RU2366441C2 - Anticoagulating biologically active complex made of siberian cedar bark - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: anticoagulant showing inhibitory activity with respect to thrombin and Xa factor represents a biologically active complex made of Siberian cedar bark by nonpolar-solvent extraction of ground cedar bark followed by ethyl acetate extraction thereof.

EFFECT: effective inhibitory activity with respect to thrombin and Xa factor.

5 tbl, 5 ex

Description

The invention relates to medicine and can be used to create an effective tool for the prevention and treatment of thrombotic conditions (heart attacks, strokes of various localization).

The direct-type anticoagulant used in our country, unfractionated heparin (UFH) is a polysaccharide with a molecular weight of 2-40 kDa, on average 15 kDa, and has a number of undesirable side effects (osteoporosis, thrombocytopenia, increased functional activity of platelets, hemorrhagic complications). In order to develop new anticoagulant drugs that are free from the side effects of heparins, sulfated polysaccharides (SP) of plant origin (isolated from algae, lichens, fungi, higher plants), as well as isolated from tissues of marine invertebrates, arthropods, and mammals are developed [1-3]. To give polysaccharides molecules the necessary properties, they are depolymerized [4], functional groups are embedded in them [5], and they are combined [conjugated] with molecules of other polysaccharides [6]. The structure of the macromolecule SP plays an important role in the manifestation of anticoagulant activity (AA). So, under the action of chitosan modified with arginine (conjugate of chitosan-arginine), conformational changes of the thrombin molecule occur [7]. The introduction of functional groups to obtain chemically modified N-propanoyl-, N-hexanoyl- and N, О-quaternary substituted chitosan sulfate leads to an increase in AA [8]. When the positive charge on the surface of a polymer biomaterial based on chitosan with side chains of polyethylene glycol and hexanal changes to neutral or negative with the addition of SO ₃ groups, platelet adhesion decreases and biocompatibility with blood increases, which positively correlates with the content of polyethylene glycol inside the polymer [9]. It is known to use cedar bark to obtain the biologically active compound resveratrol [10]. There is a method of obtaining the drug Pycnogenol from seaside pine bark, which is an effective antioxidant, anticoagulant, capillary protector, and also exhibits an antiviral effect and anti-inflammatory activity [11].

Closest to the claimed anticoatulant, in fact, is unfractionated heparin. Thus, a composition containing heparin with an average molecular weight, which is a mixture of sulfonated oligosaccharides having molecular weights from ~ 6000 to ~ 12000 Da, inhibits thrombin bound to fibrin and thrombin in the liquid phase, catalyzes antithrombin, and inhibits the formation of thrombin by catalyzing the inactivation of factor Xa by antithrombin [12].

The disadvantage of heparin is that it has a number of side effects [13] and that it is isolated from mammalian tissues.

Objectives of the invention:

1. The expansion of the raw material base for the production of plant-based anticoagulants free of viruses and prions.

2. Partial disposal of Siberian cedar bark.

To obtain a biologically active complex from cedar bark, extraction of crushed cedar bark with a non-polar solvent and subsequent extraction with ethyl acetate are used. The desalted cedar bark is extracted with ethyl acetate in a flow-type apparatus at the boiling point of the solvent.

The effect of cedar bark extract on the change in the time of coagulation of human blood plasma was studied in coagulological tests of activated partial thromboplastin time (APTT, effect on the internal blood coagulation pathway), ReaKlot NPO Renam (effect on factor Xa activity) [14]. Lyophilically dried human plasma was purchased at the Renam NGO. The inhibition of the amidolytic activity of thrombin and factor Xa by the studied samples was studied by changing the cleavage rate of chromogenic substrates S-2238 or S-2222 at λ = 405 nm. To calculate the specific antithrombin (anti-factor IIa, aIIa) and anti-factor Xa (aXa) activities, calibration curves of the 5th International Standard for Unfractionated Heparin ((NIBSC).

The possibility of carrying out the claimed invention is shown by the following examples.

Example 1. 100 g of crushed cedar bark with a moisture content of up to 5% was subjected to extraction with a non-polar solvent (hexane) in a Soxhlet type apparatus for separating coniferous wax for 6 hours. The tarred cedar bark was extracted with ethyl acetate in a Soxhlet apparatus for 10 hours at the boiling point of the solvent. Ethyl acetate was distilled off, the residue was dried in air. The product yield was 2.3%.

Example 2. To a mixture of 0.06 ml of plasma and 0.06 ml of aPTT reagent (NPO Repam) was added 0.02 ml of a solution of cedar bark extract at final concentrations of 0.2-1.0 mg / ml. After 3 minutes of incubation at 37 ° C, 0.06 ml of a 0.025 M CaCl ₂ solution was added and the time (s) for the appearance of the fibrin clot was recorded. A significant elongation of the plasma coagulation time with an increase in the concentration of cedar bark extract was noted (Table 1). The concentration at which the plasma coagulation time increased by a factor of 2 (2-PT), in comparison with the control (31.5 ± 2.3), was 0.54 ± 0.017 mg / ml (Table 5). For unfractionated heparin, selected as the standard, a similar value was 0.33 ± 0.1 μg / ml. The antithrombin activity (aIIa) calculated according to heparin was 0.2 U / mg and correlated with a value of 2-PT.

