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Sulfated modification of the polysaccharides obtained from defatted rice bran and their antitumor activities
International Journal of Biological Macromolecules 44 (2009) 211–214
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Sulfated modification of the polysaccharides obtained from defatted rice bran and their antitumor activities Li Wang a,b , Xiaoxuan Li b , Zhengxing Chen a,b,∗ a b
State Key Laboratory of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi 214122, China School of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi 214122, China
a r t i c l e
i n f o
Article history: Received 18 November 2008 Received in revised form 7 December 2008 Accepted 10 December 2008 Available online 24 December 2008 Keywords: Rice bran polysaccharide Chemical modification Degrees of sulfation Antitumor activity
a b s t r a c t Nine sulfated defatted rice bran polysaccharides (sRBPS), with various degrees of sulfation (DS) and carbohydrate content, were prepared by chlorosulfonic acid–pyridine (CSA–Pyr) method according to orthogonal test. Nine sulfated derivatives sRBPS were obtained and their antitumor activities were compared by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results showed that when DS within the scope of 0.81–1.29, carbohydrate content in the range of 41.41–78.56%, sulfated derivatives exhibit relatively strong antitumor activity in vitro. The optimum modification conditions were reaction temperature of 70 ◦ C, the ratio of chlorosulfonic acid to pyridine of 1:4 and the reaction time of 2 h. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Polysaccharides have drawn the attention of biochemical and nutritional researchers in recent years due to their various biological activities [1–3]. Many studies have demonstrated that biological activities of polysaccharides are obviously increased by chemical modification. Molecular modification and structural improvement of polysaccharide appeared to elicit excellent physiological properties in maintaining health and preventing diseases [4,5]. Sulfated polysaccharide is a kind of polysaccharide with sulfated group in its hydroxyls. Many studies have confirmed that the sulfated polysaccharides exerted potent biological properties in comparison with non-sulfated polysaccharides, such as anti-coagulant [6], anti-virus [7], anti-oxidant [8] and antitumor activities [9]. Therefore, sulfated modification could be considered as the effective way to enhance the biological activities of polysaccharides. CSA–Pyr method was the most popular method to sulfate polysaccharides. Many studies have indicated that CSA–Pyr method posses advantages of high yield, high DS and convenient manipulation. The ratio of CSA to Pyr, reaction temperature and the reaction time are the important factors in this method. Polysaccharide performed at different conditions resulted in sulfated polysaccharides with various DS and bioactivities.
Rice bran polysaccharide is one of the main effective ingredients in rice bran. In previous researches, many biologically active polysaccharides extracted from rice bran appeared to elicit excellent physiological properties in maintaining health and preventing diseases [10–13]. However, the chemical modification and antitumor activity of polysaccharides from defatted rice bran have never been studied. In our previous research, we carried out experiments to purify a novel water-soluble heteropolysaccharide RBPS2a from the defatted rice bran, the research demonstrated that RBPS2a possessed anti-complementary activities, antitumor activities and immunity effect [14,15]. To our knowledge, there is limited literature on the sulfated modification of the rice bran polysaccharides. Therefore, it is important to establish an appropriate sulfated method for rice bran polysaccharides. In this paper, nine sulfated rice bran polysaccharides (sRBPS), with various degrees of sulfation (DS) and carbohydrate content, were prepared by CSA–Pyr method according to orthogonal test. Their antitumor activities were also investigated. The purpose of this study was to probe into the probability of the improving the antitumor activity of sRBPS through the sulfated modification and optimize the sulfated reaction condition of defatted rice bran polysaccharides. It may also provide a basic understanding of the correlation of DS to bioactivities. 2. Materials and methods
∗ Corresponding author at: State Key Laboratory of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi 214122, China. Tel.: +86 510 8591 7025; fax: +86 510 8591 7025. E-mail address: zxchen [email protected] (Z. Chen). 0141-8130/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2008.12.006
2.1. Materials and reagents Defatted rice bran was obtained from Hangzhou Zhonggu Grain & Oil Co., Ltd (Zhejiang Province, China). It was squeezed
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L. Wang et al. / International Journal of Biological Macromolecules 44 (2009) 211–214
and ground to pass through 1 mm sieve, and then stored at 4 ◦ C. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). Roswell Park Memorial Institute (RPMI)-1640 medium, phosphate-buffered saline (PBS) and fetal bovine serum were purchased from Gibco (Grand Island, NY, USA). Chlorosulfonic acid (CSA) and pyridine (Pyr) were produced in Sinopharm Chemical Reagent Co., Ltd. All other chemical reagents were of analytical reagent grade. 2.2. Preparation of RBPS2a sample RBPS2a fraction was isolated and purified according to a previous method described by Wang et al. [14]. Briefly, defatted rice bran was extracted with 15 times of water at 90 ◦ C for 2 h. After removed the proteins and starch, the crude polysaccharide fraction was obtained by ethanol precipitation and dissolved with distilled water. RBPS2a was obtained by applying the water-soluble polysaccharide fraction using Q-Sepharose big beads anion exchange chromatography (Pharmacia AP, Sweden) and Sepharose CL-6B gel chromatography (Pharmacia AP, Sweden). 2.3. Sulfated modification of polysaccharide 2.3.1. Design of modification of polysaccharide In order to investigate the effects of three factors on sulfation, the molar ratio of CSA to Pyr, the reaction time and the reaction temperature were tested at different concentrations. Nine reacting conditions were designed according to orthogonal test as L9 (33 ) (Table 1). Three levels per factor were used with the ratio of CSA to Pyr of 1:4, 1:6 and 1:8, the reaction time of 1, 2 and 3 h, and the reaction temperature of 45, 70 and 95 ◦ C, respectively. 2.3.2. Preparation of sulfating reagent The sulfation reagent, complex of CSA and Pyr was prepared by slowly adding CSA into Pyr (25 ml) filled in three-necked flask, with continuous stirring and cooling in an ice bath by keeping the temperature at 4–10 ◦ C. The ratio of CSA to Pyr referred to Table 1. All determinations were completed in 40 min and nine kinds of sulfating reagents were obtained.
