Propolis extract with active fractions titrated into bioactive components and method of its production

Abstract

The invention relates to a propolis extract with active fractions titrated into bioactive components, characterized in that it consists of flavonoids and chalcones in a percentage of 47% to 78%, esters in a percentage of 3% to 18%, sesquiterpenes in a percentage of 14% to 22%, triterpenes at a rate of 2% to 4% and other components including aliphatic acids, aromatic acids and sugars at a rate of 3% to 4%. The invention also relates to a method for the production of propolis extract with active fractions titrated to bioactive components.

Description

[0001] PROPOLIS EXTRACT WITH ACTIVE FRACTIONS TITRATED INTO BIOACTIVE COMPONENTS AND METHOD OF ITS PRODUCTION

[0002] DESCRIPTION

[0003] Technical field of the invention

[0004] The invention refers to a propolis extract and its production method which is characterized by the fact that contains active fractions, titrated into bioactive components, with selective antimicrobial action related to the inhibition of the growth of pathogenic and potentially pathogenic microorganisms of the human skin, while preserving the normal microorganisms. At the same time, it has an effect against the creation of a biofilm of the bacterium Cutibacterium acnes (C. acnes), which additionally exhibits beneficial properties on intrinsic antimicrobial / anti-inflammatory mechanisms of human epidermal cells.

[0005] Technological background of the invention

[0006] Bee products represent an important class of natural products that exhibit specific health benefits. All bee products - honey, royal jelly, propolis, bee pollen, beeswax and even bee venom - have been extensively researched for their healing properties. (Denisow and Denisow-Pietrzyk, 2016; Kocot et al., 2018; Zhang et al., 2018; Munstedt and Mannle, 2019).

[0007] Propolis is a resinous substance produced by bees. It exhibits antimicrobial, immunostimulating and antioxidant activity and is used in the preparation of functional foods and cosmetics as well as in traditional medicine (Ravesteyn etal., 2017; Zabaiou et al., 2017; Kocot et al., 2018; Przybylek and Karpinski, 2019). Its composition varies depending on the flora of the foraging area of the bees and the collection season. Contains about 50% resins, 30% waxes, 10% aromatics, 5% pollen and 5% miscellaneous ingredients (Kocot et al., 2018; Anjum et al., 2019; El-Guendouz, Lyoussi and Miguel, 2019). The bioactive components of propolis are polyphenols, terpenes, as well as sugars and amino acids (Pickard et al., 2003; Saavedra et al., 2016; Aminimoghadamfarouj and Nematollahi, 2017; Popova et al., 2017; Vukovic et al., 2018; Misir et al., 2020). The main polyphenols are flavonoids and phenolic acids. The complexity of the propolis structure combined with the variable composition depending on the region and season of collection make it difficult to formulate into products for per os intake or products applied to the skin (Kapare etal., 2017; Zabaiou et al., 2017).

[0008] Propolis extraction has the disadvantage that it usually has a low yield in active concentration of components (Devequi-Nunes et al., 2018; Kubiliene et al., 2018), especially when using natural and skin-nontoxic solvents such as water or vegetable glycerin. The reason is mainly the hydrophobic nature of most propolis' active ingredients.

[0009] Also, due to the complexity of the composition of propolis, it has not been clarified which specific components are responsible for specific biological actions. Various works have assigned propolis fractions with antimicrobial and antioxidant activity (Przybylek and Karpinski, 2019; Nichitoi et al., 2021 ), no methods are generally described that accurately titrate propolis extracts into active components that are associated with specific biological actions.

[0010] In order to increase the extraction efficiency, various extraction methods have been developed (Jug, Karas and Kosalec, 2017; Cavalaro et al., 2019) as well as methods of encapsulation of its components in various carriers (Andrade et al., 2018; de Melo Silva et al., 2020).

[0011] Various propolis extracts have shown antimicrobial activity against pathogenic microorganisms such as bacteria of the genus Salmonella spp but also against microorganisms found in the microflora of the skin (microbiome) such as bacteria Staphylococcus epidermidis (Przybylek I 2019). Microorganisms (bacteria, fungi and viruses), living symbiotically on the skin, are thought to provide protection against invading pathogens by creating a natural barrier and aiding the immune defense (Byrd A 2018). Disturbances of the 'healthy' microflora have been linked to skin diseases such as acne (acne vulgaris) and atopic dermatitis (Rocha MA 2018, Kobayashi T et.al 2015). Considering the contribution of the microbiome to the integrity of the skin, products intended for direct application to the skin should aim to inhibit pathogenic or potentially (opportunistically) pathogenic microorganisms without affecting the qualitative and quantitative composition of the microbiome.

[0012] The antimicrobial action of propolis is focused on two levels, namely, a) direct action against the microorganism, inhibiting its growth and biofilm formation, and b) stimulating the host's immune system with the aim of activating the body's natural defenses (Przybylek and Karpinski, 2019) (Sforcin and Bankova, 201 1 ).

[0013] Biofilms are defined as consortia of microorganisms attached to a biotic or abiotic surface. Biofilm formation is a multistep process in which microbial cells adhere to the surface (initial reversible adhesion), while the subsequent production of an extracellular matrix (containing polysaccharides, proteins and DNA) results in a more stable attachment. Cells embedded in this matrix communicate with each other and show a coordinated group behaviour, mediated by a process called quorum sensing (QS) (Azimi S. 2020). Immobilized (biofilm-associated) cells are phenotypically and physiologically different from non-adherent (planktonic) cells, and one of the typical properties of immobilized cells is their increased resistance to antimicrobial agents. Biofilm formation is often considered the main reason why treatment with an antimicrobial agent fails, and it is estimated that 65-80% of all infections are associated with biofilm formation. (Nakamura et aL, 2020).

[0014] The prevention of the formation of biofilms and the action against the established biofilm, are a strategy for the development of new anti-pathogenic treatments. An important role in the above actions is the obstruction of the ability of bacteria to adhere as well as the inhibition of cell-to-cell communication mediated by QS systems (Pecoraro C, 2023).

[0015] The stage of bacterial attachment to the host is considered the first stage of bacterial pathogenesis as well as biofilm formation. Important factors in the fundamental stage of adhesion are bacterial virulence factors. These are bacterial molecules used by pathogens to colonize the host at the cellular level. These factors may be secreted, cytosolic, or membrane associated. Secreted factors are important tools used by bacteria to escape the host's innate and adaptive immune response. Cytosolic factors are involved in metabolic, physiological, and morphological adaptive processes, while membrane-associated virulence factors confer the bacterium the ability to adhere to host tissues and biomaterials (Dufrene, Y.F. 2020) (Parrino, 2018).

