Agent, having antitumour, anticoagulant, wound-healing, anti-inflammatory and antioxidant activity, capacity to inhibit collagenase and angiotensin converting enzyme, and method for production thereof

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to produce biologically active collagen peptides from marine hydrobionts. Patiria pectinifera starfish is dehydrated with 96% ethyl alcohol and then demineralised with 1-2 N solution of an inorganic acid with ratio of the raw material to inorganic acid of 1:(3-5) for 1-3 days. The demineralised raw material is washed from traces of acid and water-soluble impurities with distilled water, after which the material is hydrolysed with an alkali solution with ratio of raw material to the alkali solution of 1:(3-5) in order to remove non-collagen proteins and washed with distilled water at temperature of 2-4°C. The obtained starfish collagen shells are homogenised. The homogenate is diluted with distilled water; pH of the suspension is brought to a value equal to 8.0-8.5 with an alkali solution and hydrolysed with 1% collagenase solution with ratio of homogenate to enzyme of (100-200):1 and temperature of 30-40°C for 3-5 hours, pH 8.5-7.0. The enzyme is inactivated at 80-90°C for 10-15 minutes. The hydrolysate solution is filtered to remove non-hydrolysed collagen, subjected to ultra-filtration through a 30 kD membrane filter to remove the inactivated enzyme; the end product, having antitumour, anticoagulant, wound-healing, anti-inflammatory, antioxidant activity, capacity to inhibit collagenase and angiotensin converting enzyme and which is a complex of collagen peptides with a high-molecular weight component weighing 22-23 kD, is concentrated in a vacuum and lyophilised.

EFFECT: obtaining a product, having antitumour, anticoagulant, wound-healing, anti-inflammatory and antioxidant activity.

2 cl, 7 tbl, 2 ex

 

The invention relates to pharmaceutical, cosmetic and food production and for the creation of biologically active agents and food additives of marine life.

Marine organisms are a valuable source of biologically active compounds. Special interest group of biopolymers that make up the structural basis of living organisms, namely collagen and connective tissue proteins. It is known that marine invertebrates are composed of tissue proteins, not inferior to the amino acid composition of traditional power sources, being, thus, a valuable food resource. In addition, the peptides of marine origin exhibit a variety of physiological properties, namely exert antihypertensive [Kim S.K., Wijesekara I. // J.Functional Foods. 2010. No. 2. P.1-9; S.K. Kim, E. Mendis // Food Res. Int. 2006. V.39. P.383-393], anticoagulant [Kim S.K., Wijesekara I. // J.Functional Foods. 2010. No. 2. P.1-9; Hayes M., Carney C. // Food Chemistry. 2011. No. 129. P.235-244; RU 2302250 C1, 10.07.2007], antithrombotic effect [J. Wilson, M. Hayes, Camey B. // Food Chemistry. 2011. No. 129. P.235-244], as well as exhibit antimicrobial activity [Kim S.K., Wijesekara I. // J.Functional Foods. 2010. No. 2. P.1-9; S.K. Kim, E. Mendis // Food Res. Int. 2006. V.39. P.383-393; Wilson J., Hayes, M., Camey B. // Food Chemistry. 2011. No. 129. P.235-244; RU 404744 C2, 27.07.2010; EN 2396984 C2, 20.08.2010], anticancer activity [Kim S.K., Wijesekara I. // J.Functional Foods. 2010. No. 2. P.1-9; EN 2161002 C1, 27.12.2000], antioxidant activity [Kim S.K., Wijesekara I. // J.Functional Food. 2010. No. 2. P.1-9; S.K. Kim, E. Mendis // Food Res. Int. 2006. V.39. P.383-393].

Due to the fact that the structure of the marine collagens similar to the structure of warm-blooded animals, they can be used in practical medicine for the treatment of damaged skin and tissue [EN 2391399 C2, 10.06.2010, EN 2396084 C1, 10.08.2010], ophthalmology [EN 2272599 C1, 27.03.2006]cosmetology [EN 2281082 C1, 10.08.2006].

Bioactive peptides from marine organisms are a potential source of functional foods [Luo R., Neither S., Xia, J., Ren, S., Jiang X. // African Jornal of Biotechnology. 2011. V.10(62). P.13610-13616; EN 2250047 C1, 20.04.2005; EN 2335951 C2, 20.10.2008; CN 1317268, 17.10.2001].

Recently, great attention is paid to the collagen and peptides from marine mollusks, arthropods, echinoderms, including collagen peptides starfish.

A method of obtaining from starfish collagen and peptide by alkaline processing to remove Nikolayevich materials, subsequent enzymatic hydrolysis using tartaroo or citric acid as oxidant and acid protease (1% pepsin) at 5-10°C. To obtain a peptide of collagen is repeated Fermentas neutral protease [KR 20060125995 AND, 07.12.2006].

A known method of obtaining a peptide of collagen shell starfish by soaking the substrate in a dilute alkali solution, decalcomania diluted hydrochloric acid is, the extraction solution of acetic acid and acid proteinase (peptazol), centrifugation followed by treatment of the peptide solution macroporous resin and freeze-drying [CN 1740199 And, 01.03.2006].

A method of obtaining low molecular weight peptides of collagen by dip crushed stars in distilled water containing hydrogen peroxide and sodium hydroxide, filtration, extraction with distilled water containing 0.5% of citric and ascorbic acids, ultrasonic treatment, decompression and sublimation [KR 20110055126 AND, 25.05.2011]. Get nanocomposite of collagen peptides with antioxidant effect. It is proposed to use it as a cosmetic anti-wrinkle.