the equation: DS =
1.62 × S% 32 − 1.02 × S%
2.5. Cell lines Human hepatoma cell line Hep-G2 was obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science. The murine B16 melanoma tumour cell strains were provided by Doctor Dahong Wang, School of Biotechnology, Jiangnan University. HeP-G2 and B16 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 IU/ml) and streptomycin (100 mg/l) in a humidified 5% CO2 atmosphere at 37 ◦ C. 2.6. Cell proliferation assay The proliferation of HeP-G2 and B16 cells was determined using the colorimetric MTT assay as described previously by Mosmann [19]. The tumour cells were inoculated on a 96-well cultivation plate at a concentration of 3 × 103 cells/well. Each well was inoculated with 100 l Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum solution containing the tumor cells for 24 h before addition of the samples. The concentrations of the sample were 50, 100, 250 and 500 g/ml while the negative controls were treated with the medium only. Incubation was carried out for another 48 h. The tumor cells were continuously inoculated for another 4 h after 20 l MTT (5 g/L) had been added. The medium was removed, and then 150 l dimethyl sulfoxide (DMSO) was added to every well to terminate the reaction. The inhibition of cell growth survival rate of the tumor cells was assayed by measuring the optical intensity by an auto enzymelabeled meter (Multiskan MK3, Finland) at 570 nm. The sample groups were compared with control group in the absence of the tested samples. All in vitro results were expressed as the inhibition ratio of tumor cell proliferation as follows:
A − B A
× 100%
where A and B are the average number of viable tumor cells of the control group and test group, respectively. 2.7. Data handing
2.3.3. Sulfation reaction Chemical sulfation of RBPS2a was carried out by using the chlorosulfonic acid–pyridine (CSA–Pyr) method described by Yoshiada et al. [16]. 200 mg RBPS2a was suspended in 20 ml anhydrous dimethyl formamide (DMF), and the sulfation reagent was added. The mixture was performed at different conditions (Table 1). After the reaction, the compound was cooled to room temperature, neutralized with 2.5 M NaOH and precipitated with 95% ethanol. The sediment was re-dissolved with water and dialysis sack against tap water for 48 h and distilled water for 24 h to remove pyridine, salt and potential degradation products. At last, nine sulfated polysaccharides named sRBP1 , sRBP2 , sRBP3 , sRBP4 , sRBP5 , sRBP6 , sRBP7 , sRBP8 , sRBP9 were collected after lyophilizing. 2.4. Content determination of sRBPS Total carbohydrate was estimated by the phenol-sulfuric acid method mentioned by Dubois et al. [17]. The sulfur contents of derivatives were determined by Dodgson and Price’s method [18]. A calibration curve was constructed with sodium sulfate as standard. The degrees of substitution (DS) was calculated according to
Data were expressed as means ± S.D. Data in all the bioassays were statistically evaluated by Student’s t-test and P < 0.05 was considered significant. 3. Results and discussion 3.1. The yield, DS and carbohydrate content of sRBPS2a The sulfated modification conditions of orthogonal design and the yield, DS and carbohydrate content of sRBPS2a are listed in Table 1. The results indicated that sRBPS1 had the highest yield of 126 mg and next was sRBPS2 (101 mg). The lowest yield was 26 mg of sRBPS7 . The carbohydrate contents of nine sulfates studied varied from 18.34% to 78.56%. The highest carbohydrate content was sRBPS2 (78.56%), while the lowest was sRBPS7 (18.34%). The DS for nine sulfates of RBPS2a followed the order: sRBPS2 > sRBPS5 > sRBPS1 > sRBPS4 > sRBPS6 > sRBPS3 > sRBPS8 > sRBPS7 > sRBPS9 and were 1.29, 0.92, 0.85, 0.81, 0.53, 0.39, 0.16, 0.08 and 0.04, respectively. Analyses on the orthogonal array design indicated that variable A, namely, the molar ratio of CSA to Pyr had the highest R
L. Wang et al. / International Journal of Biological Macromolecules 44 (2009) 211–214
213
Table 1 Sulfation of RBPS2a. Reaction conditions
Results
Products
A CSA:Pyr
B Temperature (◦ C)
C Time (h)
Yield (mg)
Carbohydrate content (%)
DS
sRBPS1 sRBPS2 sRBPS3 sRBPS4 sRBPS5 sRBPS6 sRBPS7 sRBPS8 sRBPS9
1:4 1:4 1:4 1:6 1:6 1:6 1:8 1:8 1:8
45 70 95 45 70 95 45 70 95
1 2 3 3 1 2 2 3 1
126 101 47 85 72 63 26 61 39
52.93 78.56 29.74 44.57 41.41 36.55 18.34 31.86 24.59
0.85 1.29 0.39 0.81 0.92 0.53 0.08 0.16 0.04
value of 2.25 and exerted the largest effect on DS. The extent of the impact of variables on DS followed the order: variable C (reaction time) < B (reaction temperature) < A (molar ratio of CSA to Pyr). Many researches confirmed that the increase of reaction temperature, the prolongation of reaction time and the enhancement of the molar ratio of CSA to Pyr may contribute to high DS [20]. In this reaction, the molar ratio of CSA to Pyr and reaction temperature was the two major factors affecting DS and carbohydrate content. DS of products increased very rapidly with increasing of the molar ratio of CSA to Pyr, but if the proportion of CSA was over high, it could cause hardening, difficult stirring and uneven reaction in practical manipulation. DS and carbohydrate content could be increased when reaction temperature was gone up to 70 ◦ C from 45 ◦ C, but the DS decreased dramatically when the temperature up to 95 ◦ C. This indicated that reaction should be in the relatively mild condition. Yang et al. [21] have shown that the rate of reaction was higher in the primary stage, about 85% of the possible substitution occurs within the first hour, which was also confirmed by our experiment.
