International Journal of Biological Macromolecules 65 (2014) 573–580
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Partial characterization, antioxidant and antitumor activities of polysaccharides from Philomycusbilineatus Rongjun He a , Jiaming Ye a , Yuejun Zhao a , Weike Su b,∗ a
College of Biological and Environmental Engineering,Zhejiang University of Technology, Hangzhou 310032, PR China Key Laboratory of Pharmaceutical Engineering of Ministry of Education, College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, PR China b
a r t i c l e
i n f o
Article history: Received 12 November 2013 Received in revised form 21 December 2013 Accepted 4 January 2014 Available online 10 January 2014 Keywords: Slug (Philomycusbilineatus) Polysaccharide Antioxidant activity Antitumor activity
a b s t r a c t Four polysaccharides (PBP60-A, PBP60-B, PBP60-C and PBP60-D) were purified from slug (Philomycusbilineatus) by ion-exchange chromatography. The antioxidant activities were studied by ABTS, DPPH, hydroxyl radical, superoxide radical and reducing power assay. In vitro antitumor activities were evaluated by MTT assay. Results demonstrated that PBP60-A was mainly composed of Man, Rha, Glc, Gal, Xyl and Fuc in a mole ratio of 6.13:3.08:8.97:5.22:2.46:1.13. PBP60-B was composed of Man, GlcN, Rha, GalN, GlcU, Glc, Gal, Xyl and Fuc in a mole ratio of 0.90:0.31:1.15:0.37:0.24:1.02:3.84:0.93:1.99. PBP60-C and PBP60-D were composed of Man, GlcN, Rha, GalN, GlcU, Glc, Gal, Xyl, Fuc and an unknown monosaccharide. Antioxidant tests indicated that four polysaccharides exhibited significant antioxidant activities in a dose-dependent manner. PBP60-D presented relative stronger antioxidant activity. PBP60-C showed higher antitumor activity against A549 and MCF-7 cells in vitro. At concentration of500 g/mL, the antitumor activities of PBP60-C on theA549 and MCF-7 cells were 65.30% and 42.45%, respectively. These results indicated that polysaccharides from Philomycusbilineatus could be explored as potential natural antioxidants and cancer prevention agents. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Slug (Philomycusbilineatus) is a land molluscaphilomycidae animalisa pest in crops that is distributed widely in Europe, America and Asia. It is one of the traditional Chinese drugs which have been used for cough, stroke and Centipedebite [1]. Most of the previous studies of slug focus on the effects of environmental factors [2] and its habits and morphology [3]. Furthermore, slug was also considered as a useful model animal in neuroscience because of the simple nervous system with large neurons, higher learning capacity, and feasibility of in vitro [4,5]. Recently, several Chinese researchers reported that crude extracts from slug presented significant antitumor activities against A549 cells [6], Hela cells [7] and S-180 cells [8], indicating that slug extracts could be explored as cancer prevention agents. However, few data has been reported on the further chemical composition, structure and biologic activity of polysaccharide from slugs. Therefore, the aim of present work was to explore the chemical composition of different polysaccharide
∗ Corresponding author at: Laboratory of Pharmaceutical Engineering of Ministry of Education, College of Pharmaceutical Science, Zhejiang University of Technology, No.18 Chao Wang Rood, Hangzhou 310032, PR China. Tel.: +86 571 88320752; fax: +86 571 88320752. E-mail address: [email protected] (W. Su). 0141-8130/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2014.01.016
extracted from slugs. Moreover, their antioxidant activities and antitumor activities were also investigated.
2. Experimental 2.1. Materials and chemicals The slugs (Philomycusbilineatus) were collected in campus of Zhejiang University of Technology (Zhejiang province, China), and then they were frozen at −20 ◦ C. The human breast cancer cell line MCF -7 and human lung cancer cell line A549 were provided by the cell bank of the Shanghai Institute of Cell Biology (Shanghai, China). The DEAE Sepharose Fast Flow was obtained from GE Healthcare Life Science (Piscataway, NJ, USA). HPLC was carried out on a waters 2695 HPLC system (2695 HPLC Pump, 2414 Refractive Index Detector). Rhamnose, fucose, xylose, galactose, glucose, mannose, ferrozine, nitrobluetetrazolium (NBT), phenazinemethosulfate (PMS), reduced nicotinamide adenine dinucleotide (NADH), 2,2 -Azino-bis(3-ethylbenzthiazoline-6-sulfonic) acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), bovine serum albumin, 3-(4,5-dimetthylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), ferrozine, deoxyribose, dextrans, and trifluoroacetic acids (TFA) were obtained from Sigma-Aldrich (Sigma-Aldrich GmbH,
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Sternheim, Germany). All other chemicals used were in analytical grade and obtained from either Sigma-Aldrich or Merck. 2.2. Extraction and purification of polysaccharides The slug (Philomycusbilineatus) bodies were homogenized with distilled water and hydrolyzed by 0.4% trypsin (2000 U/mg) at 50 ◦ C for 7 h. Subsequently, it was centrifuged to remove the insoluble materials. The supernatant was deproteined by Sevag method and precipitated with ethanol that were successively fractionated by different gradient concentrations of ethanol (50% and 60%) into two fractions named PBP50 and PBP60. The crude polysaccharides were obtained by lyophylization. The PBP60 fraction was dissolved in distilled water, filtered through 0.45 m filter and applied to a DEAE-Sepharose Fast Flow column (XK 26 mm × 100 cm), successively eluted in order with distilled water, 0.1, 0.3 and 0.5 M NaCl solutions at flow rate of 3 mL/min. The relevant fractions were collected, concentrated, dialyzed and lyophilized, and determined by means of the phenolsulfuric acid assay. 2.3. Characterization of polysaccharide fractions 2.3.1. Preliminary chemical analysis Total carbohydrate content was determined by phenol-sulfuric acid method using D-glucose as the standard [9]. Protein was analyzed by Bradford method using bovine serum albumin (BSA) as the standard [10]. Uronic acid contents were analyzed colorimetrically by m-hydroxybiphenyl method using D-galacturonic acid as the standard [11]. 2.3.2. Monosaccharides composition The monosaccharide components of the polysaccharides were analyzed by HPLC method based on Harazono et al. [12] and Dai et al. [13] with some modifications. Briefly, the polysaccharide samples were hydrolyzed with 4 mL trifluoroacetic acid (2 M) in an oven at 110 ◦ C for 2 h.The hydrolyzate was vacuum evaporated with methanol to dryness, and the procedure was repeated thrice for trifluoroacetic acid to be removed. The hydrolyzate and standard monosaccharide mixtures (mannose, glucosamine, rhamnose, galactosamine, glucuronic acid, N-acetyl-glucosamine, glucose, galactose, xylose, and fucose) were dissolved in 0.5 M PMP methanolic solution (200 L). After adding 200 L of NH3 solution, the whole mixture was incubated at 70 ◦ C for 30 min and then was cooled down to room temperature with addition of 1 mL water. The solution was vacuum evaporated to dryness under 50 ◦ C. The residue was dissolved in water and chloroform (1 mL each). After vigorous shaking, organic layer was discarded. The final aqueous layer was filtered through a 0.45 m syringe filter and subsequently subjected to HPLC (Waters Symmetry ShieldTM RP 18, 5 m, 250 × 4.6 mm) analysis by UV absorbance 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.
