Unsaturated Derivatives of Polysaccharides, Method of Preparation Thereof and Use Thereof

Abstract

The invention relates to the preparation of new polysaccharide derivatives comprising a double bond in the positions 4 and 5 of the pyranose cycle. The method of preparation consists in the oxidation of OH group in the position 6 to an aldehyde, followed by the elimination to form a double CC bond in the positions 4 and 5, and the fmal reduction of the aldehyde group in the position 6 into the original alcohol. The derivatives of polysaccharides prepared according to the invention show an enhanced antioxidant activity and some of them also a selective negative influence on carcinoma cell viability. (formula) where R represents NHCOCH.sub.3 or OH.

##STR00001##

Claims

1. Unsaturated derivatives of polysaccharides comprising, in their structure, at least one heterocycle having a double bond in positions 4 and 5 according to the structural formula X, ##STR00013## where R represents NHCOCH.sub.3 or OH.

2. Unsaturated derivatives of polysaccharides according to claim 1 characterized in that their molecular weight is in the range of 5.10.sup.3 to 5.10.sup.5 g.mol.sup.1 and that the polysaccharides are selected from the group consisting of chondroitin sulfate, carrageenan, dermatan sulfate, hyaluronic acid, and keratan sulfate.

3. A method of preparation of the polysaccharide derivatives defined in claim 1 characterized in that the starting polysaccharide, comprising the fragment Y ##STR00014## where R is NHCOCH.sub.3 or OH, and R.sup.1 is SO.sub.2ONa, SO.sub.2OH or H, is in the first step oxidized to an aldehyde in the position 6, in the second step the oxidized polysaccharide is eliminated in the positions 4 and 5 of the cycle to form a double bond, and in the third step the aldehyde group is selectively reduced.

4. The method of preparation according to claim 3 characterized in that the starting polysaccharide is chondroitin sulfate, carrageenan, dermatan sulfate, hyaluronic acid, or keratan sulfate.

5. The method of preparation according to claim 3 characterized in that, in the first step, the oxidation in the position C-6 proceeds either by means of the R.sup.3-TEMPO/NaClO system, where R.sup.3 is hydrogen or N-acetyl group, in water at the temperature of 0 C. to 10 C., wherein the molar amount of NaClO is within the range of 0.3 to 0.8 eq. and the molar amount of R.sup.3-TEMPO is within the range of 0.005 to 0.2 eq., with respect to the repeating unit of the polysaccharide, or by means of 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (DMP) in DMSO at the temperature of 10 C. to 50 C., wherein the amount of DMP is within the range of 0.05 to 2 eq., with respect to the repeating unit of the polysaccharide.

6. The method of preparation according to claim 3 characterized in that the starting polysaccharide is hyaluronic acid or keratan sulfate, and that, in the second step, the oxidized polysaccharide undergoes the elimination reaction in a mixture of water/polar aprotic solvent in the presence of a base at the temperature of 30 to 80 C.

7. The method of preparation according to claim 6 characterized in that the amount of the base is 5 to 10 eq., with respect to the repeating polysaccharide unit, wherein the base is selected from the group consisting of pyridine, triethylamine, N,N-diisopropylethylamine, and Ca(OH).sub.2.

8. The method of preparation according to claim 6 characterized in that the aprotic solvent is water miscible and comprises DMSO or sulfolane, and the volume ratio of solvent/water is in the range of 3/1 to 1/2.

9. The method of preparation according to claim 6 characterized in that the second reaction step proceeds for 12 to 150 hours.

10. The method of preparation according to claim 3 characterized in that the starting polysaccharide is chondroitin sulfate, carrageenan, or dermatan sulfate, and that the oxidized polysaccharide is spontaneously eliminated in the second step directly in the reaction mixture to form an ,-unsaturated aldehyde, and the spontaneous elimination proceeds without the necessity of adding any base, organic solvent, and without increasing the reaction temperature.