As a prototype of the selected bioactive peptide of collagen from sea stars, which are suppressing the increase of glucose level in blood [JP 2009235064 And, 15.10.2009]. It is produced by sequential processing of the raw material alkali, then with acid. Next, the raw material is treated with a neutral protease with getting hydrolysate from which secrete collagen peptide starfish.

Starfish are one of the most common animals of the class Echinodermata. Among them predatory starfish - patiria grebeshkov (Patiria pectinifera) - is the main enemy mariculture gardens. At the same time, this n the commercial species of marine echinoderms can serve as a valuable source of protein. Protein content in starfish is about 20% of dry weight [Luo P., Hu, S., Xia J, Ren S., Jiang X. // African Jornal of Biotechnology. 2011. V.10(62). P.13610-13616].

The present invention is the task of creating new funds from the starfish Patiria pectinifera, possessing a wide spectrum of biological activity, and how it is retrieved from the organisms.

The problem is solved by providing a method of obtaining funds with antitumor, anticoagulant, wound healing, anti-inflammatory, antioxidant activity, the ability to inhibit collagenase and angiotensinase enzyme (ACE)inhibitors, namely, that the sea star Patiria pectinifera dehydrated in 96% ethanol, then demineralizer 1-2 N solution of mineral acid at a ratio of raw materials:mineral acid 1:(3-5) within 1-3 days. Demineralized raw materials washed from traces of acid and water-soluble impurities distilled water.

Then raw hydrolyzing an alkali solution at a ratio of raw material:the alkali solution 1:(3-5) to remove Nikolayevich proteins. Next deproteinisation the raw material is washed with distilled water at a temperature of 2-4°C, then the resulting collagen shells starfish homogenized, the homogenate was diluted with distilled water to bring the pH of the suspension to 8.0 to 8.5 with a solution of selachii hydrolyzing a 1% collagenase solution at a ratio of homogenate:enzyme (100-200):1 and a temperature of 30-40°C for 3-5 h, pH 8.5-7,0.

Then inactivate the enzyme at 80-90°C for 10-15 min, a solution of the hydrolyzate was filtered to remove non-hydrolyzed collagen, is subjected to ultrafiltration through a membrane filter 30 kDa to remove Deaktivierung enzyme, then the target product was concentrated in vacuo and lyophilizers.

As a result of implementation of this method get a new agent with antitumor, anticoagulant, wound healing, anti-inflammatory, antioxidant activity, the ability to inhibit collagenase and angiotensinase enzyme (ACE), representing a complex of collagen peptides with high molecular weight component 22-23 kDa identified when conducting electrophoresis in polyacrylamide gel.

It was established experimentally that the resulting implementation of the claimed method means it has high biological activity, namely:

1. Antitumor (inhibition of tumor growth (SRW)>56%);

2. anticoagulant (activated partial thromboplastin time (APTT) is increased 4-fold compared with control);

3. anti-inflammatory (slow inflammation by 43% compared with control);

4. antioxidant (reducing products reacting with thiobarbituric acid (TBA-RP) 46%);

5. wound healing is the composition of the ointment compositions (faster healing thermal burn on the 7th day of 163%);

6. inhibiting proteolytic enzymes (ACE 60%collagenase 80%).

Technical result provided by the invention, is to obtain new funds from the starfish Patiria pectinifera, with a wide range of physiologically active properties in comparison with the prototype. For some activities there is an increase compared with the same activities, demonstrate basic Comparators. The inventive tool can be used as a substance in the manufacture of medicines, pharmaceutical drugs, and cosmetics.

The invention also provides, on the one hand, the expansion of raw materials for the production of biologically active peptides of collagen, and on the other hand, solves the problem of disposal of such predatory echinoderms for mariculture, which is the starfish Patiria pectinifera.

The inventive method is carried out as follows.

Starfish R. pectinifera, fresh or deportirovanniy, placed on the mesh filter full flow sea water, then dehydrated in 96% ethanol. Alcohol sent for recycling. Next, raw materials demineralizer 1-2 N solution of mineral acid at a ratio of raw materials:mineral acid 1:(3-5) within 1-3 days. Demineralized raw materials washed from shadowkitty and water-soluble impurities distilled water. Collagenase proteins separated alkaline proteolysis of 0.1-0.5 N caustic soda solution at a ratio of raw material:the alkali solution 1:(3-5).

Then deproteinization raw materials washed from the soluble products of enzymatic lysis by distilled water at a temperature of 2-4°C.

The obtained collagen shells starfish homogenized, and then the homogenate was diluted with distilled water to bring the pH of the suspension to 8.0-8.5 alkaline solution and hydrolyzing 1% collagenase solution at 30-40°C for 3-5 h at a ratio of homogenate:enzyme (100-200):1. The initial pH value of 8.5-7,8, the final pH value of the process of 7.0. The regulation of pH is 5 N caustic soda solution. Inaktivirovanie enzyme is carried out by heating the hydrolyzate to 80-90°C for 10-15 minutes

Then the solution of the hydrolyzate was filtered to remove non-hydrolyzed collagen, is subjected to ultrafiltration through a membrane filter 30 kDa to remove Deaktivierung enzyme, then the target product (CCS) was concentrated in vacuo and lyophilizers. The yield of the target product is 7-10% by weight of the raw material (23-33% in terms of dry weight).

Abbreviation CCS is a complex of peptides of collagen starfish R. Pectinifera.

To confirm the protein nature of the product analysis samples of biologically active peptides of collagen starfish R. pectinifera by IR and UV-VIS spectrometry

The IR spectra of the peptides were recorded on a Fourier transform infrared spectrometer Equinox 55 (Bruker, Germany) in KBr (~1 mg/700 mg) with a resolution of 2 cm-1.

