RU2542464C1 - Composition based on phytoextracts, suppressing quorum sensing in bacteria - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to composition, suppressing quorum sensing. Composition, suppressing quorum sensing in bacteria, contains two dried water or alcohol extracts from oak bark (Quercus cortex), birch bugs (Betulae gemmae), eucalyptus leaves (Eucalyptus folia) with the specified ratio of components.

EFFECT: composition effectively suppresses quorum sensing in bacteria.

3 dwg, 1 tbl, 3 ex

Description

The invention relates to the pharmaceutical industry and relates to compositions based on plant extracts intended for the prevention, relief or treatment of pathological conditions of living organisms by affecting the physiology of a parasitic microorganism. In particular, the invention relates to biological compositions capable of suppressing collective behavior (a sense of quorum) in pathogenic, conditionally pathogenic and putrefactive bacteria.

The invention can find application in the manufacture of therapeutic, prophylactic, cosmetic and sanitary-hygienic means for controlling bacterial infections of humans and animals.

A number of herbal compositions with various therapeutic and prophylactic orientations are known from the prior art. In particular, a plant composition with anti-allergic properties is known [1 - Patent RU 2264224. An improved plant composition having anti-allergic properties and a method for its preparation], anti-ulcer composition [2 - Patent RU 2302255. Anti-ulcer plant composition, method for its preparation and use] and etc. In this case, the main argument for the formation of such compositions is the presence in each of the plant extracts of their composition of the corresponding biological activity, as well as the additive effect when used together.

The closest in technical essence and the achieved effect to the claimed invention is a herbal composition with fungicidal, antimicrobial and virucidal action [3 - Patent RU 2372096. Composition with fungicidal, antimicrobial and virucidal effect based on phytoextracts], aimed at the treatment and prevention of infections of microbial etiology and confined space infections by exposure to pathogenic forms of microorganisms. However, this method is focused only on the development of a direct antimicrobial effect, which can potentially lead to the entry of products of the destruction of microorganisms into the internal environment and the living environment of humans and animals with the formation of toxic, allergic and other undesirable effects.

As an alternative to natural and chemically synthesized direct-acting bactericides, an approach is currently being increasingly considered that consists not in destroying, but in changing the physiology of the microorganism in the direction of suppressing (decreasing) its ability to parasitize and damage biological substrates. In particular, the above refers to the search and creation of quorum sensing inhibitors in bacteria, which are assessed as an attractive alternative to traditional means of fighting infections, especially those caused by multiresistant strains of microorganisms [4 - Bhardwaj AK, Vinothkumar K., Rajpara N Bacterial Quorum sensing inhibitors: attractive alternatives for control of infectious pathogens showing multiple drug resistance // Recent Patents on Anti-Infective Drug Discovery. 2013. V.8. P.68-83].

The term “quorum feeling” in bacteria refers to their density-dependent communication mediated by small diffusing molecules (autoinductors) and, when the threshold population density is reached, leading to the functional and morphological differentiation of its constituent cells [5 - Waters C.M., Bassler B.L. Quorum-sensing: cell-to-cell communication in bacteria // Ann. Rev. Cell and Developmental Biol., 2005. V.21. P.319-346]. Moreover, in relation to the purpose of the present invention, it is fundamentally important that the physiology of the parasitic microorganism, such as the formation of virulence factors and the formation of biofilms, are controlled by a sense of quorum.

Analysis of open patent sources allows us to state that, based on the claimed approach, a whole series of inventions has been developed that over the past five years (from 2009 to 2013) have been reflected in international protection documents: WO 2009055054 A2 (2009), WO 2010033236 (2010), WO 2011001419 A1 (2011), US 20100273890 (2010), US 20100256369 (2010), US 7812134 (2010), US 7642285B2 (2010), US 8063108B2 (2011), US 7910622 (2011), US 8247443B2 (2012), US 20120135925 (2012). At the same time, the proposed technical solutions were: 1) suppression of auto-inductor synthesis; 2) its binding by specific antibodies; 3) degradation of the auto-inductor by the corresponding enzymes (lactonases or acylases); 4) covalent modification (inactivation) of receptor proteins interacting with autoinductors; 5) the use of agonists and antagonists of autoinductors capable of directly interfering with the natural signal for binding to receptor proteins.