The results of in vitro assay of RBPS2a and the nine derivatives against HeP-G2 cells and B-16 cells are summarized in Tables 2 and 3. From the results we could find that sRBPS1 and sRBPS2 presented significantly higher antitumor activity against the Hep-G2 cells and B16 cells in vitro, and the inhibition ability was dose-dependent. At 500 mg/ml, the inhibition rates of sRBPS1 on the Hep-G2 cells and B16 cells were 46.75% and 54.21%, respectively. sRBPS6 only presented obvious higher antitumor activity against the B16 cells (38.81%). sRBPS4 and sRBPS5 also showed markedly antitumor activity against the B16 cells (39.46% and 41.22%, respectively) and Hep-G2 cells (33.47% and 37.84%, respectively) at 500 mg/ml. No obvious antitumor activity was observed with sRBPS7 , sRBPS8 and sRBPS9 , indicating that they were inef-
fective in suppressing the growth of the Hep-G2 cells and B16 cells. These differences in antitumor activities may be attributed to their different parameters, such as the degree of sulfation, the carbohydrate content, the sulfation position, type of sugar, and glycosidic branching. Among them, DS and the carbohydrate content of a sulfated polysaccharide are two major factors for its biological effects [21]. It was reported that when the DS within scope of 1.5–2.0, the sulfated derivatives showed the excellent biological activity [22]. However, Huang et al. [23] found that sulfated polysaccharide from Poric cocos mycelia with the DS of 0.89 had markedly antitumor activities against Hep-G2 and Sarcoma 180 solid tumor cells. In this research, the inhibiting effects on two kinds of tumor cell lines are changed along with the DS of nine polysaccharide derivatives. It seems that polysaccharides with relatively higher DS exhibit stronger antitumor activity in vitro. sRBPS2 and sRBPS5 with the higher DS (1.29 and 0.92) presented the best activity. Furthermore, sRBPS7, sRBPS8 and sRBPS9 with lower DS (0.08, 0.16 and 0.04) showed ineffective activity. The result also indicated that the antitumor activity of sulfated polysaccharides was direct correlation with carbohydrate content, sRBPS2 and sRBPS1 with higher carbohydrate content (78.56% and 52.93%) displayed significantly efficacy, which was in accordance with the reports of Lu et al. [24]. From the results, we could find that DS and carbohydrate content of defatted rice bran polysaccharides sulfates must be within the optimum scope. When DS within the scope of 0.81–1.29, carbohydrate content in the range of 41.41–78.56%, sulfated derivatives exhibit relatively strong antitumor activity in vitro. Biological properties strongly depend on DS, while DS determined by various reaction conditions. According to antitumor activity, DS and carbohydrate content of sRBPS, the optimum sulfated conditions of defatted rice bran polysaccharide RBPS2a should be 70 ◦ C of the reaction temperature, 1:4 of CSA–Pyr ratio and 2 h of reaction time.
Table 2 Growth inhibition of S. polysaccharides at different concentrations against the human hepatoma cell line Hep-G2 in vitro.
Table 3 Growth inhibition of S. polysaccharides at different concentrations against the murine B16 melanoma tumour cell strains in vitro.
Groups
Groups
3.2. Antitumor activity of RBPS2a and nine derivatives
RBPS2a sRBP1 sRBP2 sRBP3 sRBP4 sRBP5 sRBP6 sRBP7 sRBP8 sRBP9
Concentration (g/ml) 500
250
100
50
35.09* 46.75** 43.35** 30.75* 33.47* 37.84** 21.36 13.12 8.37 10.29
26.4* 30.54* 37.53** 17.24 24.39 31.28* 19.35 10.23 12.38 9.37
19.73 26.89* 27.46* 12.13 20.74 16.32 12.59 3.29 2.28 3.97
10.98 8.24 12.93 7.39 10.37 6.24 5.38 4.97 5.69 2.43
Significant differences from the control were evaluated using Student’s t-test: * P < 0.05, ** P < 0.01.
