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Chemical characteristics and antithrombotic effect of chondroitin sulfates from sturgeon skull and sturgeon backbone
Accepted Manuscript Title: Chemical characteristics and antithrombotic effect of chondroitin sulfates from sturgeon skull and sturgeon backbone Author: Meng Gui Juyi Song Lu Zhang Shun Wang Changwei Ma Pinglan Li PII: DOI: Reference:
S0144-8617(15)00077-6 http://dx.doi.org/doi:10.1016/j.carbpol.2015.01.046 CARP 9634
To appear in: Received date: Revised date: Accepted date:
6-9-2014 20-1-2015 21-1-2015
Please cite this article as: Gui, M., Song, J., Zhang, L., Wang, S., Ma, C., and Li, P.,Chemical characteristics and antithrombotic effect of chondroitin sulfates from sturgeon skull and sturgeon backbone, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.01.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights Chondroitin sulfates from sturgeon skull and sturgeon backbone were characterized. Sturgeon skull CS was primarily composed of nonsulfated disaccharide.
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The two CSs both had anticoagulant, anti-platelet and thrombolysis activities.
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Sturgeon backbone CS showed stronger antithrombotic effect than sturgeon skull CS.
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Chemical characteristics and antithrombotic effect of chondroitin sulfates from sturgeon skull and sturgeon backbone
The authors contributed equally to this work
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✟
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Meng Gui 1✟, Juyi Song 1,2✟, Lu Zhang1, Shun Wang3,,Changwei Ma1, Pinglan Li 1*
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1 Beijing Laboratory of Food Quality and Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China
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2 Institute of Agricultural Sciences of Jiangsu Changjiang River Bank District, Rugao 226541, China
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3 Beijing Fisheries Research Institute, Beijing 100068, China
Author. Meng Gui, Mailing address: NO.17 Qinghua East Road, East Campus, China Haidian
District,
d
University,
Beijing,
100083,
China;
E-mail:
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Agricultural
[email protected]; Tel: +86 10 62737664.
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Juyi Song, Email: [email protected] Lu Zhang, Email: [email protected] Shun Wang. Email: [email protected] Changwei Ma, Email: [email protected] *Corresponding Author. Mailing address: NO.17 Qinghua East Road, East Campus, China Agricultural
University,
Haidian
District,
Beijing,
100083,
China;
E-mail:
[email protected]; Tel: +86 10 62738678; Fax: 86 10 62738678.
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Abstract
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Chondroitin sulfates (CSs) were extracted from sturgeon skull and backbone, and their chemical composition, anticoagulant, anti-platelet and thrombolysis activities were evaluated. The average molecular weights of CS from sturgeon skull and backbone were 38.5 kDa and 49.2 kDa, respectively. Disaccharide analysis indicated that the sturgeon backbone CS was primarily composed of disaccharide monosulfated in position 4 of the GalNAc (37.8%)
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disaccharide monosulfated in position 6 of the GalNAc (59.6%) while sturgeon skull CS was primarily composed of nonsulfated disaccharide (74.2%).
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Sturgeon backbone CS showed stronger antithrombotic effect than sturgeon skull CS. Sturgeon backbone CS could significantly prolong activated partial
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thromboplastin time (APTT) and thrombin time (TT), inhibited ADP-induced platelet aggregation and dissolved platelet plasma clots in vitro. The results suggested that sturgeon backbone CS can be explored as a functional food with antithrombotic function.
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Key words: sturgeon, chondroitin sulfate, anticoagulant, anti-platelet aggregation, thrombolysis
1. Introduction 3
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Chondroitin sulfate (CS) is a polymeric carbohydrate comprising alternating disaccharide units of glucuronic acid/iduronic acid and N-acetyl-galactosamine
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linked by β-(1→3) glycosidic bonds and sulfated in different hydroxyl (Im, Park & Kim, 2010). CS can form a wide range of structures by incorporating different disaccharide repeat units or being modified with different numbers/positions of sulfate groups at C-4 and/or C-6 in N-acetyl-galactosamine and and C-2 and/or C-3 in glucuronic acid or uronic acid (Pomin, 2014). Structure variation of CS may result in its functional diversity. Sturgeon is one of the most economically important freshwater-cultured fish in China. The production of sturgeon in China accounts for approximately 80% of
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total production in the world (Gui, Song, Zhang, Hui & Li, 2014). The cartilage of sturgeon, which contains high concentration of CS, is discarded. Since CS
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possesses a wide variety of bioactivities, the cartilage of sturgeon has potential to be developed into functional foods or nutraceuticals. Recently, a few studies have investigated CS from sturgeon cartilage or bone (Im, Park & Kim, 2010; Maccari, Ferrarini & Volpi, 2010). However, no study has been conducted to characterize the chemical composition of CSs from sturgeon skull and backbone separately. Therefore, the first objective of our study was to investigate these
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chemical compositions of CSs from sturgeon backbond and skull, respectively. Diseases due to dysfunction of the blood circulatory system have become the major causes of mortality in developing countries all over the world (WHO Report, 2003). Treatment mainly involves medicines with antithrombotic, anticoagulant and anti-platelet activities (Pawlaczyk et al., 2011). These therapeutics inhibit the enzymes of the coagulation pathway in the blood circulatory, or inhibit the activation and aggregation of platelets, respectively 4
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(Łopaciuk, 2002). However, functional foods with antithrombotic effect have not yet been developed. Recent literature suggests that CS can suppress blood
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coagulation and prevent vein hardening (Sugahara, Mikami, Uyama, Mizuguchi, Nomura & Kitagawa, 2003b). Moreover, it is reported that CS has been utilized as a nutraceutical in dietary supplements in Europe and the USA (Volpi, 2009; Volpi & Maccari, 2008; Sim, Im, Cho, Jang, Jo & Kim, 2007). It is also well documented that oral CS is a valuable and safe symptomatic (and structure-modifying) treatment for osteoarthritic disease (Uebelhart, 2008; Hochberg & Clegg, 2008; Sawitzke et al., 2008). Then sturgeon CS may be also orally bioavaiable and has the potencial to be functional food with antithrombotic function.
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However, the antithrombotic effects of CS from sturgeon skull and backbone have yet to be studied. Therefore, the second objective of the present study was to
2 Materials and methods
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2.1 Materials
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evaluate the anticoagulant, anti-platelet and thrombolysis activities of sturgeon CSs.
Sturgeon (Hybrid Sturgeon, Acipenser baerii×Acipenser schrenckii) was purchased from Beijing Hui Longguan wholesale market. Shark cartilage CS was obtained from Seikagaku Co. (Tokyo, Japan) and was used to compare its antithrombotic effects to those of sturgeon CSs. The characterization of shark cartilage CS has been well documented (Im, Park & Kim, 2010). Chondroitinase ABC (0.5-2 units/mg) from Proteus vulgaris was purchased from Sigma (St. 5
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Louis, MO, USA.). Chondroitin sulfate specific enzyme (16 KNPU-S/g) and alcalase® (2.4 Anson units/g) from Bacillus licheniformis were purchased from Unsaturated disaccharides [ΔDi0s (ΔUA-[1→3]-GalNAc), ΔDi6s (ΔUA-[1→3]-GalNAc-6s) and ΔDi4s
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Novozymes (Bagsvaerd, Denmark).
(ΔUA-[1→3]-GalNAc-4s) were purchased from Sigma (St. Louis, MO, USA.). Sodium carbonate, trichloroacetic acid solution, cetylpyridinium chloride and all other chemicals and reagents were analytical grade from Sinopharm Chemical Reagent (Beijing, China). New Zealand white rabbits were provided by the 309th Hospital of Chinese People Liberation Army (Beijing, China).