[0016] QS is a chemical communication between bacteria that coordinates gene expression at the community level. Recent studies have identified many natural and synthetic compounds as QS inhibitors (QQ compounds)(Rasmussen & Givskov, 2006). Quorum Quenching (QQ) respectively, is the enzymatic degradation / blocking of molecules used by bacteria to synchronize their behaviour within communities. QQ has attracted the interest of the scientific community due to its ability to inhibit biofilm formation and suppress the production of virulence factors. Several different QQ compounds have been described that vary in stability and catalytic performance. A preliminary screening for QS inhibitors revealed that propolis is a substance that exhibited qualitative QS inhibitory activity making it interesting to investigate the potential action of propolis as a QQ compound (Bovbjerg et aL, 2005).

[0017] Inhibition of the QS system and biofilm formation is an attractive target for the treatment of pathological conditions resulting from the action of the bacterium C. acnes. C. acnes is a Gram+ bacterium that is included in the normal flora of the skin in lipid-rich areas and has a proven role in the pathogenesis of acne. It has been verified that it can form biofilms and that C. acnes in the biofilm is more resistant to antibiotics than bacteria in the planktonic growth phase (Ramage et aL, 2003). C. acnes can form a biofilm both in vitro and in vivo (Bayston, Ashraf, et aL, 2007; Bayston, Nuradeen, et aL, 2007; Coenye et aL, 2007; Holmberg et aL, 2009b; Qi et aL, 2008). Lipase production, which is a putative determinant of its virulence, was found to be expressed at an increased rate during the biofilm formation phase of bacterial function (Coenye et aL, 2007).

[0018] On the skin, C. acnes can behave as an opportunistic pathogen, depending on the strain and environmental conditions. The acne-causing strains of C. acnes form biofilms within skin-gland cavities, causing inflammation and skin disorders. The essential exogenous products of C. acnes accumulate in the extracellular matrix of the biofilm, conferring essential bacterial functions to this structure. The biofilm of C. acnes acne strains consists mainly of 62.6% polysaccharides, 9.6% proteins, 4.0% DNA and 23.8% other compounds (porphyrin precursors and others) (Gannesen et aL, 2019). In the literature there are very few reports on the activity of propolis against C. acnes, while there are no reports of its activity against the biofilm of this particular bacterium. The present invention relates to the method of producing a propolis extract with a specific composition, which exhibits antimicrobial activity against pathogenic microorganisms of various anatomical areas of the human body, while at the same time it does not disturb the balance of the remaining native bacteria of the skin flora. In addition, it exhibits specific biological activity on the quorum sensing phenomenon and on the colonization ability of the C. acnes bacterium.

[0019] The process involves the quantification of specific polyphenols and terpenes from propolis of various regions based on the flora to which the bees have access, the determination of specific ratios from these propolis to achieve the specific concentration of components and the extraction of the propolis. The final extract, with the specific ratio of components shows specific bioactivity.

[0020] To create the present invention, the in vitro effects of specific proportions of propolis polyphenols, in combination with specific propolis terpenes, were investigated in order to determine:

[0021] A. The action against pathogenic microorganisms of the microflora of specific skin areas, without a harmful effect on microorganisms of the skin-friendly microflora

[0022] B. the effect against genes related to the mechanisms of attachment and colonization of the bacterium C. acnes

[0023] C. inhibition of C.acnes biofilm formation and growth and quorum sensing mechanism (Quorum Quenching)

[0024] D. Transcriptional regulation of genes associated with antimicrobial and antiinflammatory activity in human keratinocytes (NHEK)

[0025] After studying all possible combinations the optimal concentration was standardized with a specific extraction method.

[0026] The present invention provides a composition that exhibits antioxidant, antimicrobial and anti-inflammatory properties. The composition of the present invention has as its main advantage that it reduces the formation and growth of biofilms of the C. acnes bacterium and the quorum detection mechanism, it acts by reducing pathogenic microorganisms of the microflora of specific skin areas, without a harmful effect on microorganisms of the friendly skin microflora and at the cellular level regulates the expression of genes related to antimicrobial and anti-inflammatory activity in keratinocytes.

[0027] Detailed description of the invention

[0028] Figure 1 : Expression of the roxP gene following exposure of C. acnes to PTE synthesis Figure 2: Determination of the activity of the component against C. acnes biofilm formation

[0029] Figure 3: Inhibition rate by the active already pre-installed C.acnes biofilm Figure 4 : Figure 4: Analysis of the expression of genes related to antimicrobial and antiinflammatory activity in keratinocytes incubated with 0.01 % and 0.03% PTE

[0030] The invention concerns a propolis extract with active fractions titrated into bioactive components, characterized in that it consists of flavonoids and chaicones in a percentage of 47% to 78%, esters in a percentage of 3% to 18%, sesquiterpenes in a percentage of 14% to 22%, triterpenes at a rate of 2% to 4% and other components including aliphatic acids, aromatic acids and sugars at a rate of 3% to 4%.

[0031] According to other optional features of the propolis extract, it can optionally include one or more of the following characteristics alone or in combination: the flavonoids and chalcoes contain Methoxyllflavanone in a percentage of 21 % to 44% of their total, galangin in a percentage of 8% to 32% of their total, dihydroxymethoxyflavone at a rate of 8% to 1 1 % of their total, pinocembrin at a rate of 9% to 13% of their total, pinocembrin chalkone at a rate of 9% to 12% of their total, kaempferol at a rate of 8% up to 12% of their total and chaicones at a rate of 8% to 10%; the esters contain caffeate derivative in a percentage of 42% to 56% of their total, cinnamyl ester of caffeic acid in a percentage of 22% to 28% of their total, cinnamyl ester of isoferulic acid, from 16% to 22% of their total and other esters from 6% to 8% of their total, the sesquiterpenes contain gamma curcumene in a percentage of 16% to 19% of their total, curcumene in a percentage of 24% to 28% of the total, beta-gurjunene in a percentage from 40% to 48% of their total, cadinene from 7% to 15% of their total and other sesquiterpenes from 1 % to 2% of their total, the triterpenes contain ledene in a percentage greater than 85% of their total.

[0032] According to another aspect, the invention concerns a method for the production of propolis extract with active fractions titrated to bioactive components according to the invention, characterized in that the method comprises, a first stage comprising:

[0033] - the selection of types of propolis having a concentration of total polyphenols >900 mg / l gallic acid equivalents (GAE) after dissolving 10% by weight of propolis in ethanol and

[0034] - showing a peak in gas chromatography of the components Methoxylflavanone, galangin, dihydroxymethoxyflavone, pinocembrin, pinocembrin chalkone, kaempferol , caffeate derivative, cinnamyl ester of caffeic acid, cinnamyl ester of isoferulic acid, gamma curcumene, curcumene, beta-gurjunene, cadinene, ledene.

