RU2467063C1 - Method of producing highly active solid-phase antibiotic biopreparation of reindeer moss from lichen fronds - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: powder of coarsely ground lichen fronds undergoes solid-phase mechanochemical treatment in a bead mill at 1500 rpm for 3 minutes with addition of 0.5 wt % sodium bicarbonate.

EFFECT: invention improves quality, antibacterial action of the preparation and simplifies the process of producing said preparation.

3 tbl, 5 dwg

Description

The invention relates to the biotechnological (biopharmaceutical) industry, namely the production of biological products for medical use (bactericidal action). The use of mechanochemical technologies in the production of an antibiotic preparation from lichen thalli without solvents in one technological stage from dry raw materials allows the formation of β-oligosaccharides (active filler) by splitting part of the β-glycosidic bonds in lichen β-polysaccharides and the complex between the active filler and pharmacone (lichenic acids contained in the thallus of lichens, or a well-known pharmaceutical product of antibiotic action), which increases the bioavailability armakona and antibiotic activity of the preparation of 5-10. This compares favorably with the manufacturability of the proposed approach and the properties of the obtained biocomplexes from analogues produced using classical technologies.

Lichens from the cladonia (Cladonia) and cetraria (Cetraria) are medicinal plants used in folk medicine to treat many diseases. Thallus of lichens contains up to 80% of carbohydrates, half of which is represented by β-homopolysaccharide lichenin, as well as isolechinin, which have both immunomodulating, antitumor, and hepatoprotective properties.

It is known about the use of lichens as an antibiotic. So, antimicrobial activity against staphylococci, streptococci, acid-resistant microorganisms, fungi, protozoa and viruses is due to the presence of lichen acids in thalli (L. Galitsky et al. // Problems of Tuberculosis. 1997, No. 4. P.35-38). High antibiotic activity is associated primarily with the presence in the lichens of usnic acid and its derivatives (Fig. 1), also related to lichen acids (Gorshkova R.P. et al. // Bioorgan., Chemistry. 1997. V.23, No. 2. S.1324-1328). The antibiotic activity of usnic acid strongly depends on the nature of the optical rotation, and the D (+) isomer, as a rule, is more active. All three forms of usnic acid were previously studied for antimicrobial activity, their prevailing activity against gram-positive and acid-resistant microorganisms was established. The mechanism of the antibiotic action of usnic acid is associated with a breakdown in the process of oxidative phosphorylation in microorganisms (Kramarenko et al., 2003).

The usnic acid obtained from lichens in the form of sodium usninate was proposed under the name Binan for medical use and was used as an external agent for the treatment of wounds, burns, cracks and in gynecology. With the advent of synthetic and semi-synthetic antibiotics, the drug was discontinued (Velyatiev V.V. Useful Plants of Central Siberia. Irkutsk. 1987. P.21-22).

Usinic acid preparations were successfully used for the treatment and prevention of caries, while the use of well-known strong antibiotics (erythromycin, cyclophaloridin, cefuroxime, etc.) in oral therapy upset the balance of microflora in the oral cavity and caused persistent stomatitis (Litvinov, Rassadina, 1958). Despite the positive experience of using lichens in many branches of clinical medicine, there are few reports on the use of this plant in the available literature. This is due to the fact that the known methods for isolating bioactive substances from lichen thalli do not give the desired result.

An analogue, from the point of view of the method of glycosidic clathration of pharmacons, is a cycle of work under the guidance of Academician G.A. Tolstikova (Tolstikova T.G., Bryzgalova O.A., Sorokina I.V. et al. On the nature of the effect of glycosidic clathration of pharmacons // DAN. 2007. V.416, No. 1. P.133-134. Tolstikova T. G., Tolstikov A.G., Tolstikov G.A. On the way to low-dose drugs // Bulletin of the Russian Academy of Sciences. 2007. V.77. No. 10. P.867-874).

