RU2691990C2 - Alginic acid derivative - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to chemical industry and is a sodium salt of alginic acid derivative obtained by a biotechnological method having a number-average molecular weight of 3134 Da, and not forming gel in 10 % aqueous solution, has pronounced anti-inflammatory, regenerative and gastroprotective effects with average effective dose 0.1 mg per dose.

EFFECT: invention has pronounced biological effects with low concentration of the active substance.

1 cl, 4 dwg, 5 tbl, 3 ex

Description

The invention relates to medicine and can be used in pharmacology, pharmacy and biotechnology.

Alginic acid, derived from the seaweed Laminaria Saccharina, grown in the White Sea. Alginates are a family of unbranched double copolymers-residues of D-mannuronic acid (M) and L-glucuronic acid (G), connected by 1-4 glycosidic bond as shown in figure 1. The relative amount of two monomers of uronic and mannuronic acids, their sequence in The polymer chain varies widely depending on the nature of the alginate. The molecular mass of natural alginic acid range from 35-500 kDa.

Currently, there are several areas of use of alginic acid salts in medicine. The main one of them is reparants based on alginic acid salts (algipor, algimaf, Gaviscon, alginatol).

Algipor (reg. No. Р N 003535/01) 1 g of lyophilized gel contains sodium alginate 0.7276 g, calcium gluconate 0.2374 g and furatsilina 0.035 g; in porous sheets with a thickness of 5-10 mm in size 135 × 250, 150 × 160, 60 × 100 and 40 × 50 mm. Each sheet is sterile and hermetically packed in a plastic film bag.

Algimaf (reg. No. Р N 003173/01) 1 g of the preparation contains sodium alginate 0.72 g, calcium gluconate 0.234 g, acetate mafenide 0.036 g and phenosanoic acid 0.01 g; in porous sheets with a thickness of 10 mm and sizes of 50 × 50, 60 × 100, 150 × 150 and 135 × 250 mm. Each sheet is sterile and hermetically packed in a plastic film bag.

Gaviscon (reg. No. LS-002445) - round, flat tablets with beveled edges, from almost white to cream color with small splashes, with a lemon or mint smell. On one side of the tablet is a picture of a circle and a sword, on the other - G 250.

Alginatol (reg. P # 001060/01) - in one suppository contains as the active ingredient sodium alginate - 250 mg and the basis for suppositories (Witepsol) - sufficient to obtain a suppository weighing 1.28 g

The second area is the use of alginic acid salts as sorbents.

The method of producing calcium alginate patent RU 2395525. Dry powder of alginic acid or its water soluble salt is mixed with an aqueous solution of calcium salt with a concentration of at least 0.6 mol / l, the mixture is thoroughly mixed, incubated for at least 10 minutes, then the resulting calcium alginate is separated from the aqueous phase (if any) by decantation, centrifugation or filtration, and dried.

The literature also describes the sorption properties of alginates as sorbents of various ions (cesium, strontium, cadmium, lead, etc.). It is known that alginates obtained using chemical hydrolysis have the ability to firmly and effectively bind lead and cadmium ions, and the lower the molecular weight, the more effectively the sorption capacity of alginates increases [1]. With this method, chemicals are used to hydrolyze alginates and prolonged heating, which leads to the destruction and partial decarboxylation of uronic acids [2].

The present invention is the creation of a biotechnologically modified derivative of alginic acid in the form of a sodium salt. Using this method makes it possible to effectively reduce the molecular weight of alginic acid (natural alginic acid has a molecular weight of 35-500 kDa) and, replacing chemical hydrolysis at high temperature by a bittechnological process, obtain a more native alginic acid structure without decarboxylation of uronic acids. The result is a highly biologically active compound with high anti-inflammatory and regenerating properties.

Obtaining a derivative of sodium alginate is based on the method of biotechnological modification of natural alginic acid for medical purposes, obtained in accordance with FS 42-3383-97. The obtained sodium alginate derivative was compared with sodium alginate for medical purposes according to physicochemical indicators of authenticity in accordance with the requirements of FS 42-3383-97 (Table 1).