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2.2 Extraction and purification of CS from Sturgeon skull and backbone
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Sturgeon skull and backbone were cleaned by tap water separately. They were chopped into small slices and dipped in 95% (v/v) ethanol for 2 h. Then these small slices were lyophilized and ground to dried powder. CS was extracted by using the method described by Im et al with modifications (Im, Park & Kim, 2010). The powder was suspended in 50 mM sodium carbonate buffer (pH 7.0) containing CS specific enzyme (15 mg enzyme per g CS powder) followed by
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incubation at 55°C for 4 h. Then the solution was boiled for 10 min to inactivate protease. Afterwards, alcalase (50 mg enzyme per g CS powder) was added after the solution was cooled down and kept at 60°C for 2 h. The solution was boiled, filtered, and cooled down 4°C followed by adding trichloroacetic acid solution (6.1 M) to a final concentration of 5% (v/v). After incubating at 4°C for 5 h, the insoluble materials were removed by centrifugation at 10,000 g for 15 min at 4°C. Then the supematant was precipitated by ethanol (75%, v/v) at 4°C overnight and the precipitate was collected by centrifugation. The precipitate 6
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was re-dissolved in water and mixed with cetylpyridinium chloride solution (final 1% w/v) and stored for 1.5 h at 20°C. It was then centrifuged again at 10,000
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g for 15 min at 4°C to collect the precipitate. The precipitate was dissolved in 2.5 M of sodium chloride (1.6%, m/v), precipitated by ethanol (75%, v/v) and centrifuged at 10,000 g for 15 min at 4°C. The final precipitate was dissolved in water, dialyzed (MWCO 7 kDa) for 2 d at 4°C and freeze-dried. 2.3 Purity and molecular weight determination
The purity and weight-average molecular weight of the CSs were estimated using gel permeation chromatography (GPC) combined with a DAWN HELEOS-II
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multi-angle laser-light scattering detector (MALLS) (Wyatt Technology, USA) (Kohno et al., 2009). The samples (10 μL, 5 mg/mL) were injected into the
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SB-806 M HR column (ID 8×300 mm, Shodex) and eluted with 0.1 M sodium nitrate at a flow rate of 0.5 mL/min. Samples were filtered (pore size 0.45 μm) prior to injection. The refractive index increment, dn/dc, was assumed to be 0.135. Data were collected and processed using Astra (version 5.3.4.20) software (Wyatt Technology). Number-average molecular weight (Mn) and weight-average molecular weight (Mw) were directly calculated according to the definition of
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Mn and Mw using molecular weight and RI signal values at each elution volume. 2.4 Disaccharide assay
The disaccharide composition of the CSs was determined by strong anion-exchange (SAX)-HPLC using a Hypersil SAX column (4.6 × 250 mm, 5 μm, Thermo, USA) after chondroitinase ABC digestion (Im, Park & Kim, 2010). To make the sample solution, 100 μL of CS test sample (1 mg/mL) was mixed with 800 μL 7
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of Tris-acetate buffer (50 mM Trizma® base and 60 mM of sodium acetate, pH 8.0). The samples was depolymerized by adding 100 μL of chondroitinase ABC
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(1 mU/μL) for 1 h at 37°C. After heating for 5 min and filtering through 0.45 μm syringe filters, the digestion mixture (100 μL) was injected into a SAX-HPLC. After the sample injection, the column was washed with water (pH 3.5) corresponding to one column volume (CV) for 4 min and a linear gradient of NaCl (0-2.0 M) at pH of 3.5 was used. The absorbance was monitored at 232 nm and the flow rate was 1.0 mL/min. Commercially available unsaturated standard disaccharides were used for qualitative and quantitative analysis.
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2.5 1H-NMR Spectroscopy
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The 1H-NMR spectra of the two CSs were recorded using a Bruker Avance 600 operating at 600 MHz. The CS samples were lyophilized three times with D2O, and then prepared by dissolving 50 mg in 0.5 mL of D2O at a high level of deuteration (99.9%).The spectra were recorded at 298 K and at the pH of 6.5. 2.6 Anticoagulant assay
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The anticoagulant activity of sturgeon skull CS, sturgeon backbone CS and shark CS was assayed in vitro using activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) as indicators (Huang, Du, Yang & Fan, 2003). Blood for anticoagulant study was collected from rabbit by venipuncture from ears. Then the blood was mixed carefully with 3.2% sodium citrate at a proportion of 9:1. Next, the blood was centrifuged for 15 min at 3,000 g at ambient temperature. Before assay, 40 μL samples of different concentrations (1 mg/mL，3 mg/mL and 5 mg/mL) of the three kinds of CSs were added 8
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individually to 360 μL of platelet-poor rabbit plasma for 15 min. The APTT, TT or PT values of various sample concentrations were measured in an ACL
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TOP700 coagulometer (Beckman Coulter Co., USA). The sodium chloride solution (0.9%) was used as the negative control. All assays were conducted in triplicate.
2.7 Inhibition of thrombin or coagulation factor Xa by antithrombin Ⅲ and heparin cofactor Ⅱassay These assays were performed according to the method of Colliec et al. (Colliec, Fischer, Tapon-Bretaudiere, Boisson, Durand & Jozefonvicz, 1991).
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Thrombin or coagulation factor Xa and inhibitors (heparin cofactor Ⅱor antithrombin Ⅲ) were incubated in 200 μL of 50 mmol/L Tris–HCl (pH 7.4), 7.5
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mmol/L EDTA and 0.15 mol/L NaCl at 37°C with or without CS. After 5 min incubation, 150 μL of Tris–HCl buffer containing 1.5 mmol/L chromogenic substrate S-2238 for thrombin or S-2222 for coagulation factor Xa was added, and the residual thrombin or coagulation factor Xa activity was determined by measuring the change in the absorbance at 405 nm. The rate of change of absorbance was proportional to the thrombin or coagulation factor Xa activity
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remaining in the incubation. No inhibition occurred in control experiments in which thrombin or coagulation factor Xa was incubated with antithrombin Ⅲ or heparin cofactor in the absence of the CS. 2.8 ADP-induced platelet aggregation assay Blood for platelet study was collected from rabbit by venipuncture from ears. Then the rabbit blood was mixed carefully with 3.2% sodium citrate at a proportion 9
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of 9:1. For the aggregation experiment, 40 μL aliquots of different concentrations of sturgeon skull CS, sturgeon backbone CS or shark CS were added to 360 μL
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of whole blood, mixed well and incubated at 37°C for 15 min. The samples were determined by PL-11 automatic platelet analyzer (SINNOWA Medical Technology Co. Ltd., Jiangsu, China) with five measurements. When the first and second results were less than 10%, 36 μL of ADP was added to initiate the reaction before the third time and the results was detected by the mean of the other three results. The sodium chloride solution (0.9%) was used as the negative control. All assays were conducted in triplicate.
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2.9 Thrombolysis assay
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Blood for thrombolysis study was collected from rabbit by venipuncture from ears. 1) Whole blood clot preparation: 30 mL of blood was distributed in PVC pipes and incubated at 37°C for 60 min. After clot formation, serum was completely removed and the clot was pushed out by injections and chopped into twelve pieces. The pieces were dried on filter papers and weighed.
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2) Platelet-rich plasma clot preparation: 30 mL of blood was centrifuged (800 g for 10 min) and platelet-rich plasma was collected from the blood in the upper layer and platelet in middle layer and dissolved in sodium citrate solution (1:9, v/v). Then thrombin (10 U/mL) was added at a dose of 0.1 mL per mL of plasma and mixed carefully. After clot formation, serum was completely removed and the clot was chopped into twelve pieces. The pieces were dried on filter papers and weighed. 10
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3) Pure fibrinogen clot preparation: 20 mL of pure fibrinogen solution (without plasmin and plasminogen, 20 mg/mL) and 2 mL of thrombin (10 U/mL) were
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mixed in a tube. After clot formation, serum was completely removed and the clot was chopped into twelve pieces. The pieces were dried on filter papers and weighed.
To each microcentrifuge tube that contained pre-weighed whole blood clots, platelet-rich plasma clots or pure fibrinogen clots, 2 mL of different concentrations of sturgeon skull CS, sturgeon backbone CS and shark CS were added separately. The negative control received 2 mL Tris-HCl buffer (pH 7.4) instead of CSs.
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All the tubes were then incubated at 37°C for 6 h and monitored for clot lysis. After incubation, clots were recovered and the dry weight was recorded to
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calculate the percentage of clot lysis. Percentage of clot lysis is defined as: (Weight before reaction-Weight after reaction)/Weight before reaction ×100. All assays were conducted in triplicate. 2.10 Statistical analyses
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Values were expressed as mean ± standard deviation (SD) and statistical analyses were performed with the SPSS 13.0 statistics software (SPSS, Chicago, IL, USA). The efficiency of biological activity in the assay was compared by using the paired t-test. P-values of less than 0.05 (P < 0.05) were considered as significant.
3 Results and discussion 11
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3.1 Purity and molecular weight
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The yields of sturgeon skull CS and backbone CS were 19.5% and 22% (lyophilized weight), respectively. The purity and molecular weight of the purified CSs from sturgeon skull and backbone were analyzed by GPC-MALLs (Fig. 1) and the results were recorded in Table 1. The purity of CSs extracted from sturgeon skull and backbone were 94.8% and 95.3%, respectively, and the average molecular weights were 38.5, and 49.2 kDa, respectively. The polydispersity indices (Mw/Mn) of the two kinds of CSs were lower than 1.5, indicating the components of both CSs were relatively uniform (Volpi, 2007).
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3.2 Disaccharide assay
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The purified CSs were treated with chondroitinase ABC, and then the unsaturated disaccharides produced were analyzed by SAX-HPLC (Fig. 2) and the results were recorded in Table 1. The chondroitinase ABC was able to produce three unsaturated disaccharides, the nonsulfated ΔDi0s [ΔUA1→3GalNAc], the monosulfated ΔDi6s [ΔUA1→3GalNAc6 (SO4)] and the ΔDi4s [ΔUA1→3GalNAc4 (SO4)]. No disulfated or trisulfated disaccharides were found, similar to
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the disaccharide composition of Acipenser sinensis (Im, Park & Kim, 2010). The disaccharide patterns of CSs from sturgeon skull and backbone were different. As shown in Table 1, sturgeon skull CS contained abundant ΔDi0s (74.2%) whereas sturgeon backbone CS contained abundant ΔDi6s (59.6%) and ΔDi4s (37.8%). The charge density value of CS from sturgeon backbone (0.97) was higher than that of sturgeon skull (0.26). This difference might due to the presence of a higher percentage of sulfated groups in sturgeon backbone (Volpi, 2009). It was reported that CS from shark was mainly composed of 12
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mono-sulfated disaccharides ΔDi-6S (50%) and ΔDi-4S (29%), and the content of ΔDi-0S (3%) was low (Volpi, 2007). Therefore, our results suggest that the
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disaccharide pattern of CS from sturgeon backbone was similar to CS from shark. 3.3 1H-NMR 1
H-NMR spectroscopy was performed to confirm the disaccharide composition and the structural integrity of the CSs (Fig. 3). The signals at 1.982 ppm (Fig. 3a)
in the spectrum of CS from sturgeon skull, 2.016 and 2.030 ppm (Fig. 3b) in the spectrum of CS from sturgeon backbone can be readily assigned to methyl
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protons of nonsulfated, 6-sulfated and 4-sulfated CS of GalNAc, respectively (Toida, Toyoda & Imanarit, 1993). The signals at 4.224 and 4.705 ppm (Fig. 3b)
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in the spectrum of CS from sturgeon backbone were assigned to H6 of GalNAc-6SO4 and H4 of GalNAc-4SO4, respectively (Toida, Toyoda & Imanarit, 1993; Mucci, Schenetti & Volpi, 2000b). The signal at 4.185 ppm (Fig. 3a) in the spectrum of CS from sturgeon skull was assigned to H6 of GalNAc-6SO4 while no signal was found to be assigned to H4 of GalNAc-4SO4. The difference suggested that the content of 4-sulfated CS in sturgeon skull was less than that in sturgeon backbone. The result was also consistent with the composition analysis of disaccharide by HPLC (Table 1).