Figure 1 shows the IR spectrum of biologically active peptides of collagen starfish R. pectinifera. The IR spectrum in the region below 2000 cm-1there are absorption bands of Amide I (1650 cm-1), Amide II (about 1550 cm-1) and Amide III (about 1230-1250 cm-1), indicating that the protein nature of the investigated substances. [Untilize. Infrared spectra and structure of polypeptides and proteins. Nauka, M., 1965, str].

UV-VIS spectra of aqueous solutions of peptides of collagen starfish R. Pectinifera (~1 mg/ml) were recorded in quartz cuvettes with a layer thickness of 1 cm spectrophotometer UV-1601 PC (Shimadsu, Japan).

Figure 2 presents the UV-VIS spectrum of an aqueous solution of biologically active peptides of collagen starfish R. pectinifera. In the UV-VIS spectrum is the absorption band (about 275 nm), characteristic of proteins.

The complex of biologically active peptides of collagen starfish R. pectinifera was analyzed by the method of nuclear magnetic resonance in nuclei1H and13With the device "Bruker Avance III (700 MHz). Figure 31H NMR spectrum, figure 4 -13From the NMR spectrum of complex peptides starfish R. pectinifera.

Determined the amino acid composition of the target product. And what it should the resulting peptides are fragments of collagen and significantly differ in amino acid composition from the composition of peptides of fish. The results are presented in table 1.

Table 1
The amino acid composition*
No.Name the amino acidsPeptides starfish Patiria pectiniferaThe composition of the peptides fish* Theragra chalcogramma
1Aspartic acid2.691.26
2Threonine1.330.93
3Series1.480.75
4Glutamic acid5.81.38
5Hydroxyproline3.32-
6Proline 4.190.72
7GlycineAt 8.620.87
8Alanine2.140.91
9Carolin--
10Valine1.20.95
11Methionine0.160.64
12Cystine0.15
13Isoleucine0.671.18
14Leucine0.831.31
15Tyrosine0.480.65
16Phenylalanine0.220.78
17Lysine0.971.84
18Histidine0.220.42
19Arginine3.711.19

*obtained by the proposed method for the comparison of amino acid composition of peptides derived from marine organisms.

Data1H NMR spectroscopy confirmed that CCS does not contain tryptophan (complete absence of signals in the field of the protons of aromatic amino acids) (figure 3). The presence of phenylalanine (7.443-7.340 ppm) and tyrosine (7.211-6.899 ppm) confirmed the identification of signals from their chemical shifts.

Quantitative assessment of the content of tyrosine and phenylalanine obtained by integrating the number of protons indicates that the tyrosine in the composition of the CCS approximately in 2 times more than phenylalanine. This agrees well with the data of amino acid composition.

Range13With NMR CCS demonstrates the presence of signals13With the peptide bond, aromatic and aliphatic amino acids (figure 4).

Molecular weight distribution PEP is the Idov collagen starfish R. pectinifera were determined by gel chromatography on Sephadex G-75.

Figure 5 shows the elution profile of the total complex of peptides of collagen starfish R. pectinifera on Sephadex G-75.

The concentration of peptides (absorption at λ = 278 nm), the volume fractions of 3 ml;

- Inhibiting activity of fractions of peptides R. pectinifera (%) in relation to angiotensinase the enzyme.

Discovered that the protein composition is a set of peptides with different molecular weight.

The molecular weight of the peptides of collagen starfish R. pectinifera were identified by means of SDS-electrophoresis in 15% polyacrylamide gel [Laemmli U.K. // Nature 1970, v.277, p.680-685].

It is established that among the major peptides of collagen starfish have a component with a molecular mass 22-23 kDa.

Figure 6 presents SDS-electrophoresis CCS:

1 is a mixture of recombinant proteins-witnesses with a molecular weight of from 250 to 15 kDa (Gel Electrophoresis Markers Kit for Protein, "Sigma-Aldrich");

2, 3 - CCS (peptides of collagen R. pectinifera).

The invention is illustrated by the following examples.

Example 1.

Freshly caught sea stars R. pectinifera in the amount of 1.0 kg (260-270 pieces) are placed on the strainer to the full flow of sea water. Then raw pour 1 l of 96% ethanol to remove tissue and internal moisture-containing is of alcosta 1 hour The resulting alcoholic extract is drained and sent for recycling.

Then spend the demineralization of raw materials of 1.0 N solution of hydrochloric acid at a ratio of the weight of the raw material to the volume of acid solution 1:5 in the day. Demineralized raw materials washed from traces of acid and water-soluble impurities distilled water, and then hydrolyzing with 0.1 N NaOH solution at a ratio of the weight of the raw material to the volume of alkali solution 1:5 in 3 days. Received deproteinization raw materials washed from the soluble products of enzymatic lysis by distilled water at a temperature of 2-4°C.

Then the obtained collagen skeletons of sea stars are ground in a meat grinder and homogenized. The homogenate was diluted with distilled water at a ratio of 1:3, stirred until a homogeneous suspension and placed in a reactor for enzymatic lysis by. Bring the pH of the suspension to 8.5 using NaOH solution. Then the reactor was added 1% collagenase solution at a ratio of homogenate:the enzyme is 100:1. The reaction mixture is intensively stirred. The process is carried out at a temperature of 30°C for 5 h in Control of the process of enzymatic lysis by changing pH. The process is complete at pH 7.0. Inactivation of the enzyme was carried out by heating the hydrolyzate to 80°C for 15 minutes, the Product was filtered hot through a woven filter to remove non-hydrolyzed collagen. Deaktivieren the th enzyme is removed by ultrafiltration of the hydrolyzate through a membrane filter 30 kDa.