Nuclear magnetic resonance spectroscopy (NMR)

A sample of a sodium alginate derivative was analyzed using a Bruker Avance III NMR spectrometer with an operating frequency of 600 MHz for protons.

To do this, the sample was dissolved in D ₂ O, a solution of 0.6 ml was placed in a vial with a diameter of 5 mm. Heavy water was used to register the reference signal of deuterium nuclei in order to adjust the uniformity of the magnetic field of the spectrometer. The NMR spectra were recorded on ¹³ C nuclei.

The ¹³ C-NMR spectrum was recorded with proton decoupling to suppress unwanted spin-spin C-H interactions and improve the signal-to-noise ratio, as shown in Figure 2. The pulse duration was 12 μs, the delay time between pulses was 16 s. The number of savings is 4096.

Signals with chemical shifts ~ 175 ppm in both spectra correspond to carbon atoms that are part of the COOH groups (position I in figure 1). For a more accurate assignment of the remaining signals, the 13C-NMR spectrum of the glucose solution in D2O figure 3 was additionally registered.

Comparison of the spectra of the derived alginate with the spectrum of glucose and natural alginic acid (FIG. 4) makes it possible to determine that in the spectrum of the sample under study in the region of ~ 100 ppm signals are likely to be observed that correspond to the carbon atoms of the carbohydrate ring associated with two oxygen atoms (position II in figure 1). The group of signals in the region of 70-80 ppm correspond to the remaining carbon atoms of the carbohydrate ring (position III-VI in FIG. 1).

Based on the study of the NMR spectra, it was found that, unlike natural sodium alginate, an alginate derivative obtained by biotechnology in aqueous solutions, there is no signal group corresponding to the range of 60-70 ppm. This indicates the absence of intramolecular bonds that contribute to the formation of natural sodium alginate gels.

The results of studies of alginic acid derivative by the method of exclusion high-performance liquid chromatography:

The analysis was performed using an HPLC system LC-20 Prominence (Shimadzu, Japan), consisting of SIL-20A autosampler, LC-20AD dual-plunger pump, DGU-A3 vacuum degasser, STO-20A column thermostat, and RID-10A refractometric detector. The separation was carried out at a temperature of 50 ° C on columns for the analysis of water-soluble polymers MSC 300 × 8 mm with a pore size of 1000-1000000 A (PSS, Germany). An aqueous solution of potassium nitrate (0.2 mol / l) and sodium dihydrogen phosphate (0.01 mol / l) with pH8 was used as eluent. The system was calibrated using monodisperse standard samples of sodium polystyrene sulfonate (PSS, Germany) with a known molecular weight in the range from 891 to 976000 Da. Data collection and processing was carried out using WinGPC software (PSS, Germany).

The results of the calculation of the average number (Mn), weight average (Mw) and z-average (Mz) masses, as well as the polydispersity of the studied samples (Mw / Mn) are given in table 2.

Thus, the alginic acid derivative differs from the natural compound:

1. does not form a gel when dissolved in water 10% derived sodium alginate. Natural sodium alginate forms persistent, non-flowing gels at a concentration of 5%.

2. on the basis of NMR spectroscopy, the absence of a group of signals corresponding to 60-70 ppm was established, which proves the absence of intramolecular bonds responsible for the adsorption of water by the alginate molecule and the formation of a gel structure.

3. The molecular weight of the alginate derivative is significantly reduced compared with natural alginic acid (Table 2). The molecular mass of natural alginic acid is 35,000-500,000 Da.

Determination of the biological (pharmacological) activity of the alginic acid derivative

The experiment was performed on outbred rats of both sexes weighing 200 +/- 40 grams. On the model of the wound process - a planar aseptic wound, obtained by the method [3]: in animals, under conditions of ether anesthesia, on pre-grafted skin of the back, after the surgical field was treated with iodopyerone and 70% alcohol in the interscapular region with a stencil diameter of 2 cm, the scalpel formed standard plane wound depth of 2 mm.

