HRP20200178A2 - Liquid propolis extract, its formulation and its use - Google Patents

Abstract

The present invention discloses a novel standardized liquid extract of propolis and a pharmaceutical formulation based on said extract, methods for their preparation and use. <BR/> <BR/> The liquid propolis extract according to the invention is produced by extracting crude propolis with an extraction solvent consisting of a mixture of polyethylene glycol 200-600 (96.5-99.9% w / w) and lecithin or lecithin hydrolyzate (0 , 1-3.5% m / m). The extract is characterized by a standardized content of p-coumaric acid (<B> 1 </B>), trans-ferulic acid (<B> 2 </B>), caffeic acid (<B> 3 </B>) and 2- phenylethyl 3,4-dihydroxy-trans-cinnamate (<B> 4 </B>). It is used as an active ingredient in the manufacture of pharmaceutical, cosmetic, veterinary, agrochemical or functional food products. The pharmaceutical formulation according to the invention consists of 5-95% w / w of said propolis extract and up to 100% of the excipients required. for forming dosage forms: solutions, suspensions, gels, creams, ointments or sprays for oral or nasal administration. It is used for the treatment of diseases and conditions in humans and animals such as: inflammatory diseases, bacterial and fungal infections, viral, autoimmune and tumor diseases and for the treatment of burns and wound healing.The present invention discloses a novel standardized liquid extract of propolis and a pharmaceutical formulation based on said extract, methods for their preparation and use. <BR/> <BR/> The liquid propolis extract according to the invention is produced by extraction of crude propolis with an extraction solvent consisting of a mixture of polyethylene glycol 200- 600 (96.5-99.9% w / w) and lecithin or lecithin hydrolysate (0.1-3.5% w / w). The extract is characterized by a standardized content of p-coumaric acid (<B> 1 </B>), trans-ferulic acid (<B> 2 </B>), caffeic acid (<B> 3 </B> ) and 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (<B> 4 </B>). It is used as an active ingredient in the production of pharmaceutical, cosmetic, veterinary, agrochemical or functional food products. <BR/> <BR/> The pharmaceutical formulation according to the invention consists of 5-95% m / m of said propolis extract and up to 100% of the excipients required to form dosage forms: solutions, suspensions, gels, creams, ointments or sprays for oral or nasal use. It is used to treat diseases and conditions in humans and animals such as: inflammatory diseases, bacterial and fungal infections, viral, autoimmune and tumor diseases, and to treat burns and wound healing.

Description

Field of invention

The invention relates to a new liquid standardized extract of propolis, the procedure for its preparation, to a new pharmaceutical formulation based on this extract and its use.

Technical problem

Considering the fact that propolis is a mixture of beeswax with a large number of natural organic compounds, different extraction procedures yield extracts with a very different composition of active substances. By applying chemoselective extraction solvents (EO) that have the ability to selectively extract predominantly certain groups of organic propolis compounds, with the application of suitable analytical methods, it is theoretically possible to achieve a consistent, standardized composition of the resulting propolis extract.

The technical problem that is solved by the subject includes:

(i) non-alcoholic liquid propolis extract characterized by a high and standardized content of propolis phenolic acids; para-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 2-phenylethyl ester of caffeic acid, 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE) which are known multiple positive pharmacological effects on the human, animal and plant organism;

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(ii) the procedure for obtaining it;

(iii) use for the purpose of production of pharmaceutical, cosmetic, veterinary, agrochemical or food products with a known, standardized content of propolis active substances 1-4; and especially on

(iv) a pharmaceutical formulation based on said propolis extract that would be effective in the treatment of inflammatory and infectious diseases as well as diseases and conditions in which the antioxidant, anti-inflammatory and immunomodulating effects of certain active substances of propolis have an important role in the etiology of their occurrence, as indicated by certain statements in the previous state of the art.

Such an alcohol-free extract with a small content of beeswax and other ballast substances of propolis, together with a high and standardized content of the mentioned highly biologically active ingredients of propolis, represents the basis for the development and production of high-value pharmaceutical, cosmetic, veterinary, agrochemical or functional food products or nutritional supplements, which in addition the use of standard ethanolic, glycerol or 1,2-propylene glycol extracts of propolis is associated with various technical problems, such as different or unknown content of the key active ingredients of propolis or low quantitative composition of the above-mentioned valuable phenolic acids and CAPE in relation to the flavonoid ingredients of propolis.

The subject invention is based on:

(i) the use of a specific formulation of the extraction solvent (EO) used for the process of chemoselective extraction of raw propolis;

(ii) methods of quantitative determination of propolis active ingredients including key active ingredients 1-4;

(iii) standardization of the obtained liquid extract of propolis by diluting it with a certain amount of the same specific extraction solvent to the level of the desired content of active substances 1-4 according to the invention; wherein said solvent is composed of two or more food and pharmaceutical acceptable substances and in the finished product, liquid standardized extract of propolis, performs the function of carrier or solvent; you

(iv) a pharmaceutical formulation based on said standardized propolis extract according to the invention, which has proven to be an effective agent in the treatment of inflammatory and infectious diseases. Due to the wide spectrum of its pharmacological action, it can be used for the treatment of a whole range of diseases and conditions such as: inflammatory diseases, bacterial and fungal infections, viral diseases, autoimmune diseases, for mucosal regeneration and treatment of burns and wounds, as well as tumor diseases.

Prior art

Propolis is a product of plant origin that is collected by bees to serve as glue for closing smaller unwanted openings in the hives. Propolis consists of beeswax as the main ingredient and a large number of different organic compounds. Many of them have significant beneficial pharmacological effects; see for example literature reference 1:

1) S. Castaldo, F. Capasso: Propolis, and old remedy used in modern medicine, Fitoterapia 73 (2002) S1-6.

In addition to the mentioned active ingredients 1-4, propolis also contains a whole series of other natural compounds, for example, among acids it also contains trans-cinnamic acid (5) and a series of flavonoids, among which chrysin (6), pinocembrin (7), galangin ( 8), apigenin (9) and kaempferol (10):

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Traditionally, raw propolis is processed by extraction with ethanol or mixtures of ethanol and water, whereby the so-called propolis tinctures. Such liquid extracts of propolis are characterized by a number of disadvantages:

(i) the presence of a relatively aggressive solvent (ethanol);

(ii) products containing alcohol are not suitable for children, pregnant women, nursing mothers and some patients; you

(iii) relatively high content of beeswax, which results in its separation when mixing with the aqueous phase in the production of pharmaceutical and other products where such an extract is used as an active ingredient.

In addition to ethanol, glycerol, water, mixtures of glycerol and water, and other organic solvents are used as extraction solvents.

Thus, for example, Tsukada et al described the use of glycerol as an extraction solvent at a ratio of propolis:extraction solvent, 1:2 m/m, at 90-160 °C with subsequent filtration. Such glycerol extract is water-soluble and is suitable for the production of pharmaceutical products as an active pharmaceutical ingredient (API); see literature reference 2:

2) JPH05957A; T. Tsukada, W. W. Kameda, M. Ide: Production of water-soluble propolis pharmaceutical preparation; Ogawa Koryo KK, Santapuron KK (JP).

Water extracts of propolis are also described in the state of the art. Extraction with water as EO can be carried out at temperatures of 30-50 °C for 6-8 minutes, whereby after filtration a liquid extract of propolis is obtained directly or it is further processed through microencapsulation via spray drying technology with suitable carriers such as maltodextrin and gum arabic in solid extracts; see literature reference 3:

3) CN103783349A; Z. Wang, S. Shao, H. Ma, S. Wang, L. Wang, C. Zhang, X. Ma: Process for preparing honeycomb polyphenol extractive microcapsules by adopting spray; Jiangsu University (CN).

The use of surfactants as components of the extraction solvent (EO), to which lecithins can certainly be classified, is generally known in the state of the art. For example, Paradkar et al. described the propolis extraction procedure using an aqueous solution of polysorbate at 40-90 °C for 2-24 h, with the formation of a liquid extract of propolis; see literature reference 4:

4) WO2011092511A1; A. Paradkar, R. Dhumal, A. Kelly, S. Gilda: Propolis and process for the treatment thereof and end products formed therefrom; Natures Lab Ltd (GB).

Sosnowski described a method of extracting propolis with various organic solvents including 1,2-propylene glycol, polyethylene glycol (PEG) and mixtures of these solvents with water; see literature reference 5:

5) US4382886; Z. M. Sosnowski: Method for extracting propolis and water soluble dry propolis powder.

Chun et al. described a pharmaceutical formulation based, among other things, on liquid propolis extract in 1,3-butylene glycol as an extraction solvent, which is converted into a pharmaceutical dosage form containing nanoparticles with propolis extract using hydrogenated lecithin and other emulsifiers; see literature reference 6:

6) KR20130134800A; Y. J. Chun, S. B. Shim, X. Ke: Composition of nano-vesicle containing propolis and manufacturing method of it; University Chungwoon IACF (KR).

Although that document does not use lecithin as a means to enhance the extraction of active substances of propolis with the help of 1,3-butylene glycol, it certainly suggests the possibility of its use as a surface-active substance that can possibly enhance the extraction and emulsification of certain fatty active ingredients from propolis in more polar solvents.

Regarding the analysis of propolis, a large number of analytical methods are known in the state of the art for the quantitative determination of the active substances of propolis in complex extracts which in turn contain a large number of ingredients. As an example similar to the analytical method used in the subject invention, we cite the work of Chinese authors Cui-Ping and associates, who described a quantitative method for determining 12 different flavonoids and 8 phenolic acids of propolis by means of high-performance liquid chromatography (HPLC). This method determines, among others, p-coumaric acid (1), trans-ferulic acid (2) and caffeic acid (3), which are mentioned as qualitative markers of propolis; see literature reference 7:

7) Z. Cui-ping, H. Shuai, W. Wen-ting, P. Shun, S. Xiao-ge, L. Ya-jing, H. Fu-liang: Development of high-performance liquid chromatographic for quality and authenticity control of Chinese propolis, J. Food Sci. 79 (2014) C1315-C1322.

Due to the content of active substances 1-10 and others, propolis and propolis extracts show a number of very valuable and useful pharmacological effects on human, animal and plant health. In the state of the art, there is a very large number of scientific and patent documents that argue for a whole series of beneficial effects, among which the most important are: anti-inflammatory; antioxidant; immunomodulating; hepatoprotective; antimicrobial, including antibacterial, antiviral, antifungal and anti-amoebic activity; and anticancer activity; see, for example, literature references 8-12:

8) E. L. Ghisalberti: Propolis: A Review, Bee World 60 (1979) 59-84.

9) A. Banskota, Y. Tezuka, S. Kadota: Recent Progress in Pharmacological Research of Propolis, Phytother. Crisp. 15 (2001) 561-571.

10) G. A. Burdock: Review of the Biological Properties and Toxicity of Bee Propolis (Propolis), Food Chem. Toxicol. 36 (1998) 347-363.

11) J.M. Sforcin: Propolis and the immune system: a review, J. Ethnopharmacol. 113 (2007) 1-14.

12) J. W. Dobrowolski, S. B. Vohora, K. Sharma, S. A. Shah, S. A. H. Naqvi, P. C. Dandiya: Antibacterial, antifungal, antiamoebic, antiinflammatory and antipyretic studies on propolis bee products, J. Ethnopharmacol. 35 (1991) 77-82.

In addition to propolis extracts, the state of the art also describes a whole series of beneficial pharmacological effects of individual isolated active substances of propolis, for example:

(i) p-coumaric acid (1); see references 13 and 14;

(ii) trans-ferulic acid (2); see references 15 and 16;

(iii) caffeic acid (3); see references 17 and 18; as well as

(iv) 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE); see references 19 and 20; which, among other things, showed immunomodulating, anti-inflammatory and antimicrobial effects.

13) T. F. Bachiega, C. L. Orsatti, A. C. Pagliarone, J. M. Sforcin: The Effects of Propolis and its Isolated Compounds on Cytokine Production by Murine Macrophages, Phytother. Crisp. 26 (2012) 1308-1313.

14) K. Pei, J. Ou, J. Huang, S. Ou: p-Coumaric acid and its conjugates: dietary sources, pharmacokinetic properties and biological activities, J. Sci. Food Agric 96 (2016) 2956-2962.

15) N. Kumar, V. Pruthi: Potential applications of ferulic acid from natural sources, Biotechnol. Tail. 4 (2014) 86-93.

16) C. Shi, X. Zhang, Y. Sun, M. Yang, K. Song, Z. Zheng, Y. Chen, X. Liu, Z. Jia, R. Dong, L. Cui, X. Xia: Antimicrobial activity of ferulic acid against Cronobacter sakazakii and possible mechanism of action, Foodborne Pathog. Dis. 13 (2016) 196-204.

