Synthetic phenolic compounds and vegetable extracts obtained from plants that are effective in treatment and prophylaxis of chlamydium infections

FIELD: medicine, pharmaceutical industry, pharmacy.

SUBSTANCE: invention relates to compositions used for treatment and/or prophylaxis of chlamydium infections caused by C. pheumoniae. Pharmaceutical composition used for treatment and/or prophylaxis of chlamydium infection caused by C. pneumoniae comprises the taken phenolic compound, or extract, or fraction, or incomplete fraction comprising the taken phenolic compound or corresponding synthetic compound, or mixture of indicated compounds obtained from plants. An anti-chlamydium effect of phenolic compound or extract, or fraction, or incomplete fraction obtained from plants and comprising indicated compound or corresponding synthetic compound on C. pneumoniae represents the definite percent of inhibition for formation of inclusions. The composition useful for health eliciting an anti-chlamydium effect with respect to C. pneumoniae comprises the taken phenolic compound or extract, or fraction, or incomplete fraction containing indicated compound or corresponding synthetic compound, or mixture of indicated compounds obtained from plants. An anti-chlamydium effect of phenolic compound or extract, or fraction, or incomplete fraction comprising indicated compound or corresponding synthetic compound obtained from plants on C. pneumoniae represents the definite percent for inhibition in formation of inclusions. Also, invention relates to applying the composition useful for health in preparing foodstuffs or as supplements for nutrition for every day. Also, invention relates to applying phenolic compound or extract, or fraction, or incomplete fraction comprising indicated compound or corresponding synthetic compound or mixture of indicated compounds obtained from plants in manufacturing a medicinal agent used for treatment and/or prophylaxis of chlamydium infections caused by C. pneumoniae. An anti-chlamydium effect of phenolic compound or extract, or fraction, or incomplete fraction comprising indicated compound or corresponding synthetic compound obtained from plants on C. pneumoniae represents the definite percent in inhibition in formation of inclusions. Compositions promote to effective prophylaxis and treatment of chlamydium infections caused by C. pneumoniae.

EFFECT: valuable medicinal properties of compounds.

21 cl, 1 dwg, 1 tbl, 6 ex

 

The invention relates to efficient phenolic compounds of plant origin and related synthetic compounds and their derivatives and plant extracts, as well as containing compositions useful for the treatment and prevention of chlamydial infections, and to the use of phenolic compounds of plant origin and related synthetic compounds and their derivatives and plant extracts, as well as compositions containing them, for the treatment and prevention of chlamydial infections. Phenolic compounds of plant origin and the corresponding synthetic compounds and their derivatives and plant extracts can be used for the preparation of pharmaceutical preparations, compositions, dietary supplements and functional foods with health benefits.

Chlamydia is a small group of gram-negative bacteria. Due to the unique intracellular reproductive cycle, when classifying their allocated in a separate order Chlamydiales, including the genus Chlamydia. The genus Chlamydia is already the source was divided into two species: C. trachomatis and C. psittaci. The separation was based on the biological properties:

 C. trachomatisC. psittaci
The accumulation of glycogen in chlamydia what's the inclusions (staining with iodine +) +-
Sensitivity to sulfa drugs+-

Beginning with C. trachomatis considered a homogeneous group, and "C. psittaci" a very heterogeneous group. When opened chlamydial strain (later S. pneumoniae)that causes respiratory infections in the absence of contact with birds, it undoubtedly considered related to the group "C. psittaci", as it satisfies the above requirements on the basis of biochemical analysis. C. trachomatis and C. pneumoniae differ in the structure of the surface. The main component of the surface structure of C. trachomatis is the major protein of the outer membrane (MOMR), which varies in four different areas and currently forms the basis for the division of C. trachomatis almost 20 different immunotypes. In C. pneumoniae MAR very conservative, and found only one immunochip. In addition, target cells in different tissues: epithelium of the genital organs and conjunctiva in the case of C. trachomatis, and the epithelium of the respiratory tract in case of S. pneumoniae. First, except in unusual strain that causes venereal lymphogranuloma (LGV), not capable of reproduction in phagocytes and macrophages, which, in particular, are the target cells of the latter. When the penetration into the cells of S. pneumoniae uses the receptor for heparin, which is not used by the PI is s genitals, except for LGV. In addition, the transmission path and the resulting clinical picture is different: S. pneumoniae is transmitted through the respiratory tract and can spread in monocytes in the circulation system, whereas C. trachomatis mainly transmitted through sexual contact. In addition, different methods of treatment: C. trachomatis is usually treated with a single dose, whereas in the case of S. pneumoniae recommend even a three-week course of antibiotics.

Below is the list of diseases caused by or associated with these types of chlamydia:

C. trachomatis:

- conjunctivitis

- cervicitis

- urethritis

- pelvic inflammatory disease (PID)

- neonatal infections, such as pneumonia in children

- peritonitis

- perihepatitis

is reactive arthritis

S. pneumoniae:

infection of the upper respiratory tract

- bronchitis

pneumonia

- chronic obstructive pulmonary disease (COPD)

- asthma

- vasculitis

- atherosclerosis with its complications

encephalitis

- some types of multiple sclerosis

- part of late manifested symptoms of Alzheimer's disease

Already in 1989, it was shown that chlamydial strain that causes respiratory infections in the absence of contact with birds, is genetically different from both C. trachomatis and described species C. psittaci, and so obvious that its you who elili in your own With. pneumoniae. Homology of its nucleic acid with C. trachomatis is less than 10%. C. trachomatis has extrachromosomal plasmids were not detected in strains of S. pneumoniae person. The genomes of both species sequenced, and the number of genes is much higher (about 200) from S. pneumoniae than in C. trachomatis. In newly completed classification of chlamydia specified strain had already been carried to a completely different genus Chlamydophila. Thus, there is a reasonable basis to assume that all that is known relative to C. trachomatis, can not be attributed to S. pneumoniae.