11. The method of preparation according to claim 3 characterized in that the molecular weight of the starting polysaccharide is in the range of 5.10.sup.3 to 5.10.sup.5 g.mol.sup.1.

12. The method of preparation according to claim 3 characterized in that sodium borohydride is added in the third step in the amount of 0.1 to 10 equivalents, calculated with respect to the repeating polysaccharide unit, in water, at the temperature of 5-40 C., at the pH in the range of 5 to 10.

13. Use of the derivatives as defined in claim 1 for the preparation of materials having enhanced antioxidation effect.

14. Use of the derivatives as defined in claim 2, where the polysaccharide is hyaluronic acid, for the preparation of materials having an anti-cancer effect.

15. Use of the derivatives as defined in claim 2 for the preparation of materials having enhanced antioxidation effect.

16. The method of preparation according to claim 3 characterized in that the starting polysaccharide is hyaluronic acid or keratan sulfate, and that, in the second step, the oxidized polysaccharide undergoes the elimination reaction in a mixture of water/polar aprotic solvent in the presence of a base at a temperature of 50 C. to 60 C.

17. The method of preparation according to claim 6 characterized in that the amount of the base is 0.01 to 20 eq., with respect to the repeating polysaccharide unit.

18. The method of preparation according to claim 3 characterized in that sodium borohydride is added in the third step in the amount of 0.3 to 2 equivalents, calculated with respect to the repeating polysaccharide unit, in water, at a temperature of 15-25 C., at a pH in the range of 6 to 8.

Description

DETAILED DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1Effect of AHA prepared according to Example 25 on cells viability The scheme shows the development of the growth inhibition of cancer cells MDA-MB-231breast adenocarcinoma, A-549lung adenocarcinoma, HEP-G2hepatocellular carcinoma, compared to the inhibition of NHDFprimary human dermal fibroblasts.

[0053] The procedure is described in Example 27.

[0054] FIG. 2antioxidant properties of AHA and ACS materials compared to the non-modified polysaccharides HA, CS, and to the standardTrolox6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid

[0055] HAhyaluronic acid

[0056] HAhyaluronic acid dehydrated in the positions 4 and 5 (Example 22)

[0057] CSchondroitin sulfate

[0058] CSchondroitin sulfate dehydrated in the positions 4 and 5 (Example 2) tested by means of 2,2-diphenyl-1 -picrylhydrazyl (DTTH) by the procedure described in the Example 23

[0059] Statistical significance t-test* p<0,05; ** p<0,01; *** p<0,001

PREFERRED EMBODIMENTS OF THE INVENTION

[0060] DS=substitution degree=100% *(molar amount of the modified saccharide unit)/(molar amount of the repeating polysaccharide unit)

[0061] The term equivalent (eq.) as used herein refers to the repeating unit of a particular polysaccharide, if not stated otherwise. The percentage is specified as weight percent if not indicated otherwise.

[0062] The molecular weight of starting polysaccharides is weight average molecular weight determined by the SECMALLS method.

EXAMPLE 1

[0063] Oxidation and Elimination of Chondroitin Sulphate (CS)=Preparation of ,-unsaturated CS aldehyde

[0064] A sodium hypochlorite solution (0.8 eq., 11% of active chlorine) was gradually added into a 2% aqueous solution of CS (200 mg, Mw=4.10.sup.4 g.mol.sup.1) cooled to 5 C. and containing di-sodium hydrogen phosphate dodecahydrate (2.2 eq.), sodium bromide (0.8 eq.), and 4-AcNH-TEMPO (0.01 eq.). The mixture was stirred for 2 hours at the temperature of 5 C. Then ethanol (10 eq.) was added to the reaction, and the reaction was stirred for another one hour at room temperature. The product was isolated by precipitation with IPA and analysed by means of NMR.