The resulting product was concentrated in vacuo and lyophilizers. Get the target product with the release of 101 g (10% by weight of the feedstock or 33% in terms of dry raw material). The content of the peptides in the product 90%. MOLEKULYaRNAYa mass of the obtained complex is about 22 kDa. Moisture 6%, impurity-4%.

Example thorozine raw - starfish R. pectinifera in the amount of 10 kg defroster, then with 15 l of 96% ethanol for 3 hours alcohol extract Obtained is drained and sent for recycling.

Then spend the demineralization of raw materials a 2.0 N solution of phosphoric acid at the ratio of the weight of the raw material to the volume of acid solution 1:3 for 3 days. Demineralized raw materials washed from traces of acid and water-soluble impurities distilled water, and then hydrolyzing with 0.5 N KOH solution at a ratio of the weight of the raw material to the volume of alkali solution (1:3 within 2 days. Received deproteinization raw materials washed from the soluble products of enzymatic lysis by distilled water at a temperature of 2-4°C.

Then the obtained collagen skeletons of sea stars are ground in a meat grinder and homogenized. The homogenate was diluted with distilled water at a ratio of 1:4, stirred until a homogeneous suspension and placed in a reactor for enzymatic lysis by. Bring the pH of the suspension to 8.0, using a solution of KOH. Then in reactor d is billaut 1% collagenase solution at a ratio of homogenate:the enzyme is 200:1. The reaction mixture is intensively stirred. The process is carried out at a temperature of 40°C for 3 h in Control of the process of enzymatic lysis by changing pH. The process is complete at pH 7.0. Inactivation of the enzyme was carried out by heating the hydrolyzate to 90°C for 10 minutes, the Product was filtered hot through a woven filter to remove non-hydrolyzed collagen. Deactivated the enzyme is removed by ultrafiltration of the hydrolyzate through a membrane filter 30 kDa.

The resulting product was concentrated in vacuo and lyophilizers. Get the target product with a yield of 0.71 kg (7.1% of the weight of the feedstock or 23% in terms of dry raw material). The content of the peptides in the product 80%. The molecular mass of the obtained obtained complex is approximately 23 kDa. Moisture is 10%, impurity-10%

Study of biological activity of the claimed means of the starfish Patiria pectinifera

Antitumor activity

The experiments are performed on Bezmaternykh the mice CD-1 and IAS weighing 20±2 g Perepevku, maintenance of strains and assessment of the obtained results is done according to the methodology adopted in the Russian oncological scientific center of the Academy of medical Sciences of the Russian Federation [Sofina Z.P. and other Experimental evaluation of anticancer drugs in the USSR and the USA. - M.: Medicine, 1980. - 296 S.]. As a positive control, use a known protivoopujolevy is a new drug "Cyclophosphamide" (fit), Biochemistry (Russia).

The antitumor effect was evaluated by the inhibition of tumor growth (SRW)% compared with the control according to the formula:

TRO(%)=(1-T/C)×100,

where T and C are average values (in mg) weight of tumors in the experimental and control groups, respectively.

Values TRO more than 25% are considered as reliable anti-tumor effect. The results are presented in table 2.

Table 2
Antitumor activity
The name of the parameterEd. ISMDoseThe measured value TRO
Fit%20 mg/kg40,0±14,55
BCZ%10 mg/kg51,36±24,97
BCZ%5 mg/kg56,75±17,65
CCS+fit%5/20 mg/kg44,51±8,59

As shown, the CCS shows more visokoaktivniy in a much lower dose compared with cyclophosphamide. In addition, there is a synergistic effect: increased activity fits in a joint application with CCS.

Anticoagulant activity

Anticoagulant activity of the samples CCS in vitro evaluated on the following parameters: determination of prothrombin time (PT) and activated partial thromboplastin time (APTT).

The determination is performed on the automatic coagulometer Amelung (Germany) with the mechanical principle of definition. For research uses a standard set of "Techplast-test" and "APTT-test" firm "Technology standard" , Barnaul.

Tests conducted on human platelet-poor plasma, stabilized with 3.8% sodium citrate in a ratio of 9:1. The results are presented in table 3.

Table 3
Anticoagulant activity of the peptides of collagen starfish R. pectinifera
The name of the parameterEd. MEAs.RequirementsMeasured value
DosePar valueMaximum deviation
Anticoagulant activity, determination of prothrombin time (PT)
RO Controlsec11,0-15,00,0515,1±0,1
PITTSsec1 mg/ml15,00,05trend of 15.87±0,12
PITTSsec1.5 mg/ml15,00,0516,87±0,15
PITTSsec2 mg/ml15,00,0518,9±0,79
Anticoagulant activity, determination of activated partial thromboplastin time (APTT)
The APTT of the Controlsec-30-500,0536,17±0,38
The APTT BCZ sec0.1 mg/ml35-500,0544,63±0,95
The APTT BCZsec0.3 mg/ml35-500,0556,97±0,25
The APTT BCZsec0.7 mg/ml35-500,0568,93±0,05
The APTT BCZsec1 mg/ml35-500,0592,27±1,40
The APTT BCZsec1.5 mg/ml35-500,05128,1±1,01
The APTT BCZsec2 mg/ml35-500,05145,9±1,87
Heparinsec0.4 mg/ml35-500,05 205,0±1,48

The product comparison - heparin. The limits of fluctuations of PV in a healthy adult, 11-15 sec. The limits of fluctuations of the APTT in a healthy adult, 30-50 sec.

In the test of determining the APTT test specimens showed a fairly strong anticoagulant effect. Already at a dose of 0.1 and 0.3 mg/ml CCS increased the time of the formation of a clot in 1,5-2 times. Apparently, CCS acts as an anticoagulant on the inner path activation of blood coagulation. Their anticoagulant effect is comparable to that of the known anticoagulant heparin.