The most common species of chlamydia in humans are S. pneumoniae and C. trachomatis that cause important common diseases. C. psittaci is widely distributed in the animal Kingdom, but it can sometimes cause infections in humans. In addition, it is known that C. pecorum causes infection in ruminants. Is the classification of new genera and species in the order Chlamydiales.

S. pneumoniae is the most common chlamydia for the human population, and almost every infected 2-3 times during life. S. pneumoniae can easily penetrate lung tissue and multiply in macrophages and endothelium of blood vessels. The clinical picture of respiratory infections caused by C. pneumoniae, greatly varies from usually moderate infections of the upper digitalin the x ways from children to serious pneumonia in adults. 5-10% of all pneumonia caused by C. pneumoniae. S. pneumoniae is distributed as airborne infection from person to person. Obviously, effective vector control are some individuals, because infections have become more widespread only at school age. In the Nordic countries infection caused by C. pneumoniae, are in the form of two - to three-year epidemics at intervals of about six years.

Chlamydial infections are sporadic and latent nature, and obviously, the most important are their chronic late complications. Epidemiological studies show a significant link between chronic infections S. pneumoniae and atherosclerosis: many studies have also shown the Association between chlamydial infection and the occurrence of acute myocardial infarction (AMI). In addition, chronic infection with C. pneumoniae, obviously, plays a role in the development of outbreaks of asthma and chronic obstructive pulmonary disease.

Atherosclerosis is a chronic inflammatory condition, and particles S. pneumoniae can be detected in foam cells and smooth muscle cells in more than half of atherosclerotic plaques. In patients with AMI is often immune response to lipopolysaccharide (LPS) of chlamydia, testifying to an exacerbation of the infection. It is also possible to detect chlamydia in damaged with technich valves, and they are especially numerous in aneurysms of the abdominal aorta. In addition, it was found that C. pneumoniae plays a role in brain infarctions and transient seizures in cerebral ischemia. Still it is not clear what is the role of chlamydia. identified in the damaged area, in the development of the damage, but, anyway, one of the factors slow the progression of these diseases, apparently, is a chronic chlamydial infection. However, in animal models it has been shown that C. pneumoniae initiates and accelerates the development of atherosclerosis. Epidemiological and clinical studies have shown that there is a clear link between chronic infection with C. pneumoniae and atherosclerosis and acute myocardial infarction. In the most recent studies also concluded that infection with C. pneumoniae infection is a risk factor of heart disease. Infection with C. pneumoniae is often associated with cigarette Smoking, which, obviously, predisposes to chronic chlamydial infection.

It is observed that treatment with antibiotics reduces the risk of heart attacks, and can also affect General marker of inflammation, CRP and fibrinogen levels in serum, using antibiotics. In most industrialized countries, the incidence of heart disease began to decline in those cases where application is very effective is Rotel chlamydia antibiotics became widely accepted in the treatment of other infections.

C. trachomatis is the most important cause of genital infections in women. In addition, some infections of the urinary tract, negative in the test bacterial cultures in women of reproductive age caused by C. trachomatis. C. trachomatis is a common cause of chronic endometriosis and PID is the most common complication infections of C. trachomatis in women. Infection with C. trachomatis can be almost asymptomatic, and it is known that even an ectopic pregnancy, and infertility are the complications associated with the formation of impenetrable scar caused by random latent infection. About half of children born in carriers of chlamydia will be infected at birth, and about half of infected infants will develop conjunctivitis with inclusions and, as complications, pneumonia C. trachomatis. C. trachomatis also causes genital infections in men.

Chlamydia sensitive to tetracycline and erythromycin; also effective rifampicin and some of the newer fluoroquinolones. C. trachomatis, unlike, for example, from S. pneumoniae, also sensitive to sulfa drugs. Despite the sensitivity to the treatment of chlamydial infections are often recurrent, and there is also the risk that they will become chronic. Chlamydia reproduce only within the cells, and therefore new macrolide antibiotics and Salida, effectively concentrated in the cells, are currently alternative to tetracycline and erythromycin as the main drug. In the case of chlamydial infection with complications treatment may need to continue for a long period of time, for example, in case of illness Reiter, caused by chlamydia recommended treatment within three months.

To date there is no vaccine for the prevention of chlamydial infections. The nature of the immune response is not well-known, and it is associated with a tendency to hypersensitivity.

In the patent EP 0377722 describes how to assess the risk of myocardial infarction, the method for the diagnosis of diseases of the heart and blood vessels, as well as the use of drugs that are effective against chlamydia. In this publication, as appropriate medicines for the treatment or prevention of chronic heart disease caused by chlamydia described tetracyclines, erythromycin, rifampicin and fluoroquinolones.

In WO 98/50074, in particular, for the treatment of infections caused by S. pneumoniae, as described combination protivodiareynah agents, in which each of the active ingredients effective at a certain stage of the life cycle of chlamydia.

In U.S. patent 5830874 described a method for the diagnosis of arterial chlamydial granuloma caused by C. pneumoiae, a therapeutic composition for the treatment of arterial chlamydial granulomatosa. As compounds that have a therapeutic effect, indicated tetracyclines, eritromicina, claritromicina, azithromycin and quinolones, etc. that are effective against chlamydia.

In the Japan patent 10139686 described the use of 2-(3,4-dimethoxycinnamoyl)aminobenzoic acid as a therapeutically active compound in a daily dose of 100-1000 mg for the treatment of atherosclerosis caused by C. pneumoniae.