[0035] According to other optional features of the method, it can optionally include one or more of the following characteristics alone or in combination : it comprises a second stage comprising a purification of the propolis from waxes, it comprises a third stage comprising :

[0036] - an extraction of the propolis in which it is cut into small particles (<1 mm) after cooling it for 24 hours at -20°C, then

[0037] - a dispersion at a rate of 1 kg / min in the solvent system consisting of (1 .3- Propanediol) and glyceryl ether ethylhexylglycerin (Ethylhexylglycerin), in a ratio of Ethylhexylglycerin: 1 .3-Propanediol = 0 / 100 to Ethylhexylglycerin: 1 .3-Propanediol = 5 / 95,

[0038] - stirring at 500 - 1500 rpm, with a concentration of propolis from 9% to 32% by weight depending on its content of polyphenols,

[0039] - an extraction of the resulting suspension placed in an extractor assisted by a pressure of 1 bar to 9 bar at a temperature from 25°C to 35°C, including 7 to 36 compression cycles, lasting from 3' to 6' and corresponding decompression cycles lasting from 2' to 6',

[0040] - the mixture is then left in a hermetically closed container at rest at 5-7 °C for 24 hours and

[0041] - a filtration through an array of cartridge type filters with pore size 10 pm - 5 pm - 1 pm - 0.45 pm, it comprises a fourth step comprising a quantification of the components of the extract, where first of all the total polyphenol content is required to be greater than 900 mg / L GAE and a quantification of groups of components flavonoids and chaicones / esters I sesquiterpenes I triterpenes I other components by gas chromatography after the generation of silyl-derivatives, it comprises a fifth stage comprising a mixture of the extracts produced in order to achieve the desired concentration of the individual components.

[0042] According to another aspect, the invention concerns a composition comprising a propolis extract with active fractions titrated into bioactive components according to the invention.

[0043] According to other optional features of the composition, it can optionally include one or more of the following characteristics alone or in combination a use in cosmetic application, a use in pharmaceutical application, for topical pharmaceutical or cosmetic use, for topical pharmaceutical or cosmetic use after formulation as a solution, suspension, emulsion, or gel, for topical pharmaceutical or cosmetic use after formulation as a solution, suspension, emulsion, or gel and exhibits antioxidant activity of 28.60 ± 3.95 mM Trolox Equivalent and antimicrobial properties, it comprises 0.1 %, 0.3%, 0.5% of Propolis Titrated Extract (PTE), its use in the reduction in vitro free radicals and against specific pathogens on the scalp, female sensitive area, axilla, and face, while remaining friendly to microorganisms that make up the normal microbial flora of the skin in said examined skin areas, for its use in reducing the formation and growth of biofilms of the C. acnes bacterium and the quorum detection mechanism, for its use in the regulation of the expression of genes related to antimicrobial and antiinflammatory activity in keratinocyte, for its use in transcriptional regulation of genes associated with antimicrobial and antiinflammatory activity in human keratinocytes (NHEK), for its use in reducing gene transcription implying in the mechanisms of attachment and colonization of the bacterium C. acnes,

[0044] The present invention relates to a composition that includes propolis components in specific proportions. These ingredients are: a) flavonoids and chaicones b) esters c) sesquiterpenes d) triterpenes e) other ingredients, including aliphatic acids, aromatic acids, sugars and their derivatives.

[0045] To produce the invented composition, a production method is followed which includes the following stages:

[0046] Stage 1 . Selection of different types of propolis of suitable specifications

[0047] For the production of the invented propolis extract, a combination of different types of propolis with specific specifications is required, namely:

[0048] 1 . Have a concentration of total polyphenols >900 mg / l gallic acid equivalents (GAE) after dissolving 10% by weight of propolis in ethanol and subsequent measurement in a spectrophotometer using the Folin-Ciocalteau method 2. To have a peak in gas chromatography of the components:

[0049] Methoxylflavanone, galangin, dihydroxymethoxyflavone, pinocembrin, pinocembrin chalkone, kaempferol, caffeate derivative, cinnamyl ester of caffeic acid, cinnamyl ester of isoferulic acid, gamma curcumene, curcumene, beta-gurjunene, cadinene, ledene.

[0050] Propolis samples are extracted with 70% ethanol and silyl-derivatives are followed (Popova, M.P.; Graikou, K.; Chinou, L; Bankova, V.S. 2010 GC-MS Profiling of Diterpene Compounds in Mediterranean Propolis from Greece. J. Agric. Food Chem, 58, 3167-3176).

[0051] Once the second condition is met, the preparation of PTE (Propolis Titrated Extract) can begin.

[0052] Stage 2. Cleaning propolis from waxes

[0053] The propolis is cut into small particles (<1 mm) after cooling it for 24 hours at -20°C. It is then dispersed in deionized water at a temperature of 5-7°C in a ratio of 1 :1 . The system is stirred under cooling for 15 min and then the supernatant water is discarded while filtration with filter paper under vacuum follows. The propolis is then cooled for 24 hours at -80° C and then lyophilized with conditions: condenser temperature -50° C, vacuum 8.2 x 10-2mb.

[0054] Stage 3. Extraction of propolis

[0055] The wax-free propolis is re-shredded into small particles (<1 mm) after cooling for 24 hours at -20°°C. It is then dispersed at a rate of 1 kg / min in the solvent system, which is under agitation 500 - 1500 rpm. The concentration of propolis can be from 9% to 32% by weight depending on its polyphenol content. The extraction solvent system consists of (1 .3-Propanediol) and the glyceryl ether ethylhexylglycerin (Ethylhexylglycerin) in a ratio of Ethylhexylglycerin: 1 .3-Propanediol = 0 / 100 to Ethylhexylglycerin: 1 .3-Propanediol = 5 / 95.

[0056] Ethyl-hexyl-glycerine is used as a co-solvent and as an enhancer of the action against the quorum-forming mechanism of C. acnes.

[0057] The resulting suspension is then placed in a pressure assisted extractor.

[0058] The additional extraction takes place under a pressure of 1 bar to 9 bar and at a temperature of 25°C to 35°C. The process includes 7 to 36 compression cycles, lasting 3' to 6' and corresponding decompression cycles lasting 2' to 6'. Then the mixture is left in a hermetically closed container at rest at 5-7 °C for 24 hours. It is then filtered by an array of cartridge-type filters with a pore size of 10 pm - 5 pm - 1 pm - 0.45 pm. Step 4. Quantification of extract components

[0059] For the extraction process to be considered successful, it is first required that the total polyphenol content be >900 mg / L GAE.

[0060] The component groups flavonoids and chaicones I esters I sesquiterpenes I triterpenes I other components are then quantified by gas chromatography after silyl derivatization.

