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

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

 

The invention relates to biotechnology (biotech) industry, namely the production of biological preparations for medical purposes (bactericidal action). The use of mechanochemical technologies in the production of antibiotic drug from the thalli of lichens without the participation of the solvent in one technological stage of dry raw material allows the formation of β-oligosaccharides (active filler) by splitting parts of β-glycosidic linkages in lichen β-polysaccharides, and of the complex between the active filler and Farmakon (lichen acids contained in the thalli of lichens or known pharmaceuticals antibiotic action), which increases the bioavailability of Farmakon and antibiotic activity of the drug in 5-10 times. This compares favorably to the adaptability of the proposed approach and the properties of the obtained bio-complexes from analogues produced using traditional technologies.

Lichens of the genus of Cladonia (Cladonia) and tsetrarii (Cetraria) - medicinal plants used in folk medicine for treatment of many diseases. In the thalli of lichens contain up to 80% carbohydrates, half of which is represented by β-homopolysaccharides latinam and selegiline with as immunomodulatory, antitumor, and heat the sacrificial properties.

Aware of the use of lichens as antibiotic funds. So, antimicrobial activity against staphylococci, streptococci, acid-fast bacilli, fungi, protozoa and viruses due to the presence in the thalli of the lichen acids (Galitsky L.A., etc. // Problems of tuberculosis. 1997, No. 4. P.35-38). High antibiotic activity is associated primarily with the presence of lichens usninovoy acid and its derivatives (figure 1), related to lichen acids (Gorshkov R.P., etc. // Bioorgan., chemistry. 1997. Volume 23, No. 2. S-1328). The antibiotic activity of usninovoy acid strongly depends on the nature of the optical rotation, and D(+) isomer, as a rule, more active. All three forms of usninovoy acid were previously investigated for antimicrobial activity, set their predominant activity against gram-positive and acid-resistant microorganisms. The mechanism of the antibiotic action of usninovoy acid is associated with the rupture process of oxidative phosphorylation in microorganisms (Kramarenko and others, 2003).

Obtained from lichens usnic acid in the form of usniate sodium was proposed under the name of "Binan" for medical use and was used as an external tool for the treatment of wounds, burns, fractures and gynecology. With the advent of synthetic and polosi the Tethyan antibiotics drug was discontinued (Teletel CENTURIES Useful plants of Central Siberia. Irkutsk. 1987. P.21-22).

Drugs usninovoy acid was successfully used for the treatment and prevention of caries, whereas the use of oral therapy known strong antibiotics (erythromycin, cichoriin, cefuroxime and others) violated the balance of microflora in the oral cavity and caused persistent stomatitis (Litvinov, Rassadina, 1958). Despite the positive experience of the use of lichens in many sections of clinical medicine, in the available literature there are few reports of the use of this plant. This is because well-known methods of extraction of bioactive substances from the thalli of lichens do not give the desired result.

Similar, from the point of view of the method glycosidic latinoware of pharmacones is a series of works under the guidance of academician G.A. Tolstikov (Tolstikova T.G., Bryzgalova, O.A., Sorokina I.V. and others About the nature of the effect glycosidic latinoware of pharmacones // DAN. 2007. T, No. 1. S-134. Tolstikova T.G., Tolstikov A.G., Tolstikov G.A. On the way to low doses of drugs, Vestn. 2007. T. No. 10. S-874).

The closest analogue of the present invention (the prototype) is the anti-TB drug "MOSS-M" for the prevention and treatment of tuberculosis and how to obtain it (Kershengoltsa BM, Filippova, GV, Shashurin M.M. and other Ways the receiving medication LICHEN-M for the prevention and treatment of tuberculosis // RF Patent for the invention №2385159, 27.03.2010; Filippova CENTURY, Pavlov N., Shashurin M.M. and other Influence of biologically active substances from the thalli of lichens of North, extracted by various methods, the biological properties of Mycobacterium tuberculosis // Siberian journal of medicine. 2008. No. 3. S-103).

