Structure, anticoagulant and cytotoxic activity of a sulfated polysaccharide from green seaweed Chaetomorpha linum

Abstract In this article, chemical structure and conformation in an aqueous solution of a new sulfated polysaccharide, PCL, extracted from green seaweed Chaetomorpha linum were elucidated by SEC-MALL, IR, NMR and SAXS. The results indicated that the obtained polysaccharide is a sulfated arabinogalactan with a molecular weight of 223 kDa, and is mainly composed of →3,6)-α-D-Galp4S→ and →2)-α-L-Araf→ connecting together through 1→3 glycoside linkages. It has a broken rod-like conformation in solution with Rgc estimated as 0.43 nm from SAXS measurements. The polysaccharide exhibited a notable anticoagulant activity measured by the assays of activated partial thromboplastintime, thrombintime and prothrombine time as well as a significant cytotoxic activity against hepatocellular, human breast cancer, and cervical cancer cell lines. Graphical Abstract


Introduction
Cardiovascular disease is the leading cause of death worldwide.Although heparin has a strong anticoagulation activity and has widely been used as an anticoagulant for haemodialysis patients, it shows some side effects, such as haemorrhage, osteoporosis etc.In addition, sometimes contaminated virus become pathogenic because heparin is obtained from animal sources (Acquisto 2014).Another leading cause of death worldwide is cancer.Currently, cancer treatment methods simultaneously affect normal cells along with cancerous cells, resulting in undesired side effects.While bleeding can be problematic in advanced-cancer patients (Johnstone and Rich 2017), there is a strong association between tumour growth and a state of hypercoagulation (Falanga 2004;Kee et al. 2008;Neema et al. 2019).Rickles and Falanga (2001) showed in their review the danger of hypercoagulable state in cancer patients.Thromboembolic complications are accounted for a considerable fraction of the morbidity and mortality of the cancers.Therefore, it is worthwhile to find new compounds derived from natural sources and possessing both anticoagulant activity and a cytotoxic effect for better cancer treatments.However, the identification of such compounds is very limited.
Green seaweeds belonging to the Chlorophyta division are ubiquitously found in both marine and freshwater.They are rich sources of sulfated polysaccharides with anticoagulant activity (Mao et al. 2006;Shanmugam and Mody 2000;Mestechkina and Shcherbukhin 2010;Matsubara et al. 2001;Arata et al. 2016;Mao et al. 2008) and cytotoxic activity (Shao et al. 2013;Reis et al. 2020;Pagolu and Khora 2017;Thành et al. 2016).The present work is devoted to isolating and characterizing the structure of a sulfated polysaccharide from green seaweed Chaetomorpha linum by various analytical methods, and investigating their anticoagulant and cytotoxic activities Chaetomorpha is a genus of green algae in the family Cladophoraceae and known as Spaghetti algae or Green hair algae.Jebamalar and Sumathy reported that polysaccharide from Chaetomorpha antennina showed an anticoagulant activity (Jebamalar and Sumathy 2018).Rao and Ramana (1991) isolated an arabinogalactan from C. antennina and suggested that the arabinogalactan could be a promising source of anticoagulant agent.However, very few researches on structure and anticoagulant properties of polysaccharide from Chaetomorpha genus have been reported.C. linum is found in brackish water and grows as a filamentous loosely entangled mass.A polysaccharide from C. linum can be used as food additive, however, detail structure of the polysaccharide has not been investigated yet (Asma et al. 2020). Qin et al. (Qin et al. 2020) revealed anticoagulant-active sulfated arabinogalactan from C. linum, which mainly composed of →5)-α-L-Araf-(1→ and →6)-β-D-Galp-(1→ residues with a small amount of →4)-α-D-Galp-(1→).
On one hand, the biological activity of sulfated polysaccharides is closely involved with their structural factors such as sulfate content and molecular weight.On the other hand, polysaccharides are known to reveal biological functions by forming a specific conformation in solution (Gao et al. 1997;Yuguchi et al. 2016).Therefore, to get a better understanding of the structure-bioactivity relationship of the polysaccharides, in this study, we carried out an in-depth study on structural characteristics, both chemical and conformational structure, of an arabinogalactan extracted from green seaweeds C. linum.We employed (size exclusion chromatography with multiple angle laser light scattering (SEC-MALLS), IR, NMR and small angle X-ray scattering (SAXS), a powerful technique that can provide additional structural information of high-resolution structures, and determine the conformation of molecule in solution (Glatter and Kratky 1982;Grant et al. 2011).
Importantly, for the aim of developing a sole-anticoagulant drug or a dual-role agent (anticoagulant and anticancer), both anticoagulant and cytotoxic activities of the polysaccharide were investigated.The anticoagulant was measured by activated partial thrombosis time (APTT), thrombin time (TT) and prothrombin time (PT).The cytotoxicity against three human cancer cell lines including HepG2 (hepatocellular carcinoma), MCF7 (human breast cancer) and Hela (cervical cancer) was evaluated.