[0061] 5. Mixing extracts to produce PTE

[0062] In the final stage, the extracts produced by the above method are mixed in order to achieve the following specific concentrations of components: flavonoids and chaicones in a percentage from 47% to 78%, esters from 3% to 18%, sesquiterpenes from 14% to 22%, triterpenes from 2% to 4% and other components from 3% to 4%.

[0063] In the individual chemical categories, the proportions must be as follows:

[0064] A) Flavonoids and chaicones

[0065] Methoxylflavanone from 21 % to 44%, galangin from 8% to 32%, dihydroxymethoxyflavone from 8% to 1 1 %, pinocembrin from 9% to 13%, pinocembrin chalkone from 9% to 12%, kaempferol from 8% to 12% and other flavonoids and chaicones from 8% to 10%. : 21 / 32 / 1 1 / 9 / 9 / 8 / 10

[0066] B) Esters caffeate derivative from 42% to 56%, cinnamyl ester of caffeic acid from 22% to 28% cinnamyl ester of isoferulic acid from 16% to 22% and other esters from 6% to 8%.

[0067] C) Sesquiterpenes

[0068] Gamma curcumene from 16% to 19%, curcumene from 24% to 28% beta-gurjunene from 40% to 48% cadinene from 7% to 15% and other sesquiterpenes from 1 % to 2%.

[0069] D) Triterpenes

[0070] Presence of ledene >85%.

[0071] The composition of the present invention is provided for topical pharmaceutical or cosmetic use after formulation as a solution, suspension, emulsion, or gel and exhibits antioxidant activity of 28.60 ± 3.95 mM Trolox Equivalen and antimicrobial properties. These properties are mediated by the composition's ability to reduce in vitro free radicals and the composition's ability to act against specific pathogens on the scalp, female sensitive area, axilla, and face, while remaining friendly to micro-organisms that make up the normal microbial flora of the skin in said examined skin areas.

[0072] These results are unprecedented, since the interaction of the different and specific active ingredients in the composition of the present invention could not have been foreseen. Such an interaction could result in the loss of potency of the individual active ingredients and / or the occurrence of adverse effects in vitro and in vivo.

[0073] Efficacy Documentation

[0074] To assess the action of PTE against an established biofilm, an extract was prepared according to the above method which showed component ratios as listed below: Solvents: 1 ,3 propanediol / ethyl hexyl glycerine 99:1 flavonoids and chaicones 56%, esters 17%, sesquiterpenes 20% triterpenes 3% other components 4%, with proportions of the individual components which are as follows:

[0075] A) Flavonoids and chaicones methoxylflavanone 29%, galangin 30%, dihydroxymethoxyflavone 8% pinocembrin 9% pinocembrin chalkone 9%, kaempferol 7%, other flavonoids and chaicones 8%

[0076] B) Esters caffeate derivative 48%, cinnamyl ester of caffeic acid 25%, cinnamyl ester of isoferulic acid 20% and other esters 7%.

[0077] C) Sesquiterpenes amma curcumene 16%, curcumene 28%, beta-gurjunene 46%, cadinene 9% and other sesquiterpenes 1%.

[0078] D) Triterpenes ledene 97%

[0079] It is clarified that PTE was diluted to final concentrations of 0.1 %, 0.3%, 0.5% w / w.

[0080] A. Study of the action of PTE against pathogenic microorganisms of the microflora of specific skin areas, without a harmful effect on microorganisms of the skin-friendly microflora.

[0081] The study consists of preparing solutions of different concentrations of PTE with a sterile diluent and infecting this preparation with specific microorganisms, which are commonly found on the skin of the scalp, the female sensitive area, the axilla, and the forehead. After 2 hours incubation at 37°C, the test microorganisms were enumerated by decimal dilutions in appropriate media. All assays were performed in triplicate and mean values are listed in the relevant data tables.

[0082] The test design includes four (4) different phases: a) Phase 1 - initial microbiological evaluation of the test product b) Phase 2 - evaluation of the effect of the product on the growth behaviour of skin microbes c) Phase 3 - evaluation of the effect of the product on pathogenic microflora and d) Phase 4 - the effect of the product on the biodiversity of the microbiome of the specific area

[0083] Phase 1 - initial microbiological evaluation of the test product

[0084] Phase 1 involved the evaluation of the microbiological quality of the sample, as received, according to ISO 17516:2014.

[0085] Table 1 . Microbiological control of the sample to be examined, where the absence of the above pathogenic microorganisms was confirmed.

[0086] Phase 2 - evaluation of the effect of the product on the growth behaviour of skin microbes

[0087] Phase 2 consisted of evaluating the effect of the product on the growth behaviour of skin microbes commonly found on the scalp, the sensitive area of women, the armpit area and the forehead. Three (3) different concentrations of PTE solution were used, in final concentrations of 0.1 %, 0.3%, 0.5%.

[0088] Phase 2 for concentration of active substance solution 0.1% Table 2. Effect of 0.1% PTE on a panel of scalp microorganisms

[0089] Table 3. Effect of 0.1% PTE on a panel of sensitive area microorganisms

[0090] Table 4. Effect of 0.1% PTE on a panel of axillary microorganisms Table 5. Effect of 0.1% PTE on a panel of forehead area microorganisms

[0091] Phase 2 for concentration of active substance solution 0.3%

[0092] Table 6. Effect of 0.3% PTE on a panel of scalp microorganisms Table 7. Effect of 0.3% PTE on a panel of sensitive area microorganisms

[0093] Table 8. Effect of 0.3% PTE on a panel of axillary microorganisms

[0094] Table 9. Effect of 0.3% PTE on a panel of forehead area microorganisms

[0095] Phase 2 for concentration of active substance solution 0.5%

[0096] Table 10. Effect of 0.5% PTE on a panel of scalp microorganisms

[0097] Table 1 1 . Effect of 0.5% PTE on a panel of sensitive area microorganisms

[0098] Table 12. Effect of 0.5% PTE on a panel of axillary microorganism Table 13. Effect of 0.5% PTE on a panel of forehead area microorganisms

[0099] Phase 3 - Evaluation of the effect of the product on pathogenic microflora

[0100] Phase 3 consisted of evaluating the effect of the product on the growth behaviour of pathogenic skin microbes found on the selected part of the body. Phase 3 for concentration of active substance solution 0.1%

[0101] Table 14. Effect of 0.1% PTE on a panel of scalp microorganisms Table 15. Effect of 0.1 % PTE on a panel of sensitive area microorganisms

[0102] Additionally, its action was examined selectively against Gardnerella vaginalis and Mycoplasma hominis:

[0103] Table 16. Effect of 0.1% PTE on a panel of axillary microorganisms Table 17. Effect of 0.1 % PTE on a panel of forehead area microorganisms Phase 3 for concentration of active substance solution 0.3%

[0104] Table 18. Effect of 0.3% PTE on a panel of scalp microorganisms Table 19. Effect of 0.3% PTE on a panel of sensitive area microorganisms

[0105] Additionally, its action was examined selectively against Gardnerella vaginalis and Mycoplasma hominis:

[0106] Table 20. Effect of 0.3% PTE on an axillary microorganisms panel.