solution was produced by mixing ABTS aqueous solution (final concentration 7 mM) with potassium per sulphate (final concentration 2.45 mM) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. After incubation, the ABTS radical solution was diluted with PBS (pH 7.0) to an absorbance of 0.70 ± 0.02 at 734 nm. The diluted solution (2 mL) was then mixed with 0.2 mL sample solution, and the mixture solution was incubated for 6 min at room temperature. The absorbance was measured at 734 nm. The scavenging activity on ABTS radical was calculated by the following formula:
Scavenging activity (%) = 1 −
(A1 − A2 ) × 100 A0
where A0 is the absorbance of the ABTS radical solution without sample, A1 is the absorbance of the ABTS radical solution with tested samples and A2 is the absorbance of ethanol solution with tested samples. 2.4.2. DPPH radical scavenging assay DPPH radical scavenging assay was assessed according to the previous method with a modification [15]. DPPH solution (0.1 mM, in 95% ethanol) was prepared and used fresh on the day of each test. 1.0 mL of water solution of sample at concentration of 0, 0.25, 0.5, 1.0, 2.0, 4.0 mg/mL was added to 2 mL DPPH solution. The mixture was shaken vigorously and kept in dark for 30 min and the absorbance was measured at 517 nm. The ability to scavenge DPPH was calculated as a percentage according to the equation:
Scavenging activity (%) = 1 −
(A1 − A2 ) × 100 A0
where A0 is the absorbance of water instead of sample solution, A1 is the absorbance of the sample solution and A2 is the absorbance of ethanol instead of DPPH solution. 2.4.3. Hydroxyl radical scavenging assay Hydroxyl radical scavenging activity was investigated by the method of Smirnoff and Cumbes [16] with a minor modification. 0.5 mL FeSO4 (1.5 mM) was mixed with 0.35 mL H2 O2 (6 mM), 0.15 mL sodium salicylate (20 mM) and 1 mL sample solution (0.25–4 mg/mL), then incubated for 1 h at 37 ◦ C. The absorbance of the hydroxylated salicylate complex was measured at 562 nm. The antioxidant activity was calculated with the following equation:
Scavenging activity (%) = 1 −
(A1 − A2 ) × 100 A0
where A0 is the absorbance of water instead of sample solution, A1 is the absorbance of the sample solution and A2 is the absorbance of water instead of sodium salicylate solution.
2.3.3. Infrared analysis FT-IR spectra were obtained on a Nicolet 6700 FT-IR spectrometer (Thermo Scientific, USA) in a range of 4000–400 cm−1 . The samples were measured as a KBr disc.
2.4.4. Superoxide radical scavenging activity assay Superoxide radical scavenging activity was performed based on the method described by Xu et al. [17] with some modification. Each 1.0 mL of sample solution, NBT solution (78 M of NBT in 20 mM sodium phosphate buffer, pH 7.4) and NADH solution (468 Mof NADH in 20 mM sodium phosphate buffer, pH 7.4) were mixed. After addition 0.4 mL of PMS solution (60 MPMS in 20 mM sodium phosphate buffer, pH 7.4), the mixture was incubated at 25 ◦ C for 5 min. The absorbance of the mixture was measured at 560 nm. The superoxide radical scavenging activity was calculated by the follow formula:
2.4. Antioxidant activity tests in vitro
Scavenging activity (%) = 1 −
2.4.1. ABTS radical scavenging assay ABTS radical scavenging assay was carried out by the method described by Re et al. [14] with some modifications. ABTS radical
where A0 is the absorbance of water instead of sample solution, A1 is the absorbance of the sample solution and A2 is the absorbance of water instead of PMS solution.
(A1 − A2 ) × 100 A0
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2.4.5. Reducing power assay The reducing power of samples was determined according to the method reported by Lei et al. [18] with minor modification. 2.5 mL samples (0.25–4.0 mg/mL), 2.5 mL pH 6.6 phosphate buffer (0.2 M) and 2.5 mL 1% (w/v) K3 (CN)6 were incubated at 50 ◦ C for 20 min. The reaction was terminated by adding 2.5 mL trichloroacetic acid (10%, w/v), and centrifuging at 2000 rpm for 15 min. The upper layer (5.0 mL) was mixed with 5 mL water and 1.2 mL ferric chloride (0.1%, w/v). The mixture was shaken and its absorbance was measured at 700 nm . The absorbance of the reaction mixture increasing indicates an increase of reduction capability. 2.5. In vitro antitumor activity A549 cells and MCF-7 cells were provided by the Cell Bank of Shanghai Institute of Cell Biology and maintained with RPMI 1640 medium containing 10% fetal bovine serum at 37 ◦ C in a humidified atmosphere with 5% CO2 . The inhibition effects of PBP60-A,PBP60-B, PBP60-C and PBP60D on the growths of A549 cells and MCF-7cells were evaluated in vitro by MTT assay [19]. Briefly, cells in the logarithmic growth phase were seeded in 96-well plates (5 × 103 cells/well) and incubated at 37 ◦ C in a humidified atmosphere with 5% CO2 for 24 h. The non-adherent cells in well were removed by washing with PBS for three times. After that, 100 L fresh medium and test sample solution were added to each well, and the cells were incubated for 48 h. At the end of the treatment, 20 L of MTT (5 mg/mL) was added to each well and the cells were cultured for another 4 h. The medium was then discarded and 100 L of DMSO was added to each well. The absorbance was measured at 570 nm by an ELISA Reader. The inhibition rate was calculated according to the formula below:
Inhibition rate(%) =
1−
Abssample Abscontrol
× 100
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Table 1 The chemical composition of PBP60-A, PBP60-B, PBP60-C and PBP60-D. Samples
PBP60-A
PBP60-B
PBP60-C
PBP60-D
Carbohydrate (%) Protein (%) Uronic acid (%)
71.79 ± 0.37 0.78 ± 0.02 nd
51.72 ± 1.71 1.38 ± 0.01 4.70 ± 0.19
53.96 ± 0.93 5.48 ± 0.25 10.85± 0.18
46.87 ± 2.78 10.99 ± 0.30 4.15 ± 0.23
Each value is expressed as mean means ±stand deviation (n = 3). Means with different letters within a row are significantly different (p < 0.05). nd: not detected.