17) H. G. Choi, P. T. Tran, J. H. Lee, B. S. Min, J. A. Kim: Anti-inflammatory activity of caffeic acid derivatives isolated from the roots of Salvia miltiorrhiza Bunge, Arch. Pharm. Crisp. (2018) 64-70.

18) V. N. Lima, C. D. Oliveira-Tintino, E. S. Santos, L. P. Morais, S. R. Tintino, T. S. Freitas, Y. S. Geraldo, R. L. Pereira, R. P. Cruz, I. R. Menezes, H. D. Coutinho: Antimicrobial and enhancement of the antibiotic activity by phenolic compounds: Gallic acid. , caffeic acid and pyrogallol, Microb. Pathog. 99 (2016) 56-61.

19) K. Frenkel, H. Wei, R. Bhimani, J. Ye, J. A. Zadunaisky, M.-T. Huang, T. Ferraro, A.H. Conney, D. Grunberger: Inhibition of Tumor Promoter-mediated Processes in Mouse Skin and Bovine Lens by Caffeic Acid Phenethyl Ester, Cancer Res. 53 (1993) 1255-1261.

20) A. Russo, R. Longo, A. Vanella: Antioxidant activity of propolis: role of caffeic acid phenethyl ester and galangin, Fitother. 73 (2002) S21-S29.

Furthermore, the active substances of propolis are fungicidal, bactericidal, virucidal, insecticidal and nematocidal, and are used as such in the protection of plants. Due to its strong antioxidant effect, propolis strengthens plants and their resistance to abiotic stress factors and helps them with infections; see literature references 21-25.

21) C. A. Guginski-Piva, I. dos Santos, A. W. Júnior, D. W. Heck, M. F. Flores, K. Pazolini: Propolis for the control of powdery mildew and the induction of phytoalexins in cucumber, IDESIA (Chile) 33 (2015) 39- 47;

22) Z. Ararso, G. Legesse: Insecticidal action of honeybees propolis extract against larvae of lesser wax moth, Agric. Biol. J. North Am. (2015) doi:10.5251/abjna.2016.7.6.302.306.

23) L. Kumar, M. K. Mahatma, K. A. Kalariya, S. K. Bishi, A. Mann: Plant Phenolics: Important Bio-Weapon against Pathogens and Insect Herbivores, Popular Kheti 2 (2014) 149-152;

24) E. M. Noweer, M. G. Dawood: Efficiency of propolis extract on faba bean plants and its role against nematode infection, Commun. Agric. Appl. Biol. Sci. 74 (2009) 593-603;

25) K. Kulbat: The role of phenolic compounds in plant resistance, Biotechnol. Food Sci. 80 (2016) 97-108.

To the best of our knowledge, the use of a specific extraction solvent (EO) based on liquid polyethylene glycols with a low lecithin content (0.1-3.5% m/m) as a propolis extraction chemoselectivity enhancer has not been described in the state of the art. By using this specific EO according to the invention, increased chemoselectivity is achieved in the extraction of raw propolis, whereby a suitable liquid extract with a significantly increased content of valuable active substances 1-4 is obtained.

Also, the use of said specific standardized liquid extract of propolis as an active pharmaceutical substance (API) in the pharmaceutical formulation according to the invention, enables unexpected improvements in a number of different indications for use, as described in the detailed description of the invention.

The essence of invention

The subject invention is based on the unexpected efficiency and chemoselectivity of raw propolis extraction with a specific extraction solvent (EO) consisting of a mixture of liquid polyethylene glycols (PEG), such as PEG 400 and lecithin or lecithin hydrolyzate in a ratio of 96.5-99.9:0. ,1-3.5% m/m.

The mentioned extraction solvent (EO) effectively and chemoselectively extracts the active ingredients of propolis p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4; CAP). Using a suitable analytical method based on high-performance liquid chromatography (HPLC), the concentration of active ingredients 1-4 in the thus prepared primary extract is quantitatively determined. Further, the standardization of the primary liquid propolis extract is carried out by diluting it with the same EO that was used in the extraction step to the desired level of the quantitative composition of active substances 1-4 according to the invention. In this way, a liquid extract of propolis according to the invention is obtained with a known and standardized concentration of key active ingredients 1-4, which is used as such as an active pharmaceutical ingredient (API), active cosmetic ingredient (ACI), or as a food ingredient for the production of functional food and nutritional supplements.

The formulation from the present invention based on said liquid extract of propolis containing the key active substances p-coumaric acid (1; 10-1,300 μg/mL), trans-ferulic acid (2; 10-800 μg/mL), caffeic acid (3; 5-300 μg/mL), and 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4; 5-400 μg/mL) is an effective agent in the treatment of inflammatory diseases, bacterial infections, fungal infections, viral diseases, autoimmune diseases , functional gastrointestinal disorders, for regeneration of mucous membranes, treatment of burns and wound healing, and treatment of tumor diseases.

Short description of the pictures

Figure 1 shows a typical HPLC chromatogram of key propolis constituents 1-4 and supporting constituents 5-10.

Figures 2.1 – 2.4 show the results of the quantitative composition of key propolis ingredients 1-4 in propolis liquid extracts obtained by extraction with ethanol (96%) and ethanol mixtures with different types and concentrations of lecithin.

Figure 2.5 – 2.10 shows the results of the quantitative composition of accompanying ingredients 5-10 in liquid extracts of propolis obtained by extraction with ethanol (96%) and mixtures of ethanol with different types and concentrations of lecithin.

Figure 3.1 – 3.4 shows the results of the quantitative composition of key propolis ingredients 1-4 in liquid propolis extracts obtained by extraction with polyethylene glycol 400 and mixtures of polyethylene glycol 400 with different types and concentrations of lecithin.

Figure 3.5 – 3.10 shows the results of the quantitative composition of accompanying ingredients 5-10 in liquid extracts of propolis obtained by extraction with polyethylene glycol 400 and mixtures of polyethylene glycol 400 with different types and concentrations of lecithin.

Figure 4 shows the block diagram of the production procedure of the standardized liquid extract of propolis according to the present invention.

Figure 5 shows a typical HPLC chromatogram of the quantitative analysis of the pharmaceutical formulation according to the invention, the product of Example 11.

Abbreviations used

EO - extraction solvent

SL - soy lecithin (Glycine max. L.)

RL – rapeseed lecithin (Brassica napus L.)

HRL - hydrolyzed rapeseed lecithin

SUL - deoleinized sunflower lecithin

EtOH - 96% ethanol

PEG - polyethylene glycol

HPLC - high performance liquid chromatography

GC - gas chromatography

TLC - thin layer chromatography.

MIC - minimum inhibitory concentration

RPMI – Roswell Park Memorial Institute medium

TTC - 2,3,5-triphenyl-2H-tetrazolium chloride, redox indicator

PBS - phosphate buffer in physiological solution

CFU - "eng. colony forming units"; the number of living individuals of microorganisms capable of forming a colony

XTT - XTT sodium salt; 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt, redox indicator

MRSA - methicillin-resistant Staphylococcus aureus

MSSA - methicillin-susceptible Staphylococcus aureus

CLSI - Clinical and Laboratory Standards Institute

EUCAST - European Committee on Antimicrobial Susceptibility Testing

API - active pharmaceutical ingredient / substance

DER - drug-to-extract, mass ratio; a measure of the strength of the extract expressed by the mass ratio of the starting drug and the finished extract

BSS - number of somatic cells

I have a. - intramammary (application)

ATCC - Engl. American Type Culture Collection; non-profit organization that collects, stores and distributes standard reference microorganisms

CNS - coagulase negative staphylococci

Detailed description of the invention

The present invention relates to a new liquid extract of propolis standardized on the content of key active substances selected from the group consisting of: p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 2-phenylethyl ester of caffeic acid, 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4), its preparation process and its use.

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The liquid extract of propolis as a pharmaceutical, cosmetic or agrochemical ingredient or food ingredient according to the invention consists of:

(A) dry propolis extract; 0.1-10.0% m/m; and

(B) extraction solvent; 90.0-99.9% m/m;

whereby the extraction solvent consists of:

(B.1) one or more liquid polyethylene glycols (PEG) 200-600; 96.5-99.9% m/m; and

(B.2) lecithin or lecithin hydrolyzate; which are characterized by a hydrophilic-lipophilic balance (HLB) of 2-12; 0.1-3.5% m/m;

whereby said liquid extract of propolis is standardized:

(I) by the quantitative ratio of the mass of raw propolis as a drug and the finished extract (DER) in the ratio:

1 : 2 – 1 : 20 m/m; and

(II) by the quantitative content of active propolis ingredients selected from the group consisting of p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4) , where the quantitative content for at least two of the four listed key active substances of propolis is as follows:

(i) p-coumaric acid (1); 100-1,300 μg/mL;

(ii) trans-ferulic acid (2); 75-800 μg/mL;

(iii) caffeic acid (3); 25-300 μg/mL; you

(iv) 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE); 40-400 μg/mL.

In a specific variant of the subject invention, liquid polyethylene glycol (PEG) as a component of the extraction solvent (EO) is selected from the group consisting of: polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 600, or mixtures of the aforementioned substances.

Specifically, liquid polyethylene glycol (PEG) as a component of the extraction solvent (EO) is selected from the group consisting of: polyethylene glycol 200, polyethylene glycol 400, or mixtures of the aforementioned substances.

Furthermore, lecithin or lecithin hydrolyzate is selected from the group consisting of products characterized by a hydrophilic-lipophilic balance (HLB) factor of 2-12, selected from the group consisting of: soy lecithin (SL; from Glycine max. L.); sunflower lecithin (SUL; from Helianthus annuus L.); rapeseed lecithin (RL; from Brassica napus L.); canola lecithin (Brassica rapa L.); lecithin from chicken (Gallus gallus domesticus L.) eggs; deoleinized products of the said lecithins; hydrogenated lecithins from the mentioned sources; hydrolysates of said lecithins; enzymatically modified derivatives of the said lecithins; or mixtures of these substances.

Specifically, the following can be used as lecithin in the present invention: native lecithin, deoleinized, hydrogenated, hydrolyzed or enzymatically modified lecithin from soy (Glycine max. L.), sunflower (Helianthus annuus L.), rapeseed (Brassica napus L.) or canola ( Brassica rapa L.); or mixtures of these substances.

The term "enzymatically modified lecithin" is considered to be enzymatically hydrolyzed lecithin in which one residue of a higher fatty acid has been enzymatically hydrolyzed with the formation of a glycerol-mono-ester of higher fatty acids with retained phosphate and choline groups in the molecule. At the same time, the HLB factor of the lecithin hydrolyzed in this way increases significantly.

Preferably, a mixture can be used as the extraction solvent (EO) for the preparation of the liquid extract of propolis according to the invention:

(i) polyethylene glycol (PEG) 200, polyethylene glycol 300, polyethylene glycol 400 or their mixtures; from 97-99% m/m; and

(ii) native lecithins, deoleinized lecithins or hydrolyzed lecithins of soy (Glycine max. L.), sunflower (Helianthus annuus L.), oilseed rape (Brassica napus L.) or canola (Brassica rapa L.); or mixtures of said substances; from 1-3% m/m.

In a preferred version, the liquid extract of propolis according to the invention is standardized:

(I) by the quantitative ratio of the mass of raw propolis as a drug and the finished extract (DER) in the ratio: 1 : 3 - 1 : 5 m/m; you

(II) by the quantitative content of active propolis ingredients selected from the group consisting of p-coumaric acid (1), ferulic acid (2), caffeic acid (3) and 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4), at for which at least two of the four listed key assets correspond to the quantitative content as follows:

(i) p-coumaric acids <1>; 500-1,300 μg/mL;

(ii) trans-ferulic acid <2>; 300-800 μg/mL;

(iii) caffeic acid <3>; 100-300 μg/mL; you

(iv) 2-phenylethyl 3,4-dihydroxy-trans-cinnamate <4; CAPE>; 100-400 μg/mL.

Analysis of active ingredients of propolis in liquid extracts

As a precondition for the development of a new standardized liquid extract of propolis according to the invention was the development of a suitable analytical method for the quantitative determination of:

(i) key active substances of propolis selected from the group consisting of the above: p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (CAPE; 4); you

(ii) accompanying active substances selected from the group consisting of: trans-cinnamic acid (5), chrysin (6), pinocembrin (7), galangin (8), apigenin (9) and kaempferol (10).

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For the purpose of the initial tests of the aforementioned analytical method, it was necessary to prepare model liquid extracts of propolis. Model extracts of propolis in ethanol (96%) and polyethylene glycol were used, prepared by the standard extraction method, maceration at room temperature for 24 h, after which the undissolved residue was separated by filtration, and the clear filtrate was used as a model liquid extract of propolis for further testing. Polyethylene glycol 400 (PEG 400) was used as a model liquid polyethylene glycol. Procedures for obtaining model liquid extracts of propolis in classic extraction solvents, ethanol (96%) and polyethylene glycol 400 are described in Example 1 (96% ethanol) and Example 2 (PEG 400).