According to the information above, there is an obvious need for new compounds and compositions that can be used for the treatment and prevention of chlamydial infections.

Shikimate or compounds that are formed from the shikimic acid biosynthetic path connection formed by biosynthesis pathway of acetate-malonate and compounds formed by the combination of both ways, belong to the group of phenolic compounds of plant origin. To these compounds include simple aromatic compounds, phenols, coumarins, lignans, lignins, and flavonoids and their derivatives. Simple aromatic compounds mainly include derivatives of phenylpropane derivatives phenylmethane. In nature flavonoids, which structure are phenolic compounds, form Shiro is about common group of plant pigments. Flavonoids are found in the plant Kingdom everywhere: bryophytes, in the family of weariness and other lower plants. Most of them were found in higher plants and vascular plants, and they are found in all fruits, vegetables and, along with others, in tea, as well as wines, especially red wines. In nature, flavonoids, mainly found in glycoside form, but they can also be a free phenols and sulfates in the so-called form of aglycone or connection associated with polysaccharides and proteins. In most cases, the flavonoids in its chemical structure are polyphenolic compounds. Identified more than 8,000 plant flavonoids, and they perform numerous functions. Due to the bitter taste they protect plants from harmful insects, but because of the antibiotic properties - protects plants against viruses and bacteria. According to modern views flavonoids are compounds that are important from the point of view of power, but, apparently, they have beneficial health effects. The specified action, apparently, does not depend on vitamins and minerals in the plant. Hardly anyone can avoid the intake of flavonoids, but their actions one way or another, depend on suction properties and bioavailability and, in addition, from the interaction although the NGOs received flavonoids.

Antioxidant effect of natural phenolic compounds already known for quite a long period of time, and several studies have discussed an antioxidant effect, as well as the capture of free radicals. According to this study, the ability of flavonoids to prevent the oxidation of LDL - cholesterol is considered as one of their most important properties. Oxidation of LDL - cholesterol in subendothelial blood vessel is a primary factor in atherogenesis. Many studies indicate that insufficient absorption of flavonoids from food may be the most important factor of morbidity caused by diseases of the heart and blood vessels. When research involving human subjects, related to the flavonoids, found only a few of the most important flavonoids, of which quercetin has been shown to prevent the oxidation of LDL cholesterol and thus reduces the risk of coronary disease because oxidized LDL - cholesterol is clearly associated with the stages of atherosclerosis.

The daily intake of flavonoids from food varies, for example, according to a study in the Netherlands, from 0 to 30 mg In studies conducted in Finland, noted that flavonoids exhibit a moderate protective effect against the incidence of heart disease and blood the vessels, but on the other hand, differences in the consumption of flavonoids were quite small, the total number was approximately 2-6 mg/day. In a study conducted in the Netherlands, noted an inverse relationship between dietary flavonols and flavanols and deaths caused by diseases of the heart and blood vessels. Also noted an inverse relationship between the intake of flavonols and flavones and risk of myocardial infarction. In addition, it is known that flavonoids influence inflammatory and immune responses, as well as many other cell functions. Some flavonoids and many other natural phenolic compounds can inhibit or enhance the absorption of calcium by the cell, which is also shown in table 1, below.

Drugs that block calcium channels play an important role in the treatment of diseases of the heart and blood vessels, such as chest pain as a result of anoxia of the heart, myocardial infarction, atherosclerosis and hypertension. These drugs act on calcium channels, preventing the influx of calcium into the cell, and thus expand coronary artery, reduce peripheral resistance of blood vessels, thus reducing the workload of the heart. Large scale application of calcium antagonists has led,for example, to develop screening programs to detect blocking action on calcium channels of compounds isolated from natural sources. As a medium for screening in the studies used, for example, a continuous line of cells derived from tumors of the posterior pituitary in rats (GH4C1), as well as the method of fixation potential, which at this point in time it is possible to evaluate a single calcium channels in one cell. Research compounds of natural origin and extracts was found, which compounds have a blocking or activating effect on calcium channels. These compounds are found among derived from plants of simple phenols, coumarins, flavonoids and extracts that are rich in these compounds. Some of these compounds in its blocking action comparable to verapamil, and some compounds have a tendency to increase calcium influx into the cell.

This invention relates to efficient phenolic compounds derived from plants, and related synthetic compounds and their derivatives and plant extracts, as well as containing compositions useful for the treatment and prevention of chlamydial infections, as well as the application of plant phenolic compounds and the corresponding the adequate synthetic compounds and their derivatives and plant extracts, and compositions containing them for the treatment and prevention of chlamydial infections.

Distinctive features derived from plants phenolic compounds and related synthetic compounds and their derivatives and plant extracts according to the invention, the compositions according to the invention and their use in the treatment and prevention of chlamydial infections are presented in the claims, as well as their application in the production of pharmaceuticals or healthy food suitable for the treatment and prevention of chlamydial infections.

It has been unexpectedly discovered that certain derived from plants phenolic compounds, the corresponding synthetic compounds and their derivatives and plant extracts and fractions and partial fractions containing the specified derived from plant compounds that have a strong effect similar to the action of antibiotics against chlamydia. According to the invention is obtained from a plant phenolic compounds are phenolic compounds, formed from the shikimic acid biosynthetic path, phenolic compounds, formed by the acetate-malonate path, and phenolic compounds formed as a result of a combination of both ways. These compounds, such as simple and ematicheskie connection phenols, coumarins, lignans, lignins and flavonoids, derived from products of the plant Kingdom, such as fruits and vegetables, especially citrus fruits, vegetables, berries, onions, tea, red wine, etc.