[0065] DS=23% (determined by NMR)

EXAMPLE 2

[0066] Reduction of ,-Unsaturated CS aldehyde=Preparation of ACS

[0067] A 2% solution w/v of ,-unsaturated CS aldehyde (200 mg, 0.5 mmol) in distilled water was prepared, the solution was cooled to 5 C. and then 2 equivalents of sodium borohydride were added. The reaction mixture was stirred for 4 hours at 5 C. The product was isolated by precipitation with isopropanol and analysed by means of NMR.

[0068] DS=25% (determined by NMR), Mw=2.10.sup.4 g.mol.sup.1 (determined by SECMALLS)

[0069] Spectral analysis of CS: NMR .sup.1H (500 MHz, D.sub.2O, ppm): 2.02 and 2.04 (3H; Ac-NH-; bs); 4.03 (2H; H6; bs); 4.22 (1H; H2; bs); 4.26 (1H; H3; bs); 5.06 (1H; H1; bs); 5.18 (1H; H4; bs); NMR .sup.1H-.sup.1H COSY (D.sub.2O); cross peaks; ppm: 4.22-5.06; 4.26-5.18; NMR .sup.1H-.sup.13C HSQC (D.sub.2O); cross peaks; ppm: 4.03-61.0; 4.22-50.5; 4.26-73.4; 5.06-98.3; 5.18-98.9; NMR DOSY (D.sub.2O); log D ((2.02 and 2.04; Ac-NH-); (4.03; H6); (4.22; H2); (4.26; H3); (5.06; H4); (5.18; H1)) 10.4 m.sup.2s.sup.1; log D (4.72; H.sub.2O) 8;6 m.sup.2s.sup.1; IR (KBr; cm.sup.1): 1660 (v CC st);

EXAMPLE 3

[0070] Oxidation and Elimination of Dermatan Sulfate (DeS)=Preparation of ,-Unsaturated DeS aldehyde

[0071] An aqueous solution of sodium hypochlorite (0.8 eq., 11% of active chlorine) was gradually added into a 2% aqueous solution of DeS (200 mg, 0.42 mmol) cooled to 5 C. and containing di-sodium hydrogen phosphate dodecahydrate (2.2 eq.), sodium bromide (0.8 eq.), and 4-AcNH-TEMPO (0.01 eq.), the mixture was stirred for 2 hours at 5 C. Then ethanol (10 eq.) was added to the reaction, and the reaction was stirred for another one hour at room temperature. The product was isolated by precipitation with IPA and analysed by means of NMR.

[0072] DS=20% (determined by NMR)

EXAMPLE 4

[0073] Reduction of ,-Unsaturated dermatan sulfate aldehyde=Preparation of DeS

[0074] A 2% solution w/v of ,-unsaturated DeS aldehyde (200 mg, 0.5 mmol) in distilled water was prepared. The solution was cooled to 5 C. and then sodium borohydride (2 equivalents per DeS disaccharide) were added. The reaction mixture was stirred for 4 hours at 5 C. The product was isolated by precipitation with isopropanol and analysed by means of NMR.

[0075] DS=23% (determined by NMR)

[0076] Spectra analysis of ADeS: NMR .sup.1H(500 MHz, D.sub.2O, ppm): 2.01 (3H, Ac-NH-, bs), 5.05 (1H, H1, bs), 5.17 (1H, H1, bs).

EXAMPLE 5

[0077] Oxidation and Elimination of carrageenan (KA)=Preparation of ,-Unsaturated KA aldehyde

[0078] An aqueous solution of sodium hypochlorite (0.8 eq., 11% of active chlorine) was gradually added into a 1% aqueous solution of KA (200 mg, 0.31 mmol) cooled to 10 C., containing di-sodium hydrogen phosphate dodecahydrate (2.2 eq.), sodium bromide (0.8 eq.), and 4-AcNH-TEMPO (0.01 eq.). The mixture was stirred for 2 hours at 10 C. Then ethanol (10 eq.) was added to the reaction, and the reaction was stirred for another one hour at room temperature. The product was isolated by precipitation with IPA and analysed by means of NMR.