RBPS2a sRBP1 sRBP2 sRBP3 sRBP4 sRBP5 sRBP6 sRBP7 sRBP8 sRBP9
Concentration (g/ml) 500
250
100
50
31.09* 54.21** 53.87** 23.17 39.46** 41.22** 38.81* 10.56 9.07 8.49
33.42* 44.47** 39.95** 14.18 27.38* 33.87* 31.67* 5.37 6.54 7.76
21.73 27.38* 29.87* 10.94 18.65 30.85* 9.77 2.79 4.39 5.23
13.98 19.36 10.42 4.97 8.91 11.05 4.83 2.43 2.03 2.78
Significant differences from the control were evaluated using Student’s t-test: * P < 0.05, ** P < 0.01.
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The antitumor activity of the polysaccharide and their modified derivatives were usually believed to be a consequence of the stimulation of the cell-mediated immune response. From the point of view of structure, the introduction of sulfate groups to polysaccharide should change its physicochemical characterization and chain conformation. Wang et al. [25] reported that relatively expanded and stiff chain conformation of sulfated polysaccharides is very helpful to the improvement of their antitumor effect. Further structural studies on the sulfated polysaccharides isolated from defatted rice bran will play an indispensable role in the understanding of the mechanism of antitumor activity. The detailed mechanism of antitumor action of the sulfated polysaccharide from defatted rice bran is currently in progress in our lab. 4. Conclusions The chemically sulfated polysaccharides were derived from defatted rice bran polysaccharide RBPS2a by CSA–Pyr method. sRBPS2 with highest DS (1.29) and carbohydrate content (78.56%) has shown evident growth inhibition on B16 and HeP-G2 cells in vitro. As sRBPS2 had potent antitumor properties, it would be explored as a potential adjuvant against cancer used in the food and pharmaceutical therapy. Sulfated modification could be considered as the effective way to enhance the antitumor activities of defatted rice bran polysaccharides. Acknowledgements This work was supported by National Natural Science Foundation of China (20576048) and University Graduate Student Science Research Innovation Project of Jiangsu Province (2008). We thank Doctor Dahong Wang, School of Biotechnology, Jiangnan University, for his assistance in the experiments.
References [1] F.J. Cui, W.Y. Tao, Z.H. Xu, W.J. Guo, H.Y. Xu, Z.H. Ao, J. Jin, Y.Q. Wei, Bioresour. Technol. 98 (2007) 395–401. [2] R. Snoeck, R. Pauwels, E. Clercq, Antimicrob. Agents Chem. 32 (1998) 1742– 1745. [3] Z.H. Zhao, J. Li, X.M. Wu, H. Dai, X.M. Gao, M.J. Liu, P.F. Tu, Food Res. Int. 39 (2006) 917–923. [4] S. Alban, G. Franz, Thromb. Res. 99 (2002) 377–388. [5] K.V. Coothan, J. Anthony, P.P. Sreenivasan, V. Palaninathan, Food Chem. 100 (2007) 1552–1559. [6] H.B. Ferial, E. Mostafa, S. Corinne, B.V. Catherine, Thromb. Res. 100 (2000) 453–459. [7] L.B. Talarico, C.A. Pujol, R.G.M. Zibetti, P.C.S. Faría, M.D. Noseda, M.E.R. Duarte, E.B. Damonte, Antivir. Res. 66 (2005) 103–110. [8] X.B. Yang, X.D. Gao, F. Han, R.X. Tan, BBA-General Subjects 1725 (2005) 120–127. [9] Y.L. Lin, L.N. Zhang, L. Chen, Y. Jin, F.B. Zeng, J. Jin, B. Wan, C.K. Peter, Int. J. Biol. Macromol. 34 (2004) 231–236. [10] S. Takenaka, Y. Itoyama, Life Sci. 52 (1993) 9–12. [11] M. Ghoneum, S. Gollapudi, Cancer Lett. 201 (2003) 41–49. [12] J.H. Cummings, S.A. Bingham, K.W. Heaton, M.A. Eastwood, Gastroenterology 103 (1992) 1783–1789. [13] A.A. Qureshi, H. Mo, L. Packer, D.M. Peterson, J. Agric. Food Chem. 48 (2000) 3130–3140. [14] L. Wang, H.B. Zhang, X.Y. Zhang, Z.X. Chen, Food Chem. 110 (2008) 150–155. [15] Y.R. Jiang, H.Y. Yao, Z.X. Chen, Z. Gu, J. Chinese Cereal Oil Assoc. 19 (2004) 1–7. [16] T. Yoshiada, Y. Yasuda, T. Mimura, Y. Kaneko, Carbohyd. Res. 276 (1995) 425–436. [17] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Pebers, F. Smith, Anal. Chem. 28 (1956) 350–356. [18] K.S. Dodgson, R.G. Price, Biochem. J. 84 (1962) 106–110. [19] T. Mosmann, J. Immunol. Methods 65 (1983) 55–63. [20] J. Chen, G. Wu, J. Wang, C. Lu, W. Zhang, H. Ji, Chinese Trad. Herbal Drugs 1 (2005) 43–46. [21] J.H. Yang, Y.M. Du, R.H. Huang, Y.Y. Wan, T.Y. Li, Biomacromolecules 31 (2002) 55–62. [22] C. Wang, H. Guang, Adv. Bio. Pro. 2 (2000) 3–8. [23] Q.L. Huang, L.N. Zhang, P.K. Cheung, X.T. Tan, Carbohyd. Polym. 64 (2006) 337–344. [24] Y. Lu, D.Y. Wang, Y.L. Hu, X.Y. Huang, J.M. Wang, Carbohyd. Polym. 71 (2008) 180–186. [25] Y.F. Wang, L.N. Zhang, Y.Q. Li, X.H. Hou, F.B. Zeng, Carbohyd. Res. 339 (2004) 2567–2574.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Sulfated modification of the polysaccharides obtained from defatted rice bran and their antitumor activities Li Wang a,b , Xiaoxuan Li b , Zhengxing Chen a,b,∗ a b
State Key Laboratory of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi 214122, China School of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi 214122, China
a r t i c l e
i n f o
Article history: Received 18 November 2008 Received in revised form 7 December 2008 Accepted 10 December 2008 Available online 24 December 2008 Keywords: Rice bran polysaccharide Chemical modification Degrees of sulfation Antitumor activity