PBP60-C and PBP60-D were 71.79%, 51.72%, 53.96% and 46.87%, respectively. Except for PBP60-A, there was no significant difference in total polysaccharide among the other three fractions. The protein contents of PBP60-A, PBP60-B, PBP60-C and PBP60-D were 0.78%, 1.38%, 5.48% and 10.99%, respectively. The uronic acid contents of PBP60-B, PBP60-C and PBP60-D were found to be 4.70%, 10.85% and 4.15%, respectively, indicating that PBP60-B, PBP60-C and PBP60-D were acid polysaccharides. There is no uronic acid in PBP60-A. The monosaccharide composition of PBP60s was shown in Fig. 2. PBP60-A is mainly composed of Man, Rha, Glc, Gal, Xyl and Fuc in the mole ratio of 6.13: 3.08: 8.97: 5.22: 2.46: 1.13. Man, Glc and Gal are the main sugar units, accounting for 22.70%, 33.23% and 19.35% of the total sugar, respectively. PBP60-B is made up of Man, GlcN, Rha, GalN, GlcU, Glc, Gal, Xyl and Fuc in a mole ratio of 0.90: 0.31: 1.15: 0.37: 0.24: 1.02: 3.84: 0.93: 1.99. Gal is main sugar unit, the total content of which nearly exceeded 35.73 mol% of the all sugar. PBP60-C and PBP60-D are composed of Man, GlcN, Rha, GalN, GlcU, Glc, Gal, Xyl, Fuc and an unknown monosaccharide. Except for the little unknown monosaccharide, PBP60-C is composed of Man, GlcN, Rha, GalN, GlcU, Glc, Gal, Xyl and Fuc in molar proportion of 0.69: 0.33: 1.49: 0.24: 0.25: 1.18: 3.91: 0.70: 1.51 and it is 0.70: 0.11: 0.75: 0.14: 0.08: 065: 1.67: 0.68: 0.74 for PBP60-D. These results demonstrated that four polysaccharides isolated from Philomycusbilineatus had different chemical composition.
2.6. Statistical analysis 3.3. FT-IR spectral analysis 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. 3. Results and discussion 3.1. Isolation of polysaccharides Polysaccharides from animal always contain high level of proteins, which brings difficulty for further purification and structural analysis [20]. Protease is often used to deproteinate the extracts of animal polysaccharides [21]. In this study, trypsin-hydrolysis, Sevag method and ethanol precipitation were used to prepare PBP60 from slugs (Philomycusbilineatus). Fig. 1 represents the chromatogram of crude polysaccharides (PBP60) subjected to a DEAE-Sepharose Fast Flow column. Four peaks were observed from distilled water and NaCl elution, and they were named as PBP60A (eluted with distilled water), PBP60-B (eluted with 0.1 M NaCl), PBP60-C (eluted with 0.3 M NaCl) and PBP60-D (eluted with 0.5 M NaCl). PBP60-C and PBP60-D showed single and symmetrically sharp peak on HPLC analysis (data not shown) among the four fractions. In current study, these major fractions were collected and dried for further physicochemical and biologic analysis. 3.2. Chemical properties and monosaccharide composition Chemical compositions of the four fractions were given in Table 1. The total polysaccharide contents of PBP60-A, PBP60-B,
FT-IR spectroscopy is typically used for identification of characteristic organic groups in the polysaccharides, especially O H, N H, and C O. FT-IR spectra of the four samples are shown in Fig. 3. The spectra are typical of polysaccharides in a broadly-stretched intense peak around 3400 cm−1 for O H stretching vibrations and a weak absorption peak about 2930 cm−1 for C H stretching vibrations. A relatively strong peak around 1650 cm−1 was attributed to C O stretching vibration, and absorption of 1540 cm−1 was N H bending vibration. The broader band of 1050 cm−1 was representative of C C stretching and C OH bending vibrations [22]. Furthermore, the presence of amide bands at 1650 cm−1 , 1540 cm−1 and 1410 cm−1 indicated the presence of some residual protein in the four polysaccharide fractions [23]. 3.4. In vitro antioxidant activity 3.4.1. ABTS radical scavenging activity ABTS assay is often used in evaluating total antioxidant power of compounds. Specific absorbance at 734 nm can be used in both organic and aqueous solvents as an index reflecting the antioxidant activity of the extracted polysaccharides [24]. The ABTS radical scavenging activities of the four polysaccharide fractions from Philomycusbilineatus were shown in Fig. 4(a). The figure illustrated the decrease in the concentration of ABTS radical due to the scavenging ability of polysaccharides. Their scavenging ability correlated well with increasing concentrations. Moreover, PBP60-C and PBP60-D fractions showed pronounced high radical scavenging activity; the scavenging ability of these four different fractions
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Fig. 1. Elution profiles of crude polysaccharides PBP60 on DEAE-Sepharose Fast Flow chromatography column with stepwise gradient of aqueous NaCl elute (0, 0.1, 0.3, and 0.5 M).
tested decreased in the order of PBP60-C > PBP60-D > PBP60B > PBP60-A, which were 73.12%, 68.32%, 51.62% and 31.97%, respectively. At the concentration of 4 mg/mL, all samples had strong scavenging activity for ABTS radical. 3.4.2. DPPH radical scavenging activity DPPH is a stable free radical compound, which shows a maximum absorption at 517 nm that can readily undergo scavenging by an antioxidant. It has been widely accepted as a tool for evaluating the free radical scavenging activities of natural compounds
[24,25]. Fig. 4(b) shows the DPPH radical scavenging activity of four fractions. The scavenging activity of polysaccharides PBP60-A, PBP60-B, PBP60-C, PBP60-D on inhibition of the DPPH radical was related to the concentration of the samples. Among four fractions, PBP60-D showed the strongest scavenging ability increased significantly with the concentration. At the concentration of 4.0 mg/mL, the scavenging effects of PBP60-A, PBP60-B, PBP60-C, PBP60-D on the DPPH radical were 33.73%, 43.14%, 47.39%, 66.39%, respectively. Several studies demonstrated that the content of uronic acid and protein group may were important factors which contribute to
Fig. 2. HPLC analysis of monosaccharide composition in samples (Man: mannose; GlcN: glucosamine; Rha: rhamnose; GalN: galactosamine; GlcUA: glucuronic acid; GlcNAc: N-acetyl-glucosamine; Glc: glucose; Gal; galactose; Xyl: xylose; Fuc: fucose and Unknown: unknown monosaccharide).
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Fig. 3. FT-IR spectra of four polysaccharides. (a)PBP60-A, (b) PBP60-B, (c) PBP60-C and (d) PBP60-D.
the promotion of DPPH radical scavenging activity of polysaccharide [26,27]. It indicated that the stronger scavenging activity of PBP60-D on DPPH radical may be partially due to the higher content of proteinous substance and uronic acid.
3.4.3. Hydroxyl radical scavenging activity Hydroxyl radical is considered to be the most reactive radical, which can react with most biomacromolecules in living cells and induce severe damage to the adjacent biomolecules [28,29]. In this experiment, hydroxyl radical is generated from well-known Fenton reaction (Fe2+ + H2 O2 → Fe3+ + •OH + OH), salicylic acid captures hydroxyl radical to form 2,3-dihydroxyl benzoic acid, and it had maximum absorbance at 510 nm. Antioxidant can compete with salicylic acid to reduce the generation of 2,3-dihydroxyl benzoic acid [26]. Fig. 4(c) described the scavenging ability on hydroxyl radical of four fractions; all the samples exhibited weak scavenging ability at low concentration, with only about 10% at the concentration of 0.25 mg/mL, but increasing quickly with higher concentration. At 4 mg/mL, the hydroxyl scavenging abilities of PBP60-A, PBP60-B, PBP60-C and PBP60-D were 48.34%, 55.15%, 59.57% and 66.39%, respectively. All samples showed obvious hydroxyl radical scavenging activity in a dose-dependent pattern. The hydroxyl radical scavenging ability was attributed to various mechanisms, such as suppression against hydroxyl radical generation, decomposition of peroxides and prevention of continued hydrogen abstraction [28]. The possibility of scavenging hydroxyl radical by polysaccharide was that polysaccharide could supply of hydrogen and combine with radical to terminate the radical chain reaction [30].