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(a)
3.4 Anticoagulant assay
The anticoagulant activities of CSs from sturgeon skull, backbone and shark were investigated by the classical coagulation assays APTT, PT and TT. Saline was used as the negative control. As shown in Table 2, CS from sturgeon backbone exhibited stronger anticoagulant activity than CSs from shark and sturgeon skull 13
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at all concentrations in APTT and TT experiments. With increasing concentrations of CS, the anticoagulant activities of CSs from sturgeon backbone,
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sturgeon skull and shark increased. This pattern was especially clear for the CS from sturgeon backbone. The anticoagulant activities of CS were affected by polysaccharide’s monosaccharide composition, sulphation, species and location of substituent group (Majdoub, Mansour, Chaubet, Roudesli & Maaroufi, 2009; Chaidedgumjorn et al., 2002; Fonseca, Oliveira, Pomin, Mecawi, Araujo & Mourão, 2008). Since both sturgeon backbone CS and skull CS were composed of mono-sulfated disaccharides ΔDi-6S, ΔDi-4S and ΔDi-0S, the higher mono-sulfated disaccharide percentage in sturgeon skull CS, might contribute to its
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stronger anticoagulant activity compared to sturgeon skull CS. (Mulloy, Mourao & Gray, 2000).
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None of the CSs extracted from sturgeon skull, sturgeon backbone and shark cartilage showed significant differences in modulating prothrombin time in the PT assay. PT assay is used to evaluate extrinsic coagulation factors and the results we obtained suggest that CS extracted from these three bone tissues do not have effect on modulating extrinsic coagulation factors. In contrast, APTT assay is used to evaluate intrinsic blood coagulation factors and TT is a simple
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screening test for the fibrin polymerization process (Ye, Xu & Li, 2012; Ekanayake, Nikapitiya, De Zoysa, Whang, Kim & Lee, 2008). The results of APTT and TT assays suggest that CS extracted from these samples has the ability to modulate intrinsic blood coagulation factors and thrombin activity of controlling the degree of conversion fibrinogen to fibrin. 3.5 Inhibition of coagulation factor Xa or thrombin by antithrombin Ⅲ and heparin cofactor Ⅱ in the presence of the sturgeon CSs 14
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The effects of CSs from sturgeon skull and backbone on coagulation factor Xa or thrombin activity were investigated (Fig. 4). Sturgeon skull CS and sturgeon
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backbone CS showed weak inhibitory effect on coagulation factor Xa in the absence of antithrombin Ⅲ. However, the two sturgeon CSs had a significant (P < 0.05) inhibitory effect on coagulation factor Xa in the presence of antithrombin Ⅲ, and the CS from sturgeon backbone exhibited greater inhibitory effect than that from sturgeon skull. Moreover, the two kinds of sturgeon CSs exhibited no significant inhibitory effect on coagulation factor Xa in the presence of heparin cofactor Ⅱ(data not shown). Meanwhile, no inhibition of thrombin was observed by the two kinds of sturgeon CSs either in the presence or absence of
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antithrombin Ⅲ or heparin cofactor Ⅱ (data not shown). Therefore, CSs extracted from sturgeon skull and sturgeon backbone did not have direct inhibition
3.6 Anti-platelet effect
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of coagulation factor Xa but were mediated by antithrombin Ⅲ.
Anti-platelet activities of CSs from sturgeon skull, backbone and shark were tested on platelet aggregation assay using rabbit whole blood (Table 3). The platelet
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inhibition rate (%) of CS extracted from sturgeon skull, sturgeon backbone, and shark increased in a dose-dependent manner. In comparison with the control, significant inhibition of the aggregation was observed from three kinds of CSs even at low concentration (1mg/mL) (P < 0.05). Sturgeon skull CS showed lower anti-platelet activity than sturgeon backbone CS and shark CS at all concentrations. The anti-platelet activity of shark CS was comparable to that of sturgeon backbone CS at the concentrations of 1 mg/mL and 3 mg/mL. However, sturgeon backbone CS exhibited higher anti-platelet activity at the concentration of 5 15
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mg/mL. Similarly, a polyphenolic-polysaccharide preparation isolated from medicinal plant Erigeron canadensis L. also has anti-aggregatory effect on
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platelets (Pawlaczyk et al., 2011). In contrast, other polysaccharides stimulate instead of preventing platelet aggregation (Li & Lian, 1998; Farias, Nazareth & Mourão, 2001). 3.7 Thrombolysis assays
Thrombolysis effects of CSs from sturgeon skull, backbone and shark were tested on whole blood clot dissolution rate, platelet plasma clot dissolution rate and
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pure fibrin clot dissolution rate using rabbit blood (Table 4). CSs from sturgeon backbone and shark exhibited strong effects on dissolving platelet plasma clots,
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while they exhibited weak effects on dissolving whole blood clot and pure fibrin clots. The results suggested that the major thrombolysis effect of CSs from sturgeon backbone and shark was reflected in dissolving platelet plasma clot. Sturgeon backbone CS exhibited a stronger effect on dissolving platelet plasma clot than shark CS with the increase of the concentration, which was consistent with the results of anti-platelet effect assay. However, sturgeon skull CS
4 Conclusions
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exhibited weak effects on dissolving all three kinds of clots.
In this study, it is concluded that CSs from sturgeon skull and backbone were composed of three kinds of disaccharides including the nonsulfated Δdi0s [ΔUA1→3GalNAc], the monosulfated Δdi6s [ΔUA1→3GalNAc6 (SO4)], and the Δdi4s [ΔUA1→3GalNAc4 (SO4)]. Sturgeon backbone CS was composed of 16
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~37.8% ΔDi4s, ~59.6% ΔDi6s and small proportions of ΔDi0s (2.6%) while sturgeon skull CS was composed of large proportions of ΔDi0s (74.2%) and small
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proportions of ΔDi4s (8.1%) and ΔDi6s (17.7%). Meanwhile, sturgeon backbone CS exhibited stronger anticoagulant, anti-platelet and thrombolysis activity than sturgeon skull CS, which might be attributed to a higher percentage of mono-sulfated disaccharides, especially ΔDi-4S in sturgeon backbone CS. However, the detailed mechanisms of the antithrombotic effect of CSs are not clear and need to be further studied. The comprehensive antithrombotic effect of sturgeon
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backbone CS suggested that it should be explored as a functional food with antithrombotic function. 5 Acknowledgement
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This work was supported by the Beijing Innovation Team of China Agriculture Research Center System (SCGWZJ20131105-2) and Major Program(s) of Beijing Municipal Science & Technology Commission (D121100003712003). 6 References
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Łopaciuk S. Zakrzepy i zatory. Warszawa: Wyd. Lekarskie PZWL; 2002. Maccari, F., Ferrarini, F., & Volpi, N. (2010). Structural characterization of chondroitin sulfate from sturgeon bone. Carbohydrate Research, 345(11), 1575-1580.
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Majdoub, H., Mansour, M. B., Chaubet, F., Roudesli, M. S., & Maaroufi, R. M. (2009). Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis. Biochimica et Biophysica Acta (BBA)-General Subjects, 1790(10), 1377-1381. Mucci, A., Schenetti, L., & Volpi, N. (2000a). 1 H and 13 C nuclear magnetic resonance identification and characterization of components of chondroitin sulfates of various origin. Carbohydrate Polymers, 41(1), 37-45. 19
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Mucci, A., Schenetti, L., & Volpi, N. (2000b). 1 H and 13 C nuclear magnetic resonance identification and characterization of components of chondroitin
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sulfates of various origin. Carbohydrate Polymers, 41(1), 37-45.
Mulloy, B., Mourao, P., & Gray, E. (2000). Structure/function studies of anticoagulant sulphated polysaccharides using NMR. Journal of Biotechnology, 77(1), 123-135.
Pawlaczyk, I., Czerchawski, L., Kuliczkowski, W., Karolko, B., Pilecki, W., Witkiewicz, W., & Gancarz, R. (2011). Anticoagulant and anti-platelet activity
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of polyphenolic-polysaccharide preparation isolated from the medicinal plant Erigeron canadensis L. Thrombosis Research, 127(4), 328-340.
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Pomin, V. H. (2014). NMR Chemical Shifts in Structural Biology of Glycosaminoglycans. Analytical Chemistry, 86(1), 65-94. Sawitzke, A. D., et al. (2008). The effect of glucosamine and/or chondroitin sulfate on the progression of knee osteoarthritis : a report from the glucosamine/chondroitin arthritis intervention trial. Arthritis Rheum, 58(10), 3183-3191.