Derived from plants phenolic compounds, the corresponding synthetic compounds and their derivatives and extracts and fractions and partial fractions containing these compounds can be used as such or in mixtures thereof, optionally in combination with sulfur compounds contained in garlic, such as allicin or allicin derivatives.

Preferred are obtained from plants of natural phenolic compounds are flavonoids and derivatives phenylmethane and phenylpropane, phenolic acids, triterpenes, coumarins and catharinae, and extracts and partial fractions containing these compounds, and the fractions containing simple phenols, flavonoids and their derivatives, polyphenols, determinedly and determinine and factions from which you removed tannin and diterpene. Preferred also are the corresponding synthetic compounds and their derivatives and pharmaceutically acceptable salts, esters and derivatives of the above compounds.

Preferred compounds and their extracts are compounds and extracts with protivolednym there is a Vij (inhibition of the formation of inclusions), equal to or greater than 30%, and especially preferred are compounds and extracts with protivolednym action equals or exceeds 90% of the actions defined in the examples. The preferred group of compounds and extracts:

- Flavones such as apigenin, luteolin, a flavone

- Flavonols, such as quercetin, rhamnetin, Maureen

- Flavanone, such as naringin

- Isoflavones, such as genistein

Connection derivatives phenylmethane, such as metalsalt, propylgallate, octisalate, dodecylsulfate, isopropylmalate

Connection, phenylpropane derivatives, such as:

Coumarins, such umbelliferon, scopoletin, methoxypsoralen, ksantotoksina and coumarin

The flavan-3-Ola, like (-)-epigallocatechin, (-)-epicatechin, (+)-catechin and (-)-epicatechine

- Synthetic compounds such as flavonoids and coumarins such as coumarin 106, 2'-methoxy-α -naphtoflavone, 6,2'-dimethoxyflavone, 6-methylcoumarin, alpha naphthoflavone, rotenone, 7 diethylamino-3-tenormin

- Natural plant extracts, such as extracts of Mentha longifolia, Mentha arvensis, Galeopsis speciosa, Salvia officinalis, Thymus vulgaris, Rumex acetocella, Rosa rugosa, Veronica longifolia, Symphytum asperum, Artemisia vulgaris, Convallaria majalis, Quercus robur, Daucus carota, Fragaria iinumae, Brassica oleracea, Brassica napus, Medicago sativa, Citrus sinensis, Phloem flour, Vaccinum myrtillus.

Derived from plants final the passed connection containing extracts and partial fractions, can accordingly be obtained from natural plants or their parts using standard extraction and excretion of substances. The upper part of the plants, roots or leaves accordingly distil water and which macerate or only distil water in order to obtain the desired extract, which then can be cleaned using any standard purification methods known to the person skilled in the art. The corresponding synthetic compounds and their derivatives are usually substances that are available from commercial sources or can be obtained by using known methods of synthesis.

In the following the following table 1 presents some preferred derived from plants phenolic compounds according to this invention, the effect of compounds on calcium influx into the cell, antioxidant activity and chemical structure of the compounds.

The chemical structure of the compounds presented in table 1, is shown in the following diagrams a, b, C, D and E. In the diagram And the flavones, flavonols, the diagram In - flavanones, in scheme With the isoflavones, the diagram D is derived phenylmethane, and the diagram E - phenylpropane derivatives. The substituents R1-7refer to the appropriate functional groups, preveden the m in table 1.

Protivodiareynoe action derived from plants phenolic compounds quercetin, Morin, rhamnetin and octisalate on C. pneumoniae and C. trachomatis investigated in examples 1-4. All these compounds inhibited growth of S. pneumoniae at a concentration of 0.5 to 50 μm, and it is shown that quercetin, Morin and rhamnetin particularly effective against C. trachomatis in the case when used pre-treated cells-owners. Thus, the preferred compounds for the treatment and prevention of infections caused by C. pneumoniae, are, for example, phenolic compounds quercetin, Morin, rhamnetin and ACTIGALL isolated from natural materials, and for the treatment and prevention of infections caused by C. trachomatis, in turn, quercetin, Morin and rhamnetin and containing extracts and partial fractions.

Protivodiareynoe action obtained from other plant phenolic compounds, some synthetic flavonoids and coumarins, and mixtures derived from plants phenolic compounds, garlic when using a concentration of 50 μm, is also investigated in example 5, which shows a noticeable inhibitory effect. Similarly investigated p is hotelkeeping action of allicin, contained in garlic, S. pneumoniae, obtaining similar results.

Protivodiareynoe effect of natural plant extracts and extracts of edible plants investigated in example 6, showing excellent inhibitory effect.

The preferred compounds and extracts and fractions are those who have protivodiareynoe action (inhibition of the formation of inclusions), equal to or greater than 30%, and especially preferred those who have protivodiareynoe action, equal to or greater than 90% compared with the action specified in the examples.

The connection according to this invention, is usually effective against chlamydia, optionally an antioxidant and has an impact on the uptake of CA2+cell.

ACTIGALL is a compound widely used as a food additive.