The technical effect of the invention is the ecological purity of the proposed method to obtain because there is no extraction or hydrolysis stages of processing biosera, the process takes place without the participation of the solvent in one technological stage that provides the reduction of resource and energy consumption in the process. Antibacterial action of the drug received the MOSS is 5-10 times higher activity against pathogenic and conditionally pathogenic microorganisms in comparison with antibiotic drugs obtained by standard methods, without mechanical activation. Minimum inhibitory concentration of the drug MOSS in 2 times less than 40% alcoholic extract of lichen mechanically activated (Moss-M)taken as a prototype.

The effect is achieved by the fact that the use of mechanochemical treatment of lichen material leads in one technological stage to the formation of β-oligosaccharides (active filler) by splitting parts of β-glycosidic linkages in lichen β-polysaccharides, and sets the KSA between the active filler and Farmakon (lichen acids, contained in the thalli of lichens or known pharmaceuticals antibiotic action). While β-oligosaccharides manifest themselves as synergene component in the complex with the natural antibiotic substances steps - with lichen acids or active substances known formproperties antibiotic action. Such weak intermolecular interactions lead to the formation of complex defiling nature, thereby creating optimal conditions for the diffusion process, increasing 5 to 10 times the bioavailability of Farmakon, which increases its bioactivity.

The essence of the invention is that the shock-abrasive impact with the addition of solid-phase reagent sodium bicarbonate to 0.5% by mass, followed, along with the destruction of cell walls, changes in the chemical composition of the components of plant materials and their transition in a bioavailable form in the gap of a number of chemical bonds (such as steady as a β-glycoside) and by the occurrence of a mechanical, solid-phase reactions of transition phenolic groups lichen acids in phenolate, with their subsequent synergetik intermolecular interactions (figure 2).

The invention can be implemented as follows.

Powder coarsely ground thalli of lichens is placed in the camera ball is alinity (for example, AGO-2 or TSEM-7-80) and subjected to mechanochemical activation for 3 minutes at 1500 rpm the Resulting preparation MOSS is being tested for antibacterial activity against pathogenic and pathogenic strains of microorganisms.

The method has the embodiments, namely, that in the chamber ball mill in the powder mixture known antibiotic pharmaceuticals (Pharmacon), such as Cefazolin, in a mass ratio of lichen: Farmakon 10:1. The activity of Farmakon also increased 5-10 times.

Antibacterial properties of the drug LICHEN was determined in vitro on cultures of bacterial strains four conditionally pathogenic and pathogenic microorganisms by standard and modified methods.

Comparative study of antibacterial action of lichen thalli coarse without mechanochemical processing (powder No. 1) and mechanically activated drug LICHEN on the following cultures by the standards of the turbidity of the cultures at 5 and 10 units:

1. Staphylococcus aureus - 6538-p

2. Escherichia coli - H-257

3. Pseudomonas aeruginosa - 33105

4. Klebsiella pneumoniae

Used standard culture medium: medium Endo, milk-yolk-salt agar medium Ploskirev, mastopathy agar (MPA), which were prepared according to standard recipe (MUK 4.2.1890-04. The definition is obsticales of microorganisms to antibiotics. Guidelines approved by the chief state sanitary doctor of the Russian Federation, 04.03.2004) adding to the culture medium of the control and the sample amount of 5.0 mg/ml. Lawn method was seeding of microorganisms and after cultivation thermostat at 37,0°C was estimated by the intensity of their growth. The evaluation results are shown in table 1.

Powder No. 1 showed either a subtle attempt to lysis, or the microorganisms were visualized in part.

Therefore, demonstrated very high efficacy MOSS in relation to conditionally pathogenic and pathogenic bacterial strains, including Staphylococus aureus, Klebsiella pneumoniae and other

Comparative study of antibacterial action of the integrated product No. 2, consisting of lichen thalli and Cefazolin in mass relations 10:1 affected joint mechanochemical treatment, and drug No. 3, consisting of a mixture of lichen thalli and Cefazolin in the same mass relations 10:1, but without mechanical activation, the obtained coarse domestic mill.