Chemical structure of P CL
The weight averaged molecular weight (Mw) and the number averaged molecular weight (Mn) of polysaccharide from C. linum (P CL ) were determined by SEC-MALLS.Like other native sulfated polysaccharides, the molecular weight distribution of the P CL is polydisperse with Mw/Mn = 1.87;Mw = 223 kDa.Chemical analysis and results estimated from SEC-MALLS measurement were summarized in Table S1.The results indicated that P CL was composed of galactose, arabinose, xylose and mannose with a mole ratio of Gal:Arab:Xyl:Man equal to 1.00:0.33:0.15:0.05 and sulfate content was 12%w.
FT-IR spectrum of P CL (Figure S1) showed bands corresponding to a typical sulfated polysaccharide.The broad stretching band at 3668 cm −1 was associated with hydrogen-bonded -OH groups.The band at 2964 cm −1 corresponded to the -CH stretching and the bands at 1461 and 1341 cm −1 corresponded to symmetrical -CH 2 and C-OH group.The absorption band at 1682 cm −1 was associated with the -OH flexural vibrations of P CL in an aqueous solution.Three absorption bands at 1148-1092 cm −1 indicated the presence of pyranose ring, while, the presence of furanose ring was indicated by the presence of two absorption bands at around 995 cm −1 .In addition, the band at 1259 cm −1 was attributed to S-O stretching vibration, the band at 838 cm −1 due to C-O-S bending vibration of sulfate in axial position of pyranose ring (Hong et al. 2021;Wu et al. 2020).
Chemical analysis and IR spectrum indicated that P CL is a sulfated arabinogalactan that contains both furanose ring and pyranose ring.
The 1 H NMR spectrum of P CL showed three anomeric proton peaks at 5.35, 5.15 and 5.10 ppm which were designated as A, B and B' , respectively.Absence of higher field signals (< 5) was the evidence that the polysaccharide contained α-configuration only.Two peaks at 5.35 and 5.15 ppm in 1 H spectrum and two peaks at 99.0 and 99.55 ppm in 13 C spectrum were assigned to H1 and C1 of α-L-arabinofuranose (A) and α-D-galactopyranose (B), respectively.The cross peaks H1/C1 in HSQC (Figure S2a) found at 5.35/99.0and 5.15/99.55ppm confirmed this assignment.The peaks at 62.6 and 66.0 ppm in 13 C spectrum were assigned to CH 2 groups of the galactopyranose and arabinofuranose residues, respectively.
Characteristic downfield shifts of C4 and C6 signals of galactose residues in comparison with those of the corresponding non-substituted units suggest that galactose residues contain glycosidic linkage at C6 and/or C4 or sulfate groups attached at C6 and/or C4.Combining with IR analysis, it points out that sulfate groups were in equatorial position; therefore, we can conclude that galactose residues have 1→6 glycoside linkage and substituted at C4.
The third proton anomeric peak at 5.10 ppm was assigned to anomeric protons of α-D-galactopyranose (B'), as branches of the main chain (1→6)-linked galactose residues.We can see the cross peaks H1(B)/H2(B) at 5.15/4.15ppm and H1(B')/H2(B) at 5.10/4.15ppm in COSY spectrum.Other signals for the branch galactose residues could not be seen because of their low intensity and overlap with the signals from the (1→6)-linked galactose residues of the main chain.
Based on all the results above, structure of polysaccharide from green seaweed C. linum can be proposed.It is a sulfated arabinogalactan, which mainly composed of →6)-α-Galp4S→ branched at C3 position and →2)-α-Araf→ connecting together through 1→3 linkages.Proposed structure of P CL is given in Figure 1.The complete NMR assignments are shown in Table S2.