A suitable analytical method was developed using the technique of high-performance liquid chromatography (HPLC), which achieved the successful separation of all 10 mentioned compounds 1-10. The method is described in detail in Example 3.

A typical HPLC chromatogram obtained according to the analytical method in question is shown in Figure 1. The retention times (tR) of compounds 1-10 are shown in Table 1.

Table 1. Retention times of active propolis substances 1-10 according to the HPLC method according to the invention.a

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a Chromatographic column: Ascentis express; C18; dimensions: 15 cm x 3.0 mm; diameter of particles in the column: 2.7 μm; mobile phase: A= 0.1% aqueous solution of formic acid, B= methanol; gradient: 0 min, 80% A, 20% B; 3 min, 70% A, 30% B; 60 min, 20% A, 80% B; 90 min, 20% A, 80% B; 100 min, 70% A, 30% B; 105 min, 80% A, 20% B; column temperature: 30 °C; flow rate: 0.25 mL/min; analysis time: 110 min; wavelength on the UV-VIS detector: for detection: 370 nm, for integration 290 nm; injection volume: 10 μL; pressure: 210-290 bar.

Examination of the influence of lecithin on the efficiency of extraction of active substances 1-4 from propolis

In the continuation of the research, the influence of different extraction solvents (EO) with the content of different types (SL, RL, HRL) and concentrations (1-30% m/m) of lecithin on the efficiency of extraction of key active substances 1-4 from raw propolis was examined; see Table 2.

Table 2. Tested extraction solvent (EO) systems for extraction of active substances 1-4 from propolis.

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Quantitative analysis of the content of key active substances 1-4 and supporting active substances 5-10 was carried out with the thus prepared liquid extracts of propolis. The results are listed in Tables 3-6.

In case of application of extraction solvents (EO) based on 96% ethanol (EtOH) and mixtures of EtOH and different types (SL, RL, HRL) and lecithin concentration (1-30% m/m in the composition of EO), primary liquid extracts of propolis as dominant active ingredients contain p-coumaric acid (1; approx. 800-1,250 μg/mL), trans-ferulic acid (2; approx. 485-770 μg/mL), caffeic acid (3; approx. 185-290 μg/mL) and 2 -phenylethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE; approx. 215-320 μg/mL); see Table 3.

Table 3. Quantitative composition of liquid propolis extracts with regard to active substances 1-4 obtained with ethanol-based extraction solvents (EO).

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At the same time, the concentrations of accompanying active ingredients 5-10 ranged at the level of: trans-cinnamic acid (5; approx. 40-65 μg/mL), chrysin (6; approx. 500-690 μg/mL), pinocembrin (7; approx. 440 -670 μg/mL), galangin (8; approx. 210-300 μg/mL), apigenin (9; approx. 90-150 μg/mL) and kaempferol (10; approx. 45-90 μg/mL); see Table 4.

In case of application of extraction solvents (EO) based on polyethylene glycol (PEG 400) and mixtures of PEG 400 and different types (SL, RL, HRL) and lecithin concentration (1-10% m/m in the composition of EO), primary liquid extracts of propolis as dominant active ingredients also contain p-coumaric acid (1; approx. 750-1,300 μg/mL), trans-ferulic acid (2; approx. 400-800 μg/mL), caffeic acid (3; approx. 140-300 μg/mL) and 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE; ca. 190-370 μg/mL); see Table 5.

Table 4. Quantitative composition of propolis liquid extracts with regard to accompanying active substances 5-10 obtained with ethanol-based extraction solvents (EO).

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Table 5. Quantitative composition of liquid extracts of propolis with regard to active substances 1-4 obtained with extraction solvents based on polyethylene glycol 400.

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In this case, the concentrations of accompanying active ingredients 5-10 ranged at the level of: trans-cinnamic acid (5; approx. 30-70 μg/mL), chrysin (6; approx. 400-830 μg/mL), pinocembrin (7; approx. 390-800 μg/mL), galangin (8; approx. 190-360 μg/mL), apigenin (9; approx. 80-170 μg/mL) and kaempferol (10; approx. 40-140 μg/mL); see Table 6.

Table 6. Quantitative composition of liquid extracts of propolis with regard to accompanying active substances 5-10 obtained with extraction solvents based on polyethylene glycol 400.

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From the obtained results it is evident that all tested lecithins (SL, RL, HRL) in combination with any pure solvent, 96% ethanol or polyethylene glycol 400 (PEG 400) when used in lower concentrations of 1-3% m/m of the extraction composition solvents (EO) significantly contribute to the increase in the chemoselectivity of the extraction of key active ingredients 1-4 compared to pure solvents.

The unexpected effect of the combination of PEG 400 and lecithin (SL, RL, HRL) is manifested in the fact that, although PEG 400, as a pure solvent, extracts active substances 1-4 significantly less than 96% ethanol (Table 3; row 1, column 2) , when used in combination with lecithins (SL, RL, HRL) in a concentration of 1-3% m/m, significantly more efficiently extracts compounds 1-4 compared to analogous combinations of 96% ethanol and these same lecithins. For example, although the concentration of p-coumaric acid (1) in the extract was obtained with the extraction solvent 100% PEG 400 at the level of 750 μg/mL (Table 5; row 1, column 2) and with EO 96% ethanol at the level of 916 μg /mL (Table 3; row 1, column 2), the application of 3% m/m SL in PEG 400 results in a concentration of 1,112 μg/mL (Table 5; row 2, column 2) compared to the level of 820 μg/mL (Table 3; row 2, column 2) in case of application of EO based on 96% ethanol with 3% m/m SL.

This typical example clearly shows the completely unexpected effect of the combination of polyethylene glycol and lecithin at a concentration of 1-3% m/m of the extraction solvent (EO), which the average expert in the field can extrapolate to an acceptable level of the range of the optimal lecithin mass fraction of 0.1- 3.5% m/m within the EO composition.

Namely, when using higher mass fractions of lecithin in the composition of the extraction solvent (EO), the beneficial effect of lecithin on the chemoselectivity of propolis extraction is lost compared to analogous ethanol-based systems. For example, when using lecithin in concentrations of 4% RL, 7.7% HRL or 10% SL in the composition of EO, lower concentrations of key active substances 1-4 were recorded compared to the use of analog EO systems based on ethanol. Typical results are shown in Tables 7 and 8 for the two most abundant active substances, p-coumaric acid (1) and trans-ferulic acid (2).

Table 7. Quantitative composition of p-coumaric acid (1) in liquid propolis extracts obtained with different extraction solvents according to the invention.

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In Table 7, a typical unexpected result of the invention is visible in row 8, column 3, where a 50% increase in the concentration of p-coumaric acid (1) was recorded when using the extraction solvent (EO) PEG 400 + RL (98.9 : 1.1 , m/m) in relation to the analogous ethanol-based EO (EtOH + RL = 98.9 : 1.1, m/m).

Table 8. Quantitative composition of trans-ferulic acid (2) in liquid propolis extracts obtained with different extraction solvents according to the invention.

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As the next typical example of an unexpected result, we cite the data from Table 8, line 12, column 3, where a 53.5% increase in the concentration of trans-ferulic acid (2) was recorded when the extraction solvent (EO) PEG 400 + HRL (97.8 : 2.2, m/m) compared to the analogous EO based on 96% ethanol.

The procedure for obtaining a standardized liquid extract of propolis according to the invention

The process of obtaining liquid extract of propolis according to the invention includes the following steps:

(i) cooling of raw propolis at –20 °C for a minimum of 1 h;

(ii) grinding of cooled propolis while passing through a sieve with pores of 1-8 mm;

(iii) extraction of raw propolis with an extraction solvent under the following conditions:

(a) mass ratio of raw propolis and extraction solvent = 1:2-1:20 m/m;

(b) extraction temperature of 10-150 °C; and

(c) extraction time from 5 minutes to 72 h;

(iv) filtration of the obtained mixture through a series of filters with a pore size of 100 μm to 5 μm, with the formation of an undissolved residue and a liquid extract of propolis;

(v) quantitative analysis of the content of the key active ingredients of propolis 1-4 by means of high-performance liquid chromatography (HPLC); and

(vi) standardization of the obtained liquid extract of propolis, in which the exact quantitative composition of the key active ingredients 1-4 was determined in step (v), through dilution with fresh extraction solvent that was also used in step (iii), to the level of the desired content of active substances 1- 4.

In the preferred version of the procedure for preparing the liquid extract of propolis according to the invention, the extraction step (iii) is carried out under the following conditions:

(a) mass ratio of raw propolis and extraction solvent = 1:3-1:5 m/m;

(b) extraction temperature of 15-70 °C; and

(c) extraction time of 1-24 h.

Also, the step of quantitative determination of key active substances 1-4 and for comparative monitoring of accompanying active substances 5-10, is carried out with the application, for this purpose, of the developed analytical method of high-performance liquid chromatography (HPLC) as follows:

(i) chromatographic column: C18; dimensions: 15 cm x 3.0 mm; diameter of particles in the column: 2.7 μm; for example, the Ascentis express column;

(ii) mobile phase: A= 0.1% aqueous solution of formic acid, B= methanol; gradient: 0 min, 80% A, 20% B; 3 min, 70% A, 30% B; 60 min, 20% A, 80% B; 90 min, 20% A, 80% B; 100 min, 70% A, 30% B; 105 min, 80% A, 20% B;

(iii) column temperature: 30 °C;

(iv) flow rate: 0.25 mL/min;

(v) analysis time: 110 min;

(vi) wavelength on the UV-VIS detector: for detection: 370 nm, for integration 290 nm;

(vii) injection volume: 10 μL;

(viii) pressure: 210-290 bar.

Experimental procedures for the preparation of liquid propolis extract according to the invention are described in Examples 4-9. Example 9 describes an optimized option for the preparation of a standardized liquid extract with a strength expressed by the drug-to-extract parameter (DER) with a mass ratio of 1:2 according to the invention.

Analytical method for quantitative determination of key active ingredients 1-4 and accompanying active ingredients 5-10 described in Example 3.

Determination of the antimicrobial efficiency of the standardized liquid extract according to the invention. Determination of the minimum inhibitory concentration (MIC) on model pathogenic microorganisms

Antimicrobial efficiency of propolis extracts was measured at the Department of Molecular Medicine of the Ruđer Bošković Institute, Zagreb, Croatia. The minimum inhibitory concentrations (MIC) of the standardized liquid extract of propolis according to the invention were determined, with the application of the product from Example 9, according to the guidelines of the CLSI (Clinical and Laboratory Standards Institute) and EUCAST (European Committee on Antimicrobial Susceptibility Testing) methods, which are described in literature references 26 -29:

26) M. Balouiri, M. Sadiki, S. K. Ibnsouda: Methods for in vitro evaluating antimicrobial activity: A review, J. Pharm. Anal. 6 (2016) 71-79.

27) CLSI, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, Approved Standard, 9th ed., CLSI document M07-A9. Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA, 2012.

28) CLSI, Reference Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts, Approved Standard, 2nd ed., NCCLS document M27-A2. CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA, 2002.

29) CLSI, Methods for Determining Bactericidal Activity of Antimicrobial Agents. Approved Guideline, CLSI document M26-A. Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA, 1998.

Antimicrobial efficiency was tested in vitro on ATCC strains of the following model pathogenic microorganisms (M):

(i) Staphylococcus aureus ATCC 29293 (M1);

(ii) methicillin-resistant Staphylococcus aureus; MRSA (MFBF collection; M2);

(iii) methicillin-susceptible Staphylococcus aureus; MSSA (MFBF collection; M3);

(iv) Enterococcus faecalis ATCC 9212 (M4);

(v) Enterococcus faecalis VRE (MFBF collection) (M5);

(vi) Escherichia coli ATCC 10536 (M6);

(vii) Acinetobacter baumanii ATCC 43498 (M7);

(viii) Pseudomonas aeruginosa ATCC 9027 (M8); and

(ix) Candida albicans ATCC 90028 (M9).

A serial microdilution procedure was performed to determine the minimum inhibitory concentrations (MIC) of the extracts. The MIK value is determined as the concentration of propolis extract at which the number of bacteria or fungi is reduced by 80% (MIK80). The minimum inhibitory concentrations (MIK80) which are shown in Table 9 in the form of dilution (%) of the liquid extract in a particular solvent. The starting liquid extract of propolis was obtained with a mass ratio of raw propolis as a drug and finished extract (DER) 1:2; the product from Example 9. The greater the dilution of the liquid extract, i.e. the lower the concentration of active substances 1-10 for MIK, the stronger the antimicrobial effect of the tested extract.