3.4.4. Superoxide radical scavenging activity Superoxide radical is a highly toxic species that is generated by metabolic process and physical irradiation, and it is considered as the primary ROS [31]. Superoxide radical can further interact with other molecules to form single oxygen, hydroxyl radical and hydrogen peroxide, which may increase lipid peroxide, cellular damage, local oxidative stress and pathological incidents [32,33]. The superoxide radical scavenging activities of the four fractions are shown in Fig. 4 (d). The scavenging activity of all samples correlated with increasing concentration. At concentration of 0.25 mg/mL, the scavenging rate of PBP60-D was up to 67.05%, however, the scavenging rate of PBP60-A, PBP60-B, PBP60-C were 26.09%, 32.01% and 46.10%, respectively. Four polysaccharides showed a relatively high level of superoxide radical scavenging ability at the concentration of 4.0 mg/mL. Among the four samples, PBP60-D showed strongest superoxide scavenging activity at all concentrations. Higher scavenging rate was found when the content of proteinous substances increased. The results were in accordance with the findings of Huang et al. [29]. But the correlation between the chemical characteristics and antioxidant activities of PBP60-D needs further investigation. 3.4.5. Reducing power The reducing power of a compound may serve as an indicator of its potential antioxidant activity. Higher absorbance value means stronger reducing power [21]. The reducing ability is generally associated with the presence of reductones, which have been shown to exert antioxidantaction by breaking the free radical chain by donating a hydrogen atom [31]. As shown in Fig. 4(e), the reducing power of all samples increased with the increase of
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Fig. 4. Antioxidant activities of PBP60-A, PBP60-B, PBP60-C and PBP60-D: (a) ABTS radical scavenging activity, (b) DPPH radical scavenging activity, (c) hydroxyl radical scavenging activity, (d) superoxide radicals scavenging activity, (e) reducing power.
concentrations. Obviously, the reducing power of PBP60-D was significantly higher than that of PBP60-A, PBP60-B and PBP60C.At concentration of 4 mg/mL; the reducing power of PBP60-A, PBP60-B, PBP60-C, PBP60-D were 0.609, 0.935, 1.196 and 1.479, respectively. The reason might be that polysaccharides from Philomycusbilineatus contain reductone-associated and hydroxide groups which can act as electron donors and react with free radicals to convert them to be more stable products. 3.5. In vitro antitumor activity Cancer is a formidable problematic disease on human health. The great majority of chemical compounds, which have high antitumor effects; however, they are also toxic to normal cells [34].
Therefore, it is important to investigate high efficiency antitumor compounds with little toxicity. Numerous polysaccharides extracted from plants, algae, fungi and animals have been proved to show significant antitumor activities with little toxicity to hosts, such as Camellia sinensispolysaccharides[17], Tricholomamatsutakepolysaccharides [35], Scolopendrasubspinipesmutilans L. Koch polysaccharides [36], etc. In this study, the antitumor activities of the four polysaccharides, PBP60-A, PBP60-B, PBP60-C and PBP60-D were tested on A549 cells and MCF-7 cells. As shown in Fig. 5 (a), PBP60-C and PBP60-D presented significant high antitumor activities against A549 cells at all concentrations, and the IC50 values of PBP60-C and PBP60-D were about 100 g/mL and 500 g/mL, respectively. However, the inhibition rates of PBP60-A and PBP60-B were low
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of 6.13: 3.08: 8.97: 5.22: 2.46: 1.13. PBP60-B is made up of Man, GlcN, Rha, GalN, GlcU, Glc, Gal, Xyl and Fuc in a mole ratio of 0.90: 0.31: 1.15: 0.37: 0.24: 1.02: 3.84: 0.93: 1.99. PBP60-C and PBP60-D are composed of Man, GlcN, Rha, GalN, GlcU, Glc, Gal, Xyl, Fuc and an unknown monosaccharide. FT-IR spectra of PBP60-A, PBP60B, PBP60-C and PBP60-D were similar, indicating the presence of some residual protein in the four polysaccharide fractions. Several assays in vitro were applied to evaluate the antioxidant activities of these polysaccharides. All the fractions showed significant free radical scavenging activities in a dose-dependent manner. PBP60-D exhibited stronger scavenging activities than the others, PBP60-A, PBP60-B and PBP60-C. Furthermore, PBP60-C showed significant inhibitory effects on growth of A549 cells and MCF-7cells. At concentration of 500 g/mL, the antitumor activities of PBP60-C on theA549 and MCF-7 cells were 65.30% and 42.45%, respectively. Acknowledgment This work was supported by Natural Science Foundation of Zhejiang Province of China (No. LY12H28007). References [1] [2] [3] [4] [5] [6] [7] [8] Fig. 5. Inhibitory effects in vitro of PBP60-A, PBP60-B, PBP60-C and PBP60-D against (a) A549 cells, (b) MCF-7cells.