Example 1. Selection of the average effective dose of the drug (Table. 3). For this purpose, solutions were prepared containing the sodium alginate derivative in the following concentrations per dose: 0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg. These drugs were applied 1 time per day for 3 days prepared histoheparates stained with hematoxylin-eosin. The counting of cells (lymphocytes, neutrophils) was performed under a MIKMED 5 microscope (Russia) under magnification × 100 in the mesh of academician G.G. Avtandilov. For the experiment, 50 laboratory animals were taken.

It was found that a single dose of 0.1 mg per use for 3 days has a pronounced anti-inflammatory effect on the wound. For further research, a single dose was 0.1 mg per application.

Medicinal products registered as medicinal products with natural alginic acid salt (algipor, algimaf, Gaviscon, alginatol) contain this salt for 1 dose in a concentration: algiport and algimaf - 700 mg, Gaviscon and alginatol - 250 mg.

Thus, biotechnological modification of alginic acid to obtain a derivative of sodium alginate can significantly increase the anti-inflammatory effect at lower concentrations of the active ingredient 0.1 mg of the derivative of alginic acid and 250 mg of natural sodium alginate.

Example 2. Regenerating ability derived sodium alginate (Table. 4).

For this experiment, 60 laboratory rats were taken with a superficial aseptic wound. They were divided into controls (30 individuals), which were not treated with anything, and 30 individuals, which were applied a wound of sodium alginate at a dose of 0.1 mg per dose (experiment) 1 time per day. Histological preparations were prepared on the 3rd, 7th and 14th day and stained with hematoxylin-eosin. Effector cells (lymphocytes, neutrophils, fibroblasts) were counted under a MIKMED 5 microscope (Russia) under magnification × 100. The number of vessels was calculated in a large square mesh Academician GG Avtandilov at magnification × 40.

On the basis of the obtained data, a significant decrease in the inflammatory reaction in the wound area was found when using the derived sodium salt of alginic acid on the 3rd day of the experiment (Table 4). At the same time, an increase in the number of fibroblasts and vessels in the wound area was established, which characterizes the development of regenerative regenerative processes. An intensive growth of new blood and lymphatic vessels occurs in a wound, blood circulation improves, hypoxia decreases, and the inflammatory response subsides. New vessels are formed in the wound, young granulation tissue ripens, which contributes to the elimination of wound tissue defects.

Example 3. Gastroprotective activity of the derivative of alginic acid in the form of sodium salt.

They took 20 outbred white male rats weighing 200 ± 40 g, who for 24 hours did not receive either food or water and were kept in cages with a cellular bottom to avoid coprophagy. 10 rats were orally administered with 0.5 ml / 200 g body weight of saline (control animals), and 10 other animals were given 1 ml / 200 g of the test drug (experiment). After 1 hour after the administration, 96% ethanol (1 ml / 200 g body weight) was injected into both control and experimental animals. After 1 hour, laboratory animals were euthanized by an overdose of medical ether, the stomachs were removed, cut along the lesser curvature. Then prepared histological preparations stained with hematoxylin-eosin. The counting of cells (lymphocytes, neutrophils) was performed under a MIKMED 5 microscope (Russia) under magnification × 100 in the mesh of academician G.G. Avtandilov.

Analysis of the data confirms the effective reduction of the inflammatory response in the area of the ulcerative defect of the gastric mucosa against the background of the use of an alginic acid derivative (Table 5)

Literature:

1. Makarov, KE, Khozhaenko, E.V., Kovalev, V.V., Podkorytova, E.A., Khotimchenko, R.Yu. Alginates with different molecular weights as sorbents of cadmium and lead ions. Materials reports at the XVIII All-Russian Congress "Ecology and human health", 8-10/10/2013.

2. Usov A.I. Alginic acids and alginates: methods of analysis, composition and determination of the structure. Successes of chemistry - 68 (11) 1999.

Claims (1)

A derivative of sodium salt of alginic acid, obtained by a biotechnological method, having a number average molecular weight of 3134 Da and not forming a gel in a 10% solution, has pronounced anti-inflammatory, regenerating and gastroprotective effects with an average effective dose of 0.1 mg per dose.

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