[0107] Table 21 . Effect of 0.3% PTE on a panel of forehead area microorganisms

[0108] Phase 3 for concentration of active substance solution 0.5% Table 22. Effect of 0.5% PTE on a panel of scalp microorganisms

[0109] Table 23. Effect of 0.5% PTE on a panel of sensitive area microorganisms

[0110] Additionally, its action was examined selectively against Gardnerella vaginalis and Mycoplasma hominis: Table 24. Effect of 0.5% PTE on a panel of axillary microorganisms Table 24. Effect of 0.5% PTE on a panel of forehead area microorganisms

[0111] Phase 4 - Evaluation of the effect of the product on the biodiversity of the microbiome for each specific area

[0112] During Phase 4, a solution of the active substance was applied to the microbiome sample from each test area of a male and a female volunteer to analyze the in vivo effect of the product (except for the sensitive area microbiome for which diluted solutions of the active substance were applied to a mixture of microorganisms of phase 2). The results in the tables correspond to the average values of three tests.

[0113] Phase 4 for concentration of active substance solution 0.1%

[0114] Table 25. Effect of 0.1 % PTE on a mixture of scalp microorganisms from two volunteers

[0115] Table 26. Effect of 0.1 % PTE on a mixture of microorganisms found in the sensitive area

[0116] Table 27. Effect of 0.1% PTE on a mixture of axillary microorganisms from two volunteers

[0117] Table 28. Effect of 0.1% PTE on a mixture of microorganisms of the forehead area from two volunteers Phase 4 for concentration of active substance solution 0.3%

[0118] Table 29. Effect of 0.3% PTE on a mixture of scalp microorganisms from two volunteers Table 30. Effect of 0.3% PTE on a mixture of microorganisms found in the sensitive area

[0119] Table 31 . Effect of 0.3% PTE on axillary microorganisms from two volunteers

[0120] Table 32. Effect of 0.3% PTE on a mixture of microorganisms of the forehead area from two volunteers

[0121] Phase 4 for concentration of active substance solution 0.5%

[0122] Table 33. Effect of 0.5% PTE on a mixture of scalp microorganisms from two volunteers Table 34. Effect of 0.5% PTE on a mixture of microorganisms found in the sensitive area Table 35. Effect of 0.5% PTE on a mixture of axillary microorganisms from two volunteers

[0123] Table 36. Effect of 0.5% PTE on a mixture of microorganisms of the forehead area from two volunteers

[0124] In conclusion, at a concentration of 0.1%, the active ingredient reduces the concentration of pathogens Streptococcus viridans, Mycoplasma hominis and Gardnerella vaginalis, at a concentration of 0.3% it reduces the concentration of pathogens: Cutibacterium acnes, Candida glabrata, Mycoplasma hominis, Gardnerella vaginalis and Streptococcus viridans , at a concentration of 0.5% it reduces the concentration of pathogens:: Candida glabrata, Cutibacterium acnes, Corynebacterium tuberculostearicum, Malassezia furfur, Streptococcus viridans, Candida tropicalis and Corynebacterium kroppenstedtii. B. Effect of PTE synthesis on genes related to the mechanisms of bacterial attachment and colonization of C. acnes

[0125] Study of the transcriptional regulation of the roxP gene related to the ability of C. acnes to adhere and colonize the skin.

[0126] The roxP gene is responsible for the synthesis of the RoxP protein of the bacterium

[0127] C. acnes, which has been shown to promote the colonization of this microorganism and its growth in aerobic conditions (Allhorn M, Arve S, 2016).

[0128] Assessment of transcriptional regulation - Methodology

[0129] To assess the effect of the test component on the transcriptional regulation of roxP, incubations of C. acnes bacteria were performed and then the roxP gene was quantified by real-time polymerase chain reaction (RT-PCR).

[0130] Cultivation of bacteria

[0131] This particular study, the strain C. acnes ATCC6919 was used. Bacteria were isolated on Columbia agar with 5% sheep blood (BioRad, Hercules, CA, USA) and cultured on plates in Brain Heart Infusion (BHI) nutrient medium (BioRad) (calf brain and bovine heart infusion) for 24 h in anaerobic conditions at 37°C to a final concentration of 107cells / mL

[0132] Gene expression analysis

[0133] C. acnes cultures were exposed to PTE at concentrations of 0.1 %, 0.3% and 0.5%. for 72 hours. For total RNA isolation as well as cDNA synthesis, Monarch® Total RNA Miniprep Kit (Hitchin, UK) and PrimeScript-RT Reagent Kit (Takara Bio, Otsu, Japan) were used, respectively. Equal amounts of cDNA were used for real-time polymerase chain reaction (RT-PCR).

[0134] Results

[0135] (Figure 1 ) In C. acnes bacteria incubated with PTE at a concentration of 0.5%, a significant difference in roxP gene expression was observed compared to bacteria whose culture medium did not contain the composition (N / T). In particular, a 5-fold decrease in roxP gene transcription levels was observed.

[0136] In conclusion, PTE synthesis reduces the expression levels of the roxP gene which significantly contributes to the adherence and colonization ability of C. acnes.

[0137] C. Inhibition of the formation and growth of C.acnes bacteria and the quorum quenching mechanism

[0138] Determination of activity against biofilm formation

[0139] The quantification of biofilm formation is carried out using an appropriately optimized methodology of biofilm formation, staining and measurement in microtiter plates, which has been described in the literature (Crystal Violet assay), in various formats and for a variety of microorganisms (Carme et aL, 2001 ; Djordjevic et aL, 2002; Holmberg et aL, 2009a; Melo et aL, 2013; Sandasi et aL, 2010; Stepanovic et aL, 2000). Briefly, C. acnes strain ATCC (Cutibacterium acnes Scholz and Kilian 6919™), a well- characterized strain with proven biofilm-forming and acne-inducing ability, was resuscitated (RCA, TSB) and cultured for 72 h in TSB, under anaerobic conditions and diluted with TSB to an appropriate concentration (OD600 - 1 x108cfu / ml). 100 pL of this culture was then added to wells of a sterile 96-well flat bottom plastic plate (Nunclon Surface F; Nunc A / S). An additional 100 pL of TSA was added to each well as a reference (CTRL Biofilm) as is or containing PTE at final concentrations of 0.1 %, 0.3%, 0.5% to each well. In addition, 100 pL of TSA containing oxytetracycline was added to separate wells containing 100 pL of the initial culture so that the final concentration was 6 pg / ml. Finally, 200pL TSA was placed in separate wells as a sterility and background control during the final measurement. Each concentration was tested in duplicate and the procedure was repeated 2 times. The microplate was incubated for 72 hours, in anaerobic conditions before biofilm staining. After incubation, in each well, the medium was removed, followed by gentle washing (three times) with 200 pL of phosphate-buffered saline (PBS). This was followed by drying for 30 minutes at 45°C and then staining with 200 pL of Crystal Violet (0.1 % (w / v) in water) for 15 minutes. The stain was appropriately removed, and the wells were washed with 200 pL of PBS three times. The dye was then extracted with 200 pL of acetone-ethanol (20:80 (v / v)) for 15 min. From each well after extraction, 100 pL was transferred to new clean wells and the absorbance was measured at 595 nm in an ELISA reader (Bio-Control). The absorbance of the negative control was subtracted from the absorbance of the contents in each well prior to statistical evaluation:

