three observations.
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Fig. 5. Scavenging activity of xyloglucan (), sulfated xyloglucan () and xyloglucan selenious ester () against hydroxyl radical measured with the system of o-Phenanthroline Fenton. Values are expressed in terms of mean ± S.D. for three observations.
Fig. 6. Inhibition by xyloglucan (), sulfated xyloglucan () and xyloglucan selenious ester () of FeSO4 induced lipid peroxidation of egg vitellose. Values are expressed in terms of mean ± S.D. for three observations.
Furthermore, superoxides are also known to indirectly initiate lipid peroxidation as a result of H2 O2 formation, creating precursors of hydroxyl radicals [3,16,17]. These results clearly suggested that the sulfation and selenition could increase their abilities to scavenge superoxides. Among the reactive oxygen species, hydroxyl radical is the most reactive in chemistry. It can abstract hydrogen atoms from biological thiol molecules and form sulfur radicals, which are capable to combine with oxygen to generate oxysulfur radicals and damage biological molecules. The results of hydroxyl radical scavenging activity of the samples were shown in Fig. 5. Xyloglucan selenious ester demonstrated the higher activity of hydroxyl radical scavenging, while sulfated xyloglucan displayed lower hydroxyl radical scavenging activity. The scavenging rate of xyloglucan selenious ester against hydroxyl radical at 1500 g/ml was 74.8%, which was much higher than the data (48.1%) observed with sulfated xyloglucan. Xyloglucan displayed no superoxide and hydroxyl radical scavenging activity. The scavenging activity of hydroxyl radicals was not due to direct scavenging but inhibition of hydroxyl radical generation by chelating ions such as Fe2+ and Cu+ [3,16]. Fe2+ has also been shown to produce oxyradicals and lipid peroxidation, and reduction of Fe2+ concentrations in the Fenton reaction would protect against oxidative damage [18,19]. In this study, the hydroxyl radical scavenging activity of selenious and sulfated derivatives are probably related to the specific chelating groups (selenite and sulfate) within the molecule due to their high nucleophilic character.
The initiation of lipid peroxidation is carried out mostly by free radicals, such as superoxide, hydroxyl radicals, etc., and other reactive oxygen species. Lipid peroxidation causes cellular injury by inactivation of the enzymes and receptors in membrane, and depolymerization of DNA/RNA as well as protein cross-linking and fragmentation. The effect of xyloglucan derivatives on nonenzymatic peroxidation was shown in Fig. 6. The sulfated xyloglucan and xyloglucan selenious ester inhibited lipid peroxidation. The inhibitory rates of lipid peroxidation were 30% and 68.6% for sulfated xyloglucan and xyloglucan selenious ester, respectively, at a concentration of 600 g/ml. The mechanism of inhibiting effect may be relative to the polysaccharide–membrane interactions. In recent years there has been growing interest in water-soluble antioxidants from natural sources, special polysaccharide derivatives. We therefore felt it worthwhile to undertake the study of the antioxidant activity of the polysaccharide selenious ester and sulfated polysaccharide, since preliminary experiments in our laboratory had indicated the antioxidant ability of this material. The results of antioxidant assay indicated that xyloglucan scavenged free radicals in a concentration-dependent manner. The antioxidant activity of xyloglucan selenious ester was higher than those of sulfated xyloglucan. It suggested that introduction of selenious groups into xyloglucan could afford more effective protection against damage. The results in this study suggested that xyloglucan selenious ester, possessing pronounced free radical scavenging activity, could be of considerable preventive significance to some life-threatening health problems such as cancer, atherogenesis and Alzheimer’s
Fig. 7. Antitumor activity in vitro of xyloglucan (), sulfated xyloglucan () and xyloglucan selenious ester () against human liver cancer cell lines. Values are expressed in terms of mean ± S.D. for three observations.
Y. Cao, I. Ikeda / International Journal of Biological Macromolecules 45 (2009) 231–235
disease, which pathologically initiated by the presence of free radicals leading to the inevitable peroxidation of important biomolecules [18,19]. Demonstration of antioxidant activity of this material would enhance potential applications in health foods, in cosmetics, and in pharmaceutics. In the mechanism of polysaccharide antioxidants, the ability of a molecule to donate hydrogen atoms to radicals and the propensity of hydrogen donation is the critical factor affecting free radical scavenging activity [18,19]. Xyloglucan selenious ester appeared to function as good hydrogen atom donors and were able to terminate radical chain reactions by converting free radicals to more stale products. Hence, xyloglucan selenious ester showed more pronounced antioxidant activity than sulfated xyloglucan, implying that selenious group in the xyloglucan derivatives plays a positive role in enhancing antioxidant activity of functionalized xyloglucan.