a b s t r a c t Nine sulfated defatted rice bran polysaccharides (sRBPS), with various degrees of sulfation (DS) and carbohydrate content, were prepared by chlorosulfonic acid–pyridine (CSA–Pyr) method according to orthogonal test. Nine sulfated derivatives sRBPS were obtained and their antitumor activities were compared by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results showed that when DS within the scope of 0.81–1.29, carbohydrate content in the range of 41.41–78.56%, sulfated derivatives exhibit relatively strong antitumor activity in vitro. The optimum modification conditions were reaction temperature of 70 ◦ C, the ratio of chlorosulfonic acid to pyridine of 1:4 and the reaction time of 2 h. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Polysaccharides have drawn the attention of biochemical and nutritional researchers in recent years due to their various biological activities [1–3]. Many studies have demonstrated that biological activities of polysaccharides are obviously increased by chemical modification. Molecular modification and structural improvement of polysaccharide appeared to elicit excellent physiological properties in maintaining health and preventing diseases [4,5]. Sulfated polysaccharide is a kind of polysaccharide with sulfated group in its hydroxyls. Many studies have confirmed that the sulfated polysaccharides exerted potent biological properties in comparison with non-sulfated polysaccharides, such as anti-coagulant [6], anti-virus [7], anti-oxidant [8] and antitumor activities [9]. Therefore, sulfated modification could be considered as the effective way to enhance the biological activities of polysaccharides. CSA–Pyr method was the most popular method to sulfate polysaccharides. Many studies have indicated that CSA–Pyr method posses advantages of high yield, high DS and convenient manipulation. The ratio of CSA to Pyr, reaction temperature and the reaction time are the important factors in this method. Polysaccharide performed at different conditions resulted in sulfated polysaccharides with various DS and bioactivities.
Rice bran polysaccharide is one of the main effective ingredients in rice bran. In previous researches, many biologically active polysaccharides extracted from rice bran appeared to elicit excellent physiological properties in maintaining health and preventing diseases [10–13]. However, the chemical modification and antitumor activity of polysaccharides from defatted rice bran have never been studied. In our previous research, we carried out experiments to purify a novel water-soluble heteropolysaccharide RBPS2a from the defatted rice bran, the research demonstrated that RBPS2a possessed anti-complementary activities, antitumor activities and immunity effect [14,15]. To our knowledge, there is limited literature on the sulfated modification of the rice bran polysaccharides. Therefore, it is important to establish an appropriate sulfated method for rice bran polysaccharides. In this paper, nine sulfated rice bran polysaccharides (sRBPS), with various degrees of sulfation (DS) and carbohydrate content, were prepared by CSA–Pyr method according to orthogonal test. Their antitumor activities were also investigated. The purpose of this study was to probe into the probability of the improving the antitumor activity of sRBPS through the sulfated modification and optimize the sulfated reaction condition of defatted rice bran polysaccharides. It may also provide a basic understanding of the correlation of DS to bioactivities. 2. Materials and methods
∗ Corresponding author at: State Key Laboratory of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi 214122, China. Tel.: +86 510 8591 7025; fax: +86 510 8591 7025. E-mail address: zxchen [email protected] (Z. Chen). 0141-8130/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2008.12.006
2.1. Materials and reagents Defatted rice bran was obtained from Hangzhou Zhonggu Grain & Oil Co., Ltd (Zhejiang Province, China). It was squeezed
212
L. Wang et al. / International Journal of Biological Macromolecules 44 (2009) 211–214
and ground to pass through 1 mm sieve, and then stored at 4 ◦ C. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). Roswell Park Memorial Institute (RPMI)-1640 medium, phosphate-buffered saline (PBS) and fetal bovine serum were purchased from Gibco (Grand Island, NY, USA). Chlorosulfonic acid (CSA) and pyridine (Pyr) were produced in Sinopharm Chemical Reagent Co., Ltd. All other chemical reagents were of analytical reagent grade. 2.2. Preparation of RBPS2a sample RBPS2a fraction was isolated and purified according to a previous method described by Wang et al. [14]. Briefly, defatted rice bran was extracted with 15 times of water at 90 ◦ C for 2 h. After removed the proteins and starch, the crude polysaccharide fraction was obtained by ethanol precipitation and dissolved with distilled water. RBPS2a was obtained by applying the water-soluble polysaccharide fraction using Q-Sepharose big beads anion exchange chromatography (Pharmacia AP, Sweden) and Sepharose CL-6B gel chromatography (Pharmacia AP, Sweden). 2.3. Sulfated modification of polysaccharide 2.3.1. Design of modification of polysaccharide In order to investigate the effects of three factors on sulfation, the molar ratio of CSA to Pyr, the reaction time and the reaction temperature were tested at different concentrations. Nine reacting conditions were designed according to orthogonal test as L9 (33 ) (Table 1). Three levels per factor were used with the ratio of CSA to Pyr of 1:4, 1:6 and 1:8, the reaction time of 1, 2 and 3 h, and the reaction temperature of 45, 70 and 95 ◦ C, respectively. 2.3.2. Preparation of sulfating reagent The sulfation reagent, complex of CSA and Pyr was prepared by slowly adding CSA into Pyr (25 ml) filled in three-necked flask, with continuous stirring and cooling in an ice bath by keeping the temperature at 4–10 ◦ C. The ratio of CSA to Pyr referred to Table 1. All determinations were completed in 40 min and nine kinds of sulfating reagents were obtained.
