Sulfated modification of arabinogalactans from Larix principis-rupprechtii and their antitumor activities

Carbohydrate Polymers 215 (2019) 207–212

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Sulfated modification of arabinogalactans from Larix principis-rupprechtii and their antitumor activities Shuo Tang, Ting Wang, Caoxing Huang, Chenhuan Lai, Yimin Fan, Qiang Yong

T

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Co-Innovation Center for Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Larix principis-rupprechtii Arabinogalactan Sulfated modification Antitumor activity Apoptosis

A highly branched arabinogalactan isolated from Larix principis-rupprechtii and subjected to sulfation derivatization to promote their antitumor bioactivity. Several structural features of the sulfated arabinogalactans (SLAG) were investigated: molecular weight, monosaccharide constitution, and chemical structures. Spectral analysis indicated that sulfate groups were successfully introduced on arabinogalactan. Sulfated products showed different degrees of substitution (DS) ranging from 0.61 to 0.80, and different Mw ranging from 19.24 to 22.03 kDa. Monosaccharide composition before and after sulfation indicated some level of derivatization selectivity. In vitro cancer cell tests demonstrated that S-LAGs were effective inhibitors to cancer cell growth depending on their dosage. The toxicity mechanisms were further investigated, and assay results revealed that SLAGs mainly induced cancer cellular apoptosis to promote atrophy and inhibit cell proliferation. The results obtained in this work offer strong demonstration of modified arabinogalactans as a potential medical substance for treating different forms of cancer.

1. Introduction Polysaccharides from plants have attracted a great deal of attention because of their biosafety, biological activity and physiological activity (Xie, Jin et al., 2016). Arabinogalactans (AGs) are one particularly interesting class of polysaccharides which can be found in a range of plants such as radish (Shimoda et al., 2014), tamarillo (do Nascimento et al., 2015), green tea (Wang, Shi, Bao, Li, & Wang, 2015), and gum ghatti (Ghosh, Ray, Ghosh, & Ray, 2015). However, there exist uniquely high quantities of AGs in the Larix trees, such as L. occidentalis (Prescott, Groman, & Gulyas, 1997), L. dahurica (Odonmaiig, Ebringerova, Machova, & Alfijldi, 1994), L. laricina (Goellner, Utermoehlen, Kramer, & Classen, 2011), and L. principis-rupprechtii (Tang et al., 2018). Larch arabinogalactan (LAG) is a water-soluble glycan with a high level of molecular branching. It is chemically mainly composed of two monosaccharide residues, D-galactose and L-arabinose, with traces of uronic acid. The inter-residue linkages are founded upon a (1→3)-β-D-galactopyranan main chain with side (1→6)-linked groups of varying length (Goellner et al., 2011; Tang et al., 2018). LAGs have been examined in literature for their immunological activity, their regulatory upon fecal microbial population, and for a variety of ocular benefits (Burgalassi et al., 2007; Currier, Lejtenyi, & Miller, 2003; Grieshop, Flickinger, & Fahey, 2002). However, it does not exhibit possible

antitumor activity. To extend the use of biomass-derived polysaccharides into more application, many studies have been focused on the biological properties of polysaccharide derivatives, especially those which have been sulfated, phosphated, acetylated, or carboxymethylated (Huang & Huang, 2017). In particular, sulfated polysaccharides have been studied extensively due to a variety of reports demonstrating potential health benefits. Sulfated groups change the molecular features of native polysaccharide, changes which significantly influence biological activities (Liu et al., 2015). Some reports on biological properties include demonstration of sulfated polysaccharide's antioxidant (Li, Chi, Yu, Jiang, & Liu, 2017), anticoagulant (Li, Liu et al., 2017) and antiviral activities (Godoi et al., 2014). Furthermore, sulfated polysaccharides were demonstrated as being especially effective at fighting tumors compared to their respective unmodified carbohydrates (Jung, Bae, Lee, & Lee, 2011). Sulfated hyperbranched mushroom polysaccharides were obtained by treatment with chlorosulfonic acid, and the sulfated derivatives exhibited higher antitumor activities against human hepatic cancer cell line (HepG2) (Tao, Zhang, & Cheung, 2006). The sulfated derivatives from Gynostemma pentaphyllum Makino polysaccharide with Mw (8.96 kDa) and DS (1.2) inhibited the growth of HepG2 cells and Hela cells in vitro significantly (Chen et al., 2011). There is no obvious antitumor activity in Radix hedysari polysaccharides, but its sulfated

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Corresponding author. E-mail addresses: [email protected] (S. Tang), [email protected] (T. Wang), [email protected] (C. Huang), [email protected] (C. Lai), [email protected] (Y. Fan), [email protected], [email protected] (Q. Yong). https://doi.org/10.1016/j.carbpol.2019.03.069 Received 19 January 2019; Received in revised form 25 February 2019; Accepted 19 March 2019 Available online 21 March 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.