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For the first time, it was found that selenition of xyloglucan significantly increases its antitumor activity and antioxidant capacity. The results are helpful to understand the bioactivities of xyloglucan derivatives and seek out new lead compound from the polysaccharides, special containing selenium. Hence, it is expected that polysaccharide derivatives with selenious ester groups have new bioactivities and various applications. Acknowledgements This research was supported by the Japan Society for the Promotion of Science (JSPS, P05151). The authors also wish to thank Dr. Satoshi Terada and Dr. Shinji Sugihara, Department of Applied Chemistry & Biotechnology, Graduate School of Engineering, University of Fukui, for MTT assays, reagents, suggestions, and invaluable scientific discussions.
3.3. Antitumor activity of xyloglucan derivatives References The antitumor activity of the derivatives was investigated by an MTT assay. Fig. 7 shows the survival rate of cells and the inhibitive rate of xyloglucan derivatives. These results indicate that the derivatives can restrain the growth of cancer cells. Xyloglucan selenious ester displayed relatively higher antitumor activity against HepG2 in vitro than those of sulfated xyloglucan, but xyloglucan showed no bioactivity effect. Antitumor activity was found even at 0.1 g/ml. The results indicated that the antitumor activity was due to the sulfation and selenition of polysaccharides. Polysaccharides containing selenium have a number of compounds with high bioactivities, such as Se-enriched polysaccharide and selenocarrageenan. We identified that xyloglucan selenious ester exhibited the higher antitumor activity against HepG2 in vitro than those of sulfated xyloglucan. It has been reported that sulfated polysaccharide with a homogenous composition exert their antitumor activity through inhibiting the synthesis of viral proteins inside the host cells rather than direct inhibition of tumor cell growth [20]. Although the mode of cytotoxicity of the xyloglucan derivatives is not yet established, the chain stiffness resulted from derivative groups with negative charge is considered to be important in the expression of antitumor activity. Further attempts will be made to investigate the possible cellular and molecular mechanisms of the antiproliferative activity of xyloglucan selenious ester by examining how it affects tumor cell membrane protein activity and gene expression. 4. Conclusion The present research investigated the antioxidant activities and antitumor activity of the sulfated xyloglucan and its selenious ester.
[1] G.O. Aspinal, The Polysaccharides, Academic Press, New York, 1982. [2] T. Coviello, P. Matricardi, F. Alhaique, Expert Opinion on Drug Delivery 3 (2006) 395–404. [3] A. Kardosova, E. Machova, Fitoterapia 77 (2006) 367–373. [4] D.G. Barceloux, Journal of Toxicology—Clinical Toxicology 37 (1999) 145–172. [5] P. Brenneisen, H. Steinbrenner, H. Sies, Molecular Aspects of Medicine 26 (2005) 256–267. [6] Q. Cui, D. Shang, X. Zou, Zhongguo Shenghua Yaowu Zazhi 24 (2003) 155–157. [7] P.Cui. Cultivation of Se-rich auricularia auricula and extraction of Sepolysaccharides from auricularia auricula, P.R. China. 1004-5902[1810082], CN, Ref Type: Patent, 2006, 6 pp. [8] A.H.S. Munoz, K. Kubachka, K. Wrobel, J.F. Gutierrez Corona, S.K.V. Yathavakilla, J.A. Caruso, K. Wrobel, Journal of Agricultural Food Chemistry 54 (2006) 3440–3444. [9] A. Chaidedgumjorn, H. Toyoda, E.R. Woo, K.B. Lee, Y.S. Kim, T. Toida, T. Imanari, Carbohydrate Research 337 (2002) 925–933. [10] R.G. Schweiger, Carbohydrate Research 21 (1972) 219–228. [11] H.D. Revanasiddappa, T.N. Kumar, Analytical Sciences: The International Journal of the Japan Society for Analytical Chemistry 17 (2001) 1309–1312. [12] K. Shimada, K. Fujikawa, K. Yahara, T. Nakamura, Journal of Agricultural Food Chemistry 40 (1992) 945–948. [13] Y. Yan, L. Wan-Shun, H. Bao-Qin, S. Hai-Zhou, Nutrition Research 26 (2006) 369–377. [14] D. Gerard-Monnier, I. Erdelmeier, K. Regnard, N. Moze-Henry, J.C. Yadan, J. Chaudiere, Chemical Research in Toxicology 11 (1998) 1176–1183. [15] S.H. Yoo, E.J. Yoon, J. Cha, H.G. Lee, International Journal of Biological Macromolecules 34 (2004) 37–41. [16] H. Tapiero, D.M. Townsend, K.D. Tew, Biomedecine & Pharmacotherapy 57 (2003) 134–144. [17] H. Yuan, W. Zhang, X. Li, X. Lu, N. Li, X. Gao, J. Song, Carbohydrate Research 340 (2005) 685–692. [18] B. Ou, D. Huang, M. Hampsch-Woodill, J.A. Flanagan, E.K. Deemer, Journal of Agricultural Food Chemistry 50 (2002) 3122–3128. [19] G.S. Cetkovic, S.M. Djilas, J.M. Canadanovic-Brunet, V.T. Tumbas, Food Research International 37 (2004) 643–650. [20] S. Koyanagi, N. Tanigawa, H. Nakagawa, S. Soeda, H. Shimeno, Biochemical Pharmacology 65 (2003) 173–179.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Antioxidant activity and antitumor activity (in vitro) of xyloglucan selenious ester and surfated xyloglucan Yu Cao ∗ , Isao Ikeda ∗∗ Department of Applied Chemistry & Biotechnology, Graduate School of Engineering, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan
a r t i c l e
i n f o
Article history: Received 19 March 2009 Accepted 20 May 2009 Available online 27 May 2009 Keywords: Xyloglucan selenious ester Sulfated xyloglucan Antioxidant Antitumor Selenition
a b s t r a c t Two kinds of xyloglucan derivatives (xyloglucan selenious ester and sulfated xyloglucan) were prepared and evaluated on antioxidant activity and antitumor activity. Compared with xyloglucan, xyloglucan derivatives have new bioactivity against oxidative damage and tumor. Furthermore, xyloglucan selenious ester is more potent than sulfated xyloglucan at antioxidant activity and antitumor activity in vitro. The current data suggest for the first time that selenition of xyloglucan significantly increases its bioactivity and the chemical modification of polysaccharide may allow the preparation of derivatives with new properties and a variety of applications. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Polysaccharide, distributed in nature extensively, plays an important role in life. With the development of polysaccharide science and technology, polysaccharide and its derivatives have a prospective application in many fields especially in biomedicine. Recently, the research on antioxidant activity and antitumor activity of polysaccharide and its derivatives has attracted much attention [1–3]. Xyloglucan is a neutral, nontoxic polysaccharide, and a major heteropolysaccharide in the primary cell walls of higher plants. Xyloglucan is used as skin patches, oral or rectal delivery of drugs, and for intraperitoneal injections. Xyloglucan provides stronger bioavailability of the relevant drug and longer residence times than previous commercial suppositories. Importantly there was no apparent tissue damage implying that xyloglucan is a biocompatible material that can be implanted noninvasively by injection in medical applications [2]. The biological role of selenium (Se) has received considerable attention since early times. Its biological functions are at least in part associated with its antioxidant activity. As known, many biological molecules such as glutathione peroxidase, and/or thioredoxin reductasaes, as well as numerous small organic molecules show promising antioxidant activity [4,5]. However, to date, the
∗ Corresponding author. Present address: College of Chemistry, Central China Normal University, 152#, Luoyu Road, Wuhan, Hubei 430079, PR China. Tel.: +86 27 61311087; fax: +86 27 67867141. ∗∗ Corresponding author. Tel.: +81 776 278637; fax: +81 776 278747. E-mail addresses: [email protected] (Y. Cao), [email protected] (I. Ikeda). 0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2009.05.007
antioxidant activities and anticarcinogenic activities of polysaccharides containing Se have received little attention. Partly, it is due to very low Se content (<1%) in commercial selenopolysaccharide [6–8]. Based on this thinking, xyloglucan selenious ester and sulfated xyloglucan were prepared and determined on antioxidant activity and antitumor activity. 2. Materials and methods 2.1. Materials Xyloglucan (commercially available as Glyloid from Dainippon Pharmaceutical, Osaka, Japan) was purified by precipitation from its aqueous solution into 2-propanol. The supernatant was lyophilized. Sources of all chemicals/materials were analytical grade unless otherwise stated. 2.2. Preparation of sulfated xyloglucan [9] Briefly, 1 g of xyloglucan was suspended in 150 ml of N,Ndimethylformamide (DMF) and was then stirred for 4 h at room temperature under N2 . A needed excess (over 10 mol equiv of available hydroxyl group in polysaccharide) of pyridine–sulfur trioxide complex was added to the sample suspension, and the mixture was stirred at 50 ◦ C for 10 h under N2 . After the reaction was interrupted by addition of 100 ml of H2 O, the mixture was cooled and neutralized. The product was isolated by extensive dialysis against distilled water for 3 days. The dialyzate was concentrated and freeze-dried to give xyloglucan sulfate.