the equation: DS =
1.62 × S% 32 − 1.02 × S%
2.5. Cell lines Human hepatoma cell line Hep-G2 was obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science. The murine B16 melanoma tumour cell strains were provided by Doctor Dahong Wang, School of Biotechnology, Jiangnan University. HeP-G2 and B16 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 IU/ml) and streptomycin (100 mg/l) in a humidified 5% CO2 atmosphere at 37 ◦ C. 2.6. Cell proliferation assay The proliferation of HeP-G2 and B16 cells was determined using the colorimetric MTT assay as described previously by Mosmann [19]. The tumour cells were inoculated on a 96-well cultivation plate at a concentration of 3 × 103 cells/well. Each well was inoculated with 100 l Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum solution containing the tumor cells for 24 h before addition of the samples. The concentrations of the sample were 50, 100, 250 and 500 g/ml while the negative controls were treated with the medium only. Incubation was carried out for another 48 h. The tumor cells were continuously inoculated for another 4 h after 20 l MTT (5 g/L) had been added. The medium was removed, and then 150 l dimethyl sulfoxide (DMSO) was added to every well to terminate the reaction. The inhibition of cell growth survival rate of the tumor cells was assayed by measuring the optical intensity by an auto enzymelabeled meter (Multiskan MK3, Finland) at 570 nm. The sample groups were compared with control group in the absence of the tested samples. All in vitro results were expressed as the inhibition ratio of tumor cell proliferation as follows:
A − B A
× 100%
where A and B are the average number of viable tumor cells of the control group and test group, respectively. 2.7. Data handing
2.3.3. Sulfation reaction Chemical sulfation of RBPS2a was carried out by using the chlorosulfonic acid–pyridine (CSA–Pyr) method described by Yoshiada et al. [16]. 200 mg RBPS2a was suspended in 20 ml anhydrous dimethyl formamide (DMF), and the sulfation reagent was added. The mixture was performed at different conditions (Table 1). After the reaction, the compound was cooled to room temperature, neutralized with 2.5 M NaOH and precipitated with 95% ethanol. The sediment was re-dissolved with water and dialysis sack against tap water for 48 h and distilled water for 24 h to remove pyridine, salt and potential degradation products. At last, nine sulfated polysaccharides named sRBP1 , sRBP2 , sRBP3 , sRBP4 , sRBP5 , sRBP6 , sRBP7 , sRBP8 , sRBP9 were collected after lyophilizing. 2.4. Content determination of sRBPS Total carbohydrate was estimated by the phenol-sulfuric acid method mentioned by Dubois et al. [17]. The sulfur contents of derivatives were determined by Dodgson and Price’s method [18]. A calibration curve was constructed with sodium sulfate as standard. The degrees of substitution (DS) was calculated according to
Data were expressed as means ± S.D. Data in all the bioassays were statistically evaluated by Student’s t-test and P < 0.05 was considered significant. 3. Results and discussion 3.1. The yield, DS and carbohydrate content of sRBPS2a The sulfated modification conditions of orthogonal design and the yield, DS and carbohydrate content of sRBPS2a are listed in Table 1. The results indicated that sRBPS1 had the highest yield of 126 mg and next was sRBPS2 (101 mg). The lowest yield was 26 mg of sRBPS7 . The carbohydrate contents of nine sulfates studied varied from 18.34% to 78.56%. The highest carbohydrate content was sRBPS2 (78.56%), while the lowest was sRBPS7 (18.34%). The DS for nine sulfates of RBPS2a followed the order: sRBPS2 > sRBPS5 > sRBPS1 > sRBPS4 > sRBPS6 > sRBPS3 > sRBPS8 > sRBPS7 > sRBPS9 and were 1.29, 0.92, 0.85, 0.81, 0.53, 0.39, 0.16, 0.08 and 0.04, respectively. Analyses on the orthogonal array design indicated that variable A, namely, the molar ratio of CSA to Pyr had the highest R
L. Wang et al. / International Journal of Biological Macromolecules 44 (2009) 211–214
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Table 1 Sulfation of RBPS2a. Reaction conditions
Results
Products
A CSA:Pyr
B Temperature (◦ C)
C Time (h)
Yield (mg)
Carbohydrate content (%)
DS
sRBPS1 sRBPS2 sRBPS3 sRBPS4 sRBPS5 sRBPS6 sRBPS7 sRBPS8 sRBPS9
1:4 1:4 1:4 1:6 1:6 1:6 1:8 1:8 1:8
45 70 95 45 70 95 45 70 95
1 2 3 3 1 2 2 3 1
126 101 47 85 72 63 26 61 39
52.93 78.56 29.74 44.57 41.41 36.55 18.34 31.86 24.59
0.85 1.29 0.39 0.81 0.92 0.53 0.08 0.16 0.04
value of 2.25 and exerted the largest effect on DS. The extent of the impact of variables on DS followed the order: variable C (reaction time) < B (reaction temperature) < A (molar ratio of CSA to Pyr). Many researches confirmed that the increase of reaction temperature, the prolongation of reaction time and the enhancement of the molar ratio of CSA to Pyr may contribute to high DS [20]. In this reaction, the molar ratio of CSA to Pyr and reaction temperature was the two major factors affecting DS and carbohydrate content. DS of products increased very rapidly with increasing of the molar ratio of CSA to Pyr, but if the proportion of CSA was over high, it could cause hardening, difficult stirring and uneven reaction in practical manipulation. DS and carbohydrate content could be increased when reaction temperature was gone up to 70 ◦ C from 45 ◦ C, but the DS decreased dramatically when the temperature up to 95 ◦ C. This indicated that reaction should be in the relatively mild condition. Yang et al. [21] have shown that the rate of reaction was higher in the primary stage, about 85% of the possible substitution occurs within the first hour, which was also confirmed by our experiment.