[0079] DS=10% (determined by NMR)

EXAMPLE 6

[0080] Reduction of ,-Unsaturated carrageenan aldehyde=Preparation of KA

[0081] A 2% solution w/v of ,-unsaturated KA aldehyde (200 mg, 0.5 mmol) in distilled water was prepared. The solution was cooled to 5 C. and then sodium borohydride (2 equivalents per KA disaccharide) were added. The reaction mixture was stirred for 4 hours at 5 C. The product was isolated by precipitation with isopropanol and analysed by means of NMR.

[0082] DS=13% (determined by NMR)

[0083] NMR .sup.1H(500 MHz, D.sub.2O, ppm): 5.07 (1H, H1, bs), 5.18 (1H, H1, bs),

EXAMPLE 7

[0084] Oxidation of keratan sulfate (KS)=Preparation of KS-aldehyde

[0085] An aqueous solution of NaClO (0.3 eq.) was gradually, under nitrogen, added into a 1% aqueous solution of KS (1 g, 2.10.sup.4 g.mol.sup.1) containing NaCl 1%, KBr 1%, TEMPO (0,01 eq.), and NaHCO.sub.3 (20 eq.). The mixture was stirred for 24 hat 0 C., then 0.1 g of sodium thiosulfate was added and the mixture was stirred for further 10 minutes. The resulting solution was diluted with distilled water to 0.2% and dialyzed against the mixture of (0.1% NaCl, 0,1% NaHCO.sub.3) 5 litres 3 times (once a day), and against distilled water, 5 litres 7 times (2 times a day). The resulting solution was then evaporated and analysed.

[0086] DS 3% (determined by NMR)

EXAMPLE 8

[0087] Elimination of KS-aldehyde=Preparation of ,-unsaturated KS aldehyde

[0088] 6.7 ml of DMSO and DIPEA base (5 eq.) were added into a 3% KS-aldehyde solution (0.1 g, oxidation degree of DS=3%, example 7) in water. The mixture was stirred for 72 hours at the temperature of 60 C. Then the resulting solution was precipitated by isopropanol/hexane mixture and the solid part was vacuum dried.

[0089] DS 2% (determined by NMR),

[0090] .sup.1H NMR (D.sub.2O) 9.22 (s, 1H, CHO), 6.32 (m, 1H, CHCCHO)

EXAMPLE 9

[0091] Reduction of ,-unsaturated KS aldehyde=Preparation of KS

[0092] Sodium borohydride (2 eq.) was added into a 2% solution of ,-unsaturated KS aldehyde (200 mg, Example 8) in distilled water. The reaction mixture was stirred for 3 hours at 5 C., then precipitated with isopropanol, and analysed by NMR.

[0093] DS=2% (determined by NMR)

[0094] .sup.1H NMR (D.sub.2O) 5.05 (1H, H1, bs), 5.17 (1H, H4, bs)

EXAMPLE 10

[0095] Oxidation of hyaluronic acid (HA)=Preparation of HA-aldehyde

[0096] An aqueous solution of NaClO (0.5 eq.) was gradually, under nitrogen, added into a 1% aqueous solution of HA (1 g, 2.10.sup.5 g.mol.sup.1) containing NaCl 1%, KBr 1%, TEMPO (0,01 eq.), and NaHCO.sub.3 (20 eq.). The mixture was stirred for 12 h at 0 C., then 0.5 ml of ethanol was added and the mixture was stirred for another 1 hour. The resulting solution was diluted with distilled water to 0.2% and dialyzed against the mixture of (0.1% NaCl, 0,1% NaHCO.sub.3), 5 litres 3 times (once a day), and against distilled water, 5 litres 7 times (2 times a day). Then the resulting solution was evaporated and analysed.