A detailed description of the experimental procedure for MIC determination is described in Example 10, and the results are shown in Table 9.

Table 9. Results of determining the minimum inhibitory concentration (MIC; %) in the form of dilution of the liquid extract of propolis, product from Example 9, in relation to liquid extracts obtained with 96% ethanol (Example 1) or polyethylene glycol 400 (Example 2) as extraction solvents.

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Tables 10-15 list the mass concentrations of active substances 1-10 in effective dilutions of propolis extracts in each of the three tested extraction solvent systems, obtained using:

(i) 96% ethanol; product from Example 1),

(ii) polyethylene glycol 400 (PEG 400); product from Example 2; and

(iii) mixtures of PEG 400 (97% w/w) and soy lecithin (3% w/w); product from Example 9;

that is, the mass concentrations of individual active substances 1-10 at which the individual liquid extract reached the MIK. It is determined from the primary liquid extract where the DER is 1:2, divided by the dilution at which the MIC was achieved.

Table 10. Mass concentrations (γ) in [μg/mL] for propolis active substances 1-4 in effective dilutions of propolis liquid extract obtained with 96% ethanol; product from Example 1.

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Table 11. Mass concentrations (γ) in [μg/mL] for active propolis substances 5-10 in effective dilutions of propolis liquid extract obtained with 96% ethanol; product from Example 1.

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Table 12. Mass concentrations (γ) in [μg/mL] for propolis active substances 1-4 in effective dilutions of propolis liquid extract obtained with polyethylene glycol 400 (PEG 400); product from Example 2.

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Table 13. Mass concentrations (γ) in [μg/mL] for active propolis substances 5-10 in effective dilutions of propolis liquid extract obtained with polyethylene glycol 400 (PEG 400); product from Example 2.

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Table 14. Mass concentrations (γ) in [μg/mL] for propolis active substances 1-4 in effective dilutions of propolis liquid extract obtained with a mixture of polyethylene glycol 400 (PEG 400; 97% m/m) and soy lecithin (3% m/ m); product from Example 9.

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Table 15. Mass concentrations (γ) in [μg/mL] for active propolis substances 5-10 in effective dilutions of propolis liquid extract obtained with a mixture of polyethylene glycol 400 (PEG 400; 97% m/m) and soy lecithin (3% m/ m); product from Example 9.

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The total antimicrobial effect (MIC) of each of the tested propolis liquid extracts is achieved by the synergistic effect of several active substances 1-10. What is interesting and contrary to expectations is that the effect of a mixture of active substances 1-10 of low concentration is more significant than a higher concentration of a single active substance 1-10.

The formulation of the present invention is most effective against Gram-positive microorganisms such as Staphylococcus spp., but as can be seen from Table 9, in higher concentrations it also works against some Gram-negative bacteria: E. coli and A. baumanii, and the fungus C. albicans.

Use of a standardized liquid extract of propolis according to the invention

The liquid extract of propolis as a pharmaceutical, cosmetic or agrochemical ingredient or food ingredient according to the invention contains a standardized concentration of highly biologically active substances:

(i) p-coumaric acid (1); 100-1,300 μg/mL;

(ii) trans-ferulic acid (2); 75-800 μg/mL;

(iii) caffeic acid (3); 25-300 μg/mL; you

(iv) 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE); 40-400 μg/mL.

Considering the standardized content of key active substances 1-4, the liquid extract of propolis according to the invention represents an active pharmaceutical ingredient (API) for use in humans or animals, an active cosmetic ingredient (ACI), i.e. a functional food ingredient for humans or animals, characterized by the following useful pharmacological effects:

(i) anti-inflammatory; see literature references 1, 8, 9, 11, 12, 13, 17;

(ii) antioxidant; see literature references 8, 9, 14, 15, 20;

(iii) immunomodulating; see literature references 1, 8, 11, 13, 15, 17, 18, 19, 20;

(iv) hepatoprotective; see literature reference 9;

(v) antimicrobial; including antibacterial, antiviral, antimycotic and antiamoebic; see literature references 9, 10, 12, 15, 16, 18;

(vi) antitumor; see literature references 9, 10, 11, 14, 19; you

(vii) anticarcinogenic; see literature references 11, 14.

Among the other valuable effects of the liquid extract of propolis according to the invention, fungicidal, bactericidal, virucidal, insecticidal and nematocidal effects in the protection of plants should be highlighted. Due to its strong antioxidant effect, the liquid extract of propolis according to the invention indirectly strengthens plants and their resistance to abiotic stress factors and helps them with infections. This is why it is also used as a herbal enhancer; see literature references 21-25.

The liquid propolis extract according to the invention is used as:

(i) active pharmaceutical ingredient or auxiliary substance for the production of pharmaceutical products selected from the group consisting of: drugs, medicinal products or auxiliary medicinal agents;

(ii) active cosmetic ingredient or auxiliary substance for the production of cosmetic products;

(iii) food ingredient for the production of functional food products, food supplements or food for special dietary needs;

(iv) active pharmaceutical ingredient or auxiliary substance for the production of veterinary products selected from the group consisting of: veterinary medical products; feed for animals; nutritional supplements for animals; or auxiliary medicinal products for use in veterinary medicine; or

(v) active agrochemical ingredient or auxiliary substance for the production of agrochemical products selected from the group consisting of: fungicides, bactericides, virucides, insecticides, nematocides and plant enhancers; which is especially suitable in ecological cultivation of plant crops.

Pharmaceutical formulation based on the described standardized liquid extract of propolis according to the invention

Furthermore, the present invention discloses a pharmaceutical formulation based on said standardized liquid extract of propolis as an active pharmaceutical ingredient (API). The pharmaceutical formulation according to the invention consists of:

(I) liquid propolis extract according to the invention; from 5-95% m/m; and,

(II) one or more pharmaceutical excipients necessary for the formation of the final dosage form selected from the group consisting of: solution, suspension, gel, cream, ointment, spray for oral or nasal administration; up to 100% m/m of the finished formulation;

where said formulation is characterized by quantitative content for at least two of the four key active substances of propolis within the following values:

(i) p-coumaric acid (1); from 10-1,300 μg/g;

(ii) trans-ferulic acid (2); from 10-800 μg/g;

(iii) caffeic acid (3); from 5-300 μg/g; and,

(iv) 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE); from 5-400 μg/g.

The pharmaceutical excipients (excipients) are selected from the following groups: fillers, humectants, preservatives, chelating agents, antioxidants, thickeners, emollients, emulsifiers, tonicity agents and pH control agents.

The filler is a pharmaceutically acceptable liquid selected from the group consisting of: purified water; ethanol; glycerol; 1,2-propylene glycol; liquid polyethylene glycols (PEG) such as PEG 200, PEG 400 or PEG 600; or mixtures of these substances.

The humectant is selected from the group consisting of: glycerol, sorbitol, 1,2-propylene glycol, or mixtures of these substances.

The preservative is selected from the group consisting of: parabens such as methyl 4-hydroxybenzoate, ethyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate, butyl 4-hydroxybenzoate; 4-chloro-m-cresol; triclosan; benzyl alcohol; 2-phenoxyethanol; benzoic acid and its salts such as sodium benzoate; sorbic acid and its salts such as potassium sorbate; dehydroacetic acid (3-acetyl-2-hydroxy-6-methyl-4H-pyran-4-one); chlorhexidine and its salts such as chlorhexidine digluconate; quaternary ammonium salts such as benzalkonium chloride or cetrimonium bromide; or mixtures of these substances.

The chelating agent is selected from the group consisting of: sodium or potassium salts of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminopentaacetic acid (DTPA), nitrilotriacetic acid (NTA); soluble citrate salts such as trisodium citrate dihydrate (Na3C6H5O7•2H2O); or mixtures of these substances. A typical chelating agent is disodium edetate dihydrate (Na2EDTA•2H2O).

The antioxidant is selected from the group consisting of: α-tocopherol and its esters such as α-tocopheryl succinate; ascorbic acid and its salts such as sodium ascorbate; 2,6-di-tert-butyl-4-methylphenol (BHT); tert-butyl-anisole (BHA); or mixtures of these substances.

The thickener is selected from the group consisting of: cellulose gums such as hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), methyl cellulose (MC), sodium carboxymethyl cellulose (NaCMC); synthetic polymers such as polyvinyl alcohol (PVA), polyacrylic acid (PAA) and its copolymers, polyvinyl pyrrolidone (PVP); various gums such as gum arabic, xanthan gum, tragacanth; alginic acid and its salts such as sodium alginate; metal salts of higher fatty acids such as aluminum monostearate, aluminum distearate, aluminum tristearate; or mixtures of these substances.

The emollient is chosen from the group consisting of: vaseline; mineral oil; vegetable oils such as almond, sunflower or olive oil; medium chain triglycerides; natural or synthetic esters of higher fatty acids and monovalent alcohols such as isopropyl myristate or jojoba oil; waxes such as beeswax; silicone oil; more fatty acids such as oleic or stearic acid; higher fatty alcohols such as cetyl alcohol; or mixtures of these substances.

The emulsifier is selected from the group consisting of: lanolin; ethoxylated lanolin; lanolin alcohols; ethoxylated lanolin alcohols; lecithin; hydrolyzed lecithin; mono- and diesters of glycerol and higher fatty acids such as glycerol monostearate; sorbitan esters with higher fatty acids such as sorbitan monostearate; ethoxylated higher fatty alcohols such as polyoxyethylene(23) lauryl ether or polyoxyethylene(2) oleate where the number 23 or 2 represents the number of ethylene oxide units; esters of ethoxylated sorbitan esters such as polysorbate 60; water-soluble soaps such as sodium stearate; water-soluble salts of sulfates of higher fatty alcohols such as sodium lauryl sulfate; water-soluble fatty alcohol phosphate salts such as potassium cetyl phosphate; or mixtures of these substances.

The agent for achieving tonicity is used primarily in pharmaceutical dosage forms that are applied to mucous membranes, for example the nasal mucosa, and are selected from the group consisting of: sodium chloride (NaCl), glycerol, 1,2-propylene glycol, or mixtures of the above substances.

The agent for controlling the pH value consists of pharmaceutically acceptable acids and bases for lowering or raising the pH value, as well as buffer systems and is chosen from the group consisting of: hydrochloric acid (HCl), sulfuric acid (H2SO4), phosphoric acid (H3PO4), citric acid, acetic acid, sodium hydroxide (NaOH), potassium hydroxide (KOH), ammonium hydroxide (NH4OH), sodium dihydrogen phosphate (NaH2PO4), sodium hydrogen phosphate (Na2HPO4), sodium dihydrogen citrate (NaH2C6H5O7), sodium hydrogen citrate (Na2HC6H5O7), sodium citrate (Na3C6H5O7) , or mixtures of these substances.

Preparation of the pharmaceutical formulation according to the invention

The pharmaceutical formulation according to the invention is prepared according to a procedure that includes the following steps:

(i) adding the standardized liquid extract of propolis according to the invention to the filler and their homogenization;

(ii) addition of one or more other excipients; and their homogenization;

wherein steps (i) and (ii) are carried out at a temperature of 10-100 °C, preferably at a temperature of 20-60 °C, for 1-15 minutes; and then, in the case of preparing a dosage form:

(iii.a) solutions or spray solutions; filtration of the finished solution is performed, including, if necessary, sterile filtration;

(iii.b) gel or suspension; addition of thickener and its homogenization are carried out;

(iii.c) creams; is done:

- preparation of the fat phase by mixing emollients and emulsifiers and their homogenization at a temperature of 50-80 °C, for 1-15 minutes, and then

- adding the solution from step (ii) heated to 50-80 °C, and then,

- emulsification using a homogenizer with a high number of revolutions (for example 1,000-3,000 rpm or more) mixing element or high pressure, at a temperature of 50-80 °C, preferably 55-65 °C, for 1-30 minutes, with,

- subsequent homogenization at temperatures of 65-20 °C, for 10-120 minutes; or at

(iii.d) fats;

- the solution from step (ii) is mixed with the previously melted mixture of emollients and possibly emulsifiers at a temperature of 50-70 °C, for 5-30 minutes, with

- subsequent homogenization at temperatures of 70-20 °C, for 10-120 minutes.

The procedures for preparing the pharmaceutical formulation according to the invention may also include different variants of all the usual technological procedures for the production of said dosage forms, as is known to the average expert in the field of pharmaceutical technology.

Representative examples of the preparation of the pharmaceutical formulation according to the invention are described in Examples 11-16.

As a special example, we single out the preparation of the dosage form of the solution for intramammary administration, which is described in Example 11. In this case, the primary standardized liquid extract of propolis, the preparation of which is described in Example 9, is only diluted with the extraction solvent according to the invention, in this case with a mixture of polyethylene glycol 400 ( 97% m/m) and soy lecithin (3% m/m), which here functions as a filler. The finished solution for intramammary application provides a quantitative content of the key active substances 1-4 within the above-defined framework; see Table 16.

