Antioxidant and antitumor activities in vitro of polysaccharides from E. sipunculoides

International Journal of Biological Macromolecules 78 (2015) 56–61

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Antioxidant and antitumor activities in vitro of polysaccharides from E. sipunculoides Rongjun He, Yuejun Zhao, Ruina Zhao, Peilong Sun ∗ Department of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310032, PR China

a r t i c l e

i n f o

Article history: Received 13 November 2014 Received in revised form 22 March 2015 Accepted 23 March 2015 Available online 31 March 2015 Keywords: E. sipunculoides Polysaccharide Antioxidant Antitumor

a b s t r a c t Three polysaccharides (SAP30, SAP60 and SAP80) were separated from the body of Edwardsia sipunculoides by tissue homogenate and papain hydrolysis. Total soluble sugar contents, monosaccharide compositions, antioxidant and antitumor activities in vitro of those polysaccharides were investigated, respectively. Results showed that the total soluble sugar contents of SAP composed of Man, GlcN, Rha, GalN, GlcUA, Glc, Gal, Xyl and Fuc were more than 85% estimated by the phenol-sulfuric acid assay. In addition, SAP had potential antioxidant and antitumor activities. SAP30 has the most significant antitumor effect. This study suggested that SAP could be a potential natural antioxidants and antitumor agents. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In recent years, marine resources have attracted worldwide attentions in searching for bioactive substance to develop new drugs and healthy foods [1], because of their relatively low toxicity and high bioactivities. Edwardsia sipunculoides (E. sipunculoides), one of the sea anemone, belongs to Cnidaria, Anthozoa, Actiniaria. It is one kind of the most ancient predatory animals, inhabiting in intertidal mudflats [2]. E. sipunculoides produces various bioactive substances, mainly of proteins and polysaccharides [3]. At present, many experts dedicated to the study on the toxins from sea anemone, like structure [4–7] and antitumor [8–11] activities, but the important value of polysaccharide has been overlooked. The main purpose of this study was to investigate the bioactivities of polysaccharides from E. sipunculoides (SAP) especially for its antioxidant and antitumor activities. This might prompt to explore the potential economic values of the polysaccharides from E. sipunculoides. 2. Materials and methods 2.1. Materials E. sipunculoides were captured from coastal Taizhou, Zhejiang Province, PR China, and frozen at −20 ◦ C. RPMI 1640 medium

∗ Corresponding author. Tel.: +86 571 8832 0388; fax: +86 571 8832 0604. E-mail address: peilong [email protected] (P. Sun). http://dx.doi.org/10.1016/j.ijbiomac.2015.03.030 0141-8130/© 2015 Elsevier B.V. All rights reserved.

(1×), PBS (1×) and 0.25% Trypsin & 0.02% EDTA bought from Gino Biological Pharmaceutical Co. DMSO and MTT were obtained from Shanghai Ze Scale Biotechnology Co. Fetal Calf Serum came from Zhejiang Biotechnology Co. All cells (A549, S180 and Du145) were obtained from Shanghai Cell Bank of Chinese Academy of Science. d-Mannose, d-galactose, l-fucose, d-glucose, d-glucosamine· HCl, d-galactosamine hydrochloride, N-acetyl-d-glucosamine, l-rhamnose, d-xylose and 2,2-azino-bis(3-ethylbenzthiazoline6-sulfonic) acid (ABTS) were purchased from Sigma–Aldrich (Sigma–Aldrich GmbH, Sternheim, Germany). Other chemicals were all of analytical reagents and purchased from Sigma–Aldrich and Merck. HPLC was carried out on a Waters 2695 system (2695 HPLC Pump, 2487 detector).