Among such solutions, the closest to the claimed invention is the search for natural substances of plant origin, in vivo serving to suppress the feeling of quorum in phytopathogens, and when transferred to other living systems that can cause a similar effect with respect to pathogenic and conditionally pathogenic microflora of humans and animals [ 6 - Koh C.-L., Sam C.-K., Yin W.-F., Tan LY, Krishnan T., Chong YM, Chan K.-G .. Plant-derived natural products as sources of anti- quorum sensing compounds // Sensors. 2013. V.13. P.6217-6228]. In particular, a known solution is WO2009114810 A2 [7 - Mathee K., Adonizio A.L., Ausubel F., Clardy J., Bennett B., Downum K. Use of ellagitaimins as inhibitors of bacterial quorum sensing. 2009], the formula of which stipulates the possibility of suppressing the feeling of the quorum of pathogenic bacteria using the components of medicinal plants - ellagitanins (vescalagin and castalagin).

At the same time, the source of the production of ellagitanins proposed by the authors, the plant Conocarpus erectus L. (Combretaceae), is listed in the International Red Book [IUCN Red List of Threatened Species. Version 2013.1] and has a relatively small distribution area in the tropical belt of America and West Africa, which creates resource limitations for the implementation of patent WO 2009114810 A2. In addition, the proposed use of only one plant source requires additional purification and isolation of biologically active substances from it, because the ballast components of this non-food raw material are potentially capable of adversely affecting the human and animal body.

In this regard, the objective of this invention is to develop a composition of phytoextracts of medicinal plants used in traditional (folk) East European medicine and exhibiting an additive effect in regulating the quorum of pathogenic and putrefactive bacteria.

In the present invention, this problem is solved by the combined use of aqueous or alcoholic extracts or similarly obtained and subsequently dried extracts from oak bark (Quercus cortex), birch buds (Betulae gemmae) and eucalyptus leaves (Eucalypti folia). In accordance with the present invention, a regulatory preparation (composition) based on them contains from 33.3 to 50.0 wt.% Of each of the phytoextracts, and a similar ratio and form of its use (in the form of liquids, powders, applications, etc. ) are determined based on the features of the localization of the infectious process and the individual characteristics of the patient.

In particular, oak bark extract is included in compositions intended for the treatment and prevention of intestinal infections, wound infections, inflammatory skin diseases, oral infections, as well as inflammatory diseases of the female genital organs. In turn, a contraindication to the inclusion of oak bark in the composition is hypersensitivity to this drug.

Birch bud extract is included in compositions intended for the treatment of infectious and inflammatory diseases of the ENT organs, wound infections, inflammatory skin diseases, as well as intestinal infections. Contraindications to the use of this phytoextract are the period of pregnancy and pathology of the urinary system (renal failure and acute diseases of the urinary tract).

Eucalyptus leaf extract is included in compositions intended for the treatment of infectious diseases of the ENT organs and upper respiratory tract, wound infections, infections of the oral cavity, intestinal infections, as well as inflammatory diseases of the female genital organs. Contraindications to the use of this phytoextract are hypersensitivity to this drug, as well as atrophy of the mucous membrane of the upper respiratory tract, bronchial asthma, bronchospasm, whooping cough.

The phytoextracts used were prepared on the basis of dry plant raw materials, carrying out water or alcohol extracts from it, and then, if necessary, additional drying of the obtained extracts was carried out.

When preparing aqueous extracts from the coarse parts of plants (in particular, oak bark), 6 grams of crushed plant material, previously sterilized in an autoclave at 1 atmosphere for 30 minutes, was poured with sterile distilled water in a volume of 50 ml. The next step was boiling in a water bath for 30 minutes, cooling - 10 minutes, after which the resulting broth was filtered through a sterile cotton-gauze filter in a glass dish. In the preparation of water extracts from the soft parts of plants (birch buds and eucalyptus leaves), a similar sequence of actions was used, except that boiling in a water bath was carried out for 15 minutes, and infusion for 45 minutes.

In the preparation of alcoholic extracts, the crushed plant material was extracted with 40% ethanol at room temperature (22-24 ° C) for 24 h, after which the extract was filtered through a sterile cotton-gauze filter in a glass dish.

If necessary, the obtained extracts were evaporated in a water bath until a thicker consistency was obtained, after which they were completely dried to obtain a powder.