Table 16. Results of quantitative analysis of active substances 1-10 by means of high-performance liquid chromatography (HPLC) in the pharmaceutical formulation of the present invention, dosage form of solution for intramammary change, product of Example 11, characterized by a mass ratio of drug-to-extract (DER) of 1 :2, diluted 10x for HPLC analysis.a

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A typical HPLC chromatogram from the quantitative analysis of the subject formulation is shown in Figure 5.

Studies of the pharmacological effects of the pharmaceutical formulation according to the invention

Selected pharmacological effects of the formulation of the subject invention in the dosage form of a solution, the product from Example 11, were tested in clinical studies in therapy:

(i) mastitis, udder inflammation in cows;

(ii) mastitis in goats; and,

(iii) wound healing in horses.

A study in the therapy of mastitis in cows

The study of the treatment of mastitis in cows was carried out on five farms of dairy cattle of the Holstein breed. 86 dairy cows, or 339 quarters, were included in the research, where the udder quarter was used as a statistical unit. The animals were kept freely on deep bedding, and were fed with a standard mixture for dairy cows without the addition of antibiotics. The research was approved by the Veterinary Ethics Committee. The study included animals, i.e. quarters without clinical symptoms of mastitis, i.e. healthy, uninfected quarters with a somatic cell count (BSS) below 200,000/mL, and infected quarters with a BSS count greater than 200,000/mL. A randomized cross-over clinical study of the safety and efficacy of the intramammary (i.mammary) solution of the formulation from the subject invention was conducted on cows in field conditions. The formulation of the subject invention from Example 11 was applied three times in all four quarters of the cow's udder: during morning milking, evening milking, and the next day after morning milking. The detailed implementation procedure is described in Example 17, while Table 17 shows the bacteriological cure for each causative agent that was identified in a certain number of districts before the first i.mam. applications.

Table 17. Presentation of the success rate of bacteriological cure of mastitis in cows after i.mam. application of the formulation of the subject invention from Example 11.a

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With the triple (two-day) application of the formulation from Example 11, 100% bacteriological cure was achieved in just 7 days.

For comparison, 66% of bacteriological cure is achieved with the cephalosporin antibiotic ceftiofur, but only after daily i.mam. therapy lasting 8 days, while after 5 days of daily application, healing is achieved in only 54% of cases; see literature reference 30. In subclinical mastitis caused by S. uberis, two-day therapy with pirlimycin led to healing in 58.1% of cases, while therapies lasting 5 and 8 days led to healing in 68.8% and 80% of cases; see literature reference 30:

30) S. P. Oliver, B. E. Gillespie, S. J. Headrick, H. Moorehead, P. Lunn, H. H. Dowlen, D. J. Johnson, K. C. Lamar, S. T. Chester, W. M. Moseley: Efficacy of extended Ceftiofur intramammary therapy for treatment of subclinical mastitis in lactating dairy cows. J. Dairy Sci. 87 (2004) 2393-2400.

Alternatively, in the therapy of mastitis in cows, the formulation of the subject invention in the dosage form of a suspension for intramammary administration as described in Example 12 can be used with equal success.

A study in the therapy of mastitis in goats

The study of the treatment of mastitis in goats was carried out at the alpine goat farm owned by OPG Matijašec from Sigetec Ludbreški, Croatia, on 25 goats diagnosed with subclinical mastitis in the left and right half of the goat's udder. Goats whose milk was positive on the microbiological examination were divided into two groups: one group was administered the formulation of the subject invention from Example 11, while the other group was administered an intramammary suspension of amoxicillin with clavulanic acid (Klavuxil®; Genera, Croatia) three times. In parallel, the tolerability and bacteriological cure of the halves after i.mam were monitored. formulations from Example 11. With the triple (two-day) application of the formulation from Example 11 in just 7 days, bacteriological cure was achieved in a total of 75% of the infected halves, and after 14 days in 85% of the halves. This proved to be more effective than intramammary antibiotics, which led to bacterial cure in 73.3% of cases. The results of the research showed that the treatment of mastitis with the formulation from Example 11 in goats can lead to a bacteriological cure in time without the use of antibiotics.

The detailed procedure of conducting the study is described in Example 18, while the results of bacteriological cure are shown in Table 18.

Table 18. Presentation of the success rate of bacteriological cure of mastitis in goats after i.mam. application of the formulation of the subject invention from Example 11 in comparison with an antibiotic (fixed combination of amoxicillin and clavulanic acid).a

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By comparing the action of i.mam. application of amoxicillin, as a broad-spectrum antibiotic that is suitable for the treatment of mastitis pathogens found in our milk samples, and the formulation from Example 11, its beneficial effect on the pathogens has been proven. While in 7 days after the first application i.mam. preparation of amoxicillin, only 60% of the halves were bacteriologically cured, this result for the formulation from Example 11 is 75%. 14 days after the first application, the total percentage of bacteriologically cured halves when using antibiotics was 73.3%, and for the tested formulation 85%.

From the above results, high efficiency in the anti-inflammatory and antimicrobial action of the formulation of the subject invention in the treatment of mastitis is evident. The effectiveness is higher than the effectiveness of classic therapy such as the one mentioned with a fixed combination of amoxicillin and clavulanic acid as a broad-spectrum antibiotic.

Alternatively, in the therapy of mastitis in goats, the formulation of the subject invention in the dosage form of a suspension for intramammary administration as described in Example 12 can be used with equal success.

It can be concluded that the formulation from the subject invention, in addition to other properties, is characterized by antimicrobial action against a number of bacteria and fungi, whereby the antimicrobial effectiveness compared to classic antibiotics is greater in vivo than in vitro conditions. It is not only an antimicrobial agent, but also an anti-inflammatory and immunomodulatory agent.

The immunomodulatory effects of propolis are closely related to the antioxidant effects, and oxidative stress is an integral part of the pathogenesis of mastitis; see for example literature reference 31:

31) O. Atakisi, H. Oral, E. Atakisi, O. Merhan, S. metin Pancarci, A. Ozcan, S. Marasli, B. Polat, A. Colak, S. Kaya: Subclinical mastitis causes alterations in nitric oxide , total oxidant and antioxidant capacity in cow milk, Res. Vet. Sci. 89 (2010) 10-13.

Presentation of a case of wound healing in a horse

A case of healing a wound on a mare's track is presented. It was a deep wound in the area of the path caused by catching up during landing. Before applying the formulation from Example 11, the wound was unsuccessfully treated conservatively with various preparations for two weeks.

The wound was then washed with saline solution, dried and treated with the formulation from Example 11 once a day for 5 days. Improvement in the form of epithelialization was visible after 48 h, and complete healing occurred after 96 h. The mare clearly limped only during the first few days and after that no signs of lameness were noticed. The recovery was eventually complete. A detailed description of the case is described in Example 19.

Although for some more precise conclusions it is necessary to conduct a more extensive clinical study on a larger number of animals, it is clear to the average expert in the field that the formulation in question works effectively as an agent that promotes wound healing. Since it is known from the literature that anti-inflammatory, antioxidant and epithelizing action, which includes the stimulation of collagen biosynthesis, is important for the wound healing process, we can conclude that the formulation of the subject invention, in addition to its proven antimicrobial action, is characterized by precisely this spectrum of pharmacological effects; for comparison, see literature reference 32:

32) S. Marinotti, E. Ranzato: Propolis: a new frontier for wound healing, Burns Trauma (2015) 3:9, doi 10.1186/s41038-015-0010-z.

Use of the pharmaceutical formulation according to the invention

The pharmaceutical formulation according to the invention is used for the treatment of diseases and conditions in humans and animals, which are selected from the group consisting of: inflammatory diseases; bacterial infections; fungal infections; viral diseases; autoimmune diseases; functional gastrointestinal disorders; for regeneration of mucous membranes, treatment of burns and wound healing; and tumor diseases.

Inflammatory diseases and conditions are selected from the group consisting of: gingivitis, periodontitis, laryngitis, gastritis, colitis, hemorrhoidal disease, dermatitis, inflammation of the external ear, sinusitis, rhinitis, vaginitis and mastitis.

The pharmaceutical formulation according to the invention is used for the treatment of bacterial infections caused by bacteria selected from the group consisting of:

(i) Gram-positive bacteria: Staphylococcus spp.: Staphylococcus aureus, MRSA (methicillin-resistant Staphylococcus aureus), MSSA (methicillin-susceptible Staphylococcus aureus), Staphylococcus intermedius, Staphylococcus pseudintermedius; coagulase-negative staphylococci: Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus hyicus; Streptococcus spp.: Streptococcus uberis, Streptococcus bovis, Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus canis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus oralis, Streptococcus thermophilus; Peptostreptococcus spp.; Corynebacterium spp.: Corynebacterium bovis; Trueperella pyogenes, Nocardia spp.; Bacillus subtilis; Bacillus cereus; Enterococci: Enterococcus faecium, Enterococcus faecalis; vancomycin-resistant enterococci (VRE): Enterococcus casseliflavus; and,

(ii) Gram-negative bacteria: Escherichia coli; Acinetobacter baumannii; Pseudomonas aeruginosa; Haemophilus influenzae; Salmonella enterica; Yersinia enterocolitica; Enterobacter spp. (Enterobacter cloacae); Klebsiella spp.: Klebsiella pneumoniae, Klebsiella oxytoca; Shigella flexneri; Burkholderia cepacia; Proteus mirabilis; Proteus vulgaris; Aggregatibacter actinomycetemcomitans; Actinomyces israelii; Bacteroides fragilis; Helicobacter pylori; Campylobacter coli; Campylobacter jejuni; Porphyromonas gulae; Porphyromonas salivosa; Porphyromonas denticanis; Prevotellaintermedia; Treponema spp.; Bacteroides splanchnicus.

Furthermore, the subject pharmaceutical formulation is used for the treatment of fungal infections caused by fungi selected from the group consisting of: Candida spp. (Candida albicans, Candida dubliniensis, Candida glabrata, Candida kruzei, Candida tropicalis, Candida parapsilosis); Aspergillus spp. (Aspergillus niger, Aspergillus versicolor); Penicillium pinophilum; Paecilomyces variotii; Trichoderma virens; Chaetomium globosum; Malassezia pachydermatis.

Also, the formulation according to the invention is used for the treatment of viral diseases caused by viruses selected from the group consisting of: Herpes simplex virus (HSV); Human papilloma virus (HPV); Epstein-Barr virus (EBV); Cytomegalovirus (CMV); poliovirus; influenza viruses A and B; retroviruses; Vaccinia virus; cold viruses: rhinovirus, picornavirus, human parainfluenza virus (HPIV), human metapneumovirus (HMPV), coronavirus, adenoviruses, human respiratory syncytial virus (HRSV), enteroviruses.

Alternatively, the pharmaceutical formulation according to the invention is also used for the treatment of autoimmune diseases selected from the group consisting of: psoriasis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, celiac disease and multiple sclerosis.

Furthermore, pharmaceutical formulations according to the invention are used for the treatment of tumor diseases selected from the group consisting of: skin and mucous membrane cancer, tumors of the digestive system, colorectal cancer.

The pharmaceutical formulation from the present invention is used for the treatment of functional gastrointestinal disorders as follows: disorders of the esophagus, stomach, duodenum, small and large intestine, such as, for example, irritable colon or irritable bowel syndrome (IBS), centrally mediated gastrointestinal pain, gallbladder and sphincter of Oddi disorders , anorectal disorders, gastrointestinal disorders specific to children and adolescents.

Specifically, the formulation according to the invention is used for the treatment of mastitis in animals.

Examples of embodiments of the invention

General notes

Raw propolis, poplar type, from the company Hedera d.o.o. was used as raw propolis. (HR). Ethanol (96%) and polyethylene glycols 200, 400 and 600 and soy lecithin (SL) in powder were purchased from the company Fagron Hrvatska d.o.o. (HR). Rapeseed lecithin (RL) and hydrolyzed rapeseed lecithin (HRL) were obtained from Pfannenschmidt (DE). Deoleinized sunflower lecithin (SUL) was purchased from Barentz (NL). Aluminum distearate was purchased from Sigma-Aldrich (US). All other raw materials were obtained from local suppliers.

Samples of chemically pure compounds for analytical purposes: p-coumaric acid (1; ≥98% HPLC), trans-ferulic acid (2; 99%), caffeic acid (3; ≥98% HPLC), 2-phenylethyl 3,4- dihydroxy-trans-cinnamate (CAPE; 4; ≥97% HPLC), trans-cinnamic acid (5; ≥96.5% GC), chrysin (6; ≥98% HPLC), pinocembrin (7; ≥95% TLC), galangin (8; ≥95% HPLC), apigenin (9; ≥99% HPLC) and kaempferol (10; ≥90% HPLC), which served as quantitative analytical standards for determining their quantitative content in propolis liquid extracts, were purchased from the company Sigma-Aldrich (US).