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2.2. Preparation of sulfated LAG

derivatives inhibited proliferation of lung cancer cells and gastric cancer cells by inducing cell apoptosis (Wei, Wei, Cheng, & Zhang, 2012). Recent studies have suggested that sulfated polysaccharides inhibited cancer cell proliferation (Wang, Zhang, Yu, & Cheung, 2009). This cytostatic effect may be due to the induction of apoptosis (Zhong et al., 2015). A large number of new drugs and nutraceuticals based on the bioactivities of sulfated polysaccharides are currently under development (Scott & Alyssa, 2013). Therefore, the development of more types of sulfated polysaccharides is gaining more attention. Methods for sulfation of polysaccharides includes the chlorosulfonic acid-pyridine (CSA-Pyr) method, SO3-pyridine (SO3-Pyr) method and concentrated sulfuric acid method. Among them, CSA-Pyr method has been elucidated as superior over recent years due to its advantageously high yield, degree of substitution (DS), and convenient handling (Raveendran, Yoshida, Maekawa, & Kumar, 2013). It was reported that DS and Mw are the critical parameters influencing the potency of biological activities exhibited by sulfated polysaccharides (Wang, Li, & Chen, 2009). Mw depends on the structure of the glycan itself as well as the methods of sulfation. Different methods can cause cleavage at various points of polysaccharide molecules, and the extent of cleavage is shown to wildly vary using the same modification method across different polysaccharides. Li, Chi et al. (2017) reveal that sulfated derivatives of the two different Mw polysaccharides were synthesized by CSA-Pyr method, and the DS of SPEP and SLEP were 0.57 and 0.81, respectively. In another work, the polysaccharides from the leaves of C. paliurus were subjected to sulfation with SO3-Pyr complex, producing the sulfated derivatives with different DS (0.12–0.55) (Xie, Jin et al., 2016). Therefore, a variety of modification methods provide a way to prepare a family of new polysaccharide derivatives with different structural characteristics, which can be used for structure/property studies. In our previous research, we successfully isolated and purified an arabinogalactan from L. principis-rupprechtii. Starting from this same material, in this work we have performed sulfation on LAG understanding that this sort of work has not yet been performed. Furthermore, we sought to advance the state of knowledge concerning the structure-function relationship between sulfated polysaccharides and antitumor activity. For this reason, four sulfated arabinogalactans (S-LAGs), with various DS, Mw and carbohydrate contents, were prepared using the CSA-Pyr method. The chemical structures of the S-LAGs were next exhaustively characterized using a variety of techniques to establish a baseline understanding of their chemical structure and intersample differences. Furthermore, antitumor activities of S-LAGs were investigated by CCK-8 assays. Any relationship between the obtained assay responses and the structural properties of S-LAGs were identified. The intent behind this work was two-fold: to further develop the value of LAGs and specifically S-LAGs, while attempting to further the understanding of which S-LAG properties most improve antitumor activities.