[0140] Action of PTE against established biofilm and inhibition of its growth.

[0141] A biofilm of C. acnes was allowed to preform, before the addition of the extract at the predetermined concentrations (0.1 %, 0.3%, 0.5%) and the antibiotic. Biofilm formation was achieved by spreading 100 pl of a standard C. acnes culture in a 96-well microtiter plate. The microplate was incubated anaerobically at 37°C for 3 days to allow cell attachment. After incubation, 100 pl of TSA with appropriate concentration of PTE was added so that the final concentration was 0.1 %, 0.3%, 0.5% respectively. An additional 100 pl of fresh TSA was added to the control wells and an equal volume of oxytetracycline to the positive control wells. Finally, pure TSA was added accordingly to check the sterility of the procedure. The plate was further incubated for 48 h before staining assays were performed with Crystal Violet solution.

[0142] Action of PTE against C. acnes biofilm

[0143] The analysis showed that there is an in vitro quorum quenching activity of the strain C. acnes ATCC 6919, by the substance under examination, varying according to its concentration. (Figure 2)

[0144] The substance under examination had an effect both in inhibiting the creation of biofilm from the beginning (24.79-82.63%), and in pre-established biofilms (13.97 - 89.07%) compared to the control and depending on the concentration. (Figure 3) The minimum concentration of the substance that has an effect of more than 50% in both tests is 0.3%. However, the 0.5% concentration had significantly better results.

[0145] D. Transcriptional regulation of genes related to antimicrobial and antiinflammatory activity in keratinocytes (NHEK).

[0146] Following the assessment of the selective antimicrobial activity of the substance under examination, its activity was studied in terms of the regulation of gene expression of human skin cells with a role in the defense against pathogens and the suppression of any hyper-inflammatory reactions that have been observed to accompany this type of skin infection by pathogens . Following a literature review, the expression profile of the following genes was examined after the administration of the active substance in human keratinocyte cultures, in amounts proportional (sub-tenfold) to the two (2) concentrations (0.1 % and 0.3%, i.e. in cell culture 0.01 %, 0.03% and 0.05 % that were deemed friendly to the normal human skin microbiome. The concentration of 0.05% in the culture of human keratinocytes showed toxicity with the image of cell lysis during the 24-hour incubation (10,000cells / ml), so no RNA sample was isolated for the subsequent experiments.

[0147] Interleukin-4 (IL-4) lnterleukin-4 is characterized as an anti-inflammatory cytokine that functions mainly by suppressing the pro-inflammatory environment (Chatterjee et aL, 2014). IL-4 helps polarize antigen-stimulated T-helper (Th) cells to effector Th2 cells as well as propagate Th2 responses by binding its receptor, IL-4Ra, and activating the STAT6 factor of six signaling pathways (le Gros et al ., 1990; Shimoda et aL, 1996). STAT6, through the induction of a zinc ring transcription factor-GATA3, can directly suppress Th1 cell development by silencing interferon-y (IFNy) expression (Takeda et aL, 1996). Recent studies also show that IL-4 enhances Th2 immunity by inhibiting Th1 responses through suppression of IL-12 signaling (Ouyang et aL, 1998). Several studies implicate IL-4 in the development and maintenance of regulatory T (Treg) cells. IL-4 signaling through STAT6 is important for FoxP3 mRNA expression and protein production, essential for the proper function of Tregs (Bettelli et aL, 2006; Pillemer et aL, 2009; Skapenko et aL, 2005 ; Wei et aL, 2007). IL-4 also stimulates the formation of inducible Tregs from naive CD4+ T cells. Thus, IL-4 not only mediates Th2 cell function but also plays a role in regulating Tregs by limiting tissue damage in immune responses to microbes and autoimmunity by inhibiting sustained T-cell activation. It is also known to regulate the development of T-cytotoxic cell responses, as well as B cell development and immunoglobulin secretion (Silva-Filho et aL, 2014). In vivo studies have confirmed that IL-4 has positive effects on the outcome of infectious diseases, contributing to tissue healing (Gause et aL, 2013).

[0148] B-defensin 1 (DEF1 B)

[0149] Human b-defensin 1 (DEF1 B), is a protein that exhibits microbicidal activity mainly against Gram-negative bacteria by interfering with the synthesis of their cell wall and causing microbial death in a manner similar to that of antibiotics (Vera et aL, 2010). It has been shown in vitro that [3-defensins are also involved in skin healing processes by stimulating the secretion of matrix metalloproteinase-9 (MMP-9) from keratinocytes, thus favoring keratinocyte migration for wound closure (Bucekova et aL, 2017 ). - defensins are key players in innate and adaptive immunity and have recently been shown to limit the production of pro-inflammatory cytokines in macrophages. In particular, they are Toll-like Receptor 4 (TLR4) ligand molecules and inhibit the transcription of pro-inflammatory genes downstream of the TLR4 pathway, such as those responsible for the activation of NFK-B (Semple et aL, 201 1 ). In addition, they have been shown to chemotactically attract different types of immune cells, through their interaction with TLR4 and other receptors (CCR6, CCR2, melanocortin receptors 5, CXCR4) that promote the maturation of dendritic and other immune cell subtypes, thus enhancing the first line of defense against of infections (Lai & Gallo, 2009). [32-integrins (ITG-B)