The term room temperature means a temperature interval of 20-25 °C.

The abbreviation "min" stands for minutes. Recovery (% of theoretical yield) is expressed as mass (% m/m) percentage of the isolated liquid extract of propolis in relation to the starting mass of the extraction solvent (ethanol, PEG, ethanol + lecithin, PEG + lecithin).

Quantitative compositions of active ingredients 1-10 in liquid propolis extracts are expressed as mass concentration (γ) in micrograms per milliliter [μg/mL], while their quantitative composition in the pharmaceutical formulation according to the invention is expressed in micrograms per gram of finished product [μg/g ].

Example 1. Preparation of liquid extract of propolis using 96% ethanol as extraction solvent

Processing of raw propolis before extraction: A sample of raw propolis (poplar-type; 1 kg) was left to cool in a freezer at -20 °C for a minimum of 1 h and was then crushed in a mill.

Extraction with 96% ethanol: Chopped propolis (30.00 g) was poured with ethanol (96%; 70.00 g). The resulting mixture was left to stand at room temperature for 72 hours with occasional stirring. After that, the mixture was filtered through filter paper (black ribbon). 60.00 g (85.7%) of liquid propolis extract was obtained in the form of a dark brown liquid with a slightly intense propolis smell.

For the purpose of testing the minimum inhibitory concentration (MIC) of the propolis alcoholic extract described in Example 10, the same procedure was repeated with a drug-to-extract ratio (DER) of 1:2.

Example 2. Preparation of liquid extract of propolis using polyethylene glycol 400 as extraction solvent

The crushed propolis obtained by the pretreatment described in Example 1 (30.00 g) was covered with polyethylene glycol 400 (PEG 400; 70.00 g). The resulting mixture was left to stand at room temperature for 72 hours with occasional stirring. After that, the mixture was filtered through filter paper (black ribbon). 55.00 g (78.6%) of liquid propolis extract was obtained in the form of a viscous dark brown liquid with a slightly intense propolis smell.

For the purpose of testing the minimum inhibitory concentration (MIC) of the polyethyleneglycol extract of propolis described in Example 10, the same procedure was repeated with a drug-to-extract ratio (DER) of 1:2.

Example 3. HPLC analytical method for quantitative determination of active ingredients 1-10 in liquid extracts of propolis

Quantitative analyzes by high-performance liquid chromatography (HPLC) were carried out using a method that was developed specifically for the purpose of monitoring key active substances 1-4, and accompanying active ingredients 5-10 from propolis.

Samples of commercially available standards of active substances 1-10 were prepared for analysis by diluting with a mixture of ethanol:water, 75:25, V/V, to a concentration of 100 μg/mL.

Samples of liquid extracts of propolis (100 μL) according to the invention were diluted with a mixture of ethanol:water, 75:25, V/V (900 μL), in a ratio of 1:10 m/m (10x dilution) before analysis.

Analyzes were performed on a Shimadzu LC201CHT instrument equipped with an autosampler, pump, degasser, column oven and UV-VIS detector under the following conditions:

(i) chromatographic column: Ascentis express; C18; dimensions: 15 cm x 3.0 mm; diameter of particles in the column: 2.7 μm;

(ii) mobile phase: A= 0.1% aqueous solution of formic acid, B= methanol; gradient: 0 min, 80% A, 20% B; 3 min, 70% A, 30% B; 60 min, 20% A, 80% B; 90 min, 20% A, 80% B; 100 min, 70% A, 30% B; 105 min, 80% A, 20% B;

(iii) column temperature: 30 °C;

(iv) flow rate: 0.25 mL/min;

(v) analysis time: 110 min;

(vi) wavelength on the UV-VIS detector: for detection: 370 nm; for integration 290 nm;

(vii) injection volume: 10 μL;

(viii) pressure: 210-290 bar.

Under the stated conditions, key active substances 1-4 and supporting active substances 5-10 have the following retention times (tR):

(i) tR [p-coumaric acid (1)]= 14.13 min; tR [trans-ferulic acid (2)]= 15.31 min; tR [caffeic acid (3)]= 10.57 min; tR [2-phenylethyl 3,4-dihydroxy-trans-cinnamate; CAPE (4)]= 48.62 min;

(ii) tR [trans-cinnamic acid (5)]= 29.76 min; tR [chrysin (6)]= 44.68 min; tR [pinocembrin (7)]= 47.39 min; tR [galangin (8)]= 49.94 min; tR [apigenin (9)]= 37.72 min; tR [kaempferol (10)]= 36.87 min.

Retention times of the key active ingredients of propolis 1-4 and accompanying active ingredients 5-10 are shown in Table 1.

Example 4. Preparation of standardized liquid extracts of propolis using an extraction solvent based on a mixture of ethanol and lecithin

The crushed propolis obtained by the pretreatment described in Example 1 (30.00 g) was poured with the extraction solvent of the following composition (experiments P1-8):

P1: 96% ethanol (67.00 g; 95.7%) and soy lecithin (SL; 3.00 g; 4.3%);

P2: 96% ethanol (60.00 g; 85.7%) and soy lecithin (SL; 10.00 g; 14.3%);

P3: 96% ethanol (50.00 g; 71.4%) and soy lecithin (SL; 20.00 g; 28.6%);

P4: 96% ethanol (40.00 g; 57.1%) and soy lecithin (SL; 30.00 g; 42.9%);

P5: 96% ethanol (67.00 g; 98.9%) and rapeseed lecithin (RL; 3.00 g of 25% preparation; 0.75 g lecithin; 1.1%); theoretical yield of experiment 5 = 67 g ethanol + 0.75 g lecithin = 67.75 g;

P6: 96% ethanol (60.00 g; 96.0%) and rapeseed lecithin (RL; 10.00 g of 25% preparation; 2.50 g lecithin; 4.0%); theoretical yield of experiment 6 = 60 g ethanol + 2.50 g lecithin = 62.50 g;

P7: 96% ethanol (67.00 g; 97.8%) and hydrolyzed rapeseed lecithin (HRL; 3.00 g of 50% preparation; 1.50 g of hydrolyzed lecithin; 2.2%); theoretical yield of experiment 7 = 67 g ethanol + 1.50 g lecithin = 68.50 g;

P8: 96% ethanol (60.00 g; 92.3%) and hydrolyzed rapeseed lecithin (HRL; 10.00 g of 50% preparation; 5.00 g lecithin; 7.7%); theoretical yield of experiment 8 = 60 g ethanol + 5.00 g lecithin = 65.00 g.

The resulting mixture was stirred at room temperature for 1 h and left to stand at room temperature for 72 h with occasional stirring. After that, the mixture was filtered through filter paper (black ribbon). 50-60 g (71.0-85.7%) of liquid propolis extract was obtained in the form of a viscous dark brown liquid, with a slightly intense smell of propolis.

The thus prepared primary liquid propolis extracts were subjected to a quantitative analysis of the content of key active substances 1-4 and accompanying active substances 5-10 according to the analytical method described in Example 3, and the results are shown in Tables 3 and 4.

Thus prepared primary liquid extracts of propolis in extraction solvents (EO) described in experiments P1-P8 with known quantitative compositions of active ingredients 1-4, were standardized by dilution with the same EO that was also used in the extraction step to the desired level of quantitative composition of said active ingredients 1 -4 according to the invention.

Example 5. Preparation of standardized liquid extracts of propolis using an extraction solvent based on a mixture of polyethylene glycol 400 and lecithin

The crushed propolis obtained by the pretreatment described in Example 1 (30.00 g) was poured with the extraction solvent of the following composition (experiments P1-6):

P1: polyethylene glycol 400 (PEG 400; 67.00 g; 97%) and soy lecithin (SL; 3.00 g; 3%);

P2: polyethylene glycol 400 (PEG 400; 60.00 g; 90%) and soy lecithin (SL; 10.00 g; 10%);

P3: polyethylene glycol 400 (PEG 400; 67.00 g; 98.9%) and rapeseed lecithin (RL; 3.00 g of 25% preparation; 0.75 g lecithin; 1.1%); theoretical yield of experiment 3 = 67 g PEG 400 + 0.75 g lecithin = 67.75 g;

P4: polyethylene glycol 400 (PEG 400; 60.00 g; 96.0%) and rapeseed lecithin (RL; 10.00 g of 25% preparation; 2.50 g lecithin; 4.0%); theoretical yield of experiment 4 = 60 g PEG 400 + 2.50 g lecithin = 62.50 g;

P5: polyethylene glycol 400 (PEG 400; 67.00 g; 97.8%) and hydrolyzed rapeseed lecithin (HRL; 3.00 g of 50% preparation; 1.50 g of hydrolyzed lecithin; 2.2%); theoretical yield of experiment 5 = 67 g PEG 400 + 1.50 g lecithin = 68.50 g;

P6: polyethylene glycol 400 (PEG 400; 60.00 g; 92.3%) and hydrolyzed rapeseed lecithin (HRL; 10.00 g of 50% preparation; 5.00 g lecithin; 7.7%); theoretical yield of experiment 6 = 60 g PEG 400 + 5.00 g lecithin = 65.00 g.

The resulting mixture was stirred at room temperature for 1 h and left to stand at room temperature for 72 h with occasional stirring. After that, the mixture was filtered through filter paper (black ribbon). 45-55 g (69.0-78.6%) of liquid propolis extract was obtained in the form of a viscous dark brown liquid, with a slightly intense smell of propolis.

The thus prepared primary liquid extracts of propolis were subjected to a quantitative analysis of the content of key active substances 1-4 and accompanying active substances 5-10 according to the analytical method described in Example 3, and the results are shown in Tables 5 and 6.

Thus prepared primary liquid extracts of propolis in extraction solvents (EO) described in experiments P1-P8 with known quantitative compositions of active ingredients 1-4, were standardized by dilution with the same EO that was also used in the extraction step to the desired level of quantitative composition of said active ingredients 1 -4 according to the invention.

Example 6. Preparation of a standardized liquid extract of propolis using an extraction solvent based on a mixture of polyethylene glycol 200 and soy lecithin

The crushed propolis obtained by the pretreatment described in Example 1 (30.00 g) was poured with the extraction solvent, which is a mixture of polyethylene glycol 200 (149.85 g; 99.9%) and soy lecithin (SL; 0.15 g; 0.1%) ). The resulting mixture was stirred at room temperature for 1 h and left to stand at room temperature for 48 h with occasional stirring. After that, the mixture was filtered through filter paper (black ribbon). 137.00 g (91.3%) of liquid propolis extract was obtained in the form of a viscous dark brown liquid, with a slightly intense smell of propolis.

After that, a quantitative HPLC analysis was performed according to the method described in Example 3, from which the quantitative proportions of the key active substances 1-4 were determined. The filtrate was diluted with the same extraction solvent, a mixture of polyethylene glycol 200 and soy lecithin (SL), 99.9 : 0.1 m/m, to the total content of active substances 1-4 according to the specification of the standardized liquid extract according to the invention.

Example 7. Preparation of a standardized liquid extract of propolis using an extraction solvent based on a mixture of polyethylene glycol 600 and deoleinized sunflower lecithin

The crushed propolis obtained by the pretreatment described in Example 1 (30.00 g) was poured with an extraction solvent that is a mixture of polyethylene glycol 600 (PEG 600; 297.00 g; 99%) and deoleinized sunflower lecithin (SUL; 3.00 g; 1, 0%). The resulting mixture was heated at 70 °C with intensive stirring for 3 h. After that, the mixture was cooled to room temperature and filtered through filter paper (black ribbon). 261.00 g (87.0%) of liquid propolis extract was obtained in the form of a viscous dark brown liquid, with a slightly intense smell of propolis.

After that, a quantitative HPLC analysis was performed according to the method described in Example 3, from which the quantitative proportions of the key active substances 1-4 were determined. The filtrate was diluted with the same extraction solvent, a mixture of polyethylene glycol 600 and deoleinized sunflower lecithin (SUL), 99:1, m/m, to the total content of active substances 1-4 according to the specification of the standardized liquid extract according to the invention.

Example 8. Preparation of a standardized liquid extract of propolis using an extraction solvent based on a mixture of polyethylene glycol 200, polyethylene glycol 600 and hydrolyzed rapeseed lecithin

The crushed propolis obtained by the pretreatment described in Example 1 (30.00 g) was poured with an extraction solvent which is a mixture of polyethylene glycol 200 (PEG 200; 150.00 g; 50%), polyethylene glycol 600 (139.50 g; 46.5%) and hydrolyzed rapeseed lecithin (HRL; 10.50 g; 3.5%). The resulting mixture was heated at 100 oC with intensive stirring for 3 h. After that, the mixture was cooled to room temperature and filtered through filter paper (black ribbon). 245.00 g (81.7%) of liquid propolis extract was obtained in the form of a viscous dark brown liquid, with a slightly intense smell of propolis.