As the active ingredients derived from plants phenolic compounds, the corresponding synthetic compounds and their derivatives and plant extracts, fractions and their mixtures can be dosed so that the daily intake in terms of the aglycone ranged from 25 mg to 3000 mg Derived from plants phenolic compounds and related synthetic compounds and their derivatives and plant extracts, fractions and their mixtures according to the of this invention can be prepared in the form of pharmaceutical preparations in the form of capsules, tablets, ointments, liquid preparations, or in other appropriate forms known to the person skilled in the art. The products contain the active ingredient so that the daily consumption ranged from 25 mg to 3000 mg in terms of the aglycone in the form of dosage forms, preferably, from 25 mg to 500 mg

Derived from plants phenolic compounds and related synthetic compounds and their derivatives and plant extracts, fractions and their mixtures also can as such be added to food and can be prepared in the form of compositions suitable for food products, such as medicines from herbs, spices, granules or the like, which can be used as such, as a Supplement to the daily diet or as functional foods with health benefits, also called health products, such as ready-to-eat foods, cereals, salad dressings, drinks, products based on milk, edible oils, frozen foods, freeze dried foods, specialized food products, potato chips, sealing sauces, etc. depending on the product. Derived from plants phenolic compounds and related synthetic compounds and their derivatives may be present in the compositions according to this invention either in the form agli is she, or in glycosidic form.

Derived from plants phenolic compounds and related synthetic compounds and their derivatives and plant extracts, fractions and their mixtures according to this invention as compounds are harmless. Compositions and preparations according to the invention can be used in the treatment of acute chlamydial infection, and dosed reception of a composition or mixture continuously and regularly with daily meals in order to prevent chlamydial infection. Because chronic coronary heart disease causes of deaths in many cases, such as AMI, and on the other hand, treatment of diseases of the heart and blood vessels is the reason for the high cost to the national economy, using the obtained from a plant phenolic compounds and related synthetic compounds and their derivatives and plant extracts, fractions and their mixtures according to this invention and containing their new compositions can largely prevent and slow the emergence and outbreaks of diseases of the heart and blood vessels, especially in risk groups. Compositions and preparations according to the invention can also be used for the treatment and prevention of acute infection with C. trachomatis, and to Dr. the prevention of late complications such as infertility, ectopic pregnancy and cervical cancer, as well as for treatment and prevention of other associated with chlamydia infections and complications.

More in detail the invention is shown in the following examples, but the invention is in no way limited by the examples. In the examples described direct protivodiareynoe action of the compounds and plant extracts according to the invention for C. pneumoniae and C. trachomatis, as well as the toxicity of the compounds and extracts in relation to the cells of the host (HL cells, lung tissue of a person, the standard line of diploid cells).

Example 1

Direct protivodiareynoe action derived from plants phenolic compounds (inhibition of the formation of inclusions)/S. pneumoniae strain C7 (clinical isolate)

Results/Concentration of 50 µm:
Connection Concentration of 50 µmInhibition (% of control DMSOInhibition (% of control DMSO (pre-processed)*
Quercetin9090
Maureen9996
Rhamnetin9959
ACTIGALL100100
DMSO00
* Cells of the host before infection were incubated for 1 day with the connection that you want to explore. In 24-hole tablets containing cells of the owners, was added to the connection that you want to explore, in 1 ml of a supportive environment in the same concentration as in the test procedure.

The aim of this study was to investigate the possible activity of a compound on the cells themselves are the masters, where the specified action can be a factor inhibiting the infection. The testing procedure was continued as described in the method definition.

Results/Concentration of 0.5 µm:
Connection Concentration of 0.5 µmInhibition (% of control DMSOInhibition (% of control DMSO (pre-processed)*
Quercetin6877
Maureen8062
Rhamnetin7350
ACTIGALL8262
DMSO00
* Cells of the host to infection were incubated for 1 day with the connection that you want to explore. In 24-hole is lansey, containing cells of the owners, was added to the connection that you want to explore, in 1 ml of a supportive environment in the same concentration as in the test procedure.

The aim of this study was to investigate the possible activity of a compound on the cells themselves are the masters, the specified action may be a factor inhibiting the infection. The testing procedure was continued as described in the method definition.

Example 2

Inhibition of infectivity/S. pneumoniae

The aim of this study was to investigate the possible activity of a compound on the cells themselves are the masters, the specified action may be a factor inhibiting the infection. The testing procedure was continued as described in the method definition.

Results/Concentration of 0.5 µm:
Connection Concentration of 0.5 µmInhibition (% of control DMSOInhibition (% of control DMSO (pre-processed)*
Quercetin580
Maureen8153
Rhamnetin10075
ACTIGALL5942
DMSO0 0
* Cells of the host to infection were incubated for 1 day with the connection that you want to explore. In 24-hole tablets containing cells of the owners, was added to the connection that you want to explore, in 1 ml of a supportive environment in the same concentration as in the test procedure.

The aim of this study was to investigate the possible activity of a compound on the cells themselves are the masters, the specified action may be a factor inhibiting the infection. The testing procedure was continued as described in the method definition.

Example 3

Direct protivodiareynoe effect on C. trachomatis

C. trachomatis, culturing the cells Msso. In the rest of the testing methodology is the same as for S. pneumoniae.

Results/Concentration of 50 µm:
Connection Concentration of 50 µmInhibition (% of control DMSOInhibition (% of control DMSO (pre-processed)*
Quercetin0100
Maureen0100
Rhamnetin0100
ACTIGALL1488
* Tile and host to infection were incubated for 1 day with the connection, want to explore. In 24-hole tablets containing cells of the owners, was added to the connection that you want to explore, in 1 ml of a supportive environment in the same concentration as in the test procedure.

The aim of this study was to investigate the possible activity of a compound on the cells themselves are the masters, the specified action may be a factor inhibiting the infection. The testing procedure was continued as described in the method definition.

Results/Concentration of 0.5 µm:
Connection Concentration of 0.5 µmInhibition (% of control DMSOInhibition (% of control DMSO (pre-processed)*
Quercetin020
Maureen0100
Rhamnetin017
OG00
* Cells of the host to infection were incubated for 1 day with the connection that you want to explore. In 24-hole tablets containing cells of the owners, was added to the connection that you want to explore, in 1 ml of a supportive environment in the same concentration as in the procedure tested the I.