at different concentrations. The inhibition rate of PBP60-B in suppressing the growth of A549 cells was lower than 6% even at a high concentration of 500 g/mL. The result showed that PBP60B exhibited no significant antitumor activities against A549 cells in vitro. As described in Fig. 5 (b), all the samples presented a dosedependent effect against the proliferation of MCF-7 cells in vitro. PBP60-A and PBP60-B exhibited a relatively lower inhibition rate against MCF-7 cells at all concentrations. PBP60-C and PBP60-D at 500 g/mL showed approximately42.45% and 37.04% inhibition rate against the proliferation of MCF-7 cells. It could be concluded that PBP60-C and PBP60-D were effective tumor cell growth inhibitors that exhibited high antitumor activities against A549 cells and MCF-7 cells, and the strongest ability of PBP60-C was probably due to its chemical components, molecular weight, monosaccharide composition, structural features [37]. Xin et al. [38] reported two acidic polysaccharides from the roots of Polygala tenuifolia that significantly inhibited the growth of A549 cells in vitro; the IC50 values were 47.8 g/mL and 41.2 g/mL, repectively. At the concentration of 250 g/mL, LpolychrousLév polysaccharide showed approximately 43.9% inhibition on MCF-7 cells [39]. Our result is in agreement with these findings. 4. Conclusion In the present study, four polysaccharide fractions (PBP60-A, PBP60-B, PBP60-C and PBP60-D) from slugs (Philomycusbilineatus) were obtained by enzyme extraction and DEAE-Sepharose Fast Flow chromatography. Results demonstrated that PBP60-A is mainly composed of Man, Rha, Glc, Gal, Xyl and Fuc in a mole ratio
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Partial characterization, antioxidant and antitumor activities of polysaccharides from Philomycusbilineatus Rongjun He a , Jiaming Ye a , Yuejun Zhao a , Weike Su b,∗ a
College of Biological and Environmental Engineering,Zhejiang University of Technology, Hangzhou 310032, PR China Key Laboratory of Pharmaceutical Engineering of Ministry of Education, College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, PR China b
a r t i c l e
i n f o
Article history: Received 12 November 2013 Received in revised form 21 December 2013 Accepted 4 January 2014 Available online 10 January 2014 Keywords: Slug (Philomycusbilineatus) Polysaccharide Antioxidant activity Antitumor activity
a b s t r a c t Four polysaccharides (PBP60-A, PBP60-B, PBP60-C and PBP60-D) were purified from slug (Philomycusbilineatus) by ion-exchange chromatography. The antioxidant activities were studied by ABTS, DPPH, hydroxyl radical, superoxide radical and reducing power assay. In vitro antitumor activities were evaluated by MTT assay. Results demonstrated that PBP60-A was mainly composed of Man, Rha, Glc, Gal, Xyl and Fuc in a mole ratio of 6.13:3.08:8.97:5.22:2.46:1.13. PBP60-B was composed of Man, GlcN, Rha, GalN, GlcU, Glc, Gal, Xyl and Fuc in a mole ratio of 0.90:0.31:1.15:0.37:0.24:1.02:3.84:0.93:1.99. PBP60-C and PBP60-D were composed of Man, GlcN, Rha, GalN, GlcU, Glc, Gal, Xyl, Fuc and an unknown monosaccharide. Antioxidant tests indicated that four polysaccharides exhibited significant antioxidant activities in a dose-dependent manner. PBP60-D presented relative stronger antioxidant activity. PBP60-C showed higher antitumor activity against A549 and MCF-7 cells in vitro. At concentration of500 g/mL, the antitumor activities of PBP60-C on theA549 and MCF-7 cells were 65.30% and 42.45%, respectively. These results indicated that polysaccharides from Philomycusbilineatus could be explored as potential natural antioxidants and cancer prevention agents. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Slug (Philomycusbilineatus) is a land molluscaphilomycidae animalisa pest in crops that is distributed widely in Europe, America and Asia. It is one of the traditional Chinese drugs which have been used for cough, stroke and Centipedebite [1]. Most of the previous studies of slug focus on the effects of environmental factors [2] and its habits and morphology [3]. Furthermore, slug was also considered as a useful model animal in neuroscience because of the simple nervous system with large neurons, higher learning capacity, and feasibility of in vitro [4,5]. Recently, several Chinese researchers reported that crude extracts from slug presented significant antitumor activities against A549 cells [6], Hela cells [7] and S-180 cells [8], indicating that slug extracts could be explored as cancer prevention agents. However, few data has been reported on the further chemical composition, structure and biologic activity of polysaccharide from slugs. Therefore, the aim of present work was to explore the chemical composition of different polysaccharide
∗ Corresponding author at: Laboratory of Pharmaceutical Engineering of Ministry of Education, College of Pharmaceutical Science, Zhejiang University of Technology, No.18 Chao Wang Rood, Hangzhou 310032, PR China. Tel.: +86 571 88320752; fax: +86 571 88320752. E-mail address: [email protected] (W. Su). 0141-8130/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2014.01.016
extracted from slugs. Moreover, their antioxidant activities and antitumor activities were also investigated.
2. Experimental 2.1. Materials and chemicals The slugs (Philomycusbilineatus) were collected in campus of Zhejiang University of Technology (Zhejiang province, China), and then they were frozen at −20 ◦ C. The human breast cancer cell line MCF -7 and human lung cancer cell line A549 were provided by the cell bank of the Shanghai Institute of Cell Biology (Shanghai, China). The DEAE Sepharose Fast Flow was obtained from GE Healthcare Life Science (Piscataway, NJ, USA). HPLC was carried out on a waters 2695 HPLC system (2695 HPLC Pump, 2414 Refractive Index Detector). Rhamnose, fucose, xylose, galactose, glucose, mannose, ferrozine, nitrobluetetrazolium (NBT), phenazinemethosulfate (PMS), reduced nicotinamide adenine dinucleotide (NADH), 2,2 -Azino-bis(3-ethylbenzthiazoline-6-sulfonic) acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), bovine serum albumin, 3-(4,5-dimetthylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), ferrozine, deoxyribose, dextrans, and trifluoroacetic acids (TFA) were obtained from Sigma-Aldrich (Sigma-Aldrich GmbH,
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Sternheim, Germany). All other chemicals used were in analytical grade and obtained from either Sigma-Aldrich or Merck. 2.2. Extraction and purification of polysaccharides The slug (Philomycusbilineatus) bodies were homogenized with distilled water and hydrolyzed by 0.4% trypsin (2000 U/mg) at 50 ◦ C for 7 h. Subsequently, it was centrifuged to remove the insoluble materials. The supernatant was deproteined by Sevag method and precipitated with ethanol that were successively fractionated by different gradient concentrations of ethanol (50% and 60%) into two fractions named PBP50 and PBP60. The crude polysaccharides were obtained by lyophylization. The PBP60 fraction was dissolved in distilled water, filtered through 0.45 m filter and applied to a DEAE-Sepharose Fast Flow column (XK 26 mm × 100 cm), successively eluted in order with distilled water, 0.1, 0.3 and 0.5 M NaCl solutions at flow rate of 3 mL/min. The relevant fractions were collected, concentrated, dialyzed and lyophilized, and determined by means of the phenolsulfuric acid assay. 2.3. Characterization of polysaccharide fractions 2.3.1. Preliminary chemical analysis Total carbohydrate content was determined by phenol-sulfuric acid method using D-glucose as the standard [9]. Protein was analyzed by Bradford method using bovine serum albumin (BSA) as the standard [10]. Uronic acid contents were analyzed colorimetrically by m-hydroxybiphenyl method using D-galacturonic acid as the standard [11]. 2.3.2. Monosaccharides composition The monosaccharide components of the polysaccharides were analyzed by HPLC method based on Harazono et al. [12] and Dai et al. [13] with some modifications. Briefly, the polysaccharide samples were hydrolyzed with 4 mL trifluoroacetic acid (2 M) in an oven at 110 ◦ C for 2 h.The hydrolyzate was vacuum evaporated with methanol to dryness, and the procedure was repeated thrice for trifluoroacetic acid to be removed. The hydrolyzate and standard monosaccharide mixtures (mannose, glucosamine, rhamnose, galactosamine, glucuronic acid, N-acetyl-glucosamine, glucose, galactose, xylose, and fucose) were dissolved in 0.5 M PMP methanolic solution (200 L). After adding 200 L of NH3 solution, the whole mixture was incubated at 70 ◦ C for 30 min and then was cooled down to room temperature with addition of 1 mL water. The solution was vacuum evaporated to dryness under 50 ◦ C. The residue was dissolved in water and chloroform (1 mL each). After vigorous shaking, organic layer was discarded. The final aqueous layer was filtered through a 0.45 m syringe filter and subsequently subjected to HPLC (Waters Symmetry ShieldTM RP 18, 5 m, 250 × 4.6 mm) analysis by UV absorbance 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.