Sulfation of LAG was performed according to (O’Neill, 2011) using the chlorosulfonic acid-pyridine (CSA-Pyr) method with some modifications. In brief, the sulfation reagent was prepared in an ice bath by adding CSA to Pyr at the ratios of 1:1, 1:3, 1:5, and 1:7 (v/v) in a dropwise fashion. At this time, the molar concentration of CSA is 7.68, 3.84, 2.56 and 1.92 mol/L, respectively. LAG powder (500 mg) was solubilized in dry formamide (50 mL) with vigorous stirring at room temperature for 24 h, after which it was completely dissolved. Next, 10.0 mL of sulfation reagent was added to the mixture followed by a continuation of stirring for another 3 h at the elevated temperature of 80 °C. After time, the reaction was terminated though addition of icewater into the system. The solution was next neutralized using 10% (w/ v) NaOH. Precipitation was induced in the neutralized solution though addition of 4x the solution volume of 95% (v/v) ethanol, and then the precipitate was re-suspended in distilled water. The solution was then dialyzed against distilled water for 72 h with several changes of water. Finally, the dialyzed solution was freeze-dried to obtain the sulfated LAG and coded as S-LAG1-1, S-LAG1-3, S-LAG1-5 and S-LAG1-7, respectively. 2.3. Characterization of sulfated AG 2.3.1. Determination of degree of substitution Degree of substitution (DS) of S-LAGs was estimated using the BaCl2-gelatin method (Dodgson & Price, 1962) with some modifications. Specifically, ˜5 mg of S-LAGs was hydrolyzed using 1.0 M HCl (2 mL) at 105 °C for 6 h. Next, 0.2 mL of S-LAGs hydrolysate was added to a test tube containing 5.8 mL of trichloroacetic acid solution (5.0%, w/v). After mixing, 2 mL of BaCl2-gelatin solution (0.5 g BaCl2, 0.5 g gelatin and 50 mL distilled water) was also added into the test tube. This mixture was shaken evenly and then allowed to rest for 15 min. The BaSO4 formed was measured turbidimetrically at 360 nm. Sample turbidity responses were calculated based upon a calibration curve generated using K2SO4 standards. DS in this work is defined as the average number of sulfate groups on per one sugar residue, and was calculated from the sulfur content using the following equation (Whistler & Spencer, 1964):

DS=

1.62×S% 32-1.02×S%

(1)

Where, S% was the mass fraction of S atom in solution. Elemental analysis was also applied to determine the sulfur content with Thermo Scientific Flash 2000 according to manufacturer’s instructions. The percentage of sulfur (%S) was used to calculate the DS according to the formula (1). 2.3.2. Chemical components quantitative analysis Total sugar content was determined calorimetrically by the phenolsulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956), using galactose as standard. To determine the monosaccharide concentration of each S-LAG preparation, ˜10 mg of the polysaccharide was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 121 °C for 1 h. After cooking, excess TFA was removed by mixing in methanol and forcing co-evaporation under vacuum using a rotary evaporator. Monosaccharides concentrations in the hydrolyzates were analyzed using a high performance anion-exchange chromatography system (Dionex ICS-5000, USA) equipped with a CarboPac™ PA10 column (2 × 250 mm) and a pulsed amperometric detector. The elution program consisted of an initial isocratic elution in 37 mM NaOH from 0 to 20 min, followed 200 mM CH3COONa from 20 to 35 min, and finally equili-brated in 37 mM NaOH from 35 to 50 min. Calibration was performed with a standard solution of monosaccharide standard.

2. Materials and methods 2.1. Materials and reagents Larch arabinogalactan (LAG) was obtained from Larix principisrupprechtii following the protocol described in our previously published report (Tang et al., 2018). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Hyclone (Logan, UT, USA). Cell Counting Kit-8 (CCK-8) were obtained from Beyotime (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit was purchased from KeyGEN BioTECH (Nangjing, China). Dextrans of different molecular weights and monosaccharide standards were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other employed reagents were analytical grade quality.

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Table 1 Degree of substitution, yield and molecular weight characterization of S-LAGs. Samples

S-LAG1-1 S-LAG1-3 S-LAG1-5 S-LAG1-7 a b

CSA:Pyra

1:1 1:3 1:5 1:7

Yieldb (%)

126.23 114.34 108.93 98.39

Mw (kDa)

22.03 20.89 19.96 19.24

Mn (kDa)

Mw/Mn

17.62 16.19 14.36 14.04

1.25 1.29 1.39 1.37

Benzidine assay

Elemental analysis

S (%)

DS

S (%)

DS

10.53 ± 0.41 9.75 ± 0.32 9.35 ± 0.25 8.71 ± 0.24

0.80 0.72 0.67 0.61

10.22 ± 0.37 9.41 ± 0.33 9.18 ± 0.18 7.98 ± 0.22

0.77 0.68 0.66 0.54

The ratio of chlorosulfonic acid to pyridine in sulfating reagent. Ratio of sulfated polysaccharide (S-LAGs) to initial polysaccharide (LAG).