The aim of this study was to investigate the possible activity of a compound on the cells themselves are the masters, the specified action may be a factor inhibiting the infection. The testing procedure was continued as described in the method definition.

Example 4

Determination of the toxicity of some of the studied samples in relation to the cells of the host

The determination was performed in the same way as in the previous tests, but in the absence of any infection. Viability was determined by staining Trifanova blue.

Samples:

Q=quercetin

M=Maureen

R=rhamnetin

OG=ACTIGALL

HL-C=cells HL only in a nutrient medium

HL-CD=HL cells with the addition of DMSO

Example 5

Direct protivodiareynoe action derived from plants phenolic compounds, some synthetic compounds and mixtures on S. pneumoniae

The used concentration was 50 μm.

Example 6

Effect of plant extracts against S. pneumoniae

Conducted initial screening 101 extract obtained from 61 material nature and food plants against S. pneumoniae. The used concentration was 40 μg/well.

Below are the results from the ATA for the most active natural plant extracts, against S. pneumoniae, calculated on the basis of 4 different definitions.

100
PlantFamilyInterevene, %Survival

ness
N48. Mentha longifoiiaLabiateae100Norma
N53. Mentha arvensisLabiateae100Norma
N44. Galeopsis speciosaLabiateae100Norma
N57. Salvia officinalisLabiateae100Norma
N57. Salvia officinalisLabiateae100Norma
N58. Thymus vulgarisLabiateae100Norma
N34. Rumex acetocellaPolvgonaceae100reduced
N30. Rosa rugosaRosaceae100Norma
N28. Veronica longifoliaScrophulariaceae100reduced
The N8. Symphytum asperumSpread over100reduced
N22. Artemisia vulgarisAsteraceae100Norma
N37. Convallaria majalisConvallariaceae100-
Quercus roburEagaceae-

Below are the results for the selected most active extracts of edible plants, which showed 100% inhibition of S. pneumoniae, calculated on the basis of 4 different definitions.

PlantFamilyInhibition, %Survival

ness
D8. Daucus carotaUmbelliferae100-
D14. Fragaria iinumaeRosaceae100reduced
D16. Brassica oleraceaCruciferae100Norma
D17. Brassica napusCruciferae100Norma
D21. Medicago sativaLeguminosae100Norma
D23. Citrus sinensisRutaceae100reduced
D25. Phloem flourPolygonaceae100Norma
D30. Vaccinum myrtillusEricaceae100Norma
D31. Vaccinum myrtillusEricaceae100Norma

No significant differences in activity between organically grown and commonly cultivated plants.

In this study, plant extracts, which showed 100% inhibition of inclusions S. pneumoniae (n=4)was considered active. Found that five of the plants that belong to the family Labiateae, were especially active. Despite the method of extraction, as distilled water and maseribane, and only distilled water extracts of Salvia officinalis, were active against S. pneumoniae.

The following is a description of the used microbiological methods.

1. CULTIVATION AND PASSAGES of CELLS HL

Passages of cell culture HL carried out at intervals of 3 days. Cells-the owners were sown on the day before infection. Cells were washed in PBS 1× 10 ml and collected by trypsinization (1:10; 1,5-2,0 ml/bottle; about 5 minutes in the box with a laminar flow or 2 min in CO2at +37°). Cell suspension was diluted to approximately 350,000 cells/ml of culture medium (RN). Cultivation was carried out at +37° WITH2(5,0%) and environment RN changed 1-2 times per week. Cells HL can samarajiva is in liquid nitrogen [1 ml of 7.5% FCS (RN)+1 ml of DMSO].

2. Clear CHLAMYDIA

ET (elementary body)=intracellular infectious form of chlamydia. The day before defrost the required amount of suspension of HL cells infected with chlamydia. The cells thoroughly suspended and incubated in an ultrasonic bath just for 2 minutes [20 seconds of sonification (amplitude 24-25) and 10 seconds cooling × 6]. Cells are destroyed, and chlamydia remain intact.

The suspension is centrifuged for 10 minutes at 1600 rpm (HD), with chlamydia are nadeshiko (residues cells HL drop). Adosados sucked off and add 5 ml of PBS, suspended and treated with ultrasound for 1 minute (10 seconds sonification 5 seconds cooling × 6).

Chlamydia can be stored frozen at -70° C.

3. TESTING METHODOLOGY CHLAMYDIA

Cultured cells HL infect ET S. pneumoniae. EB, which penetrate into the cell, become metabolically active reticular cells (RT)that undergo binary fission in vacuole-endosome or enable. After a certain period of time (approximately 72 hours) RT S. pneumoniae again condensed into THIS, then enable destroyed, cell owners are destroyed and ET are released. In this way the aim is to identify up to the destruction of host cells.

As kladionica C. pneumoniae in vitro using cells HL. As the host cells of C. trachomatis in vitro often use cells Msso.

Infection of HL cells and cells MSO

On the day prior to infection, cells-the owners were seeded in 24-well plate: cells are harvested by trypsinization, suspended in about 5 ml of culture fluid and counted in the chamber Barker. Each well seeded using concentration 250000-400000 cells/well for cultivation. Before adding cells into the hole if necessary, put a round cover glass (diameter 13 mm) for staining. Add nutritious liquid so that the volume was 1 ml/well. The next day cells infect desired bacteria.