2.2. Extraction and purification of SAP The bodies of E. sipunculoides were firstly homogenized by mechanical homogenizer for 10 min followed by extracted at 30 ◦ C for 2 h (the ratio of water to mantle tissue was 8:1). Then the mixture was centrifuged at 10,000 × g for 10 min. The residue was extracted twice. The total supernatant was concentrated to one tenth of the volume by vacuum evaporation. SAP30, SAP60 and SAP80 were obtained by a graded ethanol precipitation, at final concentrations of 30%, 60% and 80% ethanol, respectively. The precipitates were collected by centrifugation at each time. Proteins were hydrolyzed by papain at 65 ◦ C for 2 h, then removed by Sevag reagent (chloroform:n-butyl alcohol = 4:1)[12]. Finally, the

R. He et al. / International Journal of Biological Macromolecules 78 (2015) 56–61

three fractions of preliminary polysaccharides were obtained by lyophilization. 2.3. Analytical methods 2.3.1. Preliminary chemical analysis Total soluble sugar (TSS) content was measured by phenolsulfuric acid method, using glucose as the standard [13]. The concentration of total protein was estimated by the Coomassie brilliant blue method [14]. The content of total reducing sugar (TRS) was determined by the method of 3,5-dinitrosalycylic acid [15]. 2.3.2. Monosaccharides composition Monosaccharide components of the polysaccharides were analyzed by HPLC method based on Harazono et al. [16] and Dai et al. [17] with some modifications. Briefly, the polysaccharide samples were hydrolyzed with 4 mL trifluoroacetic acid (2 M) for 2 h at 110 ◦ C. The excess trifluoroacetic acid was completely removed from hydrolyzates by vacuum evaporating with methanol to dryness thrice. The hydrolysate and standard monosaccharide mixtures (l-rhamnose, d-galactose, l-fucose, d-glucose, d-xylose, d-galactosamine hydrochloride, N-acetyl-dglucosamine, d-mannose and d-glucosamine·HCl) were dissolved in 200 ␮L PMP methanolic (0.5 M) solution. After added 200 ␮L of NH3 solution, the whole mixture was incubated for 30 min at 70 ◦ C and then was cooled down to room temperature with addition of 1 mL water. The solution was dried by vacuum evaporation under 50 ◦ C. The residue was dissolved in 1 mL water and chloroform respectively. After vigorous shaking, aqueous layer was analyzed. It was filtered through a 0.45 ␮m syringe filter, subsequently, subjected to HPLC (Waters Symmetry ShieldTM RP 18, 5 ␮m, 250 mm × 4.6 mm) with UV detector at 245 nm, isocratic elution with 20 mM ammonium acetate aqueous solution (pH 5.5) and acetonitrile in a ratio of 78:22 (v/v, %) at a flow rate of 0.8 mL/min.

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50 ◦ C for 20 min. The reaction was terminated by 2.5 mL TCA (10%, w/v), and then mixed with 1.2 mL of 0.1% FeCl3 (w/v). Three test tubes have been done the same dose of experiment. After vortex oscillation, absorbance of the mixture was measured at 700 nm against a blank. Higher absorbance of the reaction mixture was an indicator of the greater reducing capability. 2.6. Cell line and culture All cells grew in RPMI 1640 medium containing 10% fetal calf serum (Gibco). Cells were maintained at 37 ◦ C in a humidified with 5% CO2 atmosphere. Exponentially growing cells were collected for the experiments. 2.7. Detection of adherent cell proliferation MTT assay was applied to observe the effect of SAP on the proliferation of different adherent cells (A549, Du145). Briefly, 5 × 103 cells in 200 ␮L per well was seeded in 96-well plates, after incubation for 24 h the supernatant was removed and the residue was washed with PBS (1×) for 2–3 times, and then added 200 ␮L of polysaccharides (31.25–250 ␮g/mL), respectively. There were 3 duplicate wells in each group, which were cultured for 48 h. At 4 h before the end of the culture, each well was added 20 ␮L of 5 mg/mL MTT, which was mixed evenly and incubated continuously for 4 h. Then the supernatant was discarded, 100 ␮L DMSO was added into each well. There were three duplicate wells in each group. The absorbance at 570 nm was recorded as Ai . A control sample containing the same amount of culture replaces the polysaccharides were measured as A0 . The inhibition rate was calculated as a percentage according to the following equation: Inhibition rate(%) =

 1 − (A − A )  0 i A0

× 100%.