[0097] DS 10% (determined by NMR)

EXAMPLE 11

[0098] Oxidation of HA=Preparation of HA-aldehyde

[0099] An aqueous solution of NaClO (0.5 eq.) was gradually, under nitrogen, added into a 1% aqueous solution of HA (1 g, 2.10.sup.5 g.mol.sup.1) containing NaCl 1%, KBr 1%, N-acetylamino-TEMPO (0,01 eq.), and NaHCO.sub.3 (20 eq.). The mixture was stirred for 12 h at 10 C., then 0.1 ml of ethanol was added and the mixture was stirred for another 1 hour. The resulting solution was diluted with distilled water to 0.2% and dialyzed against the mixture of (0.1% NaCl, 0,1% NaHCO.sub.3), 5 litres 3 times (once a day), and against distilled water, 5 litres 7 times (2 times a day). Then the resulting solution was evaporated and analysed.

[0100] DS 9% (determined by NMR)

EXAMPLE 12

[0101] Oxidation of HA=Preparation of HA-aldehyde

[0102] An aqueous solution of NaClO (0.3 eq.) was gradually, under nitrogen, added into a 1% aqueous solution of HA (1 g, 2.10.sup.5 g.mol.sup.1) containing NaCl 1%, KBr 1%, TEMPO (0,2 eq.), and NaHCO.sub.3 (20 eq.). The mixture was stirred for 48 h at 5 C., then 0.1 ml of ethanol was added and the mixture was stirred for another 1 hour. The resulting solution was diluted with distilled water to 0.2% and dialyzed against the mixture of (0.1% NaCl, 0,1% NaHCO.sub.3), 5 litres 3 times (once a day), and against distilled water, 5 litres 7 times (2 times a day). Then the resulting solution was evaporated and analysed.

[0103] DS 5% (determined by NMR)

EXAMPLE 13

[0104] Oxidation of hyaluronic acid (HA)=Preparation of HA-aldehyde

[0105] An aqueous solution of NaClO (0.7 eq.) was gradually, under nitrogen, added into a 1% aqueous solution of HA (1 g, 2.10.sup.5 g.mol.sup.1) containing NaCl 1%, KBr 1%, TEMPO (0,01 eq.), and NaHCO.sub.3 (20 eq.). The mixture was stirred for 0.5 h at 0 C., then 0.1 ml of ethanol was added and the mixture was stirred for another 1 hour. The resulting solution was diluted with distilled water to 0.2% and dialyzed against the mixture of (0.1% NaCl, 0,1% NaHCO.sub.3), 5 litres 3 times (once a day), and against distilled water, 5 litres 7 times (2 times a day). Then the resulting solution was evaporated and analysed.

[0106] DS 9% (determined by NMR)

EXAMPLE 14

[0107] Oxidation of hyaluronic acid (HA)=Preparation of HA-aldehyde

[0108] An aqueous solution of NaClO (0.5 eq.) was gradually, under nitrogen, added into 1% aqueous solution of HA (1 g, 2.10.sup.5 g.mol.sup.1) containing NaCl 1%, KBr 1%, TEMPO (0,01 eq.), and NaHCO.sub.3 (20 eq.). The mixture was stirred for 12 h at 0 C., then 0.1 ml of ethanol was added and the mixture was stirred for another 1 hour. The resulting solution was diluted with distilled water to 0.2% and dialyzed against the mixture of (0.1% NaCl, 0,1% NaHCO.sub.3), 5 litres 3 times (once a day), and against distilled water, 5 litres 7 times (2 times a day). Then the resulting solution was evaporated and analysed.

[0109] DS 10% (determined by NMR)

EXAMPLE 15

[0110] Oxidation of hyaluronic acid (HA)=Preparation of HA-aldehyde

[0111] 1.2 eq. of 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3 (1H)-one (Dess-Martin Periodinan) was added into a 1% solution of acid form of hyaluronane (1 g, 1.10.sup.5 g.mol.sup.1) in non-aqueous DMSO, and the mixture was stirred for 5 h at 20 C. The resulting solution was then diluted with distilled water to 0.2% and dialyzed against the mixture of (0.1% NaCl, 0,1% NaHCO.sub.3), 5 litres 3 times (once a day), and against distilled water, 5 litres 7 times (2 times a day). Then the resulting solution was evaporated and analysed.