The compositions were obtained by mixing aqueous or alcoholic extracts in a ratio of 33.3 wt.% Of three phytoextracts or 50.0 wt.% When mixing two phytoextracts.

Compared with the above technical solutions [3, 7], the claimed invention has a number of significant differences that meet the requirement of novelty.

1) Unlike the phytoextract composition in the patent RU 2372096, the claimed composition provides the development of not direct fungicidal, antimicrobial or virucidal effects, but has a regulatory effect on the physiology of the parasitic microorganism in the direction of suppressing the quorum feeling of pathogenic and putrefactive bacteria. As a result, effective control of bacterial pathogens is possible without the destruction of microbial cells, usually accompanied by the release of toxic and allergizing substances.

An experimental justification for the use of these phytoextracts is the data on the effect of aqueous and alcoholic extracts of 20 medicinal plants used by traditional (folk) East European medicine to control bacterial infections on the growth and manifestation of a quorum in the indicator strain Chromobacterium violaceum NCTC 13274.

The adequacy of the use of this microorganism for the stated purposes is determined by the presence of a mutation (insertion) in the cviI gene, which is responsible for the formation of the self-inducer of the C6-homoserinlactone (C6-GSL) sense quorum. As a result, this strain is incapable of developing density-dependent communication, including the blue-violet pigment violacein, which is well detected by optical methods and is manifested in the biosynthesis. On the other hand, the introduction of exogenous C6-GSL restores the quorum feeling and leads to pigment biosynthesis. Thus, C. violaceum NCTC 13274 is a convenient tool for identifying substances that interfere with the perception of autoregulatory signals in bacteria, and therefore is currently widely used in the search for quorum inhibitors: the presence of such activity is judged by the inhibition of violacein synthesis in the presence of C6- GSL.

In a series of experiments (Table 1), it was found that 4 aqueous and 8 alcoholic extracts from medicinal plants develop direct antibacterial activity against the indicator strain C. violaceum NCTC 13274, which manifested itself in the formation of detectable growth suppression zones around wells with corresponding phytoextracts. On the other hand, 6 aqueous and 11 alcoholic extracts caused the inhibition of the formation of violacein pigment by this strain, which indicated the suppression of the quorum sensory mechanisms initiated by the addition of C6-GSL. So figure 1 presents the results of testing phytoextracts: 1 - bud of a birch (Betulae gemmae), I - leaves of lingonberry (Vaccinium folia), 3 - grass of a series (Bidentis herba), 4 - leaves of bearberry (Arctostaphylos folia), 5 - leaves of eucalyptus (Eucalyptus folia), 6 - oak bark (Quercus cortex), 1 - control; where growth inhibition is fixed for

Table 1. Areas of zones of inhibition of growth and feelings of quorum (mm ² ) of the indicator strain C. violaceum NCTC 13274 with the addition of C ₆ -HSL (5 * 10 ^-7 mol / l) Type of plant Plant part Manifestations of the biological activity of phytoextracts Growth inhibition Suppression of the “quorum feeling” Water extract Alcohol extract Water extract Alcohol extract Achillea millefolium Grass 0 0 0 0 Arctostaphylos uva-ursi Leaves 207 235 153 126 Betula verucosa Kidney 0 9 113 105 Bidens tripartita Grass 0 0 0 0 Calendula officinalis Flowers 0 0 0 31 Chelidonium majus Rhizomes 9 fourteen 0 0 Comarum palustre Rhizomes 0 0 0 75 Eucalyptus viminalis Leaves 19 31 276 204 Inula helenium Rhizomes 0 9 0 85 Juniperus communis Fruit 0 0 0 19 Lieli palustris Shoots 0 19 19 25 Matricaria chamomilla Flowers 0 0 0 0 Plantago major Leaves 0 0 0 0 Quercus spp. Bark 0 0 94 94 Rosa majalis Fruit 0 0 0 fourteen Salvia officinalis Leaves 0 25 0 0 Taraxacum officinale Rhizomes 0 0 0 0 Tussilago farfara Leaves 0 0 0 0 Vaccinium vitis-idaeae Leaves 31 59 83 54 Violae tricoloris Flowers 0 0 0 0

samples 2, 4 and 5, and suppression of the formation of the pigment violacein for samples 1, 5 and 6.