2.4. ABTS radical scavenging activity assay 2,2 -Azino-bis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) assay was employed to measure the antioxidant activity of polysaccharides [18]. Briefly, ABTS + solution was prepared by 12 h of reaction of ABTS (7 mmol) with potassium persulfate (140 mmol) at 4 ◦ C in dark. The solution was diluted with phosphate buffer solution (phosphate buffer 10 mmol, pH 7.4) until the absorbance was 0.700 ± 0.005 at 734 nm. The depolarization assay started by mixing the diluted ABTS solution (3 mL) with different concentrations of polysaccharides (1 mL, 0.25–4 mg/mL). Three test tubes have been done the same dose of experiment. The mixture was allowed to react at room temperature for 10 min and the absorbance at 734 nm was recorded as Ai . A control sample containing the same amount of phosphate buffer and ABTS radical was measured as A0 . The absorbance of polysaccharides solution and phosphate buffer with the same amount of ABTS was recorded as Aj . The activity to clearance ABTS radical was calculated as a percentage according to the following equation:



ABTS clearance rate(%) =

1 − (Ai − Aj ) A0



× 100%.

2.8. Detection of suspension cell proliferation Growth inhibitory effect of SAP on suspension cell (S180) was determined by measuring the absorbance of 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) dye for living cells. Briefly, 5 × 103 cells in 100 ␮L per well were seeded in 96-well plates, then added different polysaccharides solutions (31.25–250 ␮g/mL) made up to 200 ␮L with culture medium. After incubation for 48 h, 20 ␮L of MTT (5 mg/mL) was added to each well and incubated for an additional 4 h at 37 ◦ C. Cell suspensions was centrifuged at 5000 × g for 10 min to discard the supernatant, followed by adding 100 ␮L DMSO with residue of each well. There were three duplicate wells in each group. The absorbance at 570 nm was recorded as Ai . A control sample containing the same amount of culture replaces the polysaccharides are measured as A0 . The inhibition rate was calculated as a percentage according to the following equation: Inhibition rate(%) =

 1 − (A − A )  0 i A0

× 100%.

2.5. Reducing power assay 2.9. Statistical analysis Reducing power was determined based on the method of Dorman and Hiltunen [19] with slight modifications. 2.5 mL polysaccharides solution at different concentrations of 0.25–4 mg/mL were mixed with 2.5 mL of 0.2 mol/L phosphate buffer (pH 6.6) and 2.5 mL of 1% K3 Fe(CN)6 (w/v). The mixture was then incubated at

One-way analysis of variance (ANOVA) was performed using the OriginLab (Origin Pro 8.5) software. Significant differences between means were determined by Tukey’s test at a significant level of P < 0.05.

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R. He et al. / International Journal of Biological Macromolecules 78 (2015) 56–61 Man

SAP80 SAP60 SAP30 Standard

Gal Rha

GlcN

Glc

GlcNAc GlcUA GalN

Xyl

Fuc

Standard

SAP30 SAP60 SAP80

10

5

15

20

25

30

min

35

40

45

50

55

60

Fig. 1. HPLC analysis of monosaccharide composition in samples.

Samples

SAP30

SAP60

SAP80

TSS (%) TRS (%) Protein (%)

95.611 ± 0.062a 9.084 ± 0.032a 0.063 ± 0.008a

87.060 ± 0.074c 4.784 ± 0.046b nd

88.267 ± 0.058b 1.011 ± 0.051c nd

Each value is expressed as means ± standard deviation (n = 3). Means with different letters within a row are significantly different (P < 0.05). nd: not detected.

3. Results and discussion

Absorbancy Data (Abs)

Table 1 The chemical composition of SAP30, SAP60 and SAP80.

