RU2464997C1 - Method for preparing sorption material of blastemas - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns pharmaceutical industry, in particular to a method for preparing sorption material of blastemas. The method for preparing sorption material of Cladonia blastemas to be ground, mechano-chemically activated at one stage under certain conditions.

EFFECT: sorption material prepared by the method described above shows high sorption activity.

4 dwg, 3 tbl

Description

The invention relates to the biotechnological (biopharmaceutical) industry, namely, to obtain sorption material for medical purposes (medical, preventive) from plant materials.

One of the main areas of biotechnology involves the development of sorption materials and their further use in medicine and the medical industry as irreplaceable materials for enterosorption. The method of enterosorption began to actively compete with hemosorption in connection with a number of advantages: the absence of the need for surgical intervention and the associated risk of possible complications characteristic of hemosorption; lack of direct damaging contact with biological fluids (blood, lymph); the possibility of widespread use of sorption therapy for outpatient treatment in the field and ordinary home conditions.

Analyzing the requirements for enterosorbents presented by regulatory documents and clinical practice, we can distinguish a set of properties inherent in the “ideal” enterosorbent:

- complete harmlessness and non-toxicity;

- high biocompatibility with tissues, blood and other biosubstrates of the body;

- non-damaging effect on the mucous membranes of the oral cavity, esophagus, gastrointestinal tract;

- selective sorption of medium molecular toxic metabolites;

- high adsorption capacity.

Scientists from different countries have been solving the problem of creating an effective and safe enterosorbent designed to cleanse the body of toxic substances that are produced in various diseases for many years.

Known enterosorbents can be combined into the following groups.

1. Carbon adsorbents based on activated carbon and carbon fiber materials (activated carbon, carbene, gastrosorb, Ensoral, Carbodon, actylene, etc.).

2. Ion exchange materials (cholestyramine).

3. Enterosorbents based on lignin (lignosorb, prlifepan).

4. Derivatives of polyvinylpyrrolidone (enterosorb, enterodesis).

5. Sorbents based on natural minerals (white clay, silica-containing mineral raw materials, zeolites).

6. Enterosorbents of plant origin (plantain seeds, guar gum, marshmallow mucus, chicory root, pumpkin seeds, dietary fiber, cereal bran, pectin).

Enterosorbents of the latter group have several advantages: they do not have an irritating effect on the gastrointestinal tract, do not cause side and toxic effects, are harmless when taken for a long time (for example, see RU 2389498, class A61K 36/185, 2009).

A known method of producing enterosorbent of plant origin (see RU 2391998, class A61K 36/902, 2008), representing granules containing Sphagnum fuscum obtained by grinding the dried turf Sphagnum fuscum to a particle size of 0.1-0.3 mm, s subsequent mixing with 10% aqueous medical low molecular weight polyvinylpyrrolidine, granulation, drying and re-granulation.

A common feature of the claimed invention and known analogues is the use of local plant materials. The disadvantages of analogues can be formulated as follows: low biological value of the obtained sorbent, low processability, expressed in the expensive and time-consuming process of obtaining enterosorbents, which, in particular, can consist of several stages, for example, dissolving in alkali, neutralization, washing with water, multi-stage drying, thickening suspension, etc.

The problem to which the claimed invention is directed is expressed in expanding the assortment of sorption materials of plant origin and methods for their preparation, increasing their sorption activity.

As a source for creating sorption materials, thalli of the lichens of the genera Cladonia - reindeer moss - are used.

The use of lichens as a food product has been known for a very long time, there is an assumption that the biblical manna was the lichen Aspicilia esculenta, a free-living nomadic lichen, common in desert areas. The crushed thalli of lichens slightly resemble flour, and the unleavened cakes baked from such flour are quite edible. In Japan, lichen as a delicacy is placed in a soup or salad, and in North America, many species are used for food. From thalli of lichens of the genus Cladonia, jelly and jelly were cooked and used as an additive to flour. Lichens Cladonia spp., Cetraria islandica today are used to produce alcohol.