The closest analogue to the proposed invention (prototype) is the anti-TB drug “YAGEL-M” for the prevention and treatment of tuberculosis and the method for its production (Kershengolts B.M., Filippova G.V., Shashurin M.M., etc. YAGEL-M for the prevention and treatment of tuberculosis // RF patent for the invention No. 2385159, 03/27/2010; Filippova G.V., Pavlov N.G., Shashurin M.M. et al. Effect of biologically active substances from thalli of northern lichens, extracted by various methods on the biological properties of mycobacterium tuber kuleza // Siberian Medical Journal. 2008. No. 3. P. 99-103).

The technical effect of the invention is the ecological purity of the proposed production method, since it does not have any extraction or hydrolysis stages of processing bio-raw materials, the process takes place without the participation of solvents in one technological stage, which reduces the resource and energy consumption of the technological process. The antibacterial effect of the obtained YAGEL preparation is characterized by 5-10 times increased activity against pathogenic and conditionally pathogenic microorganisms in comparison with antibiotic preparations obtained by standard methods without mechanical activation. The minimum inhibitory concentration of the YAGEL preparation is 2 times less than that of 40% alcohol extract of mechanically activated reindeer moss (Yagel-M), taken as a prototype.

The effect is achieved by the fact that the use of the mechanochemical treatment of lichen raw materials leads in one technological stage to the formation of β-oligosaccharides (active filler) due to the cleavage of part of β-glycosidic bonds in lichen β-polysaccharides, and the complex between the active filler and pharmacon (lichen acids contained in the thalli of lichens, or with a well-known pharmaceutical product of antibiotic action). At the same time, β-oligosaccharides manifest themselves as a synergistic component in combination with natural substances of antibiotic action - with lichen acids, or with active substances of known pharmaceutical preparations of antibiotic action. Such weak intermolecular interactions lead to the formation of a complex of a diphilic nature, thereby creating optimal conditions for the diffusion process, increasing the bioavailability of the pharmacon by 5–10 times, which contributes to an increase in its bioactivity.

The essence of the invention lies in the fact that the shock-abrasive effect with the addition of a solid-phase reagent of sodium bicarbonate 0.5% by weight is accompanied, along with the destruction of the cell walls, a change in the chemical composition of the components of plant materials and their transition into a bioavailable form as a result of breaking a number of chemical bonds even as strong as β-glycosidic) and the occurrence of mechanochemical, solid-phase reactions of the transition of phenolic groups of lichen acids into phenolic acids, followed by their synergistic intermolecular interaction action (figure 2).

The invention can be implemented as follows.

The powder of coarsely ground thalli of lichens is placed in a ball mill chamber (for example, AGO-2 or TsEM-7-80) and subjected to mechanochemical activation for 3 minutes at 1500 rpm. The resulting drug YAGEL is tested for antibacterial activity against opportunistic and pathogenic microorganism strains.

The method has embodiments, consisting in the fact that a known antibiotic pharmaceutical preparation (pharmacacon), for example cefazolin, is added to the powder mixture in the ball mill chamber in a mass ratio of lichen: pharmacacon 10: 1. At the same time, the activity of pharmacon also increased 5-10 times.

The antibacterial properties of YAGEL 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) and mechanically activated drug YAHEL in the following cultures were carried out according to the standards of turbidity of crops by 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. Methodical guidelines approved by the main by the State Sanitary Doctor of the Russian Federation, 04.03.2004) with the addition of a control and test sample to the nutrient medium 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, their growth rate was estimated. The evaluation results are shown in table 1.

Powder No. 1 showed either a barely noticeable attempt to lysis, or microorganisms were partially lysed.

Thus, the very high efficiency of YAGEL with respect to opportunistic and pathogenic bacterial strains, including Staphylococus aureus, Klebsiella pneumonia, etc., has been demonstrated.