solution was produced by mixing ABTS aqueous solution (final concentration 7 mM) with potassium per sulphate (final concentration 2.45 mM) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. After incubation, the ABTS radical solution was diluted with PBS (pH 7.0) to an absorbance of 0.70 ± 0.02 at 734 nm. The diluted solution (2 mL) was then mixed with 0.2 mL sample solution, and the mixture solution was incubated for 6 min at room temperature. The absorbance was measured at 734 nm. The scavenging activity on ABTS radical was calculated by the following formula:
Scavenging activity (%) = 1 −
(A1 − A2 ) × 100 A0
where A0 is the absorbance of the ABTS radical solution without sample, A1 is the absorbance of the ABTS radical solution with tested samples and A2 is the absorbance of ethanol solution with tested samples. 2.4.2. DPPH radical scavenging assay DPPH radical scavenging assay was assessed according to the previous method with a modification [15]. DPPH solution (0.1 mM, in 95% ethanol) was prepared and used fresh on the day of each test. 1.0 mL of water solution of sample at concentration of 0, 0.25, 0.5, 1.0, 2.0, 4.0 mg/mL was added to 2 mL DPPH solution. The mixture was shaken vigorously and kept in dark for 30 min and the absorbance was measured at 517 nm. The ability to scavenge DPPH was calculated as a percentage according to the equation:
Scavenging activity (%) = 1 −
(A1 − A2 ) × 100 A0
where A0 is the absorbance of water instead of sample solution, A1 is the absorbance of the sample solution and A2 is the absorbance of ethanol instead of DPPH solution. 2.4.3. Hydroxyl radical scavenging assay Hydroxyl radical scavenging activity was investigated by the method of Smirnoff and Cumbes [16] with a minor modification. 0.5 mL FeSO4 (1.5 mM) was mixed with 0.35 mL H2 O2 (6 mM), 0.15 mL sodium salicylate (20 mM) and 1 mL sample solution (0.25–4 mg/mL), then incubated for 1 h at 37 ◦ C. The absorbance of the hydroxylated salicylate complex was measured at 562 nm. The antioxidant activity was calculated with the following equation:
Scavenging activity (%) = 1 −
(A1 − A2 ) × 100 A0
where A0 is the absorbance of water instead of sample solution, A1 is the absorbance of the sample solution and A2 is the absorbance of water instead of sodium salicylate solution.
2.3.3. Infrared analysis FT-IR spectra were obtained on a Nicolet 6700 FT-IR spectrometer (Thermo Scientific, USA) in a range of 4000–400 cm−1 . The samples were measured as a KBr disc.
2.4.4. Superoxide radical scavenging activity assay Superoxide radical scavenging activity was performed based on the method described by Xu et al. [17] with some modification. Each 1.0 mL of sample solution, NBT solution (78 M of NBT in 20 mM sodium phosphate buffer, pH 7.4) and NADH solution (468 Mof NADH in 20 mM sodium phosphate buffer, pH 7.4) were mixed. After addition 0.4 mL of PMS solution (60 MPMS in 20 mM sodium phosphate buffer, pH 7.4), the mixture was incubated at 25 ◦ C for 5 min. The absorbance of the mixture was measured at 560 nm. The superoxide radical scavenging activity was calculated by the follow formula:
2.4. Antioxidant activity tests in vitro
Scavenging activity (%) = 1 −
2.4.1. ABTS radical scavenging assay ABTS radical scavenging assay was carried out by the method described by Re et al. [14] with some modifications. ABTS radical
where A0 is the absorbance of water instead of sample solution, A1 is the absorbance of the sample solution and A2 is the absorbance of water instead of PMS solution.
(A1 − A2 ) × 100 A0
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2.4.5. Reducing power assay The reducing power of samples was determined according to the method reported by Lei et al. [18] with minor modification. 2.5 mL samples (0.25–4.0 mg/mL), 2.5 mL pH 6.6 phosphate buffer (0.2 M) and 2.5 mL 1% (w/v) K3 (CN)6 were incubated at 50 ◦ C for 20 min. The reaction was terminated by adding 2.5 mL trichloroacetic acid (10%, w/v), and centrifuging at 2000 rpm for 15 min. The upper layer (5.0 mL) was mixed with 5 mL water and 1.2 mL ferric chloride (0.1%, w/v). The mixture was shaken and its absorbance was measured at 700 nm . The absorbance of the reaction mixture increasing indicates an increase of reduction capability. 2.5. In vitro antitumor activity A549 cells and MCF-7 cells were provided by the Cell Bank of Shanghai Institute of Cell Biology and maintained with RPMI 1640 medium containing 10% fetal bovine serum at 37 ◦ C in a humidified atmosphere with 5% CO2 . The inhibition effects of PBP60-A,PBP60-B, PBP60-C and PBP60D on the growths of A549 cells and MCF-7cells were evaluated in vitro by MTT assay [19]. Briefly, cells in the logarithmic growth phase were seeded in 96-well plates (5 × 103 cells/well) and incubated at 37 ◦ C in a humidified atmosphere with 5% CO2 for 24 h. The non-adherent cells in well were removed by washing with PBS for three times. After that, 100 L fresh medium and test sample solution were added to each well, and the cells were incubated for 48 h. At the end of the treatment, 20 L of MTT (5 mg/mL) was added to each well and the cells were cultured for another 4 h. The medium was then discarded and 100 L of DMSO was added to each well. The absorbance was measured at 570 nm by an ELISA Reader. The inhibition rate was calculated according to the formula below:
Inhibition rate(%) =
1−
Abssample Abscontrol
× 100
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Table 1 The chemical composition of PBP60-A, PBP60-B, PBP60-C and PBP60-D. Samples
PBP60-A
PBP60-B
PBP60-C
PBP60-D
Carbohydrate (%) Protein (%) Uronic acid (%)
71.79 ± 0.37 0.78 ± 0.02 nd
51.72 ± 1.71 1.38 ± 0.01 4.70 ± 0.19
53.96 ± 0.93 5.48 ± 0.25 10.85± 0.18
46.87 ± 2.78 10.99 ± 0.30 4.15 ± 0.23
Each value is expressed as mean means ±stand deviation (n = 3). Means with different letters within a row are significantly different (p < 0.05). nd: not detected.