[0150] The receptors of the integrin family play a vital role in cell adhesion, in the transmission of signals in the cytoplasm. Interestingly, several of these receptors are exploited by pathogens to establish contact with host cells. Therefore, microbes subvert the normal processes of eukaryotic cells to create conditions that allow their survival (Scibelli et aL, 2007). Especially for the very common skin pathogen, Staphylococcus aureus, several different adhesins have been found to form complexes with the extracellular matrix (ECM) protein fibronectin, which acts as a molecular bridge connecting S. aureus to host cell receptors. Furthermore, the binding of integrins mediated by this S. aureus -fibronectin adhesion molecule complex triggers the internalization of microbes by non-professional phagocytes such as epithelial cells, endothelial cells, keratinocytes and fibroblasts (Borisova et aL, 2013 ). Integrin receptors may represent a critical target for new therapeutic strategies. Integrins enable bacteria to anchor to host cells and activate cell signaling pathways that promote the cytoskeletal rearrangements required for their internalization. Integrin antagonists such as monoclonal antibodies, disintegrins and natural or synthetic peptides could represent a reliable tool to enhance protection against a multitude of pathogenic microorganisms. [32-integrins (ITG-B) have also been associated with several immunosuppressive functions. ITG-Bs can inhibit TLR signaling in macrophages, limit the migratory phenotype of dendritic cells (DC) and thus suppress DC-mediated T cell activation ultimately leading to immune tolerance, which is exploited by many microorganisms to evade the innate immune system (Fagerholm et aL, 2019).

[0151] C-X-C ligand motif chemokine 12 (CXCL12 q SDF-1 )

[0152] The CXC family of chemokines and their receptors are crucial for inflammation. Chemokine 12 (CXCL12 or SDF-1 ) is a pleiotropic protein that functions as an antiinflammatory chemokine in autoimmune inflammatory responses. However, many studies have shown that CXCL12 and its cognate chemokine receptor C-X-C motif type 4 (CXCR4) play an important role in the immune response and inflammatory processes. Specifically, under conditions of peripheral inflammatory pain, CXCL12- CXCR4 signaling between non-neuronal glial cells and primary neuronal nociceptors activates factors that lead to hyperstimulation of peripheral nociceptors, which contribute to the maintenance of persistent spontaneous pain and hypersensitivity (Yang et al ., 2015). CXCL12 has been found to have angiogenic activity in different systems in vitro and in vivo. It is expressed in several tissues including skin and bone marrow. Increased mRNA levels of both CXCL12 and CXCR4, as well as increased CXCL12 / SDF-1 protein levels, have been found in psoriatic skin lesions. New studies have highlighted the important role of the CXCL12 / CXCR4 axis in skin inflammation and inflammatory angiogenesis and suggest that inhibition of the CXCL12 / CXCR4 axis may serve as a novel therapeutic strategy for chronic inflammatory skin diseases (Zgraggen et aL, 2014 ).

[0153] Assessment of transcriptional regulation - Methodology

[0154] To assess the effect of the component under consideration on the transcriptional regulation of genes related to antimicrobial and anti-inflammatory activity, 24-hour incubations of human NHEK cells from an adult donor were performed and then the above genes were quantified by real-time polymerase chain reaction (RT-PCR).

[0155] Keratinocyte cultures

[0156] NHEKs from an adult donor were purchased from Lonza (Basel, Switzerland) and cultured in KGM™ Gold medium containing KGM2 growth supplements containing insulin, human epidermal growth factor, bovine pituitary extract, hydrocortisone, epinephrine, transferrin, and gentamicin / amphotericin B. Lonza . The cells were left in culture until 70-80% fullness of the flask and then experiments were carried out with their 24-hour incubations with the formulation (PTE) at concentrations of 0.01 % and 0.03%.

[0157] Gene expression analysis

[0158] For total RNA isolation (500 ng) as well as cDNA synthesis, the Nucleospin RNA kit (Macherey-Nagel, Duren, Germany) and the PrimeScript-RT reagent kit (Takara Bio, Otsu, Japan) were used, respectively. The real-time polymerase chain reaction (RT- PCR) method as well as the gene analysis procedure were performed according to the standard protocols of the Laboratory (Letsiou et aL, 2017). Glyceraldehyde 3- phosphate dehydrogenase gene (GAPDH) was chosen as a reference gene to normalize values. Two conditions are shown: NHEK cells not incubated with the active (N / T), NHEK cells incubated with the compound (PTE) at concentrations of 0.01 % and 0.03%.

[0159] Results

[0160] In NHEK cells incubated with the composition (PTE) at concentrations of 0.01 % and 0.03%, significant differences were observed in the expression of the tested genes, in relation to the cells whose culture medium did not contain the composition. Notable was the increase observed in the transcription of the IL-4 gene (18 times higher when incubated with the composition at a concentration of 0.01 % and 55 times higher when incubated with the composition at a concentration of 0.03%), but also of the gene of DEF1 B (47% higher when incubated with the composition at a concentration of 0.01 % and 6 times higher when incubated with the composition at a concentration of 0.03%), while a statistically significant decrease in the expression of ITG-B genes was observed (5 times lower when incubated with the formulation at a concentration of 0.01 % and similarly when incubated with the formulation at a concentration of 0.03%) and CLCX-12 (3 times lower when incubated with the formulation at a concentration of 0.01 % and 4 times lower when incubated with the formulation at a concentration of

[0161] O.03 %). (Figure 4)

[0162] In conclusion, the formulation induces the expression of antimicrobial peptides in keratinocytes while reducing factors associated with potential escape of pathogens from immune surveillance and factors associated with inflammatory angiogenesis and persistent inflammation.

[0163] Bibliography

[0164] Devequi-Nunes, D., Machado, B. A. S., Barreto, G. A., Rebougas Silva, J., da Silva, D. F., da Rocha, J. L. C., Brandao, H. N., Borges, V. M., & Umsza-Guez, M. A. (2018). Chemical characterization and biological activity of six different extracts of propolis through conventional methods and supercritical extraction. PloS one, 13(12), e0207676.

[0165] Jug, M., Karas, O., & Kosalec, I. (2017). The Influence of Extraction Parameters on Antimicrobial Activity of Propolis Extracts. Natural product communications, 12(1 ), 47- 50.

[0166] Cavalaro, R. L, Cruz, R. G. D., Dupont, S., de Moura Bell, J. M. L. N., & Vieira, T. M. F. S. (2019). In vitro and in vivo antioxidant properties of bioactive compounds from green propolis obtained by ultrasound-assisted extraction. Food chemistry: X, 4, 100054.

[0167] Andrade, J. K. S., Denadai, M., Andrade, G. R. S., da Cunha Nascimento, C., Barbosa,

[0168] P. F., Jesus, M. S., & Narain, N. (2018). Development and characterization of microencapsules containing spray dried powder obtained from Brazilian brown, green and red propolis. Food research international (Ottawa, Ont.), 109, 278-287 de Melo Silva, I. S., do Amorim Costa Gaspar, L. M., Rocha, A. M. O., da Costa, L. P., Tada, D. B., Franceschi, E., & Padilha, F. F. (2020). Encapsulation of Red Propolis in Polymer Nanoparticles for the Destruction of Pathogenic Biofilms. AAPS PharmSciTech, 21 (2), 49.