The results of in vitro assay of RBPS2a and the nine derivatives against HeP-G2 cells and B-16 cells are summarized in Tables 2 and 3. From the results we could find that sRBPS1 and sRBPS2 presented significantly higher antitumor activity against the Hep-G2 cells and B16 cells in vitro, and the inhibition ability was dose-dependent. At 500 mg/ml, the inhibition rates of sRBPS1 on the Hep-G2 cells and B16 cells were 46.75% and 54.21%, respectively. sRBPS6 only presented obvious higher antitumor activity against the B16 cells (38.81%). sRBPS4 and sRBPS5 also showed markedly antitumor activity against the B16 cells (39.46% and 41.22%, respectively) and Hep-G2 cells (33.47% and 37.84%, respectively) at 500 mg/ml. No obvious antitumor activity was observed with sRBPS7 , sRBPS8 and sRBPS9 , indicating that they were inef-
fective in suppressing the growth of the Hep-G2 cells and B16 cells. These differences in antitumor activities may be attributed to their different parameters, such as the degree of sulfation, the carbohydrate content, the sulfation position, type of sugar, and glycosidic branching. Among them, DS and the carbohydrate content of a sulfated polysaccharide are two major factors for its biological effects [21]. It was reported that when the DS within scope of 1.5–2.0, the sulfated derivatives showed the excellent biological activity [22]. However, Huang et al. [23] found that sulfated polysaccharide from Poric cocos mycelia with the DS of 0.89 had markedly antitumor activities against Hep-G2 and Sarcoma 180 solid tumor cells. In this research, the inhibiting effects on two kinds of tumor cell lines are changed along with the DS of nine polysaccharide derivatives. It seems that polysaccharides with relatively higher DS exhibit stronger antitumor activity in vitro. sRBPS2 and sRBPS5 with the higher DS (1.29 and 0.92) presented the best activity. Furthermore, sRBPS7, sRBPS8 and sRBPS9 with lower DS (0.08, 0.16 and 0.04) showed ineffective activity. The result also indicated that the antitumor activity of sulfated polysaccharides was direct correlation with carbohydrate content, sRBPS2 and sRBPS1 with higher carbohydrate content (78.56% and 52.93%) displayed significantly efficacy, which was in accordance with the reports of Lu et al. [24]. From the results, we could find that DS and carbohydrate content of defatted rice bran polysaccharides sulfates must be within the optimum scope. When DS within the scope of 0.81–1.29, carbohydrate content in the range of 41.41–78.56%, sulfated derivatives exhibit relatively strong antitumor activity in vitro. Biological properties strongly depend on DS, while DS determined by various reaction conditions. According to antitumor activity, DS and carbohydrate content of sRBPS, the optimum sulfated conditions of defatted rice bran polysaccharide RBPS2a should be 70 ◦ C of the reaction temperature, 1:4 of CSA–Pyr ratio and 2 h of reaction time.
Table 2 Growth inhibition of S. polysaccharides at different concentrations against the human hepatoma cell line Hep-G2 in vitro.
Table 3 Growth inhibition of S. polysaccharides at different concentrations against the murine B16 melanoma tumour cell strains in vitro.
Groups
Groups
3.2. Antitumor activity of RBPS2a and nine derivatives
RBPS2a sRBP1 sRBP2 sRBP3 sRBP4 sRBP5 sRBP6 sRBP7 sRBP8 sRBP9
Concentration (g/ml) 500
250
100
50
35.09* 46.75** 43.35** 30.75* 33.47* 37.84** 21.36 13.12 8.37 10.29
26.4* 30.54* 37.53** 17.24 24.39 31.28* 19.35 10.23 12.38 9.37
19.73 26.89* 27.46* 12.13 20.74 16.32 12.59 3.29 2.28 3.97
10.98 8.24 12.93 7.39 10.37 6.24 5.38 4.97 5.69 2.43
Significant differences from the control were evaluated using Student’s t-test: * P < 0.05, ** P < 0.01.
RBPS2a sRBP1 sRBP2 sRBP3 sRBP4 sRBP5 sRBP6 sRBP7 sRBP8 sRBP9
Concentration (g/ml) 500
250
100
50
31.09* 54.21** 53.87** 23.17 39.46** 41.22** 38.81* 10.56 9.07 8.49
33.42* 44.47** 39.95** 14.18 27.38* 33.87* 31.67* 5.37 6.54 7.76
21.73 27.38* 29.87* 10.94 18.65 30.85* 9.77 2.79 4.39 5.23
13.98 19.36 10.42 4.97 8.91 11.05 4.83 2.43 2.03 2.78
Significant differences from the control were evaluated using Student’s t-test: * P < 0.05, ** P < 0.01.