Anti-inflammatory activity

The experiments are performed on Bezmaternykh the mice CBA weighing 20±2 g

Nonspecific local inflammation induce the introduction of the Delta-carrageenan (type IV, Sigma). CCS and the comparison drug indomethacin used for medicinal dose of 10 mg per 1 kg body weight of the animal. Drugs injected intraperitoneally 1 h before the induction of inflammation by carrageenan. Control animals injected with saline.

The level of slowdown tissue granuloma is fixed at 5 h after injection of carrageenan and calculated according to equation:

slow inflammation (%)=TA-TV/TA×100,

where TC is the fabric weight of granuloma in the control group;

TV - fabric weight of granuloma in the group that received anti-inflammatory drugs. The results are presented in table 4.

Table 4Anti-inflammatory activityName of productEd. MEAs.RequirementsMeasured valuePar valueMaximum deviationDetermination of anti-inflammatory activity on carraginanous modelIndomethacin, 10 mg/kg%>25,00,0552,0±4,06CCS, 10 mg/kg%>25,00,0543,0±2,47

The peptides of collagen echinoderms exhibit high anti-inflammatory activity comparable to that of a drug indomethacin.

Wound healing activity

Use composition CCS-based baby cream ("AVANTA", Russia) - IPPC 1% and IPPC 5%. The treatment of wounds spend 4 times a day. Treatment 11 days. The measurement of areas damaged surfaces and assess what the state of the Russian Academy of Sciences carried out individually for each animal.

Wound healing activity (RA) calculated as a percentage by the formula:

PA(%)=100-(S1×100/S2),

where S1- the final size of the wound, S2- the initial size of the wound.

The results are presented in table 5.

Table 5
Wound healing activity
Name of productEd. MEAs.RequirementsMeasured value
Par valueMaximum deviationThe healing thermal burns, % (m±σ)
day 7day 9day 11
Methyluracil%>100,00,05144,5±23,2113,7±1,4to 100.4±1,2
IPPC 1%%>100,00,051632±10,1 123,43±2,1of 101.7±1,2
IPPC 5%%>100,00,05128,2±18,8115,6±4,1of 101.4±1,3

The application of a cream BCZ composition reduces the healing time of wounds, especially the difference in wound healing activity are seen on the seventh day after the induction of thermal burns (>50%), while 1%cream BCZ shows higher activity than 5%cream. CCS in the composition of the cream drugs (IPPC 1 and 5%) has a pronounced healing effect and is superior to the action of the drug methyluracil.

Antioxidant activity

Studies conducted on mice-males line IAS weighing 18-20 g with alloxan induced diabetes.

Treatment should be performed within 7 days after the induction of alloxan diabetes. As the comparison drug use glibenclamide.

After the experiment was conducted fences blood and determine the main indicators of changes in lipid and carbohydrate metabolism biochemical analyzer (ROCHE, Switzerland). Changes in carbohydrate metabolism by induction of diabetes determine glucose tolerance test using a glucometer "Satellite" LLC "Eltom", Russia) after 60 min after oral administration of glucose (4 g/kg of body weight.

The intensity of free radical processes in the blood was determined by the content of TBA-reactive products. The calculation of the level of TBA-reactive products is carried out according to the formula:

C=ΔE1,56×105(mmol/l)

With the amount of TBA-reactive products in the blood plasma;

ΔΕ is the difference of extinction experienced and blank samples;

of 1.56×105(mmol/l) is the molar coefficient of extinction. The results are presented in table 6.

Table 6
Antioxidant activity
Name of productThe measured value %
Glucose,GTT*CholesterolTriglyceridesBilirubinTBA-RP**
Glibenclamide 5 mg/kg79,6±1,52,28±0,919,9±0, 6,7±0,127,3±2,530,4±1,3
BCZ, 50 mg/kgof 80.6±1,35,9±1,8of 21.9±0,340,7±0,219,3±3,132,6±1,6
BCZ, 100 mg/kg74,6±1,88,2±3,222,1±0,184,5±0,147,8±2,346,7±2,4
GTT* - glucose tolerance test;
TBA-RP** products reacting with thiobarbituric acid

The inventive tool shows good antioxidant activity, its therapeutic effect is comparable with known drug glibenclamide.

Inhibitory effect on proteolytic enzymes, including metalloproteinases

Inhibition of collagenase activity

The method is based on the hydrolysis of type I collagen by collagenase to amino acids followed by their definition. Per unit collagenolytic activity take that amount of enzyme which, in contact with collagen type I for 30 is in at pH 7.5 and temperature (37±0,1)°C increases the optical density of a solution of 1.0 per minute at a wavelength of 570 nm.

Collagenolytic drug activity collagenase (KA) in units of milligrams calculated by the formula:

KA=D×V/0,1×C

KA1=D×V/0,1×C×C1

where D is the optical density of the solution;

V - volume of sample collagenase;

C - concentration of collagenase;

C1- concentration of the sample;

0.1 - multiplier on the number collagenolytic units.

For the final test result arithmetic mean value of the activity obtained in the analysis of two parallel portions enzyme preparation.

Inhibitory activity is calculated by the formula:

% inhibition = (1-KA1/KA)×100

The results are presented in table 7.

Study of the inhibitory activity showed the presence of high dose dependent inhibitory effect of CCS (80%).

Inhibition of angiotensin-converting enzyme

Evaluation of inhibitory activity of CCS in relation to angiotensinase enzyme (ACE) was performed according to the method of Cushman D.W., Cheung H.S. [D.W. Cushman, H.S. Cheung Spectrophotometric assay and properties of the angiotension I-converting enzyme of rabbit lung // Biochem. Pharmacol. 1971. V.20. P.1637-1648].