Table 1
The effect of cedar bark extract on the internal blood coagulation pathway in the activated partial thromboplastin time test (aPTT, s) sample Final concentration, mg / ml 0 0.2 0.33 0.4 0.5 0.67 0.8 one Cedar Bark Extract 31.5 ± 2.3 44.2 ± 3.5 * 52.9 ± 5 ** 54.4 ± 4.8 ** 58.9 ± 4.4 *** 100 ± 11 *** 189 ± 15 *** 265.9 ± 21 *** * - p <0.05, ** - p <0.01 and *** - p <0.001 significance of differences with indications at a final heparin concentration of 0 μg / ml

Example 3. To a mixture of 0.069 ml of plasma and 0.035 ml of a solution of factor Xa (ReaKlot, NPO Renam - determining the effect of the test substances on the activity of factor Xa in a coagulological test), 0.01 ml of a solution of cedar bark extract in final concentrations of 0.067-1.340 mg was added / ml After 2 min incubation at 37 ° C, 0.06 ml of a 0.035 M CaCl ₂ solution was added and the time (s) for the appearance of the fibrin clot was recorded. A significant elongation of the clotting time of plasma with increasing sample concentration was noted (Table 2). The concentration at which the plasma coagulation time increased by a factor of 2 (2ReaClot), in comparison with the control (20.9 ± 1.7 s), was 1.30 ± 0.19 mg / ml (Table 5). The activity against factor Xa (aXa) for the sample, calculated by heparin, was 0.044 U / mg and correlated with a value of 2ReaClot (Table 5).

table 2
The effect of cedar bark extract on factor Xa activity in the ReaClot test (s) sample Final concentration, mg / ml 0 0,067 0.13 0.22 0.27 0.34 0.45 0.67 1.34 Cedar Bark Extract 20.9 ± 1.7 20.6 ± 1.4 20.7 ± 1.3 20.9 ± 1.9 21 ± 1,1 22.5 ± 2.2 23.4 ± 3.5 ** 27.8 ± 2.8 ** 44.8 ± 5 *** * - p <0.05, ** - p <0.01 and *** - p <0.001 significance of differences with indications at a final heparin concentration of 0 μg / ml

Example 4. To 250 μl of a solution of antithrombin "Behring", Germany (1 IU / ml of 0.05 M Tris-Hcl buffer with 0.0075 M Na ₂ -EDTA and 0.175 M NaCl, pH 7.4) was added cedar bark extract to final concentrations of 0.042-0.060 mg / ml and incubated for 3 min at 37 ° C. Then, 100 μl of a bovine thrombin solution from Behring, Germany (2 NIH ED / ml Tris-EDTA buffer) was added. After 30 seconds, 200 μl of a solution of the chromogenic substrate for thrombin - S-2238 from Behring (2 mM Tris-EDTA buffer) was added. The change in the optical density of the solution per minute at a wavelength of 405 nm was used to judge the activity of the test sample. The sample reduced the rate of hydrolysis of the chromogenic substrate by thrombin (Table 3). The concentration of IC 50 was 0.32 ± 0.054 mg / ml. A correlation with the antithrombin activity of cedar bark extract calculated according to the heparin standard was noted.

Table 3
Inhibition of thrombin amidolytic activity by cedar bark extract (A 405 / min) sample Final concentration, mg / ml 0 0,042 0,084 0.21 0.42 0.6 Cedar Bark Extract 0.195 ± 0.023 0.202 ± 0.022 0.184 ± 0.017 0.142 ± 0.017 * 0.064 ± 0.006 *** 0.05 ± 0.007 *** * - p <0.05, ** - p <0.01 and *** - p <0.001 significance of differences with indications at a final heparin concentration of 0 μg / ml

Example 5. To 250 μl of a solution of antithrombin "Behring", Germany (1 ED / ml of 0.05 M Tris-EDTA buffer with 0.0075 M Na ₂ -EDTA and 0.175 M NaCl, pH 8.4) was added cedar bark extract to final concentrations of 0.11- 2.40 μg / ml and 250 μl of a Behring factor Xa mixture (8 U / ml Tris-EDTA buffer) with dextran sulfate from Boehringer Manhame, Austria (0.02 mg / ml) . After a three-minute incubation at 37 ° C, the residual factor Xa activity was determined by adding 50 μl of Behring chromogenic substrate-S 2222 (4 mM). Activity was determined by the change in the optical density of the solution at a wavelength of 405 nm. The inhibition of the amidolytic activity of factor Xa by the test sample is shown in Table 4. The concentration of IC ₅₀ for the sample was 1.71 ± 0.22 mg / ml (Table 5) and correlated with aXa activity calculated by heparin.