fresh medium for 24 h. After treatment, adherent cell populations were collected by trypsinization (0.25% trypsin without EDTA) and washed with ice-cold phosphate buffer saline (PBS) twice. The samples were centrifuged at 2000 rpm for 5 min between washes to separate cells and wash solution. After washing, cells were re-suspended in 0.5 mL binding buffer and 5 μL FITC Annexin V and PI were added. The cells were vortexed and incubated for 10 min at room temperature in the dark. After staining, the cells were measured by flow cytometry. A total of 10,000 cells were collected from each sample. Depending on fluorescence intensity of Annexin V-FITC and PI, the populations could be distinguished into Annexin-V positive (early apoptotic) cells, double positive (late apoptotic and necroptotic) cells and double negative (healthy) cells.

2.3.3. Molecular weight determination Average molecular weight and polydispersity of each S-LAGs preparations was estimated using GPC controlled by Agilent 1260 system (Agilent, USA) equipped with Ultrahydrogel 250 and Ultrahydrogel 2000 columns in series (7.8 × 300 mm, Waters Corp, USA). Column temperature was kept at 50 °C during elution. The sample solution (2 g/ L) was eluted with 0.05 M NaNO3 flowing at the rate of 0.6 mL/min. The average molecular weights were calculated calibrated using the retention times of different dextran standards. 2.3.4. FT-IR spectra analysis Fourier-transform infrared spectra (FT-IR) over a range of 4000–400 cm−1 were recorded on a VERTEX 80V (Bruker, Germany) spectrometer using the KBr-disk method.

2.6. Statistical analysis 2.4. Cell viability assay All experiments were carried out at least in triplicate, and results are expressed as mean ± standard deviation. Statistical analysis was performed by the Student’s t-test using SPSS 20 software. P values < 0.05 (∗) were considered statistically significant.

Several human cancer cell lines, including A549 human lung cancer cells, HepG2 hepatocellular carcinoma cells and MCF-7 human breast cancer cells, were purchased from Chinese Academy of Sciences (Shanghai, China). All cell lines were maintained in DMEM media supplemented with fetal bovine serum (10%), penicillin (100 units/ mL), and streptomycin (100 units/mL) at 37 °C in CO2 incubator (95% relative humidity, 5% CO2). The effect of S-LAGs on cell viability was evaluated using CCK-8 assay kits, taking care to carefully follow the manufacturer’s instructions. In detail, cells were seeded in a 96-well plate at a density of 5000 cells/well, and incubated in 100 μL of culture medium. After incubation for 24 h, the cells were treated with S-LAGs to reach final concentrations of 25, 50, 100, 200, 400 and 800 μg/mL. S-LAGs were formulated with fresh medium solution. The cells were incubated in the wells for another 72 h. After time, 10 μL of CCK-8 regent was added to each well and then allowed to further incubate for 1 h at 37°C, 5% CO2 and 95% humidity. Absorbance in each well was measured at 450 nm using a microplate reader (FilterMax F5, Molecular Devices, USA). The cell survival ratio was expressed as a percentage of the control using the following formula:

Cell viability (%)=

ODsamples − ODblank ODcontrol − ODblank

×100%

3. Results and discussion 3.1. Chemical analysis of S-LAGs DS of sulfated polysaccharides has been reported to be closely related to its biological activity (Jung et al., 2011). One significant finding reported was that controlling the amount of sulfation reagent in the CSA-Pyr method was a more effective means of controlling DS compared to the reaction temperature (Vogl, Paper, & Franz, 2000). Therefore, we varied the ratios of chlorosulfonic acid to formamide to produce four S-LAG preparations. These preparations are denoted based on their formulation ratio: S-LAG1-1, S-LAG1-3, S-LAG1-5 and S-LAG1-7. DS of the S-LAGs was determined by BaCl2-gelatin method and elemental analysis (Table 1). As expected, DS was controllable by varying derivatization agent ratios. Specifically, DS was shown to decrease at lower levels of chlorosulfonic acid addition. Comparing the two atomic sulfur measurement tests, the BaCl2-gelatin method indicated that sulfur content of the derivatives increased from 8.7% to 10.5% with increasing ratios of chlorosulfonic acid. This increase corresponded to DS elevating from 0.61 to 0.80. Elemental analysis revealed the same trends. the sulfur content of the derivatives increased from 8.0% to 10.2%, and the estimated DS increased from 0.54 to 0.77. Therefore, it was confirmed that the sulfated polysaccharides having various degree of substitution were successfully prepared. Our finding concerning control over DS are in agreement with what was reported in other works (Wang, Bao et al., 2018; Wang, Xie, Shen, Nie, & Xie, 2018; Xu et al., 2016). However, it is important to note that greater DS could be achieved using a different polysaccharide (Liu et al., 2009). This indicates that the maximum achievable of DS is also closely related to the structure of the polysaccharide being used. The Mw of sulfated polysaccharide is another important parameter influencing bioactivities. High-performance gel permeation