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Sim, J. S., Im, A. R., Cho, S. M., Jang, H. J., Jo, J. H., & Kim, Y. S. (2007). Evaluation of chondroitin sulfate in shark cartilage powder as a dietary supplement: Raw materials and finished products. Food Chemistry, 101(2), 532-539. Sugahara, K., Mikami, T., Uyama, T., Mizuguchi, S., Nomura, K., & Kitagawa, H. (2003a). Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate. Current Opinion In Structural Biology, 13(5), 612-620. 20
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Sugahara, K., Mikami, T., Uyama, T., Mizuguchi, S., Nomura, K., & Kitagawa, H. (2003b). Recent advances in the structural biology of chondroitin sulfate
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and dermatan sulfate. Current Opinion In Structural Biology, 13(5), 612-620.
Toida, T., Toyoda, H., & Imanarit, T. (1993). High-Resolution Proton on Chondroitin Sulfates. SCIENCES, 9. Uebelhart, D. (2008). Clinical review of chondroitin sulfate in osteoarthritis. Osteoarthritis Cartilage, 16 (Suppl. 3): S19-S21. Volpi, N. (2007). Analytical aspects of pharmaceutical grade chondroitin sulfates. Journal of Pharmaceutical Sciences, 96(12), 3168-3180.
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Volpi, N., Maccri, F. (2008). Quantitative and qualitative evaluation of chondroitin sulfate in dietary supplemens. Food Analytical Methods, 1(3), 195-204.
1271-1280.
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Volpi, N. (2009). Quality of different chondroitin sulfate preparations in relation to their therapeutic activity. Journal of Pharmacy and Pharmacology, 61(10),
2003.
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World Health Organization. WHO Report, Cardiovascular disease: Prevention and control. http://www.who.int/dietphysicalactivity/publications/facts/cvd/en
Ye, L., Xu, L., & Li, J. (2012). Preparation and anticoagulant activity of a fucosylated polysaccharide sulfate from a sea cucumber Acaudina molpadioidea. Carbohydrate Polymers, 87(3), 2052-2057.
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Figure captions
Fig. 1 GPC-MALLS of chondroitin sulfates from sturgeon skull (red) and sturgeon backbone (blue). Typical molar mass vs elution time plots are shown overlapped for sturgeon skull CS and backbone CS with traces for scattered light measured by one of 18 detectors (solid line) and molar mass (dashed line) calculated by instrument software.
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ΔDi4s, ΔUA1→3GalNAc4 (SO4).
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Fig. 2 SAX-HPLC of chondroitin sulfates from (a) sturgeon skull and (b) sturgeon backbone. ΔDi0s, ΔUA1→3GalNAc. ΔDi6s, ΔUA1→3GalNAc6 (SO4).
Fig. 3 1H NMR spectra of chondroitin sulfates from (a) sturgeon skull and (b) sturgeon backbone. Fig.4 Effect of chondroitin sulfate from sturgeon skull and sturgeon backbone on inhibition of coagulation factor Xa activity in the presence and absence of
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antithrombin Ⅲ. Coagulation factor Xa was incubated with chondroitin sulfate from sturgeon skull in the absence (◆) and in the presence (■) of antithrombin Ⅲ. Coagulation factor Xa was incubated with chondroitin sulfate from sturgeon backbone in the absence (◇) and in the presence (□) of antithrombin Ⅲ.
22
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Table(s)
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Table1
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The chemical characterization (purity, weight, polydispersity index, disaccharide ratio and charge density) of sturgeon CSs in this study and literatures.
Molecular weight (kDa)
Polydispersity index (Mw/Mn)
Sturgeon skull CS
94.8
38.5±1.0
1.36±0.06
Sturgeon backbone CS
95.3
49.2±2.6
Sturgeon cartilage CS
91.1
Sturgeon backbone CS
100>
Sturgeon bone CS Shark cartilage CS
Charge density
References
ΔDi-4S
74.2
17.7
8.1
0.97
This study
2.6
59.6
37.8
0.26
This study
8.0
4.0
7.2
88.8
Im et al, 2010
43.4
9.9
66.2
23.9
Im et al, 2010
7.0
55
38
2.3
40.8
34.9
d
ΔDi-6S
1.44±0.14
ep te 37.5
98.2
Disaccharide ratio (%)a
ΔDi-0S
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Sample
M
Purity (%)
60.6
1.34
0.93
Maccari et al, 2010
Im et al, 2010
aΔDi0s[ΔUA1→3GalNAc]; ΔDi6s [ΔUA1→3GalNAc6(SO4)]; ΔDi4s [ΔUA1→3GalNAc4(SO4)].
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Table 2
APTT(s) at different concentrations (mg/mL) 1 3 5
PT(s) at different concentrations (mg/mL) 1 3 5
Negative control Sturgeon skull CS Sturgeon backbone CS Shark cartilage CS
28.9±2.6
－
－
9.9±0.6
－
－
15.7±0.6
－
－
31.2±0.4
33.8±0.9
34.6±1.8*
9.8±0.3
10.3±0.4
10.6±0.5
16.5±0.8
17.7±0.6*
17.9±0.6*
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sample
d
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APTT, PT and TT of rabbit platelet-poor plasma containing sturgeon skull CS, sturgeon backbone CS and shark CS. TT(s) at different concentrations (mg/mL) 1 3 5
73.4±1.3*
9.6±0.1
10.8±0.1
11.0±0.9
19.5±0.3* 22.9±0.1*
26.5±0.2*
32.5±1.6* 40.8±3.4*
64.8±3.1*
10.2±0.3
10.7±0.9
11.1±1.0
20.5±0.4* 23.5±1.2*
26.7±0.2*
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36.0±1.3* 41.2±1.1*
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Note: all the data were the mean of three parallel assays.－indicated the value was not evaluated. *Significantly inhibited compared with negative control (P<0.05).
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Table 3
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Effect of sturgeon skull CS, sturgeon backbone CS and shark CS on platelet aggregation in vitro. Concentration (mg/mL)
Platelet maximal aggregation rate (%)
Platelet inhibition rate (%)
Negative control
0
39.9±0.3
0
1 3 5 1 3 5 1 3 5
37.3±0.5* 28.8±0.3** 19.8±0.9** 34.7±1.0** 24.5±1.8** 12.2±0.8** 34.1±0.9** 25.4±0.3** 16.1±1.0**
Sturgeon backbone CS
Shark CS
d
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Sturgeon skull CS
M
Sample
7.0±1.2* 28.4±0.7** 36.8±0.4** 13.4±2.5** 39.0±4.4** 69.7±1.9** 15.1±2.3** 38.1±2.5** 59.8±.2.5**
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Note: all the data were the mean of three parallel assays.
*Significantly inhibited compared with negative control (P<0.05). ** Significantly inhibited compared with negative control (P<0.01).
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Table 4
Whole blood clot dissolution rate (%) at different concentrations (mg/mL) 3 －
5 －
0.8 ±0.03
1.3±0.03
1.3±0.07
Sturgeon backbone CS
1.4±0.02
2.9±0.01
3.5±0.08
Shark cartilage CS
1.1±0.03
2.3±0.09
3.7±0.08
Pure fibrin clot dissolution rate (%) at different concentrations (mg/mL)
3 －
5 －
1 0
3 －
5 －
2.4±0.03
3.3±0.07
1.2±0.04
2.0±0.04
2.3±0.04
6.2±0.03
9.2±0.02
17.7±0.08
2.4±0.02
3.3±0.04
4.3±0.03
6.1±0.03
8.0±0.10
15.2±0.79
2.1±0.04
3.4±0.03
4.5±0.06
1.3±0.03
ep te
Negative control Sturgeon skull CS
1 0
d
1 0
Platelet plasma clot dissolution rate (%) at different concentrations (mg/mL)
M
Sample
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Effect of sturgeon skull CS, sturgeon backbone CS and shark CS on thrombolysis in vitro.
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Note: all the data were the mean of three parallel assays.－indicated the value was not evaluated.
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Figure 2
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Figure 3
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Figure 4
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S0144-8617(15)00077-6 http://dx.doi.org/doi:10.1016/j.carbpol.2015.01.046 CARP 9634
To appear in: Received date: Revised date: Accepted date:
6-9-2014 20-1-2015 21-1-2015
Please cite this article as: Gui, M., Song, J., Zhang, L., Wang, S., Ma, C., and Li, P.,Chemical characteristics and antithrombotic effect of chondroitin sulfates from sturgeon skull and sturgeon backbone, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.01.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights Chondroitin sulfates from sturgeon skull and sturgeon backbone were characterized. Sturgeon skull CS was primarily composed of nonsulfated disaccharide.
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The two CSs both had anticoagulant, anti-platelet and thrombolysis activities.
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Sturgeon backbone CS showed stronger antithrombotic effect than sturgeon skull CS.
1
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Chemical characteristics and antithrombotic effect of chondroitin sulfates from sturgeon skull and sturgeon backbone
The authors contributed equally to this work
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✟
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Meng Gui 1✟, Juyi Song 1,2✟, Lu Zhang1, Shun Wang3,,Changwei Ma1, Pinglan Li 1*
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1 Beijing Laboratory of Food Quality and Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China
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2 Institute of Agricultural Sciences of Jiangsu Changjiang River Bank District, Rugao 226541, China
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3 Beijing Fisheries Research Institute, Beijing 100068, China
Author. Meng Gui, Mailing address: NO.17 Qinghua East Road, East Campus, China Haidian
District,
d
University,
Beijing,
100083,
China;
E-mail:
te
Agricultural
[email protected]; Tel: +86 10 62737664.