Inhibitory activity of the studied peptides expressed as a percentage of the amount of the hippuric acid in the control and the samples

A(%)=ICthe ISU×100%/SPCSthe ISU,

where: ICthe ISUthe number Kippur the howling acid in the sample;

Spyship - the amount of hippuric acid in the sample;

A-ACE - inhibiting activity BCZ.

The results are presented in table 7.

Table 7
Inhibiting activity
No.The name of the parameterEd. MEAs.RequirementsThe measured value in %. inhibition
Par valueMaximum deviation
1Inhibition of collagenase activity:%>10 with respect to the control5,043,0±1,23
57,0±1,27
80,0±1,35
100 mcg
200 mcg
300 mcg
2Antihypertensive activity - inhibitory activity against angiotensin-1-converting enzyme (ACE)inhibitors:>10 with respect to the control5,020,0±1,38
30,0±1,27
60,0±1,41
100 mcg
200 mcg
300 mcg

Figure 5 shows inhibitory activity of fractions of CCS obtained by gel chromatography on Sephadex G-75, in relation to angiotensinase enzyme.

It is established that the fraction of CCS have a high activity against ACE.

1. The method of obtaining funds with antitumor, anticoagulant, wound healing, anti-inflammatory, antioxidant activity, the ability to inhibit collagenase and angiotensinase enzyme (ACE)inhibitors, namely, that the sea star Patiria pectinifera dehydrated in 96% ethanol, then demineralizer 1-2 N solution of mineral acid at a ratio of raw materials:mineral acid 1:(3-5) within 1-3 days, demineralized raw materials washed from traces of acid and water-soluble impurities distilled water, then the materials hydrolyzing an alkali solution at a ratio of raw material:the alkali solution 1:(3-5) to remove Nikolayevich proteins, then deproteinization raw materials washed with distilled water PR is the temperature of 2-4°C, the obtained collagen shells starfish homogenized, the homogenate was diluted with distilled water to bring the pH of the suspension to 8.0-8.5 alkaline solution and hydrolyzing 1% collagenase solution at a ratio of homogenate enzyme (100-200):1 and a temperature of 30-40°C for 3-5 h, pH 8.5-7,0, then inactivate the enzyme at 80-90°C for 10-15 min, then the solution of the hydrolyzate was filtered to remove non-hydrolyzed collagen, is subjected to ultrafiltration through a membrane filter 30 kDa to remove Deaktivierung enzyme, then the target product, which is a complex of collagen peptides with high molecular weight component of weight 22-23 kDa, concentrated in vacuo and lyophilizers.

2. The agent with antitumor, anticoagulant, wound healing, anti-inflammatory, antioxidant activity, the ability to inhibit collagenase and angiotensinase enzyme (ACE)inhibitors, characterized in that it is obtained by the method according to claim 1 and represents a complex of collagen peptides with high molecular weight component 22-23 kDa identified when conducting electrophoresis in polyacrylamide gel.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: peptides consist of four amino-acid residues that are used for stimulation of collagen production with fibroblast.

EFFECT: invention allows effective stimulation of collagenoses in fibroblast cells.

11 cl, 3 dwg, 7 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention discloses a medicine for treating a patient with rheumatoid arthritis on the basis of an antibody or a fragment thereof that bind the Extra Domain-A (ED-A) isoform of fibronectin. The antibody or fragment thereof contain the domain VH comprising a frame and a number of complementary-determining regions HCDR 1 -3, and the domain VL comprising a frame and a number of complementary-determining regions LCDR 1-3 with the antibody or fragment thereof being conjugated with IL-10. The invention discloses a medicine on the basis of the above antibody or fragment thereof for delivering a molecule conjugated with the antibody or fragment thereof wherein the molecule represents IL-10 to a newly formed vascular tree of the rheumatoid arthritis regions in a patient. There are disclosed method of treating rheumatoid arthritis with using the medicines under the invention, method of delivering IL-10 conjugated with the antibody or fragment thereof to the newly formed vascular tree of the rheumatoid arthritis regions in the patient, as well as a conjugate of the antibody or fragment thereof binding ED-A of fibronectin to interleukine-10. The antibody or fragment thereof may be represented by a diabody, contain one-chain Fv, present a small immunoprotein (SIP).

EFFECT: invention provides the successful treatment of rheumatoid arthritis ensured by the selective delivery of biologically active compounds conjugated on the antibody to the disease locations.

35 cl, 8 dwg, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine. The invention aims at creating biodegraded implants of the regenerated silk fibroin Bombyx mori. A method for preparing the biodegraded composite matrix of the regenerated silk fibroin Bombyx mori involves the stages: - dissolving the regenerated fibroin in a buffer containing CaCl2, C2H5OH, H2O in molar ratio 1:2:8. That is followed by dialysis of the prepared solution to the fibroin concentration min. 20 mg/ml. The fibroin solution of the concentration min. 20 mg/ml is mixed with dimethyl sulphoxide (DMSO). Gelatin or polylysine is added to the prepared mixture. The prepared mixture is frozen and thawed with an organic solvent added; what is also presented is the use of the specified biodegraded composite matrix as a base for the biodegraded implant.

EFFECT: group of inventions enables improving the biocompatibility, increasing the adhesive properties of matrix and cell proliferation in a mammalian body.

2 cl, 3 dwg, 3 ex, 1 tbl

FIELD: biotechnologies.

SUBSTANCE: invention refers to the area of biotechnology, namely to production of short peptides - stimulators of production of extracellular matrix protein in the skin, and can be used in the medicine. The peptides produced consist of four amino-acid residues that can be used separately or in combination in the method of stimulation of collagen production using fibroblast.

EFFECT: invention provides for effective collagenesis stimulation in fibroblast cells.

