RU2385159C2 - Method for making preparation of jagel-m with antituberculous action - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to a preparation with antituberculous action. The method for making the preparation with antituberculous action, characterised by that a dried blastema of sort Cladonia is mechanically treated with solid sodium alkali added. The prepared dry mixture is drawn in an aqueous-alcoholic mixture under certain conditions. The solution is separated from sediment by centrifugation and neutralised with citric acid.

EFFECT: produced preparation shows improved antituberculous action.

1 dwg

Description

The invention relates to the biopharmaceutical industry, and in particular to the production of biological preparations of anti-tuberculosis action. The use of mechanochemical technology in the technological cycle of production of an antibacterial drug from lichen thalli at the stage of processing dry raw materials and subsequent water-alcohol extraction make it possible to isolate a more complete group of natural substances with antibiotic action (PVAD).

Cladonia lichens 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 lichenin homopolysaccharide, as well as isolechinin, which have both immunomodulating, antitumor, and hepatoprotective properties

It is known about the use of lichens as an antibiotic. So, the antimicrobial antibiotic activity against staphylococci, streptococci, acid-resistant microorganisms, fungi, protozoa and viruses is due to the presence of lichen acids in thalli [Galitsky L.A. et al., Probl. tube - 1997. - No. 4. - S.35-38], primarily, usnic acid (Fig.) And its derivatives [R. Gorshkova et al. // Bioorgan. chemistry. - 1997. - T.23, No. 2. - S.1324-1328].

Fig. The general formula of usnic acids.

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 [V. Telyatiev. Useful plants of Central Siberia. - Irkutsk. - 1987. - S.21-22].

The closest analogue to the proposed invention (prototype) is an anti-tuberculosis drug with a wide spectrum of pharmacological action "Islate" (dry extract from the thalli of Icelandic cetraria), which has a tonic, anti-inflammatory, antimicrobial, antihypoxic, immunomodulating, hepatoprotective, antioxidant M. et al. The role of islacet (an extract from the thalli of Icelandic cetraria) in the complex treatment of experimental tuberculosis // Problems of Tuberculosis and Pulmonary Diseases: monthly scientific and practical journal / Russian Society of TB Physicians (M). - 2005. - N11. - S. 44-47; Pat.№2203081 of the Russian Federation The drug islacet for the prevention and treatment of tuberculosis and the method of its production. / Lesiovskaya E.E. and etc.}.

The technical effect of the invention is the antibacterial effect of the drug YAGEL-M, characterized by increased activity against pathogenic microorganisms, primarily mycobacteria (tubercle bacillus).

The effect is achieved by the fact that the use of mechanochemical treatment of lichen raw materials in the presence of alkali leads to the transfer of water-insoluble flavonoids of the usnic group and other lichen acids to a soluble state.

The essence of the invention lies in the fact that the pre-extraction of dry thalli of lichens using mechanochemical technology with the addition of dry alkali makes it possible to isolate a more complete group of PVAD due to the transition of poorly soluble aromatic phenols in water and water-alcohol mixtures (Fig.) Into highly soluble phenolates. This ensures maximum extraction of the entire structural spectrum of active substances, which also does not allow the formation of resistance reactions of microorganisms to this antibiotic complex.

The invention can be implemented as follows.

Dried thalli of the lichen of the genus Cladonid are subjected to mechanochemical treatment in a ball mill (for example, AGO-2) in the presence of solid sodium alkali at 1500 rpm. The resulting dry mixture was insisted on a 40% aqueous-alcohol mixture for 7 days, the solution was separated from the precipitate by centrifugation (pressure filtration) and neutralized to pH 6.9-7.0 with citric acid.

The antibacterial effect of the drug YAGEL-M against opportunistic, pathogenic microorganisms and Mycobacterium tuberculosis (MBT) has been proven in experiments on cultures of bacterial strains and outbred white mice by standard methods.

Investigated the effect

- a complex of lichen PVAD on cultures of bacterial strains of eight conditionally pathogenic and pathogenic microorganisms;

- extract from thalli of lichens on the biological properties of MBT in vitro, including those with drug resistance to anti-TB drugs (PTP);

- a complex of lichen PVAD for the course of experimental tuberculosis in outbred white mice.

Comparative studies of the antibacterial action of a 40% water-alcohol extract from lichen thalli were carried out — without preliminary mechanochemical treatment (extract No. 1) and YAGEL-M preparation (Table 1).