PBP60-C and PBP60-D were 71.79%, 51.72%, 53.96% and 46.87%, respectively. Except for PBP60-A, there was no significant difference in total polysaccharide among the other three fractions. The protein contents of PBP60-A, PBP60-B, PBP60-C and PBP60-D were 0.78%, 1.38%, 5.48% and 10.99%, respectively. The uronic acid contents of PBP60-B, PBP60-C and PBP60-D were found to be 4.70%, 10.85% and 4.15%, respectively, indicating that PBP60-B, PBP60-C and PBP60-D were acid polysaccharides. There is no uronic acid in PBP60-A. The monosaccharide composition of PBP60s was shown in Fig. 2. PBP60-A is mainly composed of Man, Rha, Glc, Gal, Xyl and Fuc in the mole ratio of 6.13: 3.08: 8.97: 5.22: 2.46: 1.13. Man, Glc and Gal are the main sugar units, accounting for 22.70%, 33.23% and 19.35% of the total sugar, respectively. PBP60-B is made up of Man, GlcN, Rha, GalN, GlcU, Glc, Gal, Xyl and Fuc in a mole ratio of 0.90: 0.31: 1.15: 0.37: 0.24: 1.02: 3.84: 0.93: 1.99. Gal is main sugar unit, the total content of which nearly exceeded 35.73 mol% of the all sugar. PBP60-C and PBP60-D are composed of Man, GlcN, Rha, GalN, GlcU, Glc, Gal, Xyl, Fuc and an unknown monosaccharide. Except for the little unknown monosaccharide, PBP60-C is composed of Man, GlcN, Rha, GalN, GlcU, Glc, Gal, Xyl and Fuc in molar proportion of 0.69: 0.33: 1.49: 0.24: 0.25: 1.18: 3.91: 0.70: 1.51 and it is 0.70: 0.11: 0.75: 0.14: 0.08: 065: 1.67: 0.68: 0.74 for PBP60-D. These results demonstrated that four polysaccharides isolated from Philomycusbilineatus had different chemical composition.
2.6. Statistical analysis 3.3. FT-IR spectral analysis 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. 3. Results and discussion 3.1. Isolation of polysaccharides Polysaccharides from animal always contain high level of proteins, which brings difficulty for further purification and structural analysis [20]. Protease is often used to deproteinate the extracts of animal polysaccharides [21]. In this study, trypsin-hydrolysis, Sevag method and ethanol precipitation were used to prepare PBP60 from slugs (Philomycusbilineatus). Fig. 1 represents the chromatogram of crude polysaccharides (PBP60) subjected to a DEAE-Sepharose Fast Flow column. Four peaks were observed from distilled water and NaCl elution, and they were named as PBP60A (eluted with distilled water), PBP60-B (eluted with 0.1 M NaCl), PBP60-C (eluted with 0.3 M NaCl) and PBP60-D (eluted with 0.5 M NaCl). PBP60-C and PBP60-D showed single and symmetrically sharp peak on HPLC analysis (data not shown) among the four fractions. In current study, these major fractions were collected and dried for further physicochemical and biologic analysis. 3.2. Chemical properties and monosaccharide composition Chemical compositions of the four fractions were given in Table 1. The total polysaccharide contents of PBP60-A, PBP60-B,
FT-IR spectroscopy is typically used for identification of characteristic organic groups in the polysaccharides, especially O H, N H, and C O. FT-IR spectra of the four samples are shown in Fig. 3. The spectra are typical of polysaccharides in a broadly-stretched intense peak around 3400 cm−1 for O H stretching vibrations and a weak absorption peak about 2930 cm−1 for C H stretching vibrations. A relatively strong peak around 1650 cm−1 was attributed to C O stretching vibration, and absorption of 1540 cm−1 was N H bending vibration. The broader band of 1050 cm−1 was representative of C C stretching and C OH bending vibrations [22]. Furthermore, the presence of amide bands at 1650 cm−1 , 1540 cm−1 and 1410 cm−1 indicated the presence of some residual protein in the four polysaccharide fractions [23]. 3.4. In vitro antioxidant activity 3.4.1. ABTS radical scavenging activity ABTS assay is often used in evaluating total antioxidant power of compounds. Specific absorbance at 734 nm can be used in both organic and aqueous solvents as an index reflecting the antioxidant activity of the extracted polysaccharides [24]. The ABTS radical scavenging activities of the four polysaccharide fractions from Philomycusbilineatus were shown in Fig. 4(a). The figure illustrated the decrease in the concentration of ABTS radical due to the scavenging ability of polysaccharides. Their scavenging ability correlated well with increasing concentrations. Moreover, PBP60-C and PBP60-D fractions showed pronounced high radical scavenging activity; the scavenging ability of these four different fractions
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Fig. 1. Elution profiles of crude polysaccharides PBP60 on DEAE-Sepharose Fast Flow chromatography column with stepwise gradient of aqueous NaCl elute (0, 0.1, 0.3, and 0.5 M).
tested decreased in the order of PBP60-C > PBP60-D > PBP60B > PBP60-A, which were 73.12%, 68.32%, 51.62% and 31.97%, respectively. At the concentration of 4 mg/mL, all samples had strong scavenging activity for ABTS radical. 3.4.2. DPPH radical scavenging activity DPPH is a stable free radical compound, which shows a maximum absorption at 517 nm that can readily undergo scavenging by an antioxidant. It has been widely accepted as a tool for evaluating the free radical scavenging activities of natural compounds
[24,25]. Fig. 4(b) shows the DPPH radical scavenging activity of four fractions. The scavenging activity of polysaccharides PBP60-A, PBP60-B, PBP60-C, PBP60-D on inhibition of the DPPH radical was related to the concentration of the samples. Among four fractions, PBP60-D showed the strongest scavenging ability increased significantly with the concentration. At the concentration of 4.0 mg/mL, the scavenging effects of PBP60-A, PBP60-B, PBP60-C, PBP60-D on the DPPH radical were 33.73%, 43.14%, 47.39%, 66.39%, respectively. Several studies demonstrated that the content of uronic acid and protein group may were important factors which contribute to
Fig. 2. HPLC analysis of monosaccharide composition in samples (Man: mannose; GlcN: glucosamine; Rha: rhamnose; GalN: galactosamine; GlcUA: glucuronic acid; GlcNAc: N-acetyl-glucosamine; Glc: glucose; Gal; galactose; Xyl: xylose; Fuc: fucose and Unknown: unknown monosaccharide).
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Fig. 3. FT-IR spectra of four polysaccharides. (a)PBP60-A, (b) PBP60-B, (c) PBP60-C and (d) PBP60-D.
the promotion of DPPH radical scavenging activity of polysaccharide [26,27]. It indicated that the stronger scavenging activity of PBP60-D on DPPH radical may be partially due to the higher content of proteinous substance and uronic acid.
3.4.3. Hydroxyl radical scavenging activity Hydroxyl radical is considered to be the most reactive radical, which can react with most biomacromolecules in living cells and induce severe damage to the adjacent biomolecules [28,29]. In this experiment, hydroxyl radical is generated from well-known Fenton reaction (Fe2+ + H2 O2 → Fe3+ + •OH + OH), salicylic acid captures hydroxyl radical to form 2,3-dihydroxyl benzoic acid, and it had maximum absorbance at 510 nm. Antioxidant can compete with salicylic acid to reduce the generation of 2,3-dihydroxyl benzoic acid [26]. Fig. 4(c) described the scavenging ability on hydroxyl radical of four fractions; all the samples exhibited weak scavenging ability at low concentration, with only about 10% at the concentration of 0.25 mg/mL, but increasing quickly with higher concentration. At 4 mg/mL, the hydroxyl scavenging abilities of PBP60-A, PBP60-B, PBP60-C and PBP60-D were 48.34%, 55.15%, 59.57% and 66.39%, respectively. All samples showed obvious hydroxyl radical scavenging activity in a dose-dependent pattern. The hydroxyl radical scavenging ability was attributed to various mechanisms, such as suppression against hydroxyl radical generation, decomposition of peroxides and prevention of continued hydrogen abstraction [28]. The possibility of scavenging hydroxyl radical by polysaccharide was that polysaccharide could supply of hydrogen and combine with radical to terminate the radical chain reaction [30].