After that, a quantitative HPLC analysis was performed according to the method described in Example 3, from which the quantitative proportions of the key active substances 1-4 were determined. The filtrate was diluted with the same extraction solvent, a mixture of polyethylene glycol 200, polyethylene glycol 600 and hydrolyzed rapeseed lecithin (HRL), 50 : 46.5 : 3.5 m/m/m, to the total content of active substances 1-4 according to the specification of the standardized liquid extract according to the invention.

Example 9. Preparation of a standardized liquid extract of propolis using a mixture of polyethylene glycol 400 and soy lecithin

The crushed propolis obtained by the pretreatment described in Example 1 (30.00 g) was poured with an extraction solvent (90.00 g) which is a mixture of polyethylene glycol 400 (PEG 400; 87.30 g; 97% m/m) and soy lecithin (SL ; 2.70 g; 3% w/w). The mixture was left to macerate with occasional stirring for 76 hours. After that, the mixture was filtered through filter paper (800 mesh/cm2). After filtration, 50-55 g of a dark brown viscous solution with an intense smell of propolis was obtained. The obtained product was supplemented with the same extraction solvent to a weight of 60.00 g. In this way, an extract with drug-to-extract (DER) was obtained with a ratio of 1:2, that is, the said 60 g of extract from 30 g of starting propolis.

The liquid extract of propolis prepared in this way is used for the purpose of preparing a pharmaceutical formulation according to the invention:

(I) is subjected to quantitative HPLC analysis according to the method described in Example 3 in order to determine the content of active substances 1-4, and then, considering the real mass fraction of active substances 1-4 in the thus prepared extract,

(II) standardize by diluting with a pure mixture of solvents: polyethylene glycol 400 (97% m/m) and soy lecithin (3% m/m);

up to the mass concentration level of active substances 1-4:

(i) p-coumaric acid (1); 10-1,300 μg/mL;

(ii) trans-ferulic acid (2); 10-800 μg/mL;

(iii) caffeic acid (3); 5-300 μg/mL; and,

(iv) 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE); 5-400 μg/mL;

divided by the factor X/100, where X = mass fraction %, m/m, of said standardized liquid extract of propolis in the recipe of the pharmaceutical formulation.

Example 10. Determination of the antimicrobial efficiency of the standardized liquid extract according to the invention. Determination of the minimum inhibitory concentration (MIC) on model pathogenic microorganisms

The minimum inhibitory concentrations (MIC) of the standardized liquid extract of propolis according to the invention, with the application of the product from Example 9, were determined in comparison with the analogous liquid extracts of propolis obtained with the application of 96% ethanol (product from Example 1) or PEG 400 (product from Example 2). , according to CLSI and EUCAST method guidelines; see literature references 26-29.

Antimicrobial efficiency was tested in vitro on ATCC strains of the following model pathogenic microorganisms (M): Staphylococcus aureus ATCC 29293 (M1); methicillin-resistant Staphylococcus aureus (MRSA; MFBF collection; M2); methicillin-susceptible Staphylococcus aureus (MSSA; MFBF collection; M3); Enterococcus faecalis ATCC 9212 (M4); Enterococcus faecalis VRE (MFBF collection) (M5); Escherichia coli ATCC 10536 (M6); Acinetobacter baumannii ATCC 43498 (M7); Pseudomonas aeruginosa ATCC 9027 (M8); and Candida albicans ATCC 90028 (M9).

A serial microdilution procedure was performed to determine the minimum inhibitory concentrations (MIC) of the extracts. Cell suspensions were prepared from mother cultures in PBS buffer (pH 7.4), and adjusted to 0.5 MacFarland units using a nephelometer. Testing was carried out in serial dilutions on microtiter plates with 96 wells ranging from 100 to 0.7125 µg/mL, so that 100 µL of propolis extract solution dissolved in Mueller Hinton broth was added to each well. After inoculation with 100 µL of a specific bacterial culture adjusted to 105 CFU/mL, the plates were incubated for 24 hours at 37 °C. MIC was determined by adding 10 µL of 0.5 mg/mL 2,3,5-triphenyl-2H-tetrazolium chloride (TTC; redox indicator) per well, and after incubation for 4 hours at 30 °C, the absorbance was read with a spectrophotometer at length 490 nm. The MIK value is determined as the concentration of propolis extract at which the number of bacteria is reduced by 80% (MIK80).

For fungal species, MIC was determined in RPMI medium with added glucose, according to the same scheme as for bacteria. After incubation (48 h, 37 °C, aerobically in the dark), XTT (redox reagent) was added in combination with menadione, and the absorbance was read with a spectrophotometer at a wavelength of 540 nm. The MIK value is determined as the concentration of propolis extract at which the number of bacteria or fungi is reduced by 80% (MIK80).

The negative control contained only medium and solvent (without added microorganisms and propolis), while the positive control was exposed to the action of antibiotics and antimycotics.

By determining the antimicrobial activity of propolis solutions in vitro, the minimum inhibitory concentrations (MIK80) were measured, which are shown in Table 9 in the form of dilution (%) of the liquid extract of the solution in a particular solvent. The starting liquid extract of propolis was obtained with a mass ratio of raw propolis as a drug and finished extract (DER) 1:2; the product from Example 9. The greater the dilution of the liquid extract, i.e. the lower the concentration of the biomarker for the MIC, the stronger the antimicrobial effect of the extract.

The results are presented in Table 9.

Tables 10-15 list the calculated values of the mass concentration (γ) in [μg/mL] for each individual active substance 1-10 from each of the three tested propolis liquid extracts, on which the individual liquid extract reached the MIC; extracts obtained using the following extraction solvents (EO): 96% ethanol (product from Example 1), polyethylene glycol 400 (PEG 400; product from Example 2) and a mixture of PEG 400 (97% w/w) and soy lecithin (3% w/w) m) (product from Example 9). They are determined from the primary liquid extracts where the DER is 1:2, divided by the dilution at which the MIC was achieved.

Example 11. Preparation of the formulation of the subject invention in the dosage form of a solution for intramammary administration with a minimum of 150 μg/g of active substances 1-4

Formulation (for 100 g of solution):

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(1) 50.00 g (50.00% m/m) liquid propolis extract according to the invention, product from Example 9, standardized to a minimum of 300 μg/mL of active substances 1-4

(2) 50.00 g (50.00% w/w) polyethylene glycol 400

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Preparation: Ingredients (1) and (2) were mixed and homogenized by mixing at room temperature for 5 minutes. The solution was filtered through filter paper and filled into intramammary injectors of 4-8 g each.

Composition of the solution: minimum 150 μg/g total concentration of active substances 1-4.

The results of the quantitative HPLC analysis are shown in Table 16, while the associated typical HPLC chromatogram is shown in Figure 5.

Example 12. Preparation of the formulation according to the invention in the dosage form of a suspension for intramammary administration with a minimum of 100 μg/g of active substances 1-4

Formulation (for 100 g of suspension):

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(1) 77.00 g (77.00% m/m) liquid extract of propolis according to the invention, product from Example 9, standardized to 150 μg/mL of active substances 1-4

(2) 20.00 g (20.00% w/w) polyethylene glycol 4000

(3) 3.00 g (3.00% w/w) aluminum distearate

-------------------------------------------------- ------------------

Preparation: (3) is added to the melted polyethylene glycol (2) at 60 °C and homogenized by stirring at that temperature for 15 minutes. Then the mass is cooled with stirring to room temperature, and propolis extract (1) is added at 40-45 °C, and it is additionally homogenized at room temperature for 15 minutes. A light yellow thick suspension is obtained, which is filled into intramammary injectors for 4-8 g.

Composition of the solution: minimum 100 μg/g total concentration of active substances 1-4.

Example 13. Preparation of the formulation according to the invention in the dosage form of a gel with a minimum of 250 μg/g of active substances 1-4

Formulation (for 100 g of gel):

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(1) 30.00 g (30.00% m/m) liquid extract of propolis according to the invention, product from Example 9, standardized to a minimum of 850 μg/mL of active substances 1-4

(2) 2.00 g (2.00% w/w) Carbopol 940

(3) 1.00 g (1.00% w/w) polysorbate 60

(4) 0.20 g (0.20% w/w) potassium sorbate

(5) 0.30 g (0.30% w/w) sodium benzoate

(6) 0.30 g (0.30% w/w) citric acid, anhydrous

(7) q.s. sodium hydroxide (20% solution)

(8) ad 100% purified water

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Preparation: (2) was added to 30 g of liquid extract of propolis according to the invention and homogenized by mixing at room temperature for 20 minutes. Then 50 g of purified water (8) was added to the resulting solution and homogenized by mixing at room temperature for 5 minutes. Then the ingredients (3-6) were added and dissolved by stirring for 10 minutes. Then the pH value was adjusted to the level of 5.5-6 with the application (7). The remaining amount of purified water was added to the obtained gel to a total mass of 100 g and homogenized by mixing at room temperature for 15 minutes with final deaeration of the product.

Gel composition: minimum 250 μg/g total concentration of active substances 1-4.

Example 14. Preparation of the formulation according to the invention in the dosage form of a cream with a minimum of 100 μg/g of active substances 1-4

Formulation (for 100 g of cream):

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(1) 20.00 g (20.00% m/m) liquid extract of propolis according to the invention, product from Example 9, standardized to a minimum of 500 μg/mL of active substances 1-4

(2) 5.00 g (5.00% w/w) polysorbate 60

(3) 5.00 g (5.00% w/w) lanolin, anhydrous

(4) 2.00 g (2.00% w/w) beeswax, white

(5) 8.00 g (8.00% w/w) cetyl alcohol

(6) 8.00 g (8.00% w/w) petroleum jelly, white

(7) 10.00 g (10.00% w/w) mineral oil, thick

(8) 0.20 g (0.20% w/w) 4-chloro-m-cresol

(9) ad 100% purified water

-------------------------------------------------- ------------------

Preparation: The oil phase is prepared by melting the mixture of ingredients (2-7) at 65-70 °C for 15-20 minutes until an almost colorless oily liquid is formed. The aqueous phase is prepared by dissolving (8) and (1) in purified water, with subsequent heating with stirring to 65-70 °C. Then the water phase is slowly added to the oil phase with intensive mixing, preferably with a homogenizer that enables a high number of revolutions of the mixing element, from 1,000-3,000 rpm, for 15-20 minutes. The obtained emulsion is further intensively mixed with gradual cooling at temperatures of 65-25 °C for 30 minutes.

Cream composition: minimum 100 μg/g total concentration of active substances 1-4.

Example 15. Preparation of the formulation according to the invention in the dosage form of ointment with a minimum of 500 μg/g of active substances 1-4

Formulation (for 100 g of fat):

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(1) 50.00 g (50.00% m/m) liquid extract of propolis according to the invention, product from Example 9, standardized to a minimum of 1,000 μg/mL of active substances 1-4

(2) 10.00 g (10.00% w/w) polyethylene glycol 400

(3) 40.00 g (40.00% m/m) polyethylene glycol 4000

-------------------------------------------------- ------------------

Preparation:

The ingredients (1-3) are carefully heated to 60 °C with stirring and homogenized by stirring for 5 minutes, and gradually cooled to room temperature with stirring.

Fat composition: minimum 500 μg/g total concentration of active substances 1-4.

Example 16. Preparation of the formulation according to the invention in the dosage form of a solution for application in the form of a nasal spray with a minimum of 50 μg/g of active substances 1-4

Formulation (for 100 g of spray solution):

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(1) 5.00 g (5.00% m/m) liquid extract of propolis according to the invention, product from Example 9, standardized to a minimum of 1,000 μg/mL of active substances 1-4

(2) 0.60 g (0.60% w/w) sodium chloride

(3) 0.10 g (0.10% w/w) polysorbate 60

(4) 0.10 g (0.10% w/w) potassium sorbate

(5) 0.10 g (0.10% w/w) sodium benzoate

(6) 0.20 g (0.20% w/w) citric acid, anhydrous

(7) 0.01 g (0.01% w/w) sodium edetate (Na2EDTA•2H2O)

(8) ad 100% purified water

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Preparation: Ingredients (2-7) were dissolved in 90 g of purified water (1) by stirring at room temperature for 5 minutes. Then (1) was added, and the mixture was homogenized by stirring at room temperature for 15 minutes. The obtained solution was then filtered through a sterile 0.2 μm filter and filled into suitable sterile vials with a cap equipped with a pump for nasal administration.

Composition of the solution for nasal application in the form of a spray: minimum 50 μg/g total concentration of active substances 1-4.