3.1. Chemical properties and monosaccharide composition

3.2. UV absorption peak detection Results of ultraviolet full scan indicated that the SAP30, SAP60 and SAP80 were polysaccharides with a little protein or nucleic acid, due to little absorption at 250–280 nm in UV spectrum as shown in Figs. 2–4. These results were consistent with the results in Table 1.

200

220

240

260

280

300

320

340

360

380

400

360

380

400

Wavelength (nm) Fig. 2. SAP30 ultraviolet full scan.

Absorbancy Data (Abs)

Polysaccharides of animal contain high levels of protein. In this study, the protein from extracts was removed by papain hydrolysis and Sevag method. Chemical compositions of SAP30, SAP60 and SAP80 were shown in Table 1. The formula of TSS standard curve was y = 6.2147x + 0.0159 (R2 = 0.9993). The total sugar contents of SAP30, SAP60 and SAP80 were 95.61%, 87.06% and 88.27%, respectively. All fractions were significantly high in total soluble sugar, while SAP30 was the highest. The formula of TRS standard curve was y = 0.2095x + 0.0118 (R2 = 0.9970). The reducing sugar contents of SAP30, SAP60 and SAP80 were 9.08%, 4.78% and 1.01%, respectively. The reducing sugar contents were not significant of these polysaccharides. The formula of protein standard curve was y = 0.3179x − 0.003 (R2 = 0.9956). The protein could be detected in SAP30 only. It represented that the protein had been removed largely. The monosaccharide composition of SAP was shown in Fig. 1. SAP30 was mainly composed of Man, GlcN, Rha, GalN, GlcUA, Glc, Gal, Xyl and Fuc in a mole ratio of 0.38:0.62:0.34:0.79:0.08:0.40:1.00:2.24:0.16. Man, Glc, Gal and Xyl were the main sugar units, which account for 11.95%, 10.81%, 38.46% and 12.45% of the total sugar, respectively. SAP60 was made up of Man, GlcN, and Glc in a mole ratio of 1.38:1.00:0.43. Man was the main sugar unit, which contented nearly 59.84% of all the sugars. SAP80 was composed of Man, GlcN, GalN, Glc and Gal in a mole ratio of 0.72:1.00:1.04:0.56. The main sugar units were Man, Glc and Gal, accounting for 26.39%, 33.56% and 25.68%, respectively. These results established that three polysaccharides isolated from E. sipunculoides had different chemical compositions.

200

220

240

260

280

300

320

340

Wavelength (nm) Fig. 3. SAP60 ultraviolet full scan.

3.3. Antioxidant activities in vitro 3.3.1. ABTS radical assay ABTS radical scavenging ability has been widely applied to determination of total antioxidant capacity in biological samples. ABTS free radicals after oxidation can generate stable free radical; samples can make the mixed solution of fade after ABTS free radical reaction. Its characteristic absorbance value will decline, the more the fading reaction obvious, the higher the samples total

R. He et al. / International Journal of Biological Macromolecules 78 (2015) 56–61

59

SAP30 SAP60 SAP80

Absorbancy Data (Abs)

1.0

Absorbance Date (Abs)

0.8

0.6

0.4

0.2 200

220

240

260

280

300

320

340

360

380

400

Wavelength (nm)

0.0 1

0 Fig. 4. SAP80 ultraviolet full scan.

2

3

4

Concentration (mg/mL)