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2.3. Preparation of xyloglucan selenious ester from sulfated xyloglucan Catalyzed with Ba2+ and H+ , the solution of sulfated xyloglucan was heated with the selenious acid in 45 ◦ C for 48 h. The sulfuric acid solution was dropped gradually into reaction solution on room temperature and the BaSO4 precipitated from solution in order to remove Ba2+ ion. Then reaction solution was filtrated in order to eliminate BaSO4 . The clear reaction solution was adjusted to pH 7. Ethanol was drip-fed to the supernatant and up to an ethanol concentration of 70%. The crude product was dissolved in water, and some insoluble material was removed by centrifugation at 8000 rpm. The ethanol was added to the supernatant solution, and the resulting precipitate was filtered with Whatman filter paper No. 1 and washed with acetone. The purification was re-dissolved in water and freeze-dried. 2.4. Analytical methods The sulfate content was determined according to the method of BaSO4 colorimetry [10], and the selenium content (Se%) in the selenited sample was determined by the Variamine Blue method [11]. Fourier-transform infrared (FTIR) spectra were recorded from the sample in KBr pellets on a JASCO FT/IR-620 system spectrometer (JASCO Co., Japan). UV analyses were conducted on a JASCO V-550 UV/VIS spectrophotometer (JASCO Co., Japan). 2.5. Antioxidant activity assays 2.5.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity The scavenging activity of the DPPH radical was assayed according to the method of Shimada et al. [12]. Briefly, a 0.1 mM of ethanolic DPPH solution was prepared. The initial absorbance of the DPPH in ethanol was measured at 517 nm and did not change throughout the period of assay. An aliquot (0.1 ml) of each sample (with appropriate dilution if necessary) was added to 3.0 ml of ethanolic DPPH solution. Discolorations were measured at 517 nm after incubation for 30 min at 30 ◦ C in the dark. Measurements were performed at least in triplicate. The percentage of DPPH which was scavenged was calculated by the following equation:
K(%) =
1−
Asample − Ablank Acontrol
× 100
Here ethanol (3.0 ml) plus sample solution (0.1 ml) was used as a blank and 3 ml of DPPH–ethanol solution plus ethanol (0.1 ml) was also used as a negative control. 2.5.2. Superoxide anion scavenging activity The superoxide radicals were generated in a pyrogallol system [13]. In this experiment, the Tris–HCl buffer (16 mM, pH 8.0) and samples were attempered for 20 min at 25 ◦ C. As soon as pyrogallol (0.1 mM) was added, the absorbances of the reaction mixtures were measured at 510 nm against a reagent blank every 1 min for 5 min. Superoxide anion scavenging activity (OD, %) was calculated by the following equation: OD(%) =
Ao − As × 100 Ao
where Ao was the change rate of absorbance of the pyrogallol system without sample, and As was the change rate of absorbance of samples reacted with the pyrogallol.