Example 17. Study of the anti-inflammatory effect of the formulation of the subject invention in the form of an intramammary solution from Example 11 in the treatment of mastitis in dairy cows

The study of the treatment of mastitis in cows was carried out on five farms of dairy cattle of the Holstein breed. 86 dairy cows, or 339 quarters, where the udder quarter was used as a statistical unit, were included in the research. The animals were kept freely on deep bedding, and were fed with a standard mixture for dairy cows without the addition of antibiotics. The study included animals, that is, quarters without clinical symptoms of mastitis, that is, healthy, uninfected quarters with a somatic cell count (BSS) below 200,000/mL, and infected quarters with a BSS count greater than 200,000/mL. A randomized cross-over clinical study of the safety and efficacy of the intramammary (i.mammary) solution of the formulation from the subject invention was conducted on cows in field conditions. The formulation of the subject invention from Example 11 was applied three times in all four quarters of the cow's udder: during morning milking, evening milking and the day after morning milking. First, the tolerability of the formulation according to the invention in intramammary application was tested. Changes in the behavior of the cows and in the macroscopic appearance of the udder (swelling and redness) and milk, as well as the sensitivity of the udder to touch, were monitored. A change in the number of somatic cells (BSS) in milk was recorded from the period before the first i.mam. applications of propolis solution, until the 7th day after that first application. Milk samples, i.e. quarters, were grouped depending on whether the BSS in them was higher or lower than 200,000/mL and whether the samples were positive or negative on the bacteriological examination; see literature reference 33:

33) European Medicines Agency (1992): Local Tolerance of Intramammary Preparations in Cows. Directive 81/852/EEC.

Then, a test of the effectiveness of the bacteriological cure of the propolis solution was conducted according to the guidelines of the European Medicines Agency (EMA), which is the only measure of the effectiveness of i.mam. preparations in the treatment of subclinical mastitis. It is defined as the absence of a previously proven causative agent in a milk sample collected in a certain period of time after the application of i.mam. preparation; see literary reference 34:

34) European Medicines Agency (2017): Guideline on the conduct of efficacy studies for intramammary products for use in cattle. CVMP. EMA/CVMP/344/1999-Rev.2.

Milk sampling was done using standard methodology. Microbiological examination was performed according to standard guidelines; see literature reference 35:

35) J. W. Hogan: Laboratory handbook on bovine mastitis. National Mastitis Council (1999) Madison, Wisconsin, USA.

The results of the bacteriological cure of mastitis in cows are shown in Table 17.

Example 18. Study of the anti-inflammatory effect of the formulation of the subject invention in the form of an intramammary solution from Example 11 in the treatment of mastitis in goats

The study of the treatment of mastitis in goats was carried out on 25 alpine goats diagnosed with subclinical mastitis in the left and right halves of the goats' udders. Goats whose milk was positive on the microbiological examination were divided into two groups: one group was administered the formulation of the subject invention from Example 11, while the other group was administered an intramammary suspension of amoxicillin with clavulanic acid (Klavuxil®; Genera, Croatia) three times. In parallel, the tolerability and bacteriological cure of the halves after i.mam were monitored. formulations from Example 11. The preparations were applied three times (two-day) application of said antibiotic, i.e. the formulations from Example 11. During the research, the goats stayed in barns, and were kept free on deep litter. There were 170 lactating goats on the farm with an average production of 500-600 kg of milk per goat in one lactation that lasts about 300 days. During the experiment, they were fed hay at will, minimum 2 kg per goat and concentrate containing 16% crude protein, about 1 kg per goat per day divided into two meals (morning and evening milking). 2% of the vitamin-mineral supplement Ovisan® (Sano, Croatia) was added to the mixture. Goats whose milk was positive on the microbiological examination were divided into two groups: one group (number of treated halves, N=20) was administered the formulation from Example 11, while the other group was administered an intramammary suspension of amoxicillin with clavulanic acid (Klavuxil®; Genera, Croatia) (N=15) three times.

When testing the tolerability of the formulation from Example 11, no changes in the behavior and in the macroscopic appearance of the udder (swelling and redness) and milk, as well as the sensitivity of the udder to touch, were observed in any goat. Goat milk samples from the left and right half of the udder were taken into pre-labeled sterile plastic tubes after milking the first few streams of milk. Milk samples were taken before the first application of the formulation from Example 11, 12 h after the first application, 24 h after the first application of propolis and on the 7th day after the first application of propolis. The milk samples were kept at 4 °C until the next day when they were analyzed in the Laboratory for mastitis and raw milk quality at the Croatian Veterinary Institute (HVI).

Bacteriological cure efficiency testing was conducted according to the guidelines of the European Medicines Agency (EMA); see literature reference 34. Milk sampling was done using standard methodology, while microbiological examination was carried out according to standard guidelines; see literature reference 35.

The results of the bacteriological cure of mastitis in goats are shown in Table 18.

Example 19. Presentation of a case of successful healing of a wound on a mare's track with the application of the formulation of the subject invention from Example 11 in the form of a solution

A case of healing a wound on a mare's track is presented. It was a deep wound in the area of the path caused by catching up during landing. Before applying the formulation from Example 11, the wound was unsuccessfully treated conservatively with various preparations for two weeks.

The wound was caused during the movement of the mare with a lunge in a gallop, when it "caught up" with its hind legs, that is, with the cranial part of the hoof and the horseshoe of the hind legs, it injured the area of the shin of the front leg, causing an elliptical wound with a diameter of 2x4 cm. Immediately after the injury, the mare showed pronounced signs of lameness 4/5 (American Association Equine Practitioners). Immediately after the injury, the wound was shaved and washed with plain cold water, then treated with an iodine-based preparation (povidone iodide, 0.01%) and sprayed with a silver nitrate-based spray to close the wound and prevent infection. Given that the mare was previously vaccinated against tetanus, no TAT serum was administered. The mare was then spared for two weeks, and the wound was treated daily by mechanically cleaning it and keeping it clean and dry. Every 48 hours she was treated with iodine and the silver nitrate spray was repeated. After 5 days, the wound began to heal and was better, so local coating with zinc-vitamin ointment was started, after the wound was cleaned and disinfected. The mare was brought back to work two weeks after the injury, but the wound bled as soon as the mare began to trot. The wound was again disinfected with iodine and a local antibiotic based on cephalosporin in a formulation for intramammary use (Cobactan®) was applied. Along with mechanical cleaning of the wound, the wound was treated locally with an antibiotic for the next 5 days. After that, another attempt was made to work with the mare, but the wound bled again.

The wound was then washed with physiological solution, dried and treated with the formulation from Example 11, once a day for 5 days. Improvement in the form of epithelialization was visible after 48 h, and complete healing occurred after 96 h. The mare limped markedly only during the first few days, and after that no signs of lameness were noticed. The recovery was eventually complete.

Conclusion

Experimental results showed that specific mixtures of liquid polyethylene glycols (PEG) such as PEG 400 in combination with lecithins in the amount of 0.1-3.5% of the mass of the extraction solvent (EO) unexpectedly more efficiently and chemoselectively extract the active substances of propolis, p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4), compared to pure solvents such as 96% ethanol, PEG 400 or mixtures of EtOH and of these same lecithins.

By applying a suitable analytical method for the quantitative determination of key active ingredients 1-4, and supporting active ingredients 5-10, such as the HPLC method developed according to the present invention, the exact quantitative composition of the primary liquid extract of propolis according to the invention is determined. After that, such primary extract is standardized by diluting it with the same extraction solvent (EO) that was used in the extraction step. In this way, a liquid extract of propolis according to the invention is obtained with a known and standardized concentration of the key active ingredients 1-4. The standardized extract of propolis prepared in this way is used as an active pharmaceutical ingredient (API), active cosmetic ingredient (ACI), or as a food ingredient for the production of functional foods and nutritional supplements.

The formulation from the present invention based on said liquid extract of propolis containing the key active substances p-coumaric acid (1; 10-1,300 μg/g), trans-ferulic acid (2; 10-800 μg/g), caffeic acid (3; 5-300 μg/g), and 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4; 5-400 μg/g) is an effective agent in the treatment of inflammatory diseases, bacterial infections, fungal infections, viral diseases, autoimmune diseases , functional gastrointestinal disorders, for regeneration of mucous membranes, treatment of burns and wound healing, and treatment of tumor diseases.

Industrial applicability

Considering the wide practical application of the liquid extract of propolis and the pharmaceutical formulation based on it, the industrial applicability of the subject invention is obvious.

Claims (27)

1. Liquid extract of propolis as a pharmaceutical, cosmetic or agrochemical ingredient or food ingredient, consisting of: (A) dry propolis extract; 0.1-10.0% m/m; and (B) extraction solvent; 90.0-99.9% m/m; indicated that the extraction solvent consists of: (B.1) one or more liquid polyethylene glycols <PEG> 200-600; 96.5-99.9% m/m; and (B.2) lecithin or lecithin hydrolyzate; 0.1-3.5% m/m; whereby said liquid extract of propolis is standardized: (I) by the quantitative ratio of the mass of raw propolis as a drug and the finished extract <DER> in the ratio: 1 : 2 – 1 : 20 m/m; and (II) quantitative content of propolis active ingredients selected from the group consisting of p-coumaric acid <1>, trans-ferulic acid <2>, caffeic acid <3> and 2-phenylethyl 3,4-dihydroxy-trans-cinnamate <4> : [image] where the quantitative content for at least two of the four listed key active substances of propolis is as follows: (i) p-coumaric acids <1>; 100-1,300 μg/mL; (ii) trans-ferulic acid <2>; 75-800 μg/mL; (iii) caffeic acid <3>; 25-300 μg/mL; you (iv) 2-phenylethyl 3,4-dihydroxy-trans-cinnamate <4; CAPE>; 40-400 μg/mL. 2. Liquid extract of propolis as a pharmaceutical, cosmetic or agrochemical ingredient or food ingredient according to claim 1, indicated by the fact that the liquid polyethylene glycol <PEG> is selected from the group consisting of: polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 600, or mixtures of the aforementioned substances . 3. Liquid extract of propolis as a pharmaceutical, cosmetic or agrochemical ingredient or food ingredient according to claim 2, characterized in that the liquid polyethylene glycol <PEG> is selected from the group consisting of: polyethylene glycol 200, polyethylene glycol 400, or mixtures of the aforementioned substances. 4. Liquid extract of propolis as a pharmaceutical, cosmetic or agrochemical ingredient or food ingredient according to claims 1-3, characterized in that lecithin or lecithin hydrolyzate is characterized by a hydrophilic-lipophilic balance factor <HLB> of 2-12, selected from the group consisting of: lecithin soybean <Glycine max L>; sunflower lecithin <Helianthus annuus L>; rapeseed lecithin <Brassica napus L>; canola lecithin <Brassica rapa L>; lecithin from chicken <Gallus gallus domesticus L> eggs; deoleinized products of the said lecithins; hydrogenated lecithins from the mentioned sources; hydrolysates of said lecithins; enzymatically modified derivatives of the said lecithins; or mixtures of these substances. 5. Liquid propolis extract pharmaceutical, cosmetic or agrochemical ingredient or food ingredient according to claim 4, characterized in that the lecithin or lecithin hydrolyzate is selected from the group consisting of: native lecithin, deoleinized, hydrogenated, hydrolyzed or enzymatically modified soy lecithin <Glycine max L> , sunflower <Helianthus annuus L>, oilseed rape <Brassica napus L> or canola <Brassica rapa L>; or mixtures of these substances. 6. Liquid extract of propolis as a pharmaceutical, cosmetic or agrochemical ingredient or food ingredient according to claims 1-5, indicated that the extraction solvent consists of: (i) polyethylene glycol <PEG> 200, polyethylene glycol 300, polyethylene glycol 400 or their mixtures; 97-99% m/m; and (ii) native lecithins, deoleinized lecithins or hydrolyzed lecithins of soy <Glycine max L>, sunflower <Helianthus annuus L> rape <Brassica napus L> or canola <Brassica rapa L>; or mixtures of said substances; 1-3% m/m. 7. Liquid extract of propolis as a pharmaceutical, cosmetic or agrochemical ingredient or food ingredient according to claims 1-6, indicated by the fact that said liquid extract of propolis is standardized: (I) by the quantitative ratio of the mass of raw propolis as a drug and the finished extract <DER> in the ratio: 1 : 3 - 1 : 5 m/m; you (II) by the quantitative content of active ingredients of propolis selected from the group consisting of p-coumaric acid <1>, ferulic acid <2>, caffeic acid <3> and 2-phenylethyl 3,4-dihydroxycinnamate <4>, with a minimum of two of the four listed key assets correspond to the quantitative content as follows: (i) p-coumaric acids <1>; 500-1,300 μg/mL; (ii) trans-ferulic acid <2>; 300-800 μg/mL; (iii) caffeic acid <3>; 100-300 μg/mL; you (iv) 2