Lichens of the genus Cladonia are natural medicinal and food raw materials used in folk medicine to treat many diseases. The thallus of lichens contains non-hormonal physiologically active substances (NPAFs) of various groups: carbohydrates (70-80%) in the form of lichen β-polysaccharides of lichenin and isolechenin (Fig. 1); tannins (1-2%); lichen acids (2-3%) - lecanoric, fizodovaya, usninovaya (figure 2); trace elements (in the raw materials used, the content of heavy metals is much lower than the MPC). The THF content in the thallus of lichens determines their rather widespread use in official and traditional medicine for the treatment of diseases of the gastrointestinal tract, dystrophy, general exhaustion, respiratory and lung diseases, infectious skin diseases, burns and diaper rash, thyroid disorders and other endocrine diseases , also their use as immunomodulating, antitumor, hepatoprotective and detoxification drugs. It has been established that lichenin and isolichenin inhibit the growth of certain tumors in animals (see Filippova G.V., Pavlov N.G., Shashurin M.M., Kershengolts B.M. Effect of biologically active substances from thalli of northern lichens extracted by various methods , on the biological properties of Mycobacterium tuberculosis // Siberian Medical Journal. 2008. No. 3. S.99-103).

At the same time, being β-polysaccharides, β-glycosidic bonds in which are practically not cleaved in the gastrointestinal tract, lichen carbohydrates themselves are hardly absorbed by the human body. The task is to increase the bioavailability of these components in the process of preliminary processing of lichen thalli. This problem is solved in the framework of the proposed method by mechanochemical activation.

The technical effect of the present invention is that during the mechanochemical treatment of the thallus of the reindeer moss, its sorption activity with respect to medium and low molecular weight toxins, heavy metal cations is significantly increased due to the formation of lichen β-oligosaccharides from the natural lichenine polysaccharide, while the biological activity of NPAF also increases, for example, lichenic acids (figure 2) in the mechanochemical preparation "Yagel-sorbent".

To solve this problem, the method of producing sorption material from lichen thalli by mechanochemical processing of dry lichen thalli differs in that it involves the occurrence of solid-phase mechanochemical reactions without solvents in one technological stage, while dry lichen thalli, previously crushed to 1-3 mm, are subjected to mechanochemical activation. at a speed of 1200-1500 rpm for 1-3 minutes in air in a planetary mill-activator. The resulting powder is the sorption material "Yagel-sorbent". The β-oligosaccharides formed during the mechanical activation of lichen thalli cause an increase in sorption activity for low and medium molecular toxins and heavy metal cations, and an increase in the sorption activity also contributes to an increase in the specific surface area and a decrease in the particle size of the sorbent (Fig. 3). The presence in the solid-phase preparation "Yagel-sorbent" of natural NSAIDs (lichen acids of a mild antibacterial effect, vitamins, microelements) does not at all worsen its sorption qualities, but give it additional therapeutic and preventive properties. Preliminary mechanical activation of reindeer moss with the production of solid phase powder "Yagel-sorbent" can significantly reduce the particle size to 10-1000 nm, simplify the processing of plant materials and expand the range of sorption materials from plant materials with high adsorption activity with a wide spectrum of action.

A comparative analysis of the characteristics of the claimed solution with the signs of analogues indicates the conformity of the claimed solution to the criterion of "novelty."

The essence of the invention lies in the fact that the mechanically activated drug "Yagel-sorbent" has a high specific surface area, is capable of adsorbing medium and low molecular weight toxins, which, in particular, include protein toxins and heavy metal cations. The obtained sorption material has a high sorption ability, which is associated not only with a developed surface, but also with an increase in the number of functional groups (Fig. 4), this allows you to bind and remove exogenous and endogenous toxins (cholesterol, bilirubin, mast cell products, etc.) . A mild bactericidal effect of lichen acids also occurs, which contributes to the formation of a healthy microflora due to the lysis of pathogenic and conditionally pathogenic bacterial cells and the sorption of their vital products.

The invention can be implemented as follows.

Dry thalli of lichens, previously crushed to 1-3 mm, undergo mechanochemical activation without the participation of solvents in one technological stage at a speed of 1200-1500 rpm for 1-3 minutes in air in a planetary mill activator, impact-abrasion mode whose work provides the maximum impact of grinding media on the object of study. The resulting powder is the sorption material "Yagel-sorbent".