(2)

ODsamples and ODcontrol were measurements from wells in the presence and absence of S-LAGs, and ODblank were the reading from wells containing only medium and CCK-8. 2.5. Cell cycle distribution and cell apoptotic analysis Flow cytometric analysis was performed to identify the cell cycle phase distribution and apoptosis rate. This analysis was conducted using Annexin V-FITC and propidium iodides (PI) double staining kit according to manufacturer’s instructions. Briefly, cells were seeded into six-well plates at the density of 2 × 105 cells/well. The cell seeds were allowed to grow for 24 h, and then treated with different concentrations (200, 400 and 800 μg/mL) of S-LAG that had been formulated with 209

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polysaccharides. The chemical composition of non-sulfated carbohydrates in S-LAGs are shown in Table 2, and values are compared with the composition of LAG. It is important to note that not all monosaccharide units are sulfated. Due to polysaccharide structure, the extra-chain monosaccharides are easily modified, and some are relatively difficult to modify in the chain. HPAEC-PAD was applied to analysis the monosaccharide composition of polysaccharide samples, and the results were presented in Table 2. Monosaccharide composition analysis enabled calculation of the molar ratios of galactan to arabinan. Results showed that all the sulfated derivates were composed of arabinose and galactose, with different molar ratios (S-LAG1-1, 1:11.19, S-LAG1-3, 1:7.83, S-LAG1-5, 1:7.18, S-LAG1-1, 1:6.58). These results indicated that the sulfation reaction occurs simultaneously in arabinose and galactose, but galactose and arabinose from LAG have different degrees of sulfation. Although most of the arabinose in LAG is found to comprise the side chain of LAG polysaccharides (Tang et al., 2018), our results show that it does not undergo significant degrees of sulfation. In all, these findings show that our sulfated polysaccharides have various degrees sulfation substitution. However, each preparation contains different constitutions of unreacted carbohydrate residues.

Fig. 1. HPGPC chromatogram profiles of LAG and its sulfated derivative.

chromatography was used to estimate the molecular weights of the SLAGs, with the superimposed chromatograms shown in Fig. 1. The elution profiles of LAG and the S-LAGs all appeared as comparatively symmetrical and narrow peaks, indicating a strong homogeneity amongst the starting material and its sulfated derivatives. This finding speaks to the efficiency of our LAG isolation protocol, and also suggests that no significant structural disruption takes place during the derivatization protocol used. Moving to quantitative analysis, the Mw estimate of LAG and its four sulfated derivatives is included in Table 1. Results show that the estimated Mw of the S-LAGs decreased from 22.0 to 19.2 kDa when lowering the amount of sulfation reagent used during derivatization. Compared with LAG, the sulfated LAG had higher estimated Mw. This indicated that sulfation was successful, and perhaps more importantly, no unwanted polysaccharide degradation took place during derivatization. This was already hinted at based on the shape of narrow shapes provided from the HPGPC chromatographs, but is now quantitatively demonstrated as well. In prior research conducted by others, degradation accompanied successful sulfation of polysaccharides (Xu et al., 2016). However, our molecular weight findings are in accordance with (Xie, Jin et al., 2016), who showed that use of formamide as the derivatization solvent avoids significant polysaccharide depolymerization. We also recorded the gravimetric yields of the S-LAG preparations relative to LAG mass. In another demonstration of successful sulfation, S-LAG1-1, S-LAG1-3 and S-LAG1-5 gave high yields due to the addition of sulfate groups in polysaccharide chain (Table 1). This result also indirectly illustrates the integrity of the polysaccharide chain following derivatization, as well as the success of derivatization. It is important to note that the sample workup includes washing to remove all unreacted reagents and solvents, therefore gravimetric yield measurements were significant and free of systematic error. Similar results occurred in the sulfation of polysaccharides isolated from Phellinus ribis (Liu et al., 2009). The chemical compositions of the carbohydrate comprising the SLAGs (arabinan and galactan) is displayed in Table 2. It was found that the total sugar content of S-LAG1-1 was the lowest (34.1 ± 1.7%) of the preparations, and the sample with the highest sugar content was SLAG1-7 (49.2 ± 2.0%). The content of carbohydrate in LAG was significantly higher than those of its sulfated derivatives, which was expected given the addition of multiple weighty sulfate groups onto the