[0112] DS 40% (determined by NMR)

EXAMPLE 16

[0113] Elimination of HA-aldehyde=Preparation of ,-unsaturated HA aldehyde

[0114] 6.7 ml of DMSO and DIPEA base (5 eq.) were added into a 3% solution of HA-aldehyde (0.1 g, oxidation degree DS=40%, Example 15) in water. The mixture was stirred for 72 hours at 60 C. The resulting solution was then precipitated by isopropanol/hexane mixture and the solid part was vacuum dried.

[0115] DS 20% (determined by NMR),

[0116] .sup.1H NMR (D.sub.2O) 9.24 (s, 1H, CHO), 6.32 (m, 1H, CHCCHO)

[0117] UV-Vis (D.sub.2O) 252 nm, -* transition ,-unsaturated aldehyde

EXAMPLE 17

[0118] Elimination of HA-aldehyde=Preparation of 4-unsaturated HA aldehyde

[0119] 6.7 ml of DMSO and triethylamine base (20 eq.) were added into a 3% solution of HA-aldehyde (0.1 g, oxidation degree DS=10%, Example 10) in water. The mixture was stirred for 150 hours at 30 C. The resulting solution was then precipitated by isopropanol/hexane mixture and the solid part was vacuum dried.

[0120] DS 5% (determined by NMR)

EXAMPLE 18

[0121] Elimination of HA-aldehyde=Preparation of 4-unsaturated HA aldehyde

[0122] 6.7 ml of DMSO and pyridine base (0.01 eq.) were added into a 3% solution of HA-aldehyde (0.1 g, oxidation degree DS=10%, Example 10) in water. The mixture was stirred for 12 hours at 80 C. The resulting solution was then precipitated by isopropanol/hexane mixture and the solid part was vacuum dried.

[0123] DS 3% (determined by NMR)

EXAMPLE 19

[0124] Elimination of HA-aldehyde=Preparation of ,-unsaturated HA aldehyde

[0125] 1.7 ml of DMSO and pyridine base (10 eq.) were added into a 3% solution of HA-aldehyde (0.1 g, oxidation degree DS=10%, Example 10) in water. The mixture was stirred for 48 hours at 60 C. The resulting solution was then precipitated by isopropanol/hexane mixture and the solid part was vacuum dried.

[0126] DS 4% (determined by NMR)

EXAMPLE 20

[0127] Elimination of HA-aldehyde=Preparation of ,-unsaturated HA aldehyde

[0128] 10 ml of DMSO and DIPEA base (5 eq.) were added into a 3% solution of HA-aldehyde (0.1 g, oxidation degree DS=10%, Example 10) in water. The mixture was stirred for 48 hours at 60 C. The resulting solution was then precipitated by isopropanol/hexane mixture and the solid part was vacuum dried.

[0129] DS 5% (determined by NMR)

EXAMPLE 21

[0130] Elimination of HA-aldehyde =Preparation of ,-unsaturated HA aldehyde

[0131] 6.7 ml sulfonane and DIPEA base (5 eq.) were added into a 3% solution of HA-aldehyde (0.1 g, oxidation degree DS=10%, Example 10) in water. The mixture was stirred for 72 hours at 50 C. The resulting solution was then precipitated by isopropanol/hexane mixture and the solid portion was vacuum dried.

[0132] DS 5% (determined by NMR)

EXAMPLE 22

[0133] Reduction of ,-unsaturated HA aldehyde =Preparation of AHA

[0134] Sodium borohydride (10 eq.) was added into a 2% solution of ,-unsaturated HA aldehyde (200 mg, Example 16) in distilled water, at 5 C. The reaction mixture was stirred for 1 h at 5 C., then precipitated with isopropanol and analysed by NMR.