The mechanochemical activation of lichen raw materials increases the bioavailability of components as a result of breaking part of chemical bonds (even as strong as β-glycosidic ones) and obtaining oligosaccharides from natural polysaccharides. The result of successful mechanochemical processing of bio-raw materials is an increase in the product of a more complete spectrum of NFAS in biologically accessible (water-soluble) forms (Figure 4), high adsorption activity, especially in the first 20 minutes (tables 2, 3).

To study the change in the composition of lichen thalli during solid-phase mechanochemical processing, the method of IR spectroscopy was used, and the change in the surface topography was studied by atomic force microscopy. To determine the bactericidal properties of the obtained sorption material "Yagel-sorbent" used microbiological testing. The efficiency of adsorption properties was studied by direct spectrophotometry.

The study of reindeer powder obtained by solid-state mechanochemical technology in one step without the use of solvents by IR spectroscopy showed that the nature of the IR spectra in the case of all reindeer samples is identical in the presence of characteristic functional groups (see figure 4). The set of absorption peaks does not vary with the processing conditions. At the same time, the intensity of all the observed absorption bands depends on the method of obtaining the sample of the biological product. An increase in the absorption intensity in the region of stretching vibrations of the OH group (3450-3350 cm ^-1 ) of the mechanochemical reindeer moss indicates a break in some of the strong β-glycosidic bonds in the initial insoluble polysaccharides that are part of the lichen raw material, and as a result, the formation of more bioavailable β- oligosaccharides.

The presence in the IR spectrum of a number of bands (1670-1630 cm ^-1 - bond OS of conjugated systems; 1200-1270 cm ^-1 -С-О-С - groups of atoms; 1100-1000 cm ^-1 - both pyranose cycles and -С -OS-groups of atoms; 900 cm ^-1 - deformation vibrations С-Н) are higher for a mechanically activated sample, which indicates a greater saturation with its functional groups and an increase in the content of non-ferrous alloys in the studied mechanochemical reindeer.

The antibacterial properties of the Yagel-sorbent preparation were determined in vitro on cultures of bacterial strains of four conditionally pathogenic and pathogenic microorganisms according to standard and modified methods.

Comparative studies of the antibacterial effect of coarse-grained lichen thalli without mechanochemical treatment (powder No. 1 — control) and the studied drug “Yagel-sorbent” were carried out in the following cultures according to the standards of turbidity of cultures of 5 and 10 units:

1. Staphylococcus aureus - 6538-p

2. Escherichia coli - H-257

3. Pseudomonas aeruginosa - 33105

4. Klebsiella pneumoniae

Standard nutrient media were used: Endo medium, milk-yolk-salt agar, Ploskirev medium, meat peptone agar (MPA), which were prepared according to a standard prescription (MUK 4.2.1890-04. Determination of the sensitivity of microorganisms to antibacterial drugs. Methodological guidelines approved by the main state Sanitary Doctor of the Russian Federation on March 4, 2004) with the addition of a control and test sample in an amount of 5.0 mg / ml The lawn method was used to sow cultures of microorganisms, and after cultivation in a thermostat at 37.0 ° C, the intensity of their growth was estimated. The evaluation results are shown in table 2.

Powder No. 1 showed either a barely noticeable attempt to lysis, or microorganisms were partially lysed. Thus, the Yagel-sorbent preparation was shown to be more effective against opportunistic and pathogenic bacterial strains, including Staphylococus aureus, Klebsiella pneumonia, etc.

The formation of new structures and a larger specific surface was recorded by atomic force microscopy (Fig.3b), while the unstructured surface of the coarse moss powder was distinguished by the absence of such and the presence of a smoother relief (Fig.3a).