2.5.3. Hydroxyl radical scavenging activity The scavenging activity of hydroxyl radicals was measured with the system of o-Phenanthroline Fenton [13]. Reaction mixtures, containing different samples, were incubated with oPhenanthroline (0.75 mM), H2 O2 (2 mM), FeSO4 (0.75 mM) in potassium phosphate buffer (20 mM, pH 7.4) for 60 min at 37 ◦ C. The absorbances of the mixtures were measured at 536 nm against a reagent blank. Hydroxyl radical scavenging activity (S, %) was calculated by the following equation: S(%) =
As − Ao × 100 Ac − Ao
where Ac was the absorbance of the samples, As was the absorbance of samples reacted with H2 O2 , and Ao was the absorbance of the mixture without sample. 2.5.4. Assays of lipid peroxidation using vitellose The inhibition of lipid peroxidation was determined by quantification of MDA decomposed from the lipid peroxide, which based on the egg vitellose reacting to thiobarbituric acid. The principal procedures were a modified method published [14]. For the in vitro studies, the fresh vitellose was dissected and homogenized in icecold PSB (20 mM, pH 7.4) to produce a 10% homogenate (v/v). The homogenate was centrifuged at 4000 rpm for 20 min to remove precipitation. 1 ml aliquots of the supernatant were incubated with the test samples in the presence of 5 mM FeSO4 at 37 ◦ C for 1 h. The reaction was stopped by addition of 1.0 ml trichloroacetic acid (TCA, 20%, w/v) and 1.5 ml thiobarbituric acid (TBA, 1%, w/v) in succession, and the solution was then heated at 100 ◦ C for 15 min. After centrifugation at 4000 rpm for 20 min to remove precipitated protein, the color of the complex was detected at 532 nm. The control group was run in parallel without sample under similar conditions, except that 1.0 ml trichloroacetic acid (TCA, 20%, w/v) was added before incubation. The lipid peroxidation scavenging activity (K, %) was calculated by the following equation: K(%) =
Ac − As × 100% Ac
where Ac was the absorbance of the control, and As was the absorbance of samples. 2.6. Antitumor activity in vitro The cytotoxicity of the synthesized compounds was measured against human liver cancer cell line HepG2 with the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenytetrazolium) assay [15]. Fifty microliters of the cell suspension of 1.85 × 105 cells/ml were seeded into wells of a 24-well plate. For HepG2 cells, the experiments were performed after 24 h. Cell viability was determined after the treated cells were incubated for 44 h. In brief, 10 l MTT was added to the wells, the cells were cultured for 3 h at 37 ◦ C in an incubator with an atmosphere of 5% CO2 . Then 100 l of 10% SDS was added to the wells and the cells were cultured overnight. The formation of formazan was measured at 570 nm using microplate spectrophotometer. The inhibitory rate was calculated as follows: inhibitory rate % =
Acontrol − Asample Acontrol
× 100.
2.7. Statistical analysis Data was expressed as means ± standard deviations of three replicated determinations, and followed by the Student’s t-test. Differences were considered to be statistically significant if P < 0.05.
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Fig. 3. Free radical scavenging capacity of sulfated xyloglucan () and xyloglucan selenious ester () on DPPH (1,1-diphenyl-2-picrylhydrazyl). Values are expressed in terms of mean ± S.D. for three observations.
Fig. 1. FTIR spectra of xyloglucan (a), sulfated xyloglucan (b) and xyloglucan selenious ester (c).
3. Results and discussion 3.1. The characterization of xyloglucan selenious ester in FTIR spectra and UV–visible spectra Compared with xyloglucan, Fig. 1 shows the FTIR spectral characterization of sulfated polysaccharide. The signal at 1253 cm−1 was attributed to the asymmetric stretching of S O and is wide and strong. The absorption at 808 cm−1 , because of sulfur groups, was assigned to C–O–S bond stretching. After the hydroxy sulfated, the bands near 3400 cm−1 became narrow and moved to a high wavenumber (3450 cm−1 ) and the bands near 1100 cm−1 decreased. It revealed a decrease in hydroxy groups. In xyloglucan selenious ester, the band at 818 cm−1 signaled the Se–O stretching vibration, while the band at 930 cm−1 was representative of the vibration of Se O groups. There was no absorbance peak in UV–vis spectra of xyloglucan from Fig. 2. Sulfated xyloglucan displayed an absorption band near 260 nm, as did other sulfated polysaccharides, while absorption of xyloglucan selenious ester was found to be around 200 nm. The sulfated content of the sulfated xyloglucan was 32.93%, while the selenium content is 28.2% in the xyloglucan selenious ester. Few kinds of polysaccharide containing selenium have been prepared and commercialized as a health food due to low selenium
Fig. 2. UV–visible spectra of xyloglucan (a), sulfated xyloglucan (b) and xyloglucan selenious ester (c).