Anticoagulant and cytotoxic activity of P CL
Anticoagulant activity of the P CL was measured by aPTT, PT and TT assays.Heparin and DMSO were used as a positive and negative control, respectively.P CL with the concentration of 400, 200, 100 and 50 µg/mL was adopted as an anticoagulant.It was found that P CL significantly delayed the blood clotting time in a dose-dependent manner in PT and TT assays (Table S3).TT assay is a simple screening technique for the fibrin polymerization process.It is a function of fibrinogen concentration and direct thrombin inhibitors.The P CL at the concentrations tested (50-400 μg/mL) significantly prolonged the clotting time in TT assay in a dose-dependent manner; TT of DMSO was 17.4 s, whereas that of P CL (400 μg/mL) reached up to 141.7 s.Prolongation of TT by the P CL indicates the inhibitory effect of this polysaccharide on thrombin activity or fibrin polymerization in a common pathway of the coagulation cascade of animal body (Mao et al. 2006).On the other hand, P CL also dose-dependently prolonged the clotting time in PT assay; the PT value of DMSO was 15.5 s, whereas that with 400 μg/mL of P CL was 35.3 s, although its effect was still weaker than heparin (Table S3).The prolongation of PT suggested that the P CL can inhibit the extrinsic pathway of the coagulation cascade of animal body.Significant prolongation of aPTT was observed when P CL was at a concentration in the range of 50-400 μg/mL.The aPTT value of P CL was over 180 s, similar to that of heparin, while this of DMSO was 141.9 s.This observation indicates that the polysaccharide strongly inhibits intrinsic pathway of blood coagulation.Taken all together, these results suggest that the P CL significantly inhibits intrinsic, extrinsic and common pathways of the blood coagulation cascade and the thrombin activity or conversion of fibrinogen to fibrin.
As mentioned previously, one goal of this study is to seek new compounds that serve dual role as anticoagulant and anticancer agents.Therefore, it is beneficial to investigate the cytotoxicity of P CL .In this work, cytotoxicity of P CL against three human cancer cell lines including HepG2 (Hepatocellular carcinoma), MCF7 (human breast cancer), and Hela (cervical cancer) was evaluated.Cytotoxic activities of P CL at various concentrations (0.8, 4, 20 and 100 µg/ml) against HepG2, MCF7 and Hela cancer cell lines were presented in Table S4.Each data point was obtained by making 3 independent measurements and all data were expressed as means ± S.D (standard deviation).The IC 50 values were estimated for HepG2, MCF7 and Hela cells to be 79.50 ± 3.40, 82.22 ± 3.30 and 68.74 ± 2.58 µg/ml, respectively, for the polysaccharide, and 0.38 ± 0.02, 0.41 ± 0.03 and 0.36 ± 0.05 µg/ml for reference drug Ellipticine.
Possessing both anticoagulant activity and cytotoxic effect makes P CL a notably drug candidate for cancer treatments

Conformational structure in solution of P CL
Sulfated polysaccharide's usefulness in the pharmaceutical field is related to the structural properties displayed in solution, that is, near the biologically active state.For a better understanding on the bioactive function of the sulfated polysaccharide, in this work, conformational structure in solution at nano-level of P CL was examined by SAXS method.
Figures S3 shows the Kratky plots (q 2 I(q) vs. q) for 1% P CL in water and in 0.5 M NaCl.Here I(q) is the scattering intensity, and q is the magnitude of scattering vector defined by (4π/λ)sinθ with θ and λ being a half of scattering angle and wavelength of incident X-ray, respectively.An interference peak can be found in ≈ 0.5 nm −1 of q in the normal plot (I(q) vs q) of P CL in water, due to the repulsive electrostatic interaction indicating the present of sulfate groups, this peak is screened by adding salt.
The results of SAXS were analyzed conventionally by the Guinier approximation for cross-section.Here the scattered intensity is approximately expressed in terms of the cross-sectional radius of gyration R gc as Figures S4 shows the Guinier plots for cross-section (lnqI(q) vs. q 2 ) from 1% P CL in water and in 0.5 M NaCl aqueous solution, respectively.Guinier plot yielded a good straight line, and the Rgc can be evaluated from the slope in linear region of cross-sectional Guinier plots (Glatter and Kratky 1982), the Rgc estimated as 0.42-0.43nm.The R GC value corresponds to the thickness of a single chain (Thành et al. 2002).Furthermore, the Guinier plot in NaCl aqueous solution shows linearity in more than 2 in q 2 and the upturn in initial q region, suggesting a broken rod-like structure

Conclusion
A novel polysaccharide isolated from C. linum was found to be a sulfated arabinogalactan, which was mainly composed of →3,6)-α-D-Galp-4S→ and →2)-α-L-Araf→ connecting together through 1→3 linkages.This study reports for the first time the structural conformation in solution of a sulfated polysaccharide extracted from Chaetomorpha genus.C. linum polysaccharide has a broken rod-like structure with cross-sectional radius of gyration around 0.43 nm as estimated from SAXS.
The sulfated arabinogalactan has a notable anticoagulant activity in a dose-dependent manner, suggesting its principal activity was mediated via the intrinsic coagulation pathway; furthermore, it also exhibited great cytotoxic activity.These physiological activities might be attributed to the chemical structure and/or chain conformation in solution of the polysaccharide.The sulfated arabinogalactan from C. linum could be used solely as an anticoagulant agent.It could also be a promising candidate for developing a drug to treat cancers that correlate with hypercoagulable state.Further study should be conducted to identify for which type of hypercoagulation-associated cancers the sulfated arabinogalactan from C. linum would be suitable.

Figure 1 .
Figure 1.Proposed structure of P cl .