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Juyi Song, Email: [email protected] Lu Zhang, Email: [email protected] Shun Wang. Email: [email protected] Changwei Ma, Email: [email protected] *Corresponding Author. Mailing address: NO.17 Qinghua East Road, East Campus, China Agricultural
University,
Haidian
District,
Beijing,
100083,
China;
E-mail:
[email protected]; Tel: +86 10 62738678; Fax: 86 10 62738678.
2
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Abstract
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Chondroitin sulfates (CSs) were extracted from sturgeon skull and backbone, and their chemical composition, anticoagulant, anti-platelet and thrombolysis activities were evaluated. The average molecular weights of CS from sturgeon skull and backbone were 38.5 kDa and 49.2 kDa, respectively. Disaccharide analysis indicated that the sturgeon backbone CS was primarily composed of disaccharide monosulfated in position 4 of the GalNAc (37.8%)
and
disaccharide monosulfated in position 6 of the GalNAc (59.6%) while sturgeon skull CS was primarily composed of nonsulfated disaccharide (74.2%).
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Sturgeon backbone CS showed stronger antithrombotic effect than sturgeon skull CS. Sturgeon backbone CS could significantly prolong activated partial
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thromboplastin time (APTT) and thrombin time (TT), inhibited ADP-induced platelet aggregation and dissolved platelet plasma clots in vitro. The results suggested that sturgeon backbone CS can be explored as a functional food with antithrombotic function.
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Key words: sturgeon, chondroitin sulfate, anticoagulant, anti-platelet aggregation, thrombolysis
1. Introduction 3
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Chondroitin sulfate (CS) is a polymeric carbohydrate comprising alternating disaccharide units of glucuronic acid/iduronic acid and N-acetyl-galactosamine
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linked by β-(1→3) glycosidic bonds and sulfated in different hydroxyl (Im, Park & Kim, 2010). CS can form a wide range of structures by incorporating different disaccharide repeat units or being modified with different numbers/positions of sulfate groups at C-4 and/or C-6 in N-acetyl-galactosamine and and C-2 and/or C-3 in glucuronic acid or uronic acid (Pomin, 2014). Structure variation of CS may result in its functional diversity. Sturgeon is one of the most economically important freshwater-cultured fish in China. The production of sturgeon in China accounts for approximately 80% of
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total production in the world (Gui, Song, Zhang, Hui & Li, 2014). The cartilage of sturgeon, which contains high concentration of CS, is discarded. Since CS
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possesses a wide variety of bioactivities, the cartilage of sturgeon has potential to be developed into functional foods or nutraceuticals. Recently, a few studies have investigated CS from sturgeon cartilage or bone (Im, Park & Kim, 2010; Maccari, Ferrarini & Volpi, 2010). However, no study has been conducted to characterize the chemical composition of CSs from sturgeon skull and backbone separately. Therefore, the first objective of our study was to investigate these
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chemical compositions of CSs from sturgeon backbond and skull, respectively. Diseases due to dysfunction of the blood circulatory system have become the major causes of mortality in developing countries all over the world (WHO Report, 2003). Treatment mainly involves medicines with antithrombotic, anticoagulant and anti-platelet activities (Pawlaczyk et al., 2011). These therapeutics inhibit the enzymes of the coagulation pathway in the blood circulatory, or inhibit the activation and aggregation of platelets, respectively 4
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(Łopaciuk, 2002). However, functional foods with antithrombotic effect have not yet been developed. Recent literature suggests that CS can suppress blood
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coagulation and prevent vein hardening (Sugahara, Mikami, Uyama, Mizuguchi, Nomura & Kitagawa, 2003b). Moreover, it is reported that CS has been utilized as a nutraceutical in dietary supplements in Europe and the USA (Volpi, 2009; Volpi & Maccari, 2008; Sim, Im, Cho, Jang, Jo & Kim, 2007). It is also well documented that oral CS is a valuable and safe symptomatic (and structure-modifying) treatment for osteoarthritic disease (Uebelhart, 2008; Hochberg & Clegg, 2008; Sawitzke et al., 2008). Then sturgeon CS may be also orally bioavaiable and has the potencial to be functional food with antithrombotic function.
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However, the antithrombotic effects of CS from sturgeon skull and backbone have yet to be studied. Therefore, the second objective of the present study was to
2 Materials and methods
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2.1 Materials
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evaluate the anticoagulant, anti-platelet and thrombolysis activities of sturgeon CSs.
Sturgeon (Hybrid Sturgeon, Acipenser baerii×Acipenser schrenckii) was purchased from Beijing Hui Longguan wholesale market. Shark cartilage CS was obtained from Seikagaku Co. (Tokyo, Japan) and was used to compare its antithrombotic effects to those of sturgeon CSs. The characterization of shark cartilage CS has been well documented (Im, Park & Kim, 2010). Chondroitinase ABC (0.5-2 units/mg) from Proteus vulgaris was purchased from Sigma (St. 5
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Louis, MO, USA.). Chondroitin sulfate specific enzyme (16 KNPU-S/g) and alcalase® (2.4 Anson units/g) from Bacillus licheniformis were purchased from Unsaturated disaccharides [ΔDi0s (ΔUA-[1→3]-GalNAc), ΔDi6s (ΔUA-[1→3]-GalNAc-6s) and ΔDi4s
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Novozymes (Bagsvaerd, Denmark).
(ΔUA-[1→3]-GalNAc-4s) were purchased from Sigma (St. Louis, MO, USA.). Sodium carbonate, trichloroacetic acid solution, cetylpyridinium chloride and all other chemicals and reagents were analytical grade from Sinopharm Chemical Reagent (Beijing, China). New Zealand white rabbits were provided by the 309th Hospital of Chinese People Liberation Army (Beijing, China).
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2.2 Extraction and purification of CS from Sturgeon skull and backbone
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Sturgeon skull and backbone were cleaned by tap water separately. They were chopped into small slices and dipped in 95% (v/v) ethanol for 2 h. Then these small slices were lyophilized and ground to dried powder. CS was extracted by using the method described by Im et al with modifications (Im, Park & Kim, 2010). The powder was suspended in 50 mM sodium carbonate buffer (pH 7.0) containing CS specific enzyme (15 mg enzyme per g CS powder) followed by
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incubation at 55°C for 4 h. Then the solution was boiled for 10 min to inactivate protease. Afterwards, alcalase (50 mg enzyme per g CS powder) was added after the solution was cooled down and kept at 60°C for 2 h. The solution was boiled, filtered, and cooled down 4°C followed by adding trichloroacetic acid solution (6.1 M) to a final concentration of 5% (v/v). After incubating at 4°C for 5 h, the insoluble materials were removed by centrifugation at 10,000 g for 15 min at 4°C. Then the supematant was precipitated by ethanol (75%, v/v) at 4°C overnight and the precipitate was collected by centrifugation. The precipitate 6
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was re-dissolved in water and mixed with cetylpyridinium chloride solution (final 1% w/v) and stored for 1.5 h at 20°C. It was then centrifuged again at 10,000
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g for 15 min at 4°C to collect the precipitate. The precipitate was dissolved in 2.5 M of sodium chloride (1.6%, m/v), precipitated by ethanol (75%, v/v) and centrifuged at 10,000 g for 15 min at 4°C. The final precipitate was dissolved in water, dialyzed (MWCO 7 kDa) for 2 d at 4°C and freeze-dried. 2.3 Purity and molecular weight determination
The purity and weight-average molecular weight of the CSs were estimated using gel permeation chromatography (GPC) combined with a DAWN HELEOS-II
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multi-angle laser-light scattering detector (MALLS) (Wyatt Technology, USA) (Kohno et al., 2009). The samples (10 μL, 5 mg/mL) were injected into the
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SB-806 M HR column (ID 8×300 mm, Shodex) and eluted with 0.1 M sodium nitrate at a flow rate of 0.5 mL/min. Samples were filtered (pore size 0.45 μm) prior to injection. The refractive index increment, dn/dc, was assumed to be 0.135. Data were collected and processed using Astra (version 5.3.4.20) software (Wyatt Technology). Number-average molecular weight (Mn) and weight-average molecular weight (Mw) were directly calculated according to the definition of
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Mn and Mw using molecular weight and RI signal values at each elution volume. 2.4 Disaccharide assay
The disaccharide composition of the CSs was determined by strong anion-exchange (SAX)-HPLC using a Hypersil SAX column (4.6 × 250 mm, 5 μm, Thermo, USA) after chondroitinase ABC digestion (Im, Park & Kim, 2010). To make the sample solution, 100 μL of CS test sample (1 mg/mL) was mixed with 800 μL 7
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of Tris-acetate buffer (50 mM Trizma® base and 60 mM of sodium acetate, pH 8.0). The samples was depolymerized by adding 100 μL of chondroitinase ABC
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(1 mU/μL) for 1 h at 37°C. After heating for 5 min and filtering through 0.45 μm syringe filters, the digestion mixture (100 μL) was injected into a SAX-HPLC. After the sample injection, the column was washed with water (pH 3.5) corresponding to one column volume (CV) for 4 min and a linear gradient of NaCl (0-2.0 M) at pH of 3.5 was used. The absorbance was monitored at 232 nm and the flow rate was 1.0 mL/min. Commercially available unsaturated standard disaccharides were used for qualitative and quantitative analysis.