The entire solution used for infection and containing particles of chlamydia, mix. Old nutrient liquid is sucked off. Infected planting material, which was stored at -70° C, diluted to a concentration of IFU, approximately equal to 103so that the volume of solution was at least 200 µl/well in 24-hole tablet. Cells infect centrifugation at 1600 rpm (550× g)/1 hour. The nutrient medium is sucked off and replace the supporting medium containing cycloheximide, as well as containing the analyzed compound (concentration of DMSO is 0.2%), 1 ml/well. Cells are incubated in an atmosphere of 5% the O 2at +35° C. Cells infected with C. pneumoniae, incubated for 3 days. 2-3 days a supportive environment were removed and the cells washed with 1× 1 ml PBS. In wells add 200 μl of SPG for further infection, collect the tip of a pipette and transferred into a test tube. The resulting mixture was used to infect new cells in the same way as described above to test the inhibition of infectivity (humidistatic actions).

Staining of chlamydia

Nutrient medium, which leave on top of cover glass for coloring, sucked off. Infected cells fixed on top of the glass with methanol for 10 minutes Cover glass is removed from the wells and transferred to a suitable conjugated with fluorescein monoclonal antibody on parafilm in a humid chamber side containing cells down. Top glass incubated for 30 min at +37° and twice washed with PBS and once with water. Finally the glass is dried. Cover glass is put down the side containing the cells on a glass slide containing a latch (e.g., environment Mounting). When viewed in a fluorescence microscope samples of cell culture, is not damaged by chlamydia exhibit characteristic fluorescence inclusions green Apple unlike red oppositely colored background.

Chemical substances is TBA and reagents

RN=nutrient medium with FCS: 100 ml RPMI 1640 (Sigma), which added 3.5% of L-glutamine and 10 mg streptomycin (final concentration 20 μg/ml), 7.5 ml of FCS. Complete solutions stored at +8° C.

Supportive environment: 100 ml of culture medium with FCS with the addition of 50 μg cycloheximide at a final concentration of 0.5 μg/ml of Ready-made solutions stored at +8° C.

PBS (phosphate-saline buffer at Dulbecco, Gibco), pH 7,4.

SPG=sucrose 0.2 M (37.5 g), KN2RHO43.8 mm (0.26 g), Na2HPO4×2H2O 6.7 mm (0,61 g), glutamic acid (C5H9NO4) 5 mm (0.36 g), which was mixed with 500 ml of water milli-Q. After sterilization stored at -20° C.

FCS, fetal calf serum (Gibco, Scotland), inactivate at 56° C, 30 min, filtered and stored at -70° C.

1. Pharmaceutical composition for treatment and/or prevention of chlamydial infections caused by C. pneumoniae, characterized in that the composition contains obtained from a plant phenolic compound selected from the apigenin, luteolin, a flavone, quercetin, quercitrin, rhamnetin, isorhamnetin, Morin, daidzein, genistein, caffeic acid, naringin, which is Mergellina, propylgallate, octisalate, dodecylsulfate, isopropylmalate, resveratrol, umbelliferon, scopoletin is, metoksipsoralena, ksantotoksina, coumarin, or a synthetic compound selected from coumarin 106, coumarin 102, 2'-methoxy-α-naphtoflavone, 6,2'-dimethoxyflavone, 6-methylcoumarin, α-naphtoflavone, rotenone, coumarin 30, 3 antivent(F)of coumarin and 7-diethylamino-3-tenormin, or an extract or fraction, or incomplete fraction containing the specified connection, or a corresponding synthetic compound or a mixture of these compounds, and the fact that protivodiareynoe effect on S. pneumoniae obtained from a plant phenolic compound, or an extract or fraction, or incomplete fractions containing the specified connection, or corresponding synthetic compounds, is equal to or greater than 30% inhibition of the formation of inclusions, which is defined in the examples.

2. The composition according to claim 1, characterized in that protivodiareynoe effect on S. pneumoniae obtained from a plant phenolic compound, or an extract or fraction, or incomplete fractions containing the specified connection, or corresponding synthetic compounds is equal to or greater than 90% inhibition of the formation of inclusions, which is defined in the examples.

3. The composition according to claim 1 or 2, characterized in that the composition comprises a compound selected from the apigenin, luteolin, a flavone, quercetin, Morin, rhamnetin, Mergellina, propylgallate, OCTI is gallate, dodecylsulfate, isopropylmalate, scopoletin, metoksipsoralena, ksantotoksina, coumarin 106, α-naphtoflavone, rotenone and 7 diethylamino-3-tenormin.

4. The composition according to claim 1 or 2, characterized in that the extract or fraction, or incomplete fraction represents an extract of natural plants or extracts of edible plants.

5. The composition according to claim 4, characterized in that the extract or fraction, or incomplete fraction represents an extract from Mentha longifolia, Mentha arvensis, Galeopsis speciosa, Salvia officinalis, Thymus vulgaris, Rumex acetocella, Rosa rugosa, Veronica longifolia, Symphytum asperum, Artemisia vulgaris, Convallaria majalis, Quercus robur, Daucus carota, Fragaria iinumae, Brassica oleracea, Brassica napus, Medicago sativa, Citrus sinensis, Vaccinum myrtillus or Phloem flour.

6. The composition according to claim 1, characterized in that the composition contains in terms of the aglycone from 25 to 3000 mg obtained from a plant phenolic compound, or an extract or fraction, or incomplete fractions containing the specified connection, or corresponding synthetic compounds in the form of aglycone or glycoside form, and optionally the composition comprises structurae compound derived from garlic.