[0135] DS 20% (determined by NMR)

[0136] .sup.1H NMR (D.sub.2O) 5.06 (1H, H1, bs), 5.17 (1H, H1, bs)

EXAMPLE 23

[0137] Reduction of ,-unsaturated HA aldehyde =Preparation of AHA

[0138] Sodium borohydride (0.1 eq.) was added into a 2% solution of ,-unsaturated HA aldehyde (200 mg, Example 17) in distilled water, at 5 C. The reaction mixture was stirred for 20 h at 5 C., then precipitated with isopropanol and analysed by NMR.

[0139] DS=4% (determined by NMR)

EXAMPLE 24

[0140] Reduction of ,-unsaturated HA aldehyde=Preparation of AHA

[0141] Sodium borohydride (1 eq.) was added into a 2% solution of ,-unsaturated HA aldehyde (200 mg, Example 17) in distilled water, at 40 C. The reaction mixture was stirred for 1 h at 40 C., then precipitated with isopropanol and analysed by NMR.

[0142] DS=5% (determined by NMR)

EXAMPLE 25

[0143] Reduction of ,-unsaturated HA aldehyde=Preparation of AHA

[0144] Sodium borohydride (2 eq.) was added into a 2% solution of ,-unsaturated HA aldehyde (200 mg, Example 17) in distilled water, at 20 C. The reaction mixture was stirred for 4 h at 20 C., then precipitated with isopropanol and analysed by NMR.

[0145] DS=5% (determined by NMR)

EXAMPLE 26

[0146] Determination of oxidation activity (FIG. 2)

[0147] An antioxidative activity of HA polysaccharides prepared according to the Example 22 and CS prepared according to the Example 2 was determined by means of a stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). This determination was performed according to the description in (Brand-Williams W. et al., LWTFood Science and Technology, 28, 1, 25-30, 1995), with a minor modification. Briefly, 100 L of 0.01% DPPH solution in methanol were added into 100 L of the tested substance dissolved in 50 mM of Tris pH 7.1. The decrease of the absorbance was measured after 15 min at 515 nm. Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) was used as the positive control. Data were measured in three independent experiments. T-test was used for the evaluation of the statistical significance * p<0.05, ** p<0.01, *** p<0.001 (FIG. 2).

EXAMPLE 27

[0148] Testing of cytotoxicity of the derivatives prepared according to the Example 25 (FIG. 1)

[0149] A cytotoxicity effect of the HA derivative was compared for non-cancer cellsprimary human dermal fibroblasts, and for breast carcinoma cell lines (MDA-MB-231), lung carcinoma cell lines (A-549), and hepatocellular carcinoma cell lines (HEP-G2). For the purpose of the experiments, the cells were cultured under standard conditions (37 C., 5% CO.sub.2) in a proper medium (10% FBSfetal bovine serum). After achieving an 80% confluency, the cells were passaged, counted by means of an automatic counter CASY TT, Roche, and put on 96-wells panels at the density of 5,000 cells per well in 200 l of media. After 24 hours, the medium was replaced with the solutions of the tested substances of concentrations of 1,000; 500; 100; and 10 g/mL of 10% medium. The cell viability was measured 24, 48, and 72 hours after the treatment by means of MTT test20 L of MTT solution (5 mg/mL) was added into each well, followed by an incubation for 2.5 hours and cell lysis with solubilisation solution (IPA:DMSO 1:1 s 10% Triton X-100 and 9.9% 37% HCl) for 30 minutes. Then the absorbance was measured by means of Microplate reader VERSAmax at 570 nm and 690 nm (background correction). The viability of the treated cells was evaluated by correlation with untreated control that corresponds to zero in FIG. 1. The values above zero refer to a cell activation (no cytotoxic effect of the derivatives) and the values below zero indicate a reduced cell viabilityi.e. the cytotoxic effect of the derivatives. In case of NHDF, it is obvious, in FIG. 1, that the tested derivative had no cytotoxic effect. In case of the carcinoma lines (MDA-MB-231, A-549, HEP-G2) a cytotoxic effect of the derivatives was observed. Based on the results of the presented tests, a potential anti-cancer effect of the derivative can be deduced (FIG. 1).