3.2. FT-IR spectra of S-LAGs Sulfated groups are easy to detect using infrared spectroscopy, so we utilized FT-IR spectroscopy to probe for structural changes induced to polysaccharides following sulfation modification (Sun et al., 2009). The FT-IR spectra acquired from the various S-LAG preparations, as well as the un-treated LAG material, are shown superimposed in Fig. 2. Typical of polysaccharide signals can be identified at around 3480, 2920, 1650 and 1055 cm−1, all of which were found across the five samples we have analyzed (Fleita, El-Sayed, & Rifaat, 2015). Regarding sulfation, the absorption peak at about 1240 cm−1 corresponding to S]O asymmetry stretching vibrations. Therefore, this signal is a good visual indicator of sulfate substitution. In addition, absorption peak at 820 cm−1 are typical of CeOeS stretching. Additionally, a new band (but minor) at 1725 cm−1 appeared across the S-LAG samples. This could be related to the unsaturated structure now present in the polysaccharides due to the sulfation modification (Vasconcelos et al., 2013). It can also be observed that the intensity of the CeH symmetrical stretching vibration (2920 cm−1) sharply decreased due to the substitution of OeH by eSO3− (Wang, Bao et al., 2018; Wang, Xie et al., 2018). In all, the FT-IR spectra provided further evidence of successful sulfation modification of LAG. 3.3. In vitro antitumor activity of S-LAGs Some sulfated natural polysaccharide have been shown to exhibit antitumor activity that is greater than that of an un-modified polysaccharide (Wang, Xie et al., 2018). In the present study, the growth inhibitory effects of LAG and the four sulfated derivatives created were tested against A549 cells, HepG-2 cells and MCF-7 cells. Results from all in vitro antitumor assays are shown Fig. 3. First, it can be seen that LAG did not show any ability to inhibit the growth of cancer cells. Compared with LAG, S-LAG1-1, S-LAG1-3, S-LAG1-5 and S-LAG1-7 each showed significant suppressive effects on the growth of A549, HepG-2 and MCF-

Table 2 Chemical and monosaccharide composition analysis of S-LAGs. Chemical composition

LAG

Total carbohydrate (%) 92.53 ± 1.43 Molar ratios of the neutral sugar components (arabinose as 1) Arabinose 1.00 Galactose 9.92

S-LAG1-1

S-LAG1-3

S-LAG1-5

S-LAG1-7

34.11 ± 1.72

41.28 ± 2.03

46.19 ± 1.79

49.21 ± 1.99

1.00 11.19

1.00 7.83

1.00 7.18

1.00 6.58

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apoptosis after exposure to S-LAG1-3 to gauge ability to induce apoptosis. The apoptosis of A549 cells after treatment with increasing dosages of S-LAG1-3 (200, 400 and 800 μg/mL) for 24 h is shown in Fig. 4. When the concentration of S-LAG1-3 was 200 μg/mL, the cells did not induce accumulation of A549 cells in the apoptotic phase compared to the control. A significant increase of cell population in the late apoptotic was observed from 10.84% of the control group to 27.36% for SLAG1-3 when the concentration was 400 μg/mL. Moving up to 800 μg/ mL, the late apoptotic cell count did not rise. However, the cells were found to have a tendency to develop necrosis. We base this inference on demonstration that the whole cell population shifts from late apoptosis to necrosis. Suppression of apoptosis during carcinogenesis is thought to play a central role in the development of some cancers (Kerr, Winterford, & Harmon, 1994). There are a variety of molecular mechanisms that tumor cells use to suppress apoptosis. Therefore, S-LAGs can abolish the inhibition of apoptosis by cancer cells. These results are consistent with grown inhibition results, in that low dosages of S-LAG exhibit little effect on cancer cells while greatly elevated dosages are remarkably effective at influencing cancer cell growth and life. Wang, Bao et al. (2018) have confirmed that sulfated polysaccharides have cytotoxic or cytostatic effect on various tumor cell lines in vitro by inducing cell apoptosis. In summary, the sulfated derivatives promote cancer cell apoptosis and gradually inhibit tumor cell growth.