Comparative studies of the antibacterial action of the complex preparation No. 2, consisting of lichen and cefazolin thalli in mass ratios of 10: 1, subjected to a joint mechanochemical treatment, and preparation No. 3, consisting of a mixture of lichen and thallus cefazolin in thalli in the same mass ratios of 10: 1, but without mechanochemical activation obtained by coarse grinding in a household mill.

The determination of the antibacterial activity of the test complex preparation (No. 2) and the coarsely ground mixture (No. 3) was carried out by the method of serial dilutions on solid nutrient media. As a nutrient medium used traditional nutrient agar medium for growing E. colli. The effectiveness of the antibacterial action of the drugs was determined by the timing of the appearance of lysis of bacterial culture cells. Comparison of the studied samples was carried out based on the concentration of cefazolin.

Under the action of the complex preparation No. 2 on the strain E.colli M17, a moderate bacteriostatic effect was observed in the range of cefazolin concentrations of 0.25; 0.5 and 1.0 μg / ml and bactericidal action with a cefazolin concentration in excess of 2.0 μg / ml (table 2).

Cefazolin in the composition of drug No. 3 (control) did not exhibit bactericidal and bacteriostatic effects in the field of the indicated concentrations. Therefore, cefazolin with a concentration of more than 2.0 μg / ml, which has undergone joint mechanical activation with reindeer moss, has a pronounced bacteriostatic effect on the E. colli strain, in contrast to cefazolin, which is part of the reindeer moss composite without mechanical activation.

We determined the comparative antibacterial activity of the YAGEL solid-phase preparation and the prototype representing 40% water-alcohol extract YAGEL-M t vitro in relation to the Staphylococcus aureus strain 6538-r by determining their minimum inhibitory concentration (MIC).

We obtained and tested the prototype in strict accordance with the specified invention (see RF Patent for the invention No. 2385159, 03/27/2010), i.e. in the form of an aqueous-alcoholic extract of dry thalli of lichens, previously mechanically activated with solid alkali. The prototype (YAGEL-M extract) is obtained by the sequence of the following technological steps: lichen thalli are subjected to mechanochemical activation in the presence of solid alkali to convert lichen acids of a water-insoluble phenolic nature to a water-soluble phenolate form. Next, the obtained powder, which is not the final product, is subjected to aqueous extraction, the insoluble residue is separated by filtration. From the resulting solution, phenolates are precipitated by acidification. The resulting precipitate containing lichenic acids of antibacterial action is dissolved in 40% water-ethanol mixture.

For reliable results, when comparing the antibacterial activity of the studied drug and the analogue, the concentration indicators are reduced to a single dimension (see table 3).

To determine the MIC, standard methods were used (MUK 4.2.1890-04. Determining the sensitivity of microorganisms to antibacterial drugs. Guidelines) to determine the sensitivity of microorganisms to antibacterial drugs, namely, the method of preparing serial dilutions of the antibacterial drug in agar. The solid-state preparation YAGEL, when introduced into the nutrient medium for uniform distribution, was used in the form of a 40% aqueous-alcohol suspension. Then, after two days of cultivation in a thermostat at 37.0 ° C, growth intensity was estimated depending on the content of the preparation. The evaluation results are shown in table 3. The results were taken into account visually, by counting the grown colonies on a cup. The obtained data were interpreted as follows: the absence of microbial growth (-) indicates its sensitivity to the drug. With the growth of the test culture in an amount from 0 to 25 colonies, the result was evaluated as 1+, from 25 to 50 colonies as 2+, from 50 to 100 as 3+. In the case of the formation of 100 or more colonies on a plate, the microorganism was considered resistant (4+).

Thus, reliable data shown in table 3 indicate that the minimum inhibitory concentration of the drug YAGEL in relation to the pure culture of strain St. aureus was 2.5 mg / ml, and the extract "YAGEL-M" - 5.0 mg / ml. The absence of significant differences between the samples designated as “Control 1” and “Control 2” indicates the inability of the obtained concentrations of ethyl alcohol to affect the growth rate of the above strain of microorganisms on nutrient medium “MZHSA” under standard cultivation conditions.