Determination of activity of antibacterial action of the test integrated product (No. 2) and coarsely ground mixture (No. 3) was performed by serial dilution method on nutrient dense environments. As the nutrient medium used tra the investment nutrient agar medium for the cultivation of E.colli. The effectiveness of the antibacterial action of the drugs was determined by timing of cell lysis, bacterial culture. Comparison of the samples was performed based on the concentration of Cefazolin.

Under the action of the integrated product # 2 strain E.colli M17 noted moderate bacteriostatic action in the field of concentrations anticipated of 0.25; 0.5 and 1.0 µg/ml and bactericidal concentrations of Cefazolin more than 2.0 µg/ml (table 2).

Cefazolin is in the drug composition No. 3 (control) did not show bactericidal and bacteriostatic action in the field of the above concentrations. Therefore, Cefazolin concentrations exceeding 2.0 µg/ml, the last joint mechanical activation with moss, has a strong bacteriostatic effect on the strain E.colli, unlike Cefazolin, part of the composite of moss coarsely ground without mechanical activation.

We have determined the comparative antibacterial activity of solid-phase preparation MOSS and prototype, representing 40% aqueous-alcoholic extract of the LICHEN-M t vitro against Staphylococcus aureus - 6538-p by determining their minimum inhibitory concentration (MIC).

The prototype we received and tested in strict accordance to the specified invention (see RF Patent for the invention №2385159, 27.03.2010), i.e. in the form of a water-alcohol extra is the dry thalli of lichens, pre-mechanically activated solid alkali. The prototype (the extract of the LICHEN-M) receive a sequence of the following stages: lichen thalli is subjected to mechanochemical activation in the presence of solid alkali to translate lichen acids water-insoluble phenolic nature in water-soluble phenolate form. Next, the resulting powder, which is not the final product, is subjected to aqueous extraction, the insoluble residue is separated by filtration. From the resulting solution of the reaction is precipitated by acidification. The precipitate, containing lichen acid antibacterial action, dissolved in 40% ethanol-water mixture.

For reliable results when comparing the antibacterial activity of the investigated drug and similar concentrations reduced to a single dimension (see table 3).

To determine MICK used standard methods (MUK 4.2.1890-04. Determination of the sensitivity of microorganisms to antibiotics. HOWTO) determining the sensitivity of microorganisms to antibiotics, namely, the method of preparing a serial dilution of antibiotic in the agar. Solid-phase preparation MOSS when making it into a nutrient medium for the distribution ispolzovalis the form of a 40% aqueous-alcoholic suspension. Then after two days of cultivation in a thermostat at 37,0°C was estimated growth intensity depending on the contents of the drug. The evaluation results are shown in table 3. The results were considered visually by counting the grown colonies on the Cup. The received data is interpreted as follows: the lack of growth of a microbe (-) indicates its sensitivity to the drug. With the growth of the test culture in an amount of from 0 to 25 colonies, the result was rated as 1+, 25 to 50 colonies as 2+, from 50 to 100 as 3+. In the case of education on the Cup 100 or more colonies of the microorganism was considered resistant (4+).

Thus, reliable data, are shown in table 3, indicate that the minimum inhibitory concentration of the drug MOSS against a pure culture of the strain St. aureus was 2.5 mg/ml, a extract of the LICHEN-M - 5.0 mg/ml No 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 mentioned strain of microorganism in a nutrient medium "MESA" under standard cultivation conditions.

For physico-chemical studies of the effect of increased antibacterial activity obtained intermolecular complexes of active filler (lichen β-oligos is haridev) Farmakon (lichen acids) used the methods of IR-spectroscopy, Raman (Raman) scattering and atomic force microscopy.