Fig. 2. FT-IR spectra of LAG and its sulfated derivates.

7 cells. One important observation was that when the concentration of sulfated derivatives was 25–200 μg/mL, the inhibition rate of cancer cells did not change significantly. However, when the concentration was increased to the range of 400–800 μg/mL, all cancer cell survival rates significantly decreased. This indicates that cell survival depends on the concentration of sulfated polysaccharides, and a certain amount of concentration is essential to achieve a better antitumor effect. According to the report by Chen et al. (2011), significant cancer cell inhibition is achieved when the concentration reaches 2000 μg/mL. It is reported that DS of a sulfated polysaccharide is major factors for its biological effects (Yang, Du, Huang, Wan, & Li, 2002). In our paper, for HepG-2 cells, S-LAG1-1 (DS of 0.80) proved superior to the others with an inhibition ratio of 41.1% at the concentration of 800 μg/mL. While for A549 cells and MCF-7 cells, S-LAG1-3 (DS of 0.72) was most potent with an inhibition ratio of 40.9% and 41.8% at the concentration of 800 μg/mL, respectively and it suggested a moderate DS of the sulfated derivatives was necessary for a high antitumor activity in vitro. It generally considered that sulfated polysaccharides with relatively high DS exhibited stronger inhibition effects on same tumor cells. However, this situation is not absolute, depending on the differences in the cells themselves (Chen et al., 2011). Wang, Li et al. (2009) also reported that moderately substituted sulfated polysaccharides (DS of 0.85) exhibited highest inhibition of B16 cells proliferation. These results demonstrated that sulfated modification was an effective method to improve antitumor activities of polysaccharide in vitro.

4. Conclusions Four sulfated derivatives of LAG from Larix principis-rupprechtii were prepared using an established method, with control on degree of sulfate substitution being established by varying the dosage of derivatization agent (chlorosulfonic acid). Two characteristic absorption bands (at near 1240 and 823 cm−1) appeared in the FT-IR spectra of the sulfated derivatives, indicating that the sulfated reaction had occurred. Sulfated products showed different degree of substitution (DS), ranging from 0.61 to 0.80. In addition, different constituent carbohydrate ratios were observed. This suggested that some selectivity was present in the chemical modification of the polysaccharide substrate. All of the produced sulfated polysaccharide preparations showed greater antitumor activities at elevated dosages, far better than what was exhibited by the unmodified polysaccharide. In addition, induction of apoptosis was found to be the major cell death pattern induced by the S-LAG. In summary, the sulfated arabinogalactans were demonstrated to have antitumor bioactivities, suggesting that they may have strong potential for application in the fields of medicine and food through this needs to be further investigated in vivo. However, the reaction pathway of polysaccharide modification is worthy to be explored, aiming to obtain more precise modified polysaccharides in the future. With the progress of modern pharmaceutical technology, sulfation modification of polysaccharides may present a new avenue for gaining new insight on cancer treatments.

3.4. Inducing apoptosis in A549 cells using S-LAGs Apoptosis is a physiological response from cancer cells that results in their elimination. Based on the anti-proliferation assay of S-LAG1-3, the annexin V-FITC/PI double staining method was used to detect cell

Fig. 3. Effect of LAG and its sulfated derivates on the viability of cells. (A) A549 cells; (B) HepG-2 cells; (C) MCF-7 cells. Cells were treated with LAG and its sulfated derivates at the various concentrations for 72 h and the cell growth was measured using CCK-8 assay. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with control group. 211

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Fig. 4. Induction of apoptosis by S-LAG1-3 in A549 cells. Cells were treated with 200, 400 and 800 μg/mL of S-LAG1-3 for 24 h, respectively. Quantitative analysis of apoptotic cells by Annexin V-FITC/PI double staining assay using flow cytometry. The lower left (Q4) quadrant represented proportion of normal cells, lower right (Q3) quadrant represented proportion of early apoptotic cells, and upper right (Q2) and upper left (Q1) quadrant represented proportion of late apoptotic and necrosis cells, respectively.

Acknowledgments

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