antioxidant capacity show [20]. ABTS radical scavenging activities of SAP30, SAP60 and SAP80 were tested and the results were presented in Fig. 5. The data indicated a positive correlation between the concentration and radical scavenging activities. Three kinds of polysaccharides exhibited good ability of scavenging ABTS radical. In the range of 0.25–1 mg/mL, ability of scavenging ABTS radical of the sample rose sharply with the increase of concentration. Especially, the scavenging rate can be 54.36% when SAP80 at a concentration of 0.25 mg/mL. These demonstrated that the scavenging ability of ABTS radical depends on their molecular weight. However, the ABTS scavenging ability of all components at the concentration of 1 mg/mL were the same as vitamin C (Vc), which showed that the three components had good scavenging activity of ABTS radical. 3.3.2. Reducing capability It has been reported that the antioxidant activities are positively correlated with the reducing power, prevention of chain initiation, radical scavenging and so on [21]. The reducing properties are generally associated with the presence of reductants, which is known to exert antioxidant action by breaking the free radical chain through donating a hydrogen atom. The reduction of Fe3+ to Fe2+ to measure the amounts of sample reduction ability through the sample. As shown in Fig. 6, higher absorbance value indicated stronger reducing power. Absorbance values of these three polysaccharides were increased with the increase of concentration at a good linear relationship. SAP60 exhibited less satisfactory reducing power at all tested concentrations than the others. SAP30 and SAP80

Fig. 6. Reducing power of the samples.

exhibited higher reducing power as its concentration increased. The reducing power of SAP30 and SAP80 were 0.679, 0.698 at 4 mg/mL, respectively. These two polysaccharides may contain reductoneassociated and hydroxide groups of polysaccharides which can act as electron donors and react with free radicals to convert them to be more stable products. 3.4. Antitumor activity in vitro Cancer is the leading death cause in urban and rural China [22]. The effect of chemical drug was notable, however, it also had high toxicity in some degree [23]. Naturally occurring drugs like polysaccharides which was extracted from plants, algae, fungi and animals, polysaccharides from the flower of tea plant [24], and Hyriopsis cumingii polysaccharides [25] have been proved its high tumor activity and low toxicity [24]. Thus, it is important to find a drug with stronger antitumor activity and low toxicity. 3.4.1. Growth inhibition of polysaccharides on A549 cancer cells Lung cancer is the one of the malignant tumor, the incidence is fast and the mortality rate is also high. To evaluate the effects of growth inhibition by SAP30, SAP60 and SAP80 on A549 cells, the cells were determined by MTT method with the concentration ranging from 31.25 to 250 ␮g/mL. Results in Fig. 7 indicated SAP30 SAP60 SAP80

60

100

SAP30 SAP60 SAP80 Vc

60

40

Inhibition rate (%)

Absorbance Date (Abs)

80

40

20

20

0

0 0

1

2

3

Concentration (mg/mL) Fig. 5. Scavenging effects of the samples on ABTS radical.

4

31.25

62.5

125

250

Concentration(μg/mL) Fig. 7. Inhibition effects of the samples on A549 cells.

60

R. He et al. / International Journal of Biological Macromolecules 78 (2015) 56–61

SAP30 SAP60 SAP80

60

SAP30 SAP60 SAP80

50 40

Inhibition rate (%)

Inhibition rate (%)

30

40

20

20 10 0 -10 -20

0 31.25

62.5

125

250

31.25

Concentration(μg/mL) Fig. 8. Inhibition effects of the samples on Du145 cells.