[0169] Popova, M., Giannopoulou, E., Skalicka-Wozniak, K., Graikou, K., Widelski, J., Bankova, V., Kalofonos, H., Sivolapenko, G., Gawel-Bgben, K., Antosiewicz, B., & Chinou, I. (2017). Characterization and Biological Evaluation of Propolis from Poland. Molecules (Basel, Switzerland), 22(7), 1 159.

Claims

Claim1. Propolis extract with active fractions titrated into bioactive components, characterized in that it consists of flavonoids and chaicones in a percentage of 47% to 78%, esters in a percentage of 3% to 18%, sesquiterpenes in a percentage of 14% to 22%, triterpenes at a rate of 2% to 4% and other components including aliphatic acids, aromatic acids and sugars at a rate of 3% to 4%.

2. Propolis extract with active fractions titrated in bioactive components, according to claim 1 , characterized in that the flavonoids and chalcoes contain Methoxyllflavanone in a percentage of 21% to 44% of their total, galangin in a percentage of 8% to 32% of their total, dihydroxymethoxyflavone at a rate of 8% to 1 1 % of their total, pinocembrin at a rate of 9% to 13% of their total, pinocembrin chalkone at a rate of 9% to 12% of their total, kaempferol at a rate of 8% up to 12% of their total and chaicones at a rate of 8% to 10%. :

3. Propolis extract with active fractions titrated into bioactive components, according to claim 1 or 2, characterized in that the esters contain caffeate derivative in a percentage of 42% to 56% of their total, cinnamyl ester of caffeic acid in a percentage of 22% to 28% of their total, cinnamyl ester of isoferulic acid, from 16% to 22% of their total and other esters from 6% to 8% of their total.

4. Propolis extract with active fractions titrated in bioactive components, according to the claims 1 to 3, characterized in that the sesquiterpenes contain gamma curcumene in a percentage of 16% to 19% of their total, curcumene in a percentage of 24% to 28% of the total, beta-gurjunene in a percentage from 40% to 48% of their total, cadinene from 7% to 15% of their total and other sesquiterpenes from 1 % to 2% of their total.

5. Propolis extract with active fractions titrated into bioactive components, according to the claim 1 to 4, characterized in that the triterpenes contain ledene in a percentage greater than 85% of their total.

6. A method for the production of propolis extract with active fractions titrated to bioactive components according to claims 1 to 5, characterized in that the method comprises, a first stage comprising :- the selection of types of propolis having a concentration of total polyphenols >900 mg / l gallic acid equivalents (GAE) after dissolving 10% by weight of propolis in ethanol and- showing a peak in gas chromatography of the components Methoxylflavanone, galangin, dihydroxymethoxyflavone, pinocembrin, pinocembrin chalkone, kaempferol , caffeate derivative, cinnamyl ester of caffeic acid, cinnamyl ester of isoferulic acid, gamma curcumene, curcumene, beta-gurjunene, cadinene, ledene.

7. A method for the production of propolis extract with active fractions titrated to bioactive components according to the claim 6, characterized in that, the method comprises a second stage comprising a purification of the propolis from waxes.

8. A method for the production of propolis extract with active fractions titrated to bioactive components according to the claims 6 or 7 characterized in that, the method comprises a third stage comprising :- an extraction of the propolis in which it is cut into small particles (<1 mm) after cooling it for 24 hours at -20°C, then- a dispersion at a rate of 1 kg / min in the solvent system consisting of (1 .3- Propanediol) and glyceryl ether ethylhexylglycerin (Ethylhexylglycerin), in a ratio of Ethylhexylglycerin: 1 .3-Propanediol = 0 / 100 to Ethylhexylglycerin: 1 .3-Propanediol = 5 / 95,- stirring at 500 - 1500 rpm, with a concentration of propolis from 9% to 32% by weight depending on its content of polyphenols,- an extraction of the resulting suspension placed in an extractor assisted by a pressure of 1 bar to 9 bar at a temperature from 25°C to 35°C, including 7 to 36 compression cycles, lasting from 3' to 6' and corresponding decompression cycles lasting from 2' to 6',- the mixture is then left in a hermetically closed container at rest at 5-7 °C for 24 hours and- a filtration through an array of cartridge type filters with pore size 10 pm - 5 pm - 1 pm - 0.45 pm.

9. A method for the production of propolis extract with active fractions titrated into bioactive components according to the claims 6 to 8 in that, the method comprises a fourth step comprising a quantification of the components of the extract, where first of all the total polyphenol content is required to be greater than 900 mg / L GAE and a quantification of groups of components flavonoids and chaicones I esters I sesquiterpenes I triterpenes I other components by gas chromatography after the generation of silyl-derivatives.

10. A method for the production of propolis extract with active fractions titrated in bioactive components according to the claims 6 to 9, characterized in that, the method comprises a fifth stage comprising a mixture of the extracts produced in order to achieve the desired concentration of the individual components.1 1 . A composition comprising a propolis extract with active fractions titrated into bioactive components according to the claims 1 to 5.

12. Composition according to the claim 11 for a use in cosmetic application.

13. Composition according to the claim 1 1 for a use in pharmaceutical application.

14. Composition according to the claims 1 1 to 13 for topical pharmaceutical or cosmetic use.

15. Composition according to the claims 1 1 to 14 for topical pharmaceutical or cosmetic use after formulation as a solution, suspension, emulsion, or gel.

16. Composition according to the claims 1 1 to 15 for topical pharmaceutical or cosmetic use after formulation as a solution, suspension, emulsion, or gel and exhibits antioxidant activity of 28.60 ± 3.95 mM Trolox Equivalent and antimicrobial properties17. Composition according to the claims 1 1 to 16 comprising 0.1 %, 0.3%, 0.5% of Propolis Titrated Extract (PTE).

18. Composition according to the claims 1 1 to 17 for its use in the reduction in vitro free radicals and against specific pathogens on the scalp, female sensitive area, axilla, and face, while remaining friendly to micro-organisms that make up the normal microbial flora of the skin in said examined skin areas.

19. Composition according to the claims 1 1 to 18 for its use in reducing the formation and growth of biofilms of the C. acnes bacterium and the quorum detection mechanism.

20. Composition according to the claims 1 1 to 19 for its use in the regulation of the expression of genes related to antimicrobial and anti-inflammatory activity in keratinocyte.21 . Composition according to the claims 11 to 20 for its use in transcriptional regulation of genes associated with antimicrobial and anti-inflammatory activity in human keratinocytes (NHEK).

22. Composition according to the claims 11 to 21 for its use in reducing gene transcription implying in the mechanisms of attachment and colonization of the bacterium C. acnes.

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