content (<1%) until now [6]. In our study, selenium content was up to 28.2% in pure xyloglucan selenious ester, which gave a chance to find the new medicine application of polysaccharide selenious ester. 3.2. Antioxidant activity of xyloglucan derivatives The DPPH free radical is a stable free radical, which has been widely accepted as a tool for estimating the free radical scavenging activities of antioxidants in a relatively short time. In this study, the antioxidant activities of the xyloglucan selenious ester and xyloglcan sulfate were noticeable at the tested concentration determined by DPPH free radical scavenging assay, and their scavenging effects of its derivatives increased with increasing concentration. As shown in Fig. 3, the percentage inhibition of DPPH radical by 500 g/ml xyloglucan seleninic ester was near 70%, while this ratio at this concentration for sulfated xyloglucan was found as 34.3%. Superoxide anion, which is formed in viable cells during several biochemical reactions, can be magnified by its effect on cell damage because it produces other types of free radicals and oxidizing agent. Superoxide anion scavenging activity of the polysaccharide derivatives was measured using the pyrogallol system and the inhibitory effect of xyloglucan derivatives was marked and concentration related. As shown in Fig. 4, the percentage inhibition of superoxide, generated by 400 g/ml xyloglucan selenious ester, was found about 70%, while this ratio was 52.3% for the sulfated xyloglucan. Xyloglucan was found to have no superoxide radical scavenging activity. Although superoxide is a relatively weak oxidant, it decomposes to form stronger, reactive oxidative species, such as singlet oxygen and hydroxyl radicals, which initiate peroxidation of lipids.
Fig. 4. Inhibitory effect of xyloglucan (), sulfated xyloglucan () and xyloglucan selenious ester () against superoxide radicals. Values are expressed in terms of mean ± S.D. for three observations.
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Fig. 5. Scavenging activity of xyloglucan (), sulfated xyloglucan () and xyloglucan selenious ester () against hydroxyl radical measured with the system of o-Phenanthroline Fenton. Values are expressed in terms of mean ± S.D. for three observations.
Fig. 6. Inhibition by xyloglucan (), sulfated xyloglucan () and xyloglucan selenious ester () of FeSO4 induced lipid peroxidation of egg vitellose. Values are expressed in terms of mean ± S.D. for three observations.
Furthermore, superoxides are also known to indirectly initiate lipid peroxidation as a result of H2 O2 formation, creating precursors of hydroxyl radicals [3,16,17]. These results clearly suggested that the sulfation and selenition could increase their abilities to scavenge superoxides. Among the reactive oxygen species, hydroxyl radical is the most reactive in chemistry. It can abstract hydrogen atoms from biological thiol molecules and form sulfur radicals, which are capable to combine with oxygen to generate oxysulfur radicals and damage biological molecules. The results of hydroxyl radical scavenging activity of the samples were shown in Fig. 5. Xyloglucan selenious ester demonstrated the higher activity of hydroxyl radical scavenging, while sulfated xyloglucan displayed lower hydroxyl radical scavenging activity. The scavenging rate of xyloglucan selenious ester against hydroxyl radical at 1500 g/ml was 74.8%, which was much higher than the data (48.1%) observed with sulfated xyloglucan. Xyloglucan displayed no superoxide and hydroxyl radical scavenging activity. The scavenging activity of hydroxyl radicals was not due to direct scavenging but inhibition of hydroxyl radical generation by chelating ions such as Fe2+ and Cu+ [3,16]. Fe2+ has also been shown to produce oxyradicals and lipid peroxidation, and reduction of Fe2+ concentrations in the Fenton reaction would protect against oxidative damage [18,19]. In this study, the hydroxyl radical scavenging activity of selenious and sulfated derivatives are probably related to the specific chelating groups (selenite and sulfate) within the molecule due to their high nucleophilic character.