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2.5 1H-NMR Spectroscopy
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The 1H-NMR spectra of the two CSs were recorded using a Bruker Avance 600 operating at 600 MHz. The CS samples were lyophilized three times with D2O, and then prepared by dissolving 50 mg in 0.5 mL of D2O at a high level of deuteration (99.9%).The spectra were recorded at 298 K and at the pH of 6.5. 2.6 Anticoagulant assay
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The anticoagulant activity of sturgeon skull CS, sturgeon backbone CS and shark CS was assayed in vitro using activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) as indicators (Huang, Du, Yang & Fan, 2003). Blood for anticoagulant study was collected from rabbit by venipuncture from ears. Then the blood was mixed carefully with 3.2% sodium citrate at a proportion of 9:1. Next, the blood was centrifuged for 15 min at 3,000 g at ambient temperature. Before assay, 40 μL samples of different concentrations (1 mg/mL，3 mg/mL and 5 mg/mL) of the three kinds of CSs were added 8
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individually to 360 μL of platelet-poor rabbit plasma for 15 min. The APTT, TT or PT values of various sample concentrations were measured in an ACL
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TOP700 coagulometer (Beckman Coulter Co., USA). The sodium chloride solution (0.9%) was used as the negative control. All assays were conducted in triplicate.
2.7 Inhibition of thrombin or coagulation factor Xa by antithrombin Ⅲ and heparin cofactor Ⅱassay These assays were performed according to the method of Colliec et al. (Colliec, Fischer, Tapon-Bretaudiere, Boisson, Durand & Jozefonvicz, 1991).
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Thrombin or coagulation factor Xa and inhibitors (heparin cofactor Ⅱor antithrombin Ⅲ) were incubated in 200 μL of 50 mmol/L Tris–HCl (pH 7.4), 7.5
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mmol/L EDTA and 0.15 mol/L NaCl at 37°C with or without CS. After 5 min incubation, 150 μL of Tris–HCl buffer containing 1.5 mmol/L chromogenic substrate S-2238 for thrombin or S-2222 for coagulation factor Xa was added, and the residual thrombin or coagulation factor Xa activity was determined by measuring the change in the absorbance at 405 nm. The rate of change of absorbance was proportional to the thrombin or coagulation factor Xa activity
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remaining in the incubation. No inhibition occurred in control experiments in which thrombin or coagulation factor Xa was incubated with antithrombin Ⅲ or heparin cofactor in the absence of the CS. 2.8 ADP-induced platelet aggregation assay Blood for platelet study was collected from rabbit by venipuncture from ears. Then the rabbit blood was mixed carefully with 3.2% sodium citrate at a proportion 9
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of 9:1. For the aggregation experiment, 40 μL aliquots of different concentrations of sturgeon skull CS, sturgeon backbone CS or shark CS were added to 360 μL
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of whole blood, mixed well and incubated at 37°C for 15 min. The samples were determined by PL-11 automatic platelet analyzer (SINNOWA Medical Technology Co. Ltd., Jiangsu, China) with five measurements. When the first and second results were less than 10%, 36 μL of ADP was added to initiate the reaction before the third time and the results was detected by the mean of the other three results. The sodium chloride solution (0.9%) was used as the negative control. All assays were conducted in triplicate.
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2.9 Thrombolysis assay
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Blood for thrombolysis study was collected from rabbit by venipuncture from ears. 1) Whole blood clot preparation: 30 mL of blood was distributed in PVC pipes and incubated at 37°C for 60 min. After clot formation, serum was completely removed and the clot was pushed out by injections and chopped into twelve pieces. The pieces were dried on filter papers and weighed.
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2) Platelet-rich plasma clot preparation: 30 mL of blood was centrifuged (800 g for 10 min) and platelet-rich plasma was collected from the blood in the upper layer and platelet in middle layer and dissolved in sodium citrate solution (1:9, v/v). Then thrombin (10 U/mL) was added at a dose of 0.1 mL per mL of plasma and mixed carefully. After clot formation, serum was completely removed and the clot was chopped into twelve pieces. The pieces were dried on filter papers and weighed. 10
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3) Pure fibrinogen clot preparation: 20 mL of pure fibrinogen solution (without plasmin and plasminogen, 20 mg/mL) and 2 mL of thrombin (10 U/mL) were
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mixed in a tube. After clot formation, serum was completely removed and the clot was chopped into twelve pieces. The pieces were dried on filter papers and weighed.
To each microcentrifuge tube that contained pre-weighed whole blood clots, platelet-rich plasma clots or pure fibrinogen clots, 2 mL of different concentrations of sturgeon skull CS, sturgeon backbone CS and shark CS were added separately. The negative control received 2 mL Tris-HCl buffer (pH 7.4) instead of CSs.
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All the tubes were then incubated at 37°C for 6 h and monitored for clot lysis. After incubation, clots were recovered and the dry weight was recorded to
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calculate the percentage of clot lysis. Percentage of clot lysis is defined as: (Weight before reaction-Weight after reaction)/Weight before reaction ×100. All assays were conducted in triplicate. 2.10 Statistical analyses
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Values were expressed as mean ± standard deviation (SD) and statistical analyses were performed with the SPSS 13.0 statistics software (SPSS, Chicago, IL, USA). The efficiency of biological activity in the assay was compared by using the paired t-test. P-values of less than 0.05 (P < 0.05) were considered as significant.
3 Results and discussion 11
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3.1 Purity and molecular weight
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The yields of sturgeon skull CS and backbone CS were 19.5% and 22% (lyophilized weight), respectively. The purity and molecular weight of the purified CSs from sturgeon skull and backbone were analyzed by GPC-MALLs (Fig. 1) and the results were recorded in Table 1. The purity of CSs extracted from sturgeon skull and backbone were 94.8% and 95.3%, respectively, and the average molecular weights were 38.5, and 49.2 kDa, respectively. The polydispersity indices (Mw/Mn) of the two kinds of CSs were lower than 1.5, indicating the components of both CSs were relatively uniform (Volpi, 2007).
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3.2 Disaccharide assay
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The purified CSs were treated with chondroitinase ABC, and then the unsaturated disaccharides produced were analyzed by SAX-HPLC (Fig. 2) and the results were recorded in Table 1. The chondroitinase ABC was able to produce three unsaturated disaccharides, the nonsulfated ΔDi0s [ΔUA1→3GalNAc], the monosulfated ΔDi6s [ΔUA1→3GalNAc6 (SO4)] and the ΔDi4s [ΔUA1→3GalNAc4 (SO4)]. No disulfated or trisulfated disaccharides were found, similar to
Ac
the disaccharide composition of Acipenser sinensis (Im, Park & Kim, 2010). The disaccharide patterns of CSs from sturgeon skull and backbone were different. As shown in Table 1, sturgeon skull CS contained abundant ΔDi0s (74.2%) whereas sturgeon backbone CS contained abundant ΔDi6s (59.6%) and ΔDi4s (37.8%). The charge density value of CS from sturgeon backbone (0.97) was higher than that of sturgeon skull (0.26). This difference might due to the presence of a higher percentage of sulfated groups in sturgeon backbone (Volpi, 2009). It was reported that CS from shark was mainly composed of 12
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mono-sulfated disaccharides ΔDi-6S (50%) and ΔDi-4S (29%), and the content of ΔDi-0S (3%) was low (Volpi, 2007). Therefore, our results suggest that the
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disaccharide pattern of CS from sturgeon backbone was similar to CS from shark. 3.3 1H-NMR 1
H-NMR spectroscopy was performed to confirm the disaccharide composition and the structural integrity of the CSs (Fig. 3). The signals at 1.982 ppm (Fig. 3a)
in the spectrum of CS from sturgeon skull, 2.016 and 2.030 ppm (Fig. 3b) in the spectrum of CS from sturgeon backbone can be readily assigned to methyl
ed
protons of nonsulfated, 6-sulfated and 4-sulfated CS of GalNAc, respectively (Toida, Toyoda & Imanarit, 1993). The signals at 4.224 and 4.705 ppm (Fig. 3b)
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in the spectrum of CS from sturgeon backbone were assigned to H6 of GalNAc-6SO4 and H4 of GalNAc-4SO4, respectively (Toida, Toyoda & Imanarit, 1993; Mucci, Schenetti & Volpi, 2000b). The signal at 4.185 ppm (Fig. 3a) in the spectrum of CS from sturgeon skull was assigned to H6 of GalNAc-6SO4 while no signal was found to be assigned to H4 of GalNAc-4SO4. The difference suggested that the content of 4-sulfated CS in sturgeon skull was less than that in sturgeon backbone. The result was also consistent with the composition analysis of disaccharide by HPLC (Table 1).