25 cl, 3 dwg, 7 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and concerns agents and methods based on using fibronectin domain EDA. Substance of the inventions involves using a polypeptide comprising an amino acid sequence of the fibronectin domain EDA, or its TLR4-binding fragment, or a version of specified domain; for preparing a pharmaceutical composition stimulating an antigen- specific immune response.

EFFECT: improved stimulant property.

36 cl, 3 ex, 11 dwg

FIELD: textile, paper.

SUBSTANCE: when boiling collagen, linear dimensions of leather tissue are measured before and after collagen boiling. The structure-to-structure distance is determined using difference of the sample thickness after boiling and the rated thickness of the sample before boiling, which is produced as a product of the sample thickness before boiling and a coefficient of layers number defined as a quotient from division of a lengthy sample length into the length of the sample after boiling. Invention makes it possible to realise the specified method objective.

EFFECT: method improvement.

4 ex, 3 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to low-molecular derivatives of peptides which are used for preparing a pharmaceutical agent which inhibits laminin/nidogen reaction.

EFFECT: increased effectiveness of compounds.

2 cl, 12 dwg, 2 tbl, 30 ex

FIELD: biotechnology, medicine.

SUBSTANCE: cells are contacted with protein containing fibronectin EDb-domen in presence of one or more chemical substances. As check experiment reaction between the same cells and abovementioned protein in absence of said substances. Expression of certain protein or presence of nucleic acid encoding the same makes it possible to select compounds bonding to fibronectin EDb-domen.

EFFECT: new biotechnological method.

1 tbl, 3 ex

FIELD: peptides, pharmacy.

SUBSTANCE: invention relates to low-molecular derivatives of peptides that are able to act as inhibitors in interaction between laminine and nidogen (interactions laminine/nidogen). Also, invention relates to a method for their preparing, pharmaceutical composition prepared on thereof and their using for preparing pharmaceutical agents, and for identification of inhibitors in interaction laminine/nidogen.

EFFECT: valuable properties of peptides.

5 cl, 12 dwg

The invention relates to compounds of the formula Y-(CR2)n-X-NHJ, where X represents C=O or CR2; n is an integer having a value of from 1 to 6; Y is an L(A)mor R1R2CR-, where L - metallocomplexes agent, And represents-CR2-, -NRCO-, -CONR - or polyalkyleneglycol; m is an integer having a value of from 0 to 10, where one of R1and R2is-NH(B)pZ1and the other is-CO(B)qZ2where p and q are integers having a value of from 0 to 20, and each independently selected from Q or amino acid residue, where Q is a cyclic peptide; Z1and Z2- protective groups which are biocompatible group which inhibits or suppresses the metabolism of the peptide in vivo; J and each R group is independently selected from H, C1-4the alkyl or C1-4alkoxyalkyl; provided that (i) the total number of amino acid residues in R1and R2the group does not exceed 20; (ii) if X is CR2then Y is-CRR1R2and Z2is metallocomplexes agent; (ii) if Y is-CRR1R2then at least one of R1and R2carries at least one frequent detektiruya

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to therapy and pulmonology, and can be used for selection of tactics of treating thromboembolism of pulmonary artery. For this purpose computered tomography with bolus enhancement is performed to patient, areas of affection located more distally than thrombotic embolus are examined and number of respiratory movements per minute is taken into account. Presence of occluded vessel or vessels in examined areas is identified. Occlusion of segmental branch of pulmonary artery, located more distally than embolus, is assessed in one point irrespective of degree of vessel occlusion. Occlusion of each of lobar branches in case of affection of right middle lobar, left middle- and upper lobar branches of pulmonary artery is assessed in 2 points. Occlusion of upper lobar branch of pulmonary artery on the right, lower lobar branch of pulmonary artery on the left is assessed in 3 points. Occlusion of right lower lobar branch of pulmonary artery is assessed in 4 points. Occlusion of left main pulmonary artery is assessed in 7 points. Occlusion of right main pulmonary artery is assessed in 9 points. Occlusion of both main pulmonary arteries and/or pulmonary trunk is assessed in 17 points. After that, points are summed up. If the sum of points is from 1 to 6, anticoagulation therapy is performed with heparin. If the sum of points constitutes from 7 to 10 at rate of respiratory movements (RRM) lower than 18, another anticoagulation therapy is performed, at RRM more than 18 - thrombolytic therapy is performed. If the sum of points constitutes from 11 to 17, thrombolytic therapy is performed.

EFFECT: method provides possibility of operative objective assessment of degree of pulmonary bed affection and beginning of required therapy in due time.

5 dwg, 1 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely an agent possessing anticoagulant activity. A method for preparing a dry fucus extract possessing anticoagulant action by the complex treatment of Fucus vesiculosus. The dry fucus extract possessing anticoagulant activity representing a polysaccharide complex containing fucoidan having the following composition: neutral monosaccharides - fucose, xylose, mannose, galacose, glucose, uronic acid; as well as polyphenols and sulphates in specific amount. The anticoagulant ointment containing the dry fucus extract.

EFFECT: agents described above possess pronounced anticoagulant action.

4 cl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to cyclohexyl ammonium salt of 2-[3-methyl-7-(1,1-dioxotiethanyl-3)-1-ethyl xanthenyl-8-tio]acetic acid of the following formula: .

EFFECT: obtaining a new compound that shows antithromboembolic action and can be used in medicine.

2 cl, 2 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to derivatives of oxazolopyrimidine in any of their stereoisomeric forms, or in the form of a mixture of stereoisomeric forms specified in Claim 1.

EFFECT: oxazolopyrimidine derivatives having agonistic activity in relation to Edg-1 receptor.

5 tbl, 319 ex

FIELD: chemistry.