The initiation of lipid peroxidation is carried out mostly by free radicals, such as superoxide, hydroxyl radicals, etc., and other reactive oxygen species. Lipid peroxidation causes cellular injury by inactivation of the enzymes and receptors in membrane, and depolymerization of DNA/RNA as well as protein cross-linking and fragmentation. The effect of xyloglucan derivatives on nonenzymatic peroxidation was shown in Fig. 6. The sulfated xyloglucan and xyloglucan selenious ester inhibited lipid peroxidation. The inhibitory rates of lipid peroxidation were 30% and 68.6% for sulfated xyloglucan and xyloglucan selenious ester, respectively, at a concentration of 600 g/ml. The mechanism of inhibiting effect may be relative to the polysaccharide–membrane interactions. In recent years there has been growing interest in water-soluble antioxidants from natural sources, special polysaccharide derivatives. We therefore felt it worthwhile to undertake the study of the antioxidant activity of the polysaccharide selenious ester and sulfated polysaccharide, since preliminary experiments in our laboratory had indicated the antioxidant ability of this material. The results of antioxidant assay indicated that xyloglucan scavenged free radicals in a concentration-dependent manner. The antioxidant activity of xyloglucan selenious ester was higher than those of sulfated xyloglucan. It suggested that introduction of selenious groups into xyloglucan could afford more effective protection against damage. The results in this study suggested that xyloglucan selenious ester, possessing pronounced free radical scavenging activity, could be of considerable preventive significance to some life-threatening health problems such as cancer, atherogenesis and Alzheimer’s
Fig. 7. Antitumor activity in vitro of xyloglucan (), sulfated xyloglucan () and xyloglucan selenious ester () against human liver cancer cell lines. Values are expressed in terms of mean ± S.D. for three observations.
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disease, which pathologically initiated by the presence of free radicals leading to the inevitable peroxidation of important biomolecules [18,19]. Demonstration of antioxidant activity of this material would enhance potential applications in health foods, in cosmetics, and in pharmaceutics. In the mechanism of polysaccharide antioxidants, the ability of a molecule to donate hydrogen atoms to radicals and the propensity of hydrogen donation is the critical factor affecting free radical scavenging activity [18,19]. Xyloglucan selenious ester appeared to function as good hydrogen atom donors and were able to terminate radical chain reactions by converting free radicals to more stale products. Hence, xyloglucan selenious ester showed more pronounced antioxidant activity than sulfated xyloglucan, implying that selenious group in the xyloglucan derivatives plays a positive role in enhancing antioxidant activity of functionalized xyloglucan.
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For the first time, it was found that selenition of xyloglucan significantly increases its antitumor activity and antioxidant capacity. The results are helpful to understand the bioactivities of xyloglucan derivatives and seek out new lead compound from the polysaccharides, special containing selenium. Hence, it is expected that polysaccharide derivatives with selenious ester groups have new bioactivities and various applications. Acknowledgements This research was supported by the Japan Society for the Promotion of Science (JSPS, P05151). The authors also wish to thank Dr. Satoshi Terada and Dr. Shinji Sugihara, Department of Applied Chemistry & Biotechnology, Graduate School of Engineering, University of Fukui, for MTT assays, reagents, suggestions, and invaluable scientific discussions.
3.3. Antitumor activity of xyloglucan derivatives References The antitumor activity of the derivatives was investigated by an MTT assay. Fig. 7 shows the survival rate of cells and the inhibitive rate of xyloglucan derivatives. These results indicate that the derivatives can restrain the growth of cancer cells. Xyloglucan selenious ester displayed relatively higher antitumor activity against HepG2 in vitro than those of sulfated xyloglucan, but xyloglucan showed no bioactivity effect. Antitumor activity was found even at 0.1 g/ml. The results indicated that the antitumor activity was due to the sulfation and selenition of polysaccharides. Polysaccharides containing selenium have a number of compounds with high bioactivities, such as Se-enriched polysaccharide and selenocarrageenan. We identified that xyloglucan selenious ester exhibited the higher antitumor activity against HepG2 in vitro than those of sulfated xyloglucan. It has been reported that sulfated polysaccharide with a homogenous composition exert their antitumor activity through inhibiting the synthesis of viral proteins inside the host cells rather than direct inhibition of tumor cell growth [20]. Although the mode of cytotoxicity of the xyloglucan derivatives is not yet established, the chain stiffness resulted from derivative groups with negative charge is considered to be important in the expression of antitumor activity. Further attempts will be made to investigate the possible cellular and molecular mechanisms of the antiproliferative activity of xyloglucan selenious ester by examining how it affects tumor cell membrane protein activity and gene expression. 4. Conclusion The present research investigated the antioxidant activities and antitumor activity of the sulfated xyloglucan and its selenious ester.
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