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(a)
3.4 Anticoagulant assay
The anticoagulant activities of CSs from sturgeon skull, backbone and shark were investigated by the classical coagulation assays APTT, PT and TT. Saline was used as the negative control. As shown in Table 2, CS from sturgeon backbone exhibited stronger anticoagulant activity than CSs from shark and sturgeon skull 13
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at all concentrations in APTT and TT experiments. With increasing concentrations of CS, the anticoagulant activities of CSs from sturgeon backbone,
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sturgeon skull and shark increased. This pattern was especially clear for the CS from sturgeon backbone. The anticoagulant activities of CS were affected by polysaccharide’s monosaccharide composition, sulphation, species and location of substituent group (Majdoub, Mansour, Chaubet, Roudesli & Maaroufi, 2009; Chaidedgumjorn et al., 2002; Fonseca, Oliveira, Pomin, Mecawi, Araujo & Mourão, 2008). Since both sturgeon backbone CS and skull CS were composed of mono-sulfated disaccharides ΔDi-6S, ΔDi-4S and ΔDi-0S, the higher mono-sulfated disaccharide percentage in sturgeon skull CS, might contribute to its
ed
stronger anticoagulant activity compared to sturgeon skull CS. (Mulloy, Mourao & Gray, 2000).
ce pt
None of the CSs extracted from sturgeon skull, sturgeon backbone and shark cartilage showed significant differences in modulating prothrombin time in the PT assay. PT assay is used to evaluate extrinsic coagulation factors and the results we obtained suggest that CS extracted from these three bone tissues do not have effect on modulating extrinsic coagulation factors. In contrast, APTT assay is used to evaluate intrinsic blood coagulation factors and TT is a simple
Ac
screening test for the fibrin polymerization process (Ye, Xu & Li, 2012; Ekanayake, Nikapitiya, De Zoysa, Whang, Kim & Lee, 2008). The results of APTT and TT assays suggest that CS extracted from these samples has the ability to modulate intrinsic blood coagulation factors and thrombin activity of controlling the degree of conversion fibrinogen to fibrin. 3.5 Inhibition of coagulation factor Xa or thrombin by antithrombin Ⅲ and heparin cofactor Ⅱ in the presence of the sturgeon CSs 14
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The effects of CSs from sturgeon skull and backbone on coagulation factor Xa or thrombin activity were investigated (Fig. 4). Sturgeon skull CS and sturgeon
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backbone CS showed weak inhibitory effect on coagulation factor Xa in the absence of antithrombin Ⅲ. However, the two sturgeon CSs had a significant (P < 0.05) inhibitory effect on coagulation factor Xa in the presence of antithrombin Ⅲ, and the CS from sturgeon backbone exhibited greater inhibitory effect than that from sturgeon skull. Moreover, the two kinds of sturgeon CSs exhibited no significant inhibitory effect on coagulation factor Xa in the presence of heparin cofactor Ⅱ(data not shown). Meanwhile, no inhibition of thrombin was observed by the two kinds of sturgeon CSs either in the presence or absence of
ed
antithrombin Ⅲ or heparin cofactor Ⅱ (data not shown). Therefore, CSs extracted from sturgeon skull and sturgeon backbone did not have direct inhibition
3.6 Anti-platelet effect
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of coagulation factor Xa but were mediated by antithrombin Ⅲ.
Anti-platelet activities of CSs from sturgeon skull, backbone and shark were tested on platelet aggregation assay using rabbit whole blood (Table 3). The platelet
Ac
inhibition rate (%) of CS extracted from sturgeon skull, sturgeon backbone, and shark increased in a dose-dependent manner. In comparison with the control, significant inhibition of the aggregation was observed from three kinds of CSs even at low concentration (1mg/mL) (P < 0.05). Sturgeon skull CS showed lower anti-platelet activity than sturgeon backbone CS and shark CS at all concentrations. The anti-platelet activity of shark CS was comparable to that of sturgeon backbone CS at the concentrations of 1 mg/mL and 3 mg/mL. However, sturgeon backbone CS exhibited higher anti-platelet activity at the concentration of 5 15
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mg/mL. Similarly, a polyphenolic-polysaccharide preparation isolated from medicinal plant Erigeron canadensis L. also has anti-aggregatory effect on
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platelets (Pawlaczyk et al., 2011). In contrast, other polysaccharides stimulate instead of preventing platelet aggregation (Li & Lian, 1998; Farias, Nazareth & Mourão, 2001). 3.7 Thrombolysis assays
Thrombolysis effects of CSs from sturgeon skull, backbone and shark were tested on whole blood clot dissolution rate, platelet plasma clot dissolution rate and
ed
pure fibrin clot dissolution rate using rabbit blood (Table 4). CSs from sturgeon backbone and shark exhibited strong effects on dissolving platelet plasma clots,
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while they exhibited weak effects on dissolving whole blood clot and pure fibrin clots. The results suggested that the major thrombolysis effect of CSs from sturgeon backbone and shark was reflected in dissolving platelet plasma clot. Sturgeon backbone CS exhibited a stronger effect on dissolving platelet plasma clot than shark CS with the increase of the concentration, which was consistent with the results of anti-platelet effect assay. However, sturgeon skull CS
4 Conclusions
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exhibited weak effects on dissolving all three kinds of clots.
In this study, it is concluded that CSs from sturgeon skull and backbone were composed of three kinds of disaccharides including the nonsulfated Δdi0s [ΔUA1→3GalNAc], the monosulfated Δdi6s [ΔUA1→3GalNAc6 (SO4)], and the Δdi4s [ΔUA1→3GalNAc4 (SO4)]. Sturgeon backbone CS was composed of 16
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~37.8% ΔDi4s, ~59.6% ΔDi6s and small proportions of ΔDi0s (2.6%) while sturgeon skull CS was composed of large proportions of ΔDi0s (74.2%) and small
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proportions of ΔDi4s (8.1%) and ΔDi6s (17.7%). Meanwhile, sturgeon backbone CS exhibited stronger anticoagulant, anti-platelet and thrombolysis activity than sturgeon skull CS, which might be attributed to a higher percentage of mono-sulfated disaccharides, especially ΔDi-4S in sturgeon backbone CS. However, the detailed mechanisms of the antithrombotic effect of CSs are not clear and need to be further studied. The comprehensive antithrombotic effect of sturgeon
ed
backbone CS suggested that it should be explored as a functional food with antithrombotic function. 5 Acknowledgement
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This work was supported by the Beijing Innovation Team of China Agriculture Research Center System (SCGWZJ20131105-2) and Major Program(s) of Beijing Municipal Science & Technology Commission (D121100003712003). 6 References
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Chaidedgumjorn, A., Toyoda, H., Woo, E. R., Lee, K. B., Kim, Y. S., Toida, T., & Imanari, T. (2002). Effect of (1→ 3)-and (1→ 4)-linkages of fully sulfated polysaccharides on their anticoagulant activity. Carbohydrate Research, 337(10), 925-933. Colliec, S., Fischer, A. M., Tapon-Bretaudiere, J., Boisson, C., Durand, P., & Jozefonvicz, J. (1991). Anticoagulant properties of a fucoidan fraction. Thrombosis Research, 64(2), 143-154. 17
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Ekanayake, P. M., Nikapitiya, C., De Zoysa, M., Whang, I., Kim, S. J., & Lee, J. (2008). Novel anticoagulant compound from fermented red alga
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Pachymeniopsis elliptica. European Food Research and Technology, 227(3), 897-903.
Farias, W. R. L., Nazareth, R. A., & Mourão, P. A. S. (2001). Dual effects of sulfated D-galactans from the red algae Botryocladia occidentalis preventing thrombosis and inducing platelet aggregation. Thrombosis and Haemostasis, 86(6), 1540-1546. Fonseca, R. J., Oliveira, S., Pomin, V. H., Mecawi, A. S., Araujo, I. G., & Mourão, P. A. (2008). Effects of oversulfated and fucosylated chondroitin sulfates
ed
on coagulation. Thromb Haemost, 99(5), 807-818.
ce pt
Gui, M., Song, J., Zhang, Z., Hui, P., & Li, P. (2014). Biogenic amines formation, nucleotide degradation and TVB-N accumulation of vacuum-packed minced sturgeon (Acipenser schrencki) stored at 4°C and their relation to microbiological attributes. Journal of The Science of Food And Agriculture, 94(10), 2057-2063.
S22-S24.
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Hochberg, M. C., Clegg, D. O. (2008). Potential effects of chondroitin sulfate on joint swelling: a GALT report. Osteoarthritis Cartilage. 16 (Suppl. 3),
Huang, R., Du, Y., Yang, J., & Fan, L. (2003). Influence of functional groups on the in vitro anticoagulant activity of chitosan sulfate. Carbohydrate Research, 338(6), 483-489. 18
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Im, A., Park, Y., & Kim, Y. S. (2010). Isolation and characterization of chondroitin sulfates from sturgeon (Acipenser sinensis) and their effects on growth of
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fibroblasts. Biological and Pharmaceutical Bulletin, 33(8), 1268-1273.
Kohno, M., Suzuki, S., Kanaya, T., Yoshino, T., Matsuura, Y., Asada, M., & Kitamura, S. (2009). Structural characterization of the extracellular polysaccharide produced by Bifidobacterium longum JBL05. Carbohydrate Polymers, 77(2), 351-357. Li, J. Z. & Lian, E. C. (1988). Aggregation of human platelets by acidic mucopolysaccharide extracted from Stichopus japonicus Selenka. Thrombosis and
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Haemostasis, 59(3), 435-439.
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Łopaciuk S. Zakrzepy i zatory. Warszawa: Wyd. Lekarskie PZWL; 2002. Maccari, F., Ferrarini, F., & Volpi, N. (2010). Structural characterization of chondroitin sulfate from sturgeon bone. Carbohydrate Research, 345(11), 1575-1580.