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The antitumor activity of the polysaccharide and their modified derivatives were usually believed to be a consequence of the stimulation of the cell-mediated immune response. From the point of view of structure, the introduction of sulfate groups to polysaccharide should change its physicochemical characterization and chain conformation. Wang et al. [25] reported that relatively expanded and stiff chain conformation of sulfated polysaccharides is very helpful to the improvement of their antitumor effect. Further structural studies on the sulfated polysaccharides isolated from defatted rice bran will play an indispensable role in the understanding of the mechanism of antitumor activity. The detailed mechanism of antitumor action of the sulfated polysaccharide from defatted rice bran is currently in progress in our lab. 4. Conclusions The chemically sulfated polysaccharides were derived from defatted rice bran polysaccharide RBPS2a by CSA–Pyr method. sRBPS2 with highest DS (1.29) and carbohydrate content (78.56%) has shown evident growth inhibition on B16 and HeP-G2 cells in vitro. As sRBPS2 had potent antitumor properties, it would be explored as a potential adjuvant against cancer used in the food and pharmaceutical therapy. Sulfated modification could be considered as the effective way to enhance the antitumor activities of defatted rice bran polysaccharides. Acknowledgements This work was supported by National Natural Science Foundation of China (20576048) and University Graduate Student Science Research Innovation Project of Jiangsu Province (2008). We thank Doctor Dahong Wang, School of Biotechnology, Jiangnan University, for his assistance in the experiments.
References [1] F.J. Cui, W.Y. Tao, Z.H. Xu, W.J. Guo, H.Y. Xu, Z.H. Ao, J. Jin, Y.Q. Wei, Bioresour. Technol. 98 (2007) 395–401. [2] R. Snoeck, R. Pauwels, E. Clercq, Antimicrob. Agents Chem. 32 (1998) 1742– 1745. [3] Z.H. Zhao, J. Li, X.M. Wu, H. Dai, X.M. Gao, M.J. Liu, P.F. Tu, Food Res. Int. 39 (2006) 917–923. [4] S. Alban, G. Franz, Thromb. Res. 99 (2002) 377–388. [5] K.V. Coothan, J. Anthony, P.P. Sreenivasan, V. Palaninathan, Food Chem. 100 (2007) 1552–1559. [6] H.B. Ferial, E. Mostafa, S. Corinne, B.V. Catherine, Thromb. Res. 100 (2000) 453–459. [7] L.B. Talarico, C.A. Pujol, R.G.M. Zibetti, P.C.S. Faría, M.D. Noseda, M.E.R. Duarte, E.B. Damonte, Antivir. Res. 66 (2005) 103–110. [8] X.B. Yang, X.D. Gao, F. Han, R.X. Tan, BBA-General Subjects 1725 (2005) 120–127. [9] Y.L. Lin, L.N. Zhang, L. Chen, Y. Jin, F.B. Zeng, J. Jin, B. Wan, C.K. Peter, Int. J. Biol. Macromol. 34 (2004) 231–236. [10] S. Takenaka, Y. Itoyama, Life Sci. 52 (1993) 9–12. [11] M. Ghoneum, S. Gollapudi, Cancer Lett. 201 (2003) 41–49. [12] J.H. Cummings, S.A. Bingham, K.W. Heaton, M.A. Eastwood, Gastroenterology 103 (1992) 1783–1789. [13] A.A. Qureshi, H. Mo, L. Packer, D.M. Peterson, J. Agric. Food Chem. 48 (2000) 3130–3140. [14] L. Wang, H.B. Zhang, X.Y. Zhang, Z.X. Chen, Food Chem. 110 (2008) 150–155. [15] Y.R. Jiang, H.Y. Yao, Z.X. Chen, Z. Gu, J. Chinese Cereal Oil Assoc. 19 (2004) 1–7. [16] T. Yoshiada, Y. Yasuda, T. Mimura, Y. Kaneko, Carbohyd. Res. 276 (1995) 425–436. [17] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Pebers, F. Smith, Anal. Chem. 28 (1956) 350–356. [18] K.S. Dodgson, R.G. Price, Biochem. J. 84 (1962) 106–110. [19] T. Mosmann, J. Immunol. Methods 65 (1983) 55–63. [20] J. Chen, G. Wu, J. Wang, C. Lu, W. Zhang, H. Ji, Chinese Trad. Herbal Drugs 1 (2005) 43–46. [21] J.H. Yang, Y.M. Du, R.H. Huang, Y.Y. Wan, T.Y. Li, Biomacromolecules 31 (2002) 55–62. [22] C. Wang, H. Guang, Adv. Bio. Pro. 2 (2000) 3–8. [23] Q.L. Huang, L.N. Zhang, P.K. Cheung, X.T. Tan, Carbohyd. Polym. 64 (2006) 337–344. [24] Y. Lu, D.Y. Wang, Y.L. Hu, X.Y. Huang, J.M. Wang, Carbohyd. Polym. 71 (2008) 180–186. [25] Y.F. Wang, L.N. Zhang, Y.Q. Li, X.H. Hou, F.B. Zeng, Carbohyd. Res. 339 (2004) 2567–2574.