The action of the mechanochemical sorption material "Yagel-sorbent" is proved in experiments with a marker of medium and low molecular weight toxins - methylene blue (table 2) and with the cation Co ²⁺ (table 3). The adsorption activity of reindeer moss powders according to methylene blue was determined by the method recommended in GOST 4453-74, but changes were made in it to simulate conditions close to gastric. The gastric environment was created by acidification of Hcl from the calculation of 30 mM at t = 37 ° C. A spectrometer with LAMBDA 20 software is used for research at a wavelength (λ) of 400 nm in cuvettes with a 10 mm thick light-absorbing layer relative to distilled water. In addition, changes were introduced at the centrifugation stage. In this case, centrifugation does not give a complete phase separation; therefore, phase separation was additionally carried out by filtration. For this, Buchner funnels and filter paper (GOST 12026-76) of the same size were used. Statistical processing of the research results was performed using the Microsoft Excel 2010 application software package. The data obtained were processed statistically by standard parametric methods with calculation of the average value, standard deviation, and Student's confidence interval (n = 9 <20) at a significance level of p = 0.05. The results are presented in the form M ± m, where M is the arithmetic mean value, m is the confidence interval. The sorption daily capacity of the mechanically activated sample was 48 ± 3 mg / g. It should be noted that these are very high indicators compared to well-known adsorbents, such as polyphepan and white clay, the adsorption capacity of which in methylene blue is 10.0 and 11.4 mg / g, respectively (Markelov D.A., Nitsak O.V. ., Gerashchenko II Comparative study of the adsorption activity of medical sorbents // Chemical and Pharmaceutical Journal. 2008. V.42. No. 7. P.30-33., Reshetnikov VI. Evaluation of the adsorption capacity of enterosorbents and their dosage forms / / Chemical-Pharmaceutical Journal. 2003. V.37. No. 5. S.28-32). The adsorption rate is also higher for a mechanically activated preparation; in the first 20 minutes, 92% of its daily adsorption capacity was adsorbed, in contrast to coarse moss, where over the same period the adsorption capacity was 2 times lower than that of a mechanically activated sample, and amounted to only 69 % daily activity.

It was previously shown that natural lichen β-oligosaccharides manifest themselves as complexing agents with metal cations (Anshakova V.V. Mechanochemical technologies for the preparation of biologically active substances from lichens / V.V. Anshakova, B.M. Kershengolts, E.S. Khlebny, A.A.Shein // Bulletin of the Samara Scientific Center of the Russian Academy of Sciences. 2011. V.13, No. 1. S.236-240). The adsorption capacity for salts of heavy metals was determined by the example of Co ^{2+ ions} from standard solutions of cobalt (II) chloride. From table 3 it can be seen that the sorption activity of the material "Yagel-sorbent" is 2.5 times higher than the sorption properties of the powder of coarse ground moss.

The presented sorption material based on mechanically activated thalli of lichens expands the raw material base for obtaining sorbents from plant materials and can be used to create enterosorbents in order to form healthy microflora and treat exogenous and endogenous intoxications of various etiologies.

Table 1 The antibacterial effect of powder No. 1 and sorption material "Yagel-sorbent" on the culture of opportunistic and pathogenic bacterial strains The name of the types of bacterial strains The antibacterial effect of powder No. 1 (control) Antibacterial effect of the drug "Yagel-sorbent" Staphylococcus aureus - 6538-p + ^*) ++++ Escherichia coli - H-257 + +++ Pseudomonas aeruginosa - 33105 + ++++ Klebsiella pneumoniae + ++++ Note: ^*) + - weak lysis; ++ - partial leasing; +++ - almost complete lysis; ++++ - complete lysis

table 2 Adsorption activity of reindeer powder by indicator methylene blue, mg / g Sorption material Adsorption time 20 minutes 24 hours Coarse Moss Powder 27 ± 3 39 ± 2 Powdered yagel mechanically activated "Yagel sorbent" 44 ± 4 48 ± 3

Table 3 Specific adsorption of cobalt (II) ions, mmol / kg Sorption material Co concentration ²⁺ mol / L 0.2 0.4 0.6 0.8 Coarse Moss Powder 10 fifteen 35 60 Powdered yagel mechanically activated "Yagel sorbent" 25 70 80 150

Claims (1)

A method of producing sorption material from plant materials, characterized in that the dry thallus of lichens of the genus Cladonia, crushed to 1-3 mm, is subjected to mechanochemical activation in one technological stage at a speed of 1200-1500 rpm for 1-3 minutes in air in planetary mill activator.

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