3.4.4. Superoxide radical scavenging activity Superoxide radical is a highly toxic species that is generated by metabolic process and physical irradiation, and it is considered as the primary ROS [31]. Superoxide radical can further interact with other molecules to form single oxygen, hydroxyl radical and hydrogen peroxide, which may increase lipid peroxide, cellular damage, local oxidative stress and pathological incidents [32,33]. The superoxide radical scavenging activities of the four fractions are shown in Fig. 4 (d). The scavenging activity of all samples correlated with increasing concentration. At concentration of 0.25 mg/mL, the scavenging rate of PBP60-D was up to 67.05%, however, the scavenging rate of PBP60-A, PBP60-B, PBP60-C were 26.09%, 32.01% and 46.10%, respectively. Four polysaccharides showed a relatively high level of superoxide radical scavenging ability at the concentration of 4.0 mg/mL. Among the four samples, PBP60-D showed strongest superoxide scavenging activity at all concentrations. Higher scavenging rate was found when the content of proteinous substances increased. The results were in accordance with the findings of Huang et al. [29]. But the correlation between the chemical characteristics and antioxidant activities of PBP60-D needs further investigation. 3.4.5. Reducing power The reducing power of a compound may serve as an indicator of its potential antioxidant activity. Higher absorbance value means stronger reducing power [21]. The reducing ability is generally associated with the presence of reductones, which have been shown to exert antioxidantaction by breaking the free radical chain by donating a hydrogen atom [31]. As shown in Fig. 4(e), the reducing power of all samples increased with the increase of
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Fig. 4. Antioxidant activities of PBP60-A, PBP60-B, PBP60-C and PBP60-D: (a) ABTS radical scavenging activity, (b) DPPH radical scavenging activity, (c) hydroxyl radical scavenging activity, (d) superoxide radicals scavenging activity, (e) reducing power.
concentrations. Obviously, the reducing power of PBP60-D was significantly higher than that of PBP60-A, PBP60-B and PBP60C.At concentration of 4 mg/mL; the reducing power of PBP60-A, PBP60-B, PBP60-C, PBP60-D were 0.609, 0.935, 1.196 and 1.479, respectively. The reason might be that polysaccharides from Philomycusbilineatus contain reductone-associated and hydroxide groups which can act as electron donors and react with free radicals to convert them to be more stable products. 3.5. In vitro antitumor activity Cancer is a formidable problematic disease on human health. The great majority of chemical compounds, which have high antitumor effects; however, they are also toxic to normal cells [34].
Therefore, it is important to investigate high efficiency antitumor compounds with little toxicity. Numerous polysaccharides extracted from plants, algae, fungi and animals have been proved to show significant antitumor activities with little toxicity to hosts, such as Camellia sinensispolysaccharides[17], Tricholomamatsutakepolysaccharides [35], Scolopendrasubspinipesmutilans L. Koch polysaccharides [36], etc. In this study, the antitumor activities of the four polysaccharides, PBP60-A, PBP60-B, PBP60-C and PBP60-D were tested on A549 cells and MCF-7 cells. As shown in Fig. 5 (a), PBP60-C and PBP60-D presented significant high antitumor activities against A549 cells at all concentrations, and the IC50 values of PBP60-C and PBP60-D were about 100 g/mL and 500 g/mL, respectively. However, the inhibition rates of PBP60-A and PBP60-B were low
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of 6.13: 3.08: 8.97: 5.22: 2.46: 1.13. PBP60-B is made up of Man, GlcN, Rha, GalN, GlcU, Glc, Gal, Xyl and Fuc in a mole ratio of 0.90: 0.31: 1.15: 0.37: 0.24: 1.02: 3.84: 0.93: 1.99. PBP60-C and PBP60-D are composed of Man, GlcN, Rha, GalN, GlcU, Glc, Gal, Xyl, Fuc and an unknown monosaccharide. FT-IR spectra of PBP60-A, PBP60B, PBP60-C and PBP60-D were similar, indicating the presence of some residual protein in the four polysaccharide fractions. Several assays in vitro were applied to evaluate the antioxidant activities of these polysaccharides. All the fractions showed significant free radical scavenging activities in a dose-dependent manner. PBP60-D exhibited stronger scavenging activities than the others, PBP60-A, PBP60-B and PBP60-C. Furthermore, PBP60-C showed significant inhibitory effects on growth of A549 cells and MCF-7cells. At concentration of 500 g/mL, the antitumor activities of PBP60-C on theA549 and MCF-7 cells were 65.30% and 42.45%, respectively. Acknowledgment This work was supported by Natural Science Foundation of Zhejiang Province of China (No. LY12H28007). References [1] [2] [3] [4] [5] [6] [7] [8] Fig. 5. Inhibitory effects in vitro of PBP60-A, PBP60-B, PBP60-C and PBP60-D against (a) A549 cells, (b) MCF-7cells.
at different concentrations. The inhibition rate of PBP60-B in suppressing the growth of A549 cells was lower than 6% even at a high concentration of 500 g/mL. The result showed that PBP60B exhibited no significant antitumor activities against A549 cells in vitro. As described in Fig. 5 (b), all the samples presented a dosedependent effect against the proliferation of MCF-7 cells in vitro. PBP60-A and PBP60-B exhibited a relatively lower inhibition rate against MCF-7 cells at all concentrations. PBP60-C and PBP60-D at 500 g/mL showed approximately42.45% and 37.04% inhibition rate against the proliferation of MCF-7 cells. It could be concluded that PBP60-C and PBP60-D were effective tumor cell growth inhibitors that exhibited high antitumor activities against A549 cells and MCF-7 cells, and the strongest ability of PBP60-C was probably due to its chemical components, molecular weight, monosaccharide composition, structural features [37]. Xin et al. [38] reported two acidic polysaccharides from the roots of Polygala tenuifolia that significantly inhibited the growth of A549 cells in vitro; the IC50 values were 47.8 g/mL and 41.2 g/mL, repectively. At the concentration of 250 g/mL, LpolychrousLév polysaccharide showed approximately 43.9% inhibition on MCF-7 cells [39]. Our result is in agreement with these findings. 4. Conclusion In the present study, four polysaccharide fractions (PBP60-A, PBP60-B, PBP60-C and PBP60-D) from slugs (Philomycusbilineatus) were obtained by enzyme extraction and DEAE-Sepharose Fast Flow chromatography. Results demonstrated that PBP60-A is mainly composed of Man, Rha, Glc, Gal, Xyl and Fuc in a mole ratio
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