The method of IR spectroscopy shows that the nature of the IR spectra of all samples of lichen identical (figure 3). However, the intensity of all observed absorption bands depends on the method of obtaining a sample of a biological product. The increase in absorption intensity in the region of stretching vibrations of OH-groups (3450-3350 cm-1) moss mechanochemical (MOSS) shows that the gap part of the solid β-glycosidic linkages in the source insoluble polysaccharides included in lichen raw materials, and as a consequence, education is more bioavailable β-oligosaccharides.

The presence in the IR spectrum of a number of bands (1670-1630 cm-1- C=C bond conjugated systems; 1200-1270 cm-1-C-O-C - group atoms; 1100-1000 cm-1as pyranose cycles, and-C-OC - group atoms; 900 cm-1- deformation C-h)characteristic of the sample usninovoy acid shows greater quantity in the study of mechanochemical sample. The presence of usninovoy acid in an investigational drug MOSS confirmed by the results of Raman spectroscopy (Raman) scattering.

Measurement of Raman scattering was carried out on the system NTegra Spectra. The proximity of the frequency ranges of the characteristic absorption lines of the Raman spectra (Raman) RA is seivane for usninovoy acid and mechanically activated moss (figa and 4B), also preferred fluorescence in the region of 570 nm and 720 nm in contrast to the spectra of lichen coarse (pigv), where you can see a lower radiation intensity of Raman peaks, indicates a higher content of usninovoy acid drug LICHEN, unlike lichen coarse.

The formation of new structures was recorded by atomic force microscopy (figb), while the flat surface of the powder lichen coarse differed by the absence and presence of smooth relief (figa).

Table 1
Antibacterial action of the powder No. 1 and drug LICHEN on the culture of conditionally pathogenic and pathogenic bacterial strains
Name types of bacterial strainsAntibacterial action of the powder No. 1 (control)Antibacterial action of the drug MOSS
Staphylococcus aureus - 6538-p+++++
Escherichia coli - H-257++++
Pseudomonas aeruginosa - 33105 +++++
Klebsiella pneumoniae+++++
Ex.:*)+ - weak lysis; ++ - partial lizirovania;
+++ - almost complete lysis; ++++ - complete lysis

Table 2
Antibacterial action of the complex preparation MOSS with the anticipated strain on E.colli M17
№№ p/pThe concentration of Cefazolin, ug/mlAntibacterial action
The integrated product No. 2The integrated product No. 3 (control)
10,25++-
20,5++-
31,0+++
42,0 ++++
54,0++++++
6control--
Ex.:*)+ - weak lysis; ++ - partial lizirovania;
+++ - almost complete lysis; ++++ - complete lysis;
- complete absence of lysis

Table 3
The dependence of the growth rate of the culture St. aureus concentration drugs
No. of samplesThe concentration of the drug (mg/ml)The growth rate of culture
MOSS-M (water-alcohol extraction)MOSS
115,0 mg/ml--
2of 12.5 mg/ml--
310,0 mg/ml--
47,5 mg/ml--
5of 5.0 mg/ml--
62.5 mg/ml+-
7of 1.25 mg/ml++++
80,63 mg/ml++++++
90,32 mg/ml++++++++
10Control 1 (MESA + 1 ml of 96% ethyl alcohol)++++++++
11Control 2 (MESA without additives)++++++++
Approx.: (-) - absence of colonies culture St. Aureus
(+)÷(++++) - the presence of colonies of culture St. Areus in the amount of from 25 to 100 or more colonies per Cup

A method of obtaining a solid antibiotic drug, including mechanochemical processing of dry thalli of lichens of the genus Cladonia in the chamber ball mill at 1500 rpm with the addition of solid inorganic reagent, characterized in that the mechanochemical processing of powder coarsely chopped thalli of lichens is carried out in one technological stage for 3 min, and as a solid inorganic reagent use sodium bicarbonate in the amount of 0.5 wt.%.



 

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