that SAP30, SAP60 and SAP80 inhibited the proliferation of A549 cells in a concentration-dependent manner. The IC50s of SAP30, SAP60 and SAP80 on A549 cells are 202.2 ␮g/mL, 391.3 ␮g/mL, and 390.5 ␮g/mL, respectively. These results suggested SAP30 had an acute cytotoxic effect on A549 cells while SAP60 and SAP80 presented relatively low antitumor activities. It is probably that the monosaccharide composition of SAP30 was more complex than the other two. SAP30 was composed of 8 kinds of monosaccharide. SAP60 was only 3 kinds of monosaccharide. SAP80 was a little more than SAP60, which was composed of 5 kinds of monosaccharide. The data demonstrated that SAP had a potent suppressing effect on the growth of A549 cells in vitro, especially the SAP30. When SAP30 reaches the highest concentration of 250 ␮g/mL, the inhibition rate can achieve 52.18%. 3.4.2. Growth inhibition of polysaccharides on Du145 cancer cells Prostate cancer is the most common malignant tumor of the male reproductive system. The method to evaluation of the growth inhibition effects of SAP30, SAP60 and SAP80 on Du145 cells is the same as A549 cells. As shown in Fig. 8, the inhibition rate of these three polysaccharides was increased with the increasing concentration. The IC50s of SAP30, SAP60 and SAP80 on Du145 cells are 258.7, 435, 323 ␮g/mL, respectively. Inhibitory effect of SAP30 on the Du145 was the most obviously in three kinds of polysaccharides. The maximum inhibition effect of SAP30 was obtained at 250 ␮g/mL (Inhibition rate = 46.74%). It showed that inhibitory effect of SAP30 and SAP80 both better than SAP60, while their total sugar content were also higher. It was probably still linked to the monosaccharide composition. SAP30 and SAP80 were more complex than SAP60. Therefore, the polysaccharides from E. sipunculoides (SAP) had potential value in inhibition on Du145 cells. 3.4.3. Growth inhibition of polysaccharides on S180 cancer cells S180 tumor cells were incubated with different concentrations (31.25–250 ␮g/mL) of SAP for 48 h, measured by MTT method. As shown in Fig. 9, all the polysaccharides exerted concentration dependent inhibition activities in vitro on S180 cells. The highest inhibition on S180 cell was 37.59%, suggesting that the SAP had a little significant cytotoxicity to S180 cells in vitro. The IC50s of SAP30, SAP60 and SAP80 on S180 cells, were 282.4, 383.4 and 374.7 ␮g/mL, respectively. The data demonstrated that, these three kinds of polysaccharides instead inhibitory effect on S180 cells has a promoting role at low concentrations (31.25 ␮g/mL). It had inhibitory effect on the cells when the concentration reaches 62.5 ␮g/mL.

62.5

125

250

Concentration(μg/mL) Fig. 9. Inhibition effects of the samples on S180 cells.

Therefore, the concentration of polysaccharides in restrain S180 tumor cells plays a crucial role. According to the results, it indicates that the higher antitumor and antioxidant activities of the three polysaccharides may be related to comprehensive effects of their molecular weight, monosaccharide composition and concentration. But the mechanisms between the chemical characteristics and bioactivities of polysaccharides need to be further investigated. 4. Conclusions SAP30, SAP60 and SAP80 were isolated from E. sipunculoides by tissue homogenate method and elimination albumen by enzymolysis with Sevag method. The total soluble sugar contents of three polysaccharides were all above 85%. SAP30 was mainly composed of Man, GlcN, Rha, GalN, GlcUA, Glc, Gal, Xyl and Fuc. SAP60 was made up of Man, GlcN and Glc. SAP80 was composed of Man, GlcN, GalN, Glc and Gal. The antioxidant activities were determined by ABTS radical assay and reducing power assay. It demonstrated that all of the polysaccharides exhibited antioxidant activity in a concentrationdependent manner. They had high effects on ABTS radical scavenging ability. The antitumor activities were studied by different cancer cells (A549, Du145 and S180) by MTT method. It suggested that SAP plays an important role on inhibition of tumor cell, and SAP30 has the most significant inhibition effect on tumor cell, especially on A549 tumor cell. Acknowledgments This work was supported by Natural Science Foundation of Zhejiang Province of China (No. LY12H28007). Furthermore, we are grateful to Taizhou Dongjing Aquatic Products Co., Ltd., China for providing the E. sipunculoides, and to Prof. Yuting Ding of Zhejiang University of Technology for the E. sipunculoides identification. References [1] T. Nagai, T. Yukimoto, Food Chem. 81 (2003) 327–332. [2] W. Stimpson, J. Rodgers, Proc. Acad. Natl. Sci. Phila. 7 (1855) 375–384. [3] B.N. Orlov, D.B. Gelashvili, Zootoksinologiya. Yadovitye zhivotnye i ihk yady (Zootoxinology. Venomous Animals and Their Venoms), Vysshaya Shkola, Moscow, 1985. [4] O. Castaneda, A.L. Harvey, Toxicon 54 (2009) 1119–1124.

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