7. The composition is useful for health, with protivolednym action against S. pneumoniae, characterized in that the composition contains obtained from a plant phenolic compound selected from the apigenin, luteolin, a flavone, quercetin, is verzieren, rhamnetin, naringin, which isorhamnetin, Morin, daidzein, genistein, caffeic acid, Mergellina, propylgallate, octisalate, dodecylsulfate, isopropylmalate, resveratrol, umbelliferon, scopoletin, metoksipsoralena, ksantotoksina, coumarin, or a synthetic compound selected from coumarin 106, coumarin 102, 2'-methoxy-α-naphtoflavone, 6,2'-dimethoxyflavone, 6-methylcoumarin, α-naphtoflavone, rotenone, coumarin 30, 3 antivent(F)of coumarin and 7-diethylamino-3-tenormin, or extract or faction, or incomplete fraction containing the specified connection, or a corresponding synthetic compound or a mixture of these compounds.

8. The composition is useful for health, according to claim 7, characterized in that protivodiareynoe effect on S. pneumoniae obtained from a plant phenolic compound, or an extract or fraction, or incomplete fractions containing the specified connection, or corresponding synthetic compounds, is equal to or greater than 30% inhibition of the formation of inclusions, which is defined in the examples.

9. The composition is useful for health, according to claim 7, characterized in that protivodiareynoe effect on S. pneumoniae obtained from a plant phenolic compound, or an extract or fraction, or incomplete fractions containing the specified connection, or corresponding synthetic modifications is to be placed, equal to or greater than 90% inhibition of the formation of inclusions, which is defined in the examples.

10. The composition is useful for health, according to claim 9, characterized in that the composition comprises a compound selected from the apigenin, luteolin, a flavone, quercetin, Morin, rhamnetin, Mergellina, propylgallate, octisalate, dodecylsulfate, isopropylmalate, scopoletin, metoksipsoralena, ksantotoksina, coumarin 106, α-naphtoflavone, rotenone and 7 diethylamino-3-tenormin.

11. The composition is useful for health, according to claim 9, characterized in that the extract or fraction, or incomplete fraction represents an extract of natural plants or extracts of edible plants.

12. The composition is useful for health, according to claim 11, characterized in that the extract or fraction, or incomplete fraction represents an extract from Mentha longifolia, Mentha arvensis, Galeopsis speciosa, Salvia officinalis, Thymus vulgaris, Rumex acetocella, Rosa rugosa, Veronica longifolia, Symphytum asperum, Artemisia vulgaris, Convallaria majalis, Quercus robur, Daucus carota, Fragaria iinumae, Brassica oleracea, Brassica napus, Medicago sativa, Citrus sinensis, Vaccinum myrtillus or Phloem flour.

13. The composition is useful for health, according to claim 7, characterized in that the composition contains in terms of the aglycone from 25 to 3000 mg obtained from a plant phenolic compound, or an extract or fraction, or incomplete fractions containing the specified connection, or corresponding synthetic soedineniya the form of aglycone or glycoside form, and optionally the composition comprises structurae compound derived from garlic.

14. The use of a composition, useful for health, according to claim 7 in the preparation of food products as such or as supplements to the daily diet.

15. The application of plant phenolic compounds, selected from apigenin, luteolin, a flavone, quercetin, quercitrin, rhamnetin, isorhamnetin, Morin, daidzein, genistein, caffeic acid, Mergellina, propylgallate, octisalate, dodecylsulfate, isopropylmalate, resveratrol, umbelliferon, scopoletin, metoksipsoralena, ksantotoksina, naringin, which coumarin derivatives, or synthetic compounds selected from coumarin 106, coumarin 102, 2'-methoxy-α-naphtoflavone, 6,2'-dimethoxyflavone, 6-methylcoumarin, α-naphtoflavone, rotenone, coumarin 30 3 antivent(F)of coumarin and 7-diethylamino-3-tenormin, or extract, or fraction, or incomplete fractions containing the specified connection, or corresponding synthetic compounds, or mixtures of these compounds, in the manufacture of medicinal products for the treatment and/or prevention of chlamydial infections caused by C. pneumoniae.

16. The application of clause 15, wherein protivodiareynoe effect on S. pneumoniae obtained from a plant phenolic compound, or an extract or fraction, or Nepal the second fraction, containing the specified connection, or corresponding synthetic compounds, is equal to or greater than 30% inhibition of the formation of inclusions, which is defined in the examples.

17. The application of clause 15, wherein protivodiareynoe effect on S. pneumoniae obtained from a plant phenolic compound, or an extract or fraction, or incomplete fractions containing the specified connection, or corresponding synthetic compounds, is equal to or greater than 90% inhibition of the formation of inclusions, which is defined in the examples.

18. The application 17, characterized in that the composition comprises a compound selected from the apigenin, luteolin, a flavone, quercetin, Morin, rhamnetin, Mergellina, propylgallate, octisalate, dodecylsulfate, isopropylmalate, scopoletin, metoksipsoralena, ksantotoksina, coumarin 106, α-naphtoflavone, rotenone and 7 diethylamino-3-tenormin.

19. The application 17, characterized in that the extract or fraction, or incomplete fraction represents an extract of natural plants or extracts of edible plants.

20. The application of claim 19, characterized in that the extract or fraction, or incomplete fraction represents an extract from Mentha longifolia, Mentha arvensis, Galeopsis speciosa, Salvia officinalis, Thymus vulgaris, Rumex acetocella, Rosa rugosa, Veronica longifolia, Symphytum aspemm, Artemisia vulgaris, Convallaria majalis, Quercus robur, Daucus carota, Frgaria iinumae, Brassica oleracea, Brassica napus, Medicago sativa, Citrus sinensis, Vaccinum myrtillus or Phloem flour.

21. The application of clause 15, wherein the composition contains in terms of the aglycone from 25 to 3000 mg obtained from a plant phenolic compound, or an extract or fraction, or incomplete fractions containing the specified connection, or corresponding synthetic compounds in the form of aglycone or glycoside form, and optionally the composition comprises structurae compound derived from garlic.



 

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