The full lyzing ability of YAGEL-M was shown for eight opportunistic and pathogenic bacterial strains, including Staphylococus aureus, Klebsiella pneumonia, hemolytic E.coli and others (Table 1), while extract No. 1 showed high efficacy only for Proteus vulgaris. In other cases, either a barely noticeable attempt to lysis or microorganisms were partially lysed.

In order to study the antibacterial effect of lichen extracts No. 1 and YAGEL-M on the biological properties of MBT, an experimental study was conducted on MBT clinical strains No. 691, sensitive to PTP, and No. 742 resistant to streptomycin at a concentration of 25 μg / ml, isoniazid at a concentration of 1 μg / ml, rifampicin 80 μg / ml (Tables 2 and 3).

The determination of the antibacterial activity of the tested lichen extracts was carried out by the method of serial dilutions on solid nutrient media in a ratio of 1: 1 to 1: 512. As a nutrient medium, we used traditional nutritious egg media for growing MBT - Finn-2 (F-2). The effectiveness of the antibacterial action of lichen extracts was determined by the timing of the appearance of the primary, intensive and massive growth of MBT cultures.

Under the action of extract No. 1 on clinical strains No. 691 and 742 in dilutions 1: 1, 1: 2, and 1: 4, there was a lack of growth of mycobacteria on the media (bactericidal effect). At a 1: 8 dilution, the initial and intensive growth is delayed compared to the control crop. A 1:16 dilution has a moderate bacteriostatic effect on the MBT (Tables 2 and 3).

The drug YAGEL-M in dilutions 1: 1, 1: 2 and 1: 4 also has a bactericidal effect on the MBT of clinical strain No. 691 (Table 4). A 1: 8 dilution has a bacteriostatic effect. Dilutions of 1:16 and 1:32 have a weak bacteriostatic effect with a delay in initial and intensive growth of 6 days and an increase of up to 100 CFU compared to the control. The bactericidal effect of YAGEL-M on clinical strain No. 742 was observed in dilutions of 1: 1, 1: 2, 1: 4 and 1: 8. Starting with a 1:16 dilution, the initial and intensive MBT growth is delayed by 7 and 6 days, respectively, with a meager growth of up to 20 CFU. Therefore, the drug YAGEL-M in a 1:16 dilution has a pronounced bacteriostatic effect on the MBT (Table 5).

The specific activity of the extracts during the course of experimental tuberculosis was determined on 18 outbred white mice weighing 13-16 g, obtained from the vivarium of the Yakut Republican Veterinary Testing Laboratory. Mice were infected by introducing a suspension under the skin of the back (0.1 mg of bacterial mass in 0.5 ml of physiological saline) of a three-week culture of the multidrug-resistant MBT clinical strain No. 238. The studied extracts were administered to mice inside from the day they were infected.

All experimental animals were divided into the following groups of 6 individuals in each group: 1st group — infected animals (infection control) not receiving treatment; 2nd group - infected animals receiving lichen extract No. 1 at a dilution of 1: 8; 3rd group - infected animals receiving the drug YAGEL-M at a dilution of 1: 8. The duration of the experiment was 2.5 months; surviving animals were killed by this time.

The main indicators of an animal's resistance to tuberculosis are the survival time after infection and the general condition of the animals.

Mortality of mice of the 1st group (infection control) not receiving treatment amounted to 100% during the experiment period. The death of mice was observed in the period from 20 to 73 days, while the average life expectancy was 39 days, which indicates a high virulence of the used MBT culture. The total weight loss in this group was 32%. In mice of the 2nd group, the mortality rate during the experiment was 50% with an average lifespan of 51.5 days. The total weight loss in this group was 8%. In group 3 mice treated with YAGEL-M, the mortality rate was 16.6% during the experiment (1 mouse died on the 73rd day of the experiment). The total body weight of animals in this group increased by 2.5 g (by 16-19%).

Thus, the very high efficiency of the YAGEL-M preparation with respect to opportunistic and pathogenic bacterial strains, including Staphylococus aureus, Klebsiella pneumonia, hemolytic E. coli, Salmonella enteridis, etc., and even their drug-resistant forms, as well as in relation to strains of mycobacteria that are sensitive and resistant to anti-TB drugs. The high antimycobacterial activity of YAGEL-M was established when used in the treatment of experimental tuberculosis in vivo, which manifested itself in a sharp decrease in the severity of the infection in mice infected with MBT.

Claims (1)

A method of obtaining a drug with anti-tuberculosis action, characterized in that the dried thallus of the cladine lichen genus (Cladonia) is treated mechanically in the presence of solid sodium alkali, the resulting dry mixture is infused in a 40% aqueous-alcohol mixture for 7 days, the solution is separated from the precipitate centrifuged, neutralized with citric acid.