SUBSTANCE: invention relates to N-carb(glutaminyl)oxymethylimidazo[4,5-e]benzo[1,2-c; 3,4-c']difuroxane of formula .

EFFECT: obtaining a novel compound which can be used as a medicinal preparation which inhibits thrombocyte aggregation.

1 dwg, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula (I) and/or stereoisomeric forms thereof and/or mixtures of said forms in any ratio and/or a physiologically tolerant salt of the compound of formula , where: X denotes -C(O)- or -SO2-, U denotes an oxygen atom or -(C0-C4)alkylene, A denotes an oxygen atom, -C(O)-NH-, -NH-C(O)- or -(C0-C4)alkylene, V denotes: 1) -(C2-C9)alkylene, where alkylene is unsubstituted or mono-, di- or tri-substituted, independently of each other, by an -OH group, 2) -(C3-C9)alkenylene, D denotes -(C1-C2)alkylene, Y denotes: 1) a covalent bond, 2) -(C6-C14)arylene-, or 3) Het, where Het denotes pyridyl or imidazolyl, R1 denotes: 1) a hydrogen atom, 2) -(C1-C6)alkyl, R3 denotes: 1) -(C2-C6)alkylene-NH2, 2) -(C1-C4)alkylene-SO2-(C1-C4) alkylene-NH2 or 3) -(C0-C4) alkylene-Het, where Het denotes pyridyl or piperidyl, where Het is unsubstituted or substituted with -NH2, R6 denotes: 1) a hydrogen atom, 2) -(C1-C6) alkyl, where the alkyl is unsubstituted or substituted, independently of each other, by a R16 group, 3) -(C0-C4) alkylene-Het, where Het denotes pyridyl, where -(C0-C4) alkylene and Het are unsubstituted or substituted, independently of each other, by a R16 group, 4) -(C0-C4) alkylene-phenyl, where -(C0-C4) alkylene and phenyl are unsubstituted or substituted, independently of each other, by a R16 group, or 5) -(C0-C4) alkylene-(C3-C8)cycloalkyl, R7 denotes a hydrogen atom, halogen or -(C1-C6)alkyl, R8 denotes a hydrogen atom or -(C1-C6)alkyl, R9 denotes a hydrogen atom, and R16 denotes -NH2, which are inhibitors of the active thrombin-activated fibrinolysis inhibitor, as well as a method for production thereof, a medicinal agent based thereon and use for prevention, secondary prevention and treatment of one or more disorders associated with thrombosis, embolism, hypercoagulation or fibrosis changes.

EFFECT: improved properties of compounds.

7 cl, 1 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: described are novel triazolopyridazines of general formula , stereoisomeric or tautomeric forms thereof and physiologically acceptable salts thereof, where Q1 denotes H, -C1-6alkyl, optionally substituted with fluorine, or -C3-6cycloalkyl; Q2 and Q3 independently denote H, -C1-6alkyl; R1-R3 independently denote H, -C1-6 alkyl, -C3-6cycloalkyl, -O-C3-6cycloalkyl, -O-C1-8alkyl, a heterocyclic residue etc; R4-R8 independently denote H, -C1-6 alkyl, -OH, -O-C1-8 alkyl, halogen, SF5 etc, a method for production thereof and use as medicinal agents.

EFFECT: compounds have antithrombotic activity and particularly inhibit the protease-activated receptor.

6 cl, 2 tbl, 242 ex

FIELD: medicine.

SUBSTANCE: present invention refers to medicine, particularly to pharmacology, and describes a method for correction of disturbed functional activity of platelets, consisting in using Melaxen as a corrector for the disaggregation and hyperaggregation state of the platelets to be orally administered into white non-linear mature rats in a dose of 1 mg/kg once a day within the 7-day therapeutic course.

EFFECT: invention aims at extending the range of preparations having an ability to control the aggregation properties of platelets depending on the nature of the haemostatic disorders.

4 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to anticoagulant compounds of formula (I) : and pharmaceutically acceptable salts thereof, wherein R represents C1-C8 alkyl substituted by at least one halogen specified in chlorine or fluorine.

EFFECT: invention refers to pharmaceutical compositions containing the compounds and salts thereof, as well as to using the compounds and salts thereof for treating bleeding disorders, to a method of treating, inhibiting epoxyredutase, vitamin K and coagulation factor synthesis.

31 cl, 3 tbl, 11 ex, 10 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are described new triazolium salts of general formula (I) stereo- and tautomer forms and physiologically acceptable salts thereof, wherein X- C-R1 or N, R1-hydrogen, C1-6alkyl or halogen; A- is an anion of a pharmacologically acceptable organic or inorganic acid; Q1 is hydrogn, -C1-6alkyl optionally substituted, -C3-6cycloalkyl, -C(O)-O-R11 or - C(O)-R11; R11 -C1-6alkyl; Q2 and Q3 are hydrogen; R2- R9 independently mean hydrogen, -C1-6alkyl optionally substituted, -O-(C1-8)alkyl optionally substituted, etc., and using the above compounds as a drug preparation.

EFFECT: compounds possess antithrombotic activity, particularly, they inhibit the protease-activated receptor 1 (PAR1), and may be used in treating the diseases such as myocardial infarction, angina, stroke, and others.

4 cl, 2 tbl, 86 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and represents a pharmaceutical combination for treating in pain conditions in the form of a solid dosage form for oral administration, characterised by the fact that it contains Mirtazapine 5 mg - 50 mg; Tizanidine 0.5 - 6 mg in ratio 20:1-5:1, and a pharmaceutically acceptable carrier.

EFFECT: invention provides creating an effective agent for preventing or treating a pain condition well-tolerated by patients.

6 cl, 2 ex

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