At the same time, the recorded direct antibacterial effect and inhibition of the quorum feeling did not statistically significantly correlate with each other (for water extracts, r = 0.430, P> 0.01; for alcoholic extracts, r = 0.358, P> 0.01). The aforesaid allows us to state that the development of these effects is determined by non-identical substances in phytoextracts, thereby emphasizing the fundamental difference between the known and the claimed approaches.

2) In contrast to WO2009114810, the ability to suppress the sense of quorum in bacteria was established in extracts not from the tropical plant Conocarpus erectus listed in the International Red Book, but from oak and birch, as well as eucalyptus, widely distributed in the temperate climatic zone of Eastern Europe, in significant growing in the subtropical zone of Russia, which removes resource (raw) restrictions in the production of their compositions based on them.

From the available scientific and patent literature, information on the pronounced effect of phytoextracts of oak bark (Quercus cortex), birch buds (Betulae gemmae) and eucalyptus leaves (Eucalypti folia) on the manifestations of the quorum feeling in bacteria is not known. Accordingly, the greatest severity of such an effect in the absence or with minimal direct antibacterial effect was the basis for recommending their use for a “new way of influencing the physiology of a parasitic microorganism in the direction of suppressing its density-dependent communication.

3) In a separate series of experiments, it was shown that extracts of oak bark, buds of birch and eucalyptus leaves have an additive effect in suppressing the feeling of quorum. The aforesaid was that the area of suppression of pigmentation of Chromobacterium violaceum NCTC 13274 when applying a combination of three phytoextracts exceeded the expected value of that when separately applied. In particular, as shown in FIG. 2, the use of a combination of aqueous extracts of oak bark, birch buds and eucalyptus leaves in a ratio of 1: 1: 1, indicated by the number 4, led to the formation of a quorum feeling inhibition zone, the area of which significantly exceeded that with isolated use each phytoextract: 1 - birch buds (Betulae gemmae), 2 - eucalyptus leaves (Eucalyptus folia), 3 - oak bark (Quercus cortex), and also turned out to be higher than the expected summation effect.

In another embodiment of the experimental design, illustrated in Fig. 3, the additive effect was recorded by separate application of alcohol phytoextracts, which consisted of inhibiting the biosynthesis of the violacein pigment at a distance from the place of application of two phytoextracts, exceeding that sufficient to provide a similar effect of only one phytoextract. In particular, this effect is registered in the zones marked by arrows and indicated by numbers: 4 - for oak bark (Quercus cortex) and eucalyptus leaves (Eucalyptus folia); 5 - for eucalyptus leaves (Eucalyptus folia) and birch buds (Betulae gemmae); 6 - for birch buds (Betulae gemmae) and oak bark (Quercus cortex), exceeding the quorum feeling suppression zones formed in the usual version of the experiment for: 1 - oak bark (Quercus cortex), 2 - eucalyptus leaves (Eucalyptus folia), 3 - birch buds (Betulae gemmae).

Thus, the combined use of phytoextracts of oak bark, birch buds and eucalyptus leaves is new, providing the additivity of their action on the manifestations of the quorum feeling in bacteria. On the other hand, such use reduces the possible undesirable effects of the ballast fraction of each of the phytoextracts, the concentration of which in the composition decreases by two to three times.

4) The possibility of extraction of the active components of medicinal plants with both water and alcohol is shown with a quantitatively higher efficiency of the latter method. This circumstance determines the possibility of the formation of phytoextract compositions on both an aqueous and an alcoholic basis with some limitations on the use of the latter in pediatric practice. In addition, in a separate series of experiments it was found that the drying of water and alcohol extracts, as well as the procedure for their repeated solubilization, do not change the patentable biological activity.

5) The results described above were also an experimental justification for the formation of phytoextract compositions with a content of each component from 33.3 wt.% (When creating three-component compositions) to 50.0 wt.% (When creating two-component compositions in the absence of indications or contraindications for use one of the phytoextracts). Applicants suggest that the additive effect can be achieved in a wider range of ratios, but limit the level of claims to the available experimental evidence.