Table 4
Inhibition of cedar bark extract amidolytic activity of factor XA (A 405 / min) sample Final concentration, mg / ml 0 0.11 0.55 1,1 1.9 2,4 Extract
bark
cedar 0.27 ±
0.018 0.272 ±
0.015 0.198 ±
0.024 * 0.175 ±
0.019 ** 0.132 ±
0.011 *** 0.066 ±
0.009 *** * - p <0.05, ** - p <0.01 and *** - p <0.001 significance of differences with indications at a final heparin concentration of 0 μg / ml

Table 5
Specific anticoagulant activity of cedar bark extract sample Coagulological research methods Amidolytic research methods 2 APTT, mg / ml 2 ReaK-lot, mg / ml aIIa, U / mg aXa, U / mg IC 50 aIIa, mg / ml IC 50 aXa, mg / ml aIIa, U / mg aXa, U / mg Cedar Bark Extract 0.54 ± 0.017 0.2 ± 0.05 0.0091 ± 0.0007 0.044 ± 0.006 0.32 ± 0.054 1.71 ± 0.22 0.01 ± 0.0024 1.3 ± 0.19

Thus, the investigated extract of cedar bark has anticoagulant activity in vitro. Antithrombin activity in the analysis of the effect on coagulation of human blood plasma is higher (in effective concentrations and in activities calculated according to heparin standards) than activity against factor Xa. The ability to inhibit the amidolytic activity of thrombin is higher when comparing effective concentrations and is equal to the ability to inhibit factor Xa when calculating activity according to the heparin standard. A decrease in the anticoagulant activity of the sample in amidolytic tests (a plasma inhibitor of serine proteinases - antithrombin is present), calculated according to heparin standards, indicates that for the manifestation of the antithrombin activity of the cedar bark extract, another plasma inhibitor of serine proteinases, cofactor heparin II, is also necessary.

Bibliography

1. Imberty A, Lortat-Jacob H, Perrez S Structural view of glycosaminoglycan-protein interactions Carbohydrate Research 342 (2007) 430-438.

2 Volpi N., Therapeutic applications ofglycosaminoglycans. Curr Med Chem, 2006; 13 (15): 1799-810.

3. Krishtanova N.A., Safonova M.Yu., Bolotova V.Ts., Pavlova E.D., Sakanyan E.I. Prospects for the use of plant polysaccharides as therapeutic and therapeutic agents. Bulletin of the Voronezh State University, series: Biology. Chemistry. Pharmacy. 2005, T1, pp. 212-221.

4.C. Noti, P. Seeberger. Chemical Approaches to Define the Structure-Activity Relationship of Heparin-like Glycosaminoglycans. Chemistry & Biology, Vol. 12, 731-756, 2005.

5. Martin de Kort, Rogier C. Buijsman and Constant A.A.van Boeckel Synthetic heparin derivatives as new anticoagulant drugs, DDT, V 10, No. 11, 2005.

6. J. Du, S. Zhang, R. Sun, L. Zhang, C.-D. Xiong, Y.-X. Peng, Novel Polyelectrolyte Carboxy-methyl Konjac Glucomannan-Chitosan Nanoparticles for Drug Delivery. II. Release of Albumin In Vitro. Appi Biomater 72B: 299-304, 2005.

7. Liu WG, Zhang JR, Cao ZQ, Xu FY, Yao KD. J Mater Sci Mater Med. 2004 Nov; 15 (11): 1199-203A Chitosan-argimne conjugate as a novel anticoagulation biomaterial.

8. Carbohydr Res. 2003 Mar 14; 338 (6): 483-9. Influence of functional groups on the in vitro anti-coagulant activity of chitosan sulfate. Huang R, Du Y, Yang J, Fan L.

9. Colloids Surf In Biointerfaces. 2005 May 10; 42 (2): 147-55. Chitosan based surfactant polymers designed to improve blood compatibility on biomaterials. Sagnella S, Mai-Ngam K.

10. RU 2294919, publ. 03/10/2007.

11. Pat. USA No. 4698360.

12. RU No. 2002100908 "Compositions containing heparin, which have the ability to inhibit the formation of a clot associated with coagulation factors", published September 20, 2003.

13. Holzheimer RG. Low-molecular-weight heparin (LMWH) in the treatment of thrombosis Eur J Med Res. 2004 Apr 30; 9 (4): 225-39.

14. Bates SM, Weitz L // Circulation. 2005. V 112. No. 4. P 53-60.

Claims (1)

An anticoagulant agent that has inhibitory activity against thrombin and factor Xa, characterized in that it is a biologically active complex from Siberian cedar bark, obtained by extraction of ground cedar bark with a non-polar solvent, followed by extraction with ethyl acetate, with a final concentration of 0.32-0 , 54 mg / ml is effective for inhibiting the activity of thrombin and a final concentration of 1.30-1.71 mg / ml is effective for inhibiting the activity of coagulation factor Ha.