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Majdoub, H., Mansour, M. B., Chaubet, F., Roudesli, M. S., & Maaroufi, R. M. (2009). Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis. Biochimica et Biophysica Acta (BBA)-General Subjects, 1790(10), 1377-1381. Mucci, A., Schenetti, L., & Volpi, N. (2000a). 1 H and 13 C nuclear magnetic resonance identification and characterization of components of chondroitin sulfates of various origin. Carbohydrate Polymers, 41(1), 37-45. 19
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Mucci, A., Schenetti, L., & Volpi, N. (2000b). 1 H and 13 C nuclear magnetic resonance identification and characterization of components of chondroitin
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sulfates of various origin. Carbohydrate Polymers, 41(1), 37-45.
Mulloy, B., Mourao, P., & Gray, E. (2000). Structure/function studies of anticoagulant sulphated polysaccharides using NMR. Journal of Biotechnology, 77(1), 123-135.
Pawlaczyk, I., Czerchawski, L., Kuliczkowski, W., Karolko, B., Pilecki, W., Witkiewicz, W., & Gancarz, R. (2011). Anticoagulant and anti-platelet activity
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of polyphenolic-polysaccharide preparation isolated from the medicinal plant Erigeron canadensis L. Thrombosis Research, 127(4), 328-340.
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Pomin, V. H. (2014). NMR Chemical Shifts in Structural Biology of Glycosaminoglycans. Analytical Chemistry, 86(1), 65-94. Sawitzke, A. D., et al. (2008). The effect of glucosamine and/or chondroitin sulfate on the progression of knee osteoarthritis : a report from the glucosamine/chondroitin arthritis intervention trial. Arthritis Rheum, 58(10), 3183-3191.
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Sim, J. S., Im, A. R., Cho, S. M., Jang, H. J., Jo, J. H., & Kim, Y. S. (2007). Evaluation of chondroitin sulfate in shark cartilage powder as a dietary supplement: Raw materials and finished products. Food Chemistry, 101(2), 532-539. Sugahara, K., Mikami, T., Uyama, T., Mizuguchi, S., Nomura, K., & Kitagawa, H. (2003a). Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate. Current Opinion In Structural Biology, 13(5), 612-620. 20
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Sugahara, K., Mikami, T., Uyama, T., Mizuguchi, S., Nomura, K., & Kitagawa, H. (2003b). Recent advances in the structural biology of chondroitin sulfate
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and dermatan sulfate. Current Opinion In Structural Biology, 13(5), 612-620.
Toida, T., Toyoda, H., & Imanarit, T. (1993). High-Resolution Proton on Chondroitin Sulfates. SCIENCES, 9. Uebelhart, D. (2008). Clinical review of chondroitin sulfate in osteoarthritis. Osteoarthritis Cartilage, 16 (Suppl. 3): S19-S21. Volpi, N. (2007). Analytical aspects of pharmaceutical grade chondroitin sulfates. Journal of Pharmaceutical Sciences, 96(12), 3168-3180.
ed
Volpi, N., Maccri, F. (2008). Quantitative and qualitative evaluation of chondroitin sulfate in dietary supplemens. Food Analytical Methods, 1(3), 195-204.
1271-1280.
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Volpi, N. (2009). Quality of different chondroitin sulfate preparations in relation to their therapeutic activity. Journal of Pharmacy and Pharmacology, 61(10),
2003.
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World Health Organization. WHO Report, Cardiovascular disease: Prevention and control. http://www.who.int/dietphysicalactivity/publications/facts/cvd/en
Ye, L., Xu, L., & Li, J. (2012). Preparation and anticoagulant activity of a fucosylated polysaccharide sulfate from a sea cucumber Acaudina molpadioidea. Carbohydrate Polymers, 87(3), 2052-2057.
21
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Figure captions
Fig. 1 GPC-MALLS of chondroitin sulfates from sturgeon skull (red) and sturgeon backbone (blue). Typical molar mass vs elution time plots are shown overlapped for sturgeon skull CS and backbone CS with traces for scattered light measured by one of 18 detectors (solid line) and molar mass (dashed line) calculated by instrument software.
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ΔDi4s, ΔUA1→3GalNAc4 (SO4).
ed
Fig. 2 SAX-HPLC of chondroitin sulfates from (a) sturgeon skull and (b) sturgeon backbone. ΔDi0s, ΔUA1→3GalNAc. ΔDi6s, ΔUA1→3GalNAc6 (SO4).
Fig. 3 1H NMR spectra of chondroitin sulfates from (a) sturgeon skull and (b) sturgeon backbone. Fig.4 Effect of chondroitin sulfate from sturgeon skull and sturgeon backbone on inhibition of coagulation factor Xa activity in the presence and absence of
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antithrombin Ⅲ. Coagulation factor Xa was incubated with chondroitin sulfate from sturgeon skull in the absence (◆) and in the presence (■) of antithrombin Ⅲ. Coagulation factor Xa was incubated with chondroitin sulfate from sturgeon backbone in the absence (◇) and in the presence (□) of antithrombin Ⅲ.
22
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Table(s)
us
Table1
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The chemical characterization (purity, weight, polydispersity index, disaccharide ratio and charge density) of sturgeon CSs in this study and literatures.
Molecular weight (kDa)
Polydispersity index (Mw/Mn)
Sturgeon skull CS
94.8
38.5±1.0
1.36±0.06
Sturgeon backbone CS
95.3
49.2±2.6
Sturgeon cartilage CS
91.1
Sturgeon backbone CS
100>
Sturgeon bone CS Shark cartilage CS
Charge density
References
ΔDi-4S
74.2
17.7
8.1
0.97
This study
2.6
59.6
37.8
0.26
This study
8.0
4.0
7.2
88.8
Im et al, 2010
43.4
9.9
66.2
23.9
Im et al, 2010
7.0
55
38
2.3
40.8
34.9
d
ΔDi-6S
1.44±0.14
ep te 37.5
98.2
Disaccharide ratio (%)a
ΔDi-0S
Ac c
Sample
M
Purity (%)
60.6
1.34
0.93
Maccari et al, 2010
Im et al, 2010
aΔDi0s[ΔUA1→3GalNAc]; ΔDi6s [ΔUA1→3GalNAc6(SO4)]; ΔDi4s [ΔUA1→3GalNAc4(SO4)].
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Table 2
APTT(s) at different concentrations (mg/mL) 1 3 5
PT(s) at different concentrations (mg/mL) 1 3 5
Negative control Sturgeon skull CS Sturgeon backbone CS Shark cartilage CS
28.9±2.6
－
－
9.9±0.6
－
－
15.7±0.6
－
－
31.2±0.4
33.8±0.9
34.6±1.8*
9.8±0.3
10.3±0.4
10.6±0.5
16.5±0.8
17.7±0.6*
17.9±0.6*
M
sample
d
an
APTT, PT and TT of rabbit platelet-poor plasma containing sturgeon skull CS, sturgeon backbone CS and shark CS. TT(s) at different concentrations (mg/mL) 1 3 5
73.4±1.3*
9.6±0.1
10.8±0.1
11.0±0.9
19.5±0.3* 22.9±0.1*
26.5±0.2*
32.5±1.6* 40.8±3.4*
64.8±3.1*
10.2±0.3
10.7±0.9
11.1±1.0
20.5±0.4* 23.5±1.2*
26.7±0.2*
ep te
36.0±1.3* 41.2±1.1*
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Note: all the data were the mean of three parallel assays.－indicated the value was not evaluated. *Significantly inhibited compared with negative control (P<0.05).
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Table 3
an
Effect of sturgeon skull CS, sturgeon backbone CS and shark CS on platelet aggregation in vitro. Concentration (mg/mL)
Platelet maximal aggregation rate (%)
Platelet inhibition rate (%)
Negative control
0
39.9±0.3
0
1 3 5 1 3 5 1 3 5
37.3±0.5* 28.8±0.3** 19.8±0.9** 34.7±1.0** 24.5±1.8** 12.2±0.8** 34.1±0.9** 25.4±0.3** 16.1±1.0**
Sturgeon backbone CS
Shark CS
d
ep te
Sturgeon skull CS
M
Sample
7.0±1.2* 28.4±0.7** 36.8±0.4** 13.4±2.5** 39.0±4.4** 69.7±1.9** 15.1±2.3** 38.1±2.5** 59.8±.2.5**
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Note: all the data were the mean of three parallel assays.
*Significantly inhibited compared with negative control (P<0.05). ** Significantly inhibited compared with negative control (P<0.01).
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Table 4
Whole blood clot dissolution rate (%) at different concentrations (mg/mL) 3 －
5 －
0.8 ±0.03
1.3±0.03
1.3±0.07
Sturgeon backbone CS
1.4±0.02
2.9±0.01
3.5±0.08
Shark cartilage CS
1.1±0.03
2.3±0.09
3.7±0.08
Pure fibrin clot dissolution rate (%) at different concentrations (mg/mL)
3 －
5 －
1 0
3 －
5 －
2.4±0.03
3.3±0.07
1.2±0.04
2.0±0.04
2.3±0.04
6.2±0.03
9.2±0.02
17.7±0.08
2.4±0.02
3.3±0.04
4.3±0.03
6.1±0.03
8.0±0.10
15.2±0.79
2.1±0.04
3.4±0.03
4.5±0.06
1.3±0.03
ep te
Negative control Sturgeon skull CS
1 0
d
1 0
Platelet plasma clot dissolution rate (%) at different concentrations (mg/mL)
M
Sample
an
Effect of sturgeon skull CS, sturgeon backbone CS and shark CS on thrombolysis in vitro.
Ac c
Note: all the data were the mean of three parallel assays.－indicated the value was not evaluated.
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Figure 3
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Figure 4
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