The composition of the patented invention also includes the use of a composition based on phytoextracts of oak bark, buds of birch and eucalyptus leaves to suppress the feeling of quorum in pathogenic and conditionally pathogenic bacterial microflora involved in the development of infectious diseases of plants, animals and humans. To this end, this composition can be introduced into the body to provide a systemic effect, and also be applied topically to affect certain areas (for example, as part of dressings for treating wounds, when treating an operative field, etc.). The phytoextract composition can be used in the form of aqueous or alcohol solutions, solids (powders), as well as applied to various carriers.

The invention is illustrated, but not limited to the following examples.

Example 1

A composition that suppresses a sense of quorum in bacteria, for example, in the treatment of wound infections and infectious and inflammatory skin diseases. The composition contains tinctures of oak bark (Quercus cortex), birch buds (Betulae gemmae) and eucalyptus leaves (Eucalypti folia). Tinctures are prepared according to the general scheme: 6 grams of crushed plant material is extracted with 40% ethyl alcohol at room temperature (22-24 ° C) for 24 hours, after which the extracts are filtered through a sterile cotton-gauze filter in a glass dish. The resulting tinctures are mixed in the following ratio of components, wt.%:

- tincture of oak bark (Quercus cortex) - 33.3;

- tincture of birch buds (Betulae gemmae) - 33.3;

- tincture of eucalyptus leaves (Eucalypti folia) - 33.4.

The composition can be used by lotions, applications or irrigation.

Example 2. A composition that suppresses a sense of quorum in bacteria, for example, in the treatment of infectious and inflammatory diseases of the ENT organs and upper respiratory tract. The composition contains infusions of eucalyptus leaves (Eucalypti folia) and birch buds (Betulae gemmae). Infusions are prepared according to the general scheme: 6 grams of crushed plant material pre-sterilized in an autoclave at 1 atmosphere for 30 minutes, poured with sterile distilled water in a volume of 50 ml. The next step is boiling in a water bath for 15 minutes, infusion - 45 minutes, after which the resulting infusion is filtered through a sterile cotton-gauze filter in a glass dish. The obtained hoods are mixed in the following ratio of components, wt.%:

- infusion of eucalyptus leaves (Eucalypti folia) - 50.0;

- infusion of birch buds (Betulae gemmae) - 50.0.

The composition can be used in the form of solutions for rinsing or inhalation.

Example 3. A composition that suppresses a sense of quorum in bacteria, for example, in the treatment and prevention of intestinal infections. The composition contains dried extracts of oak bark (Quercus cortex), birch buds (Betulae gemmae) and eucalyptus leaves (Eucalypti folia). Extracts of oak bark are prepared according to the following scheme: 6 grams of crushed plant material, previously sterilized in an autoclave at 1 atmosphere for 30 minutes, is poured with sterile distilled water in a volume of 50 ml. The next step is boiling in a water bath for 30 minutes, cooling - 10 minutes, after which the resulting broth is filtered through a sterile cotton-gauze filter in a glass dish. Extraction of birch buds and eucalyptus leaves was prepared according to the general scheme: 6 grams of crushed plant material, previously sterilized in an autoclave at 1 atmosphere for 30 minutes, was poured with sterile distilled water in a volume of 50 ml. The next step is boiling in a water bath for 15 minutes, cooling - 45 minutes, after which the resulting infusions are filtered through a sterile cotton-gauze filter in a glass dish. At the next stage, the broth and infusions are evaporated in a water bath until a thicker consistency is obtained, after which they are completely dried to obtain a powder. The resulting dry phytoextracts are mixed in the following ratio of components, wt.%:

- extract of oak bark (Quercus cortex) - 33.3;

- extract of birch buds (Betulae gemmae) - 33.3;

- extract of eucalyptus leaves (Eucalypti folia) - 33.4.

The composition can be used as powders and other solid forms for oral administration, as well as applied to various carriers.

Claims (1)

A composition that suppresses the feeling of quorum in bacteria, containing at least two dried water or alcohol extracts from oak bark (Quercus cortex), birch buds (Betulae gemmae), eucalyptus leaves (Eucalyptus folia) in the following ratio of components, during the formation of a three-component composition, wt .%:
- dry extract of oak bark (Quercus cortex) - 33.3;
- dry extract of birch buds (Betulae gemmae) - 33.3;
- dry extract of eucalyptus leaves (Eucalyptus folia) - 33.4;
when forming a two-component composition, the content of each dry extract is 50 wt.%.

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