For physicochemical studies of the effect of increased antibacterial activity of the obtained intermolecular complexes of the active filler (lichen β-oligosaccharides) with pharmacon (lichen acids), IR spectroscopy, Raman scattering and atomic force microscopy were used.

The method of IR spectroscopy showed that the nature of the IR spectra in the case of all samples of reindeer moss is identical (figure 3). 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 mechanicochemical reindeer moss (YAGEL) indicates the breaking of some 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 - C = C bonds of conjugated systems; 1200-1270 cm ^-1 -COC- groups of atoms; 1100-1000 cm ^-1 - both pyranose cycles and -C- OC - groups of atoms; 900 cm ^-1 - deformation vibrations of CH.), Characteristic of the usnic acid sample, indicates its greater number in the studied mechanochemical sample. The presence of usnic acid in the investigated YAGEL preparation is also confirmed by the results of Raman spectroscopy.

Raman scattering was measured on an NTegra Spectra system. The proximity of the frequency ranges of the characteristic absorption lines of Raman spectra for usnic acid and mechanically activated reindeer moss (Figs. 4a and 4b) is also predominantly fluorescence in the 570 nm and 720 nm regions, in contrast to the coarse ground moss spectrum (Fig. 4c), where one can notice a lower radiation intensity of Raman peaks, which indicates a higher content of usnic acid in the YAGEL preparation, in contrast to the coarse moss.

The formation of new structures was recorded by atomic force microscopy (Fig.5b), while the unstructured surface of the coarse moss powder was distinguished by the absence of such and the presence of a smoother relief (Fig.5a).

Table 1 The antibacterial effect of powder No. 1 and YAGEL on cultures of opportunistic and pathogenic bacterial strains The name of the types of bacterial strains The antibacterial effect of powder No. 1 (control) The antibacterial effect of the drug YAGEL 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 The antibacterial effect of the complex preparation YAGEL with cefazolin on E. colli M17 strain №№ Cefazolin concentration, mcg / ml Antibacterial action Complex preparation No. 2 Complex preparation No. 3 (control) one 0.25 ++ ^∗ - 2 0.5 ++ - 3 1,0 ++ + four 2.0 +++ + 5 4.0 ++++ ++ 6 the control - - Note: ^*) + - weak lysis; ++ - partial leasing; +++ - almost complete lysis; ++++ - complete lysis; - complete absence of lysis

Table 3 The dependence of the growth rate of culture St. aureus from drug concentration No. of samples The concentration of the drug (mg / ml) Culture growth rates YAGEL-M (water-alcohol extraction) REINDEER MOSS one 15.0 mg / ml - - 2 12.5 mg / ml - - 3 10.0 mg / ml - - four 7.5 mg / ml - - 5 5.0 mg / ml - - 6 2,5 mg / ml + - 7 1.25 mg / ml +++ + 8 0.63 mg / ml ++++ ++ 9 0.32 mg / ml ++++ ++++ 10 Control 1 (MLA + 1 ml - 96% ethyl alcohol) ++++ ++++ eleven Control 2 (MLA without additives) ++++ ++++ Note: (-) - the absence of colonies of culture St. Aureus (+) ÷ (++++) - the presence of colonies of culture St. Aureus in an amount of 25 to 100 or more colonies per cup

Claims (1)

A method for producing a solid-phase antibiotic preparation, including mechanochemical processing of dry thalli of lichens of the genus Cladonia in a ball mill chamber at 1500 rpm with the addition of a solid inorganic reagent, characterized in that the mechanochemical processing of powder of coarsely ground thalli of lichens is carried out in one technological stage for 3 minutes, and as a solid inorganic reagent, sodium bicarbonate is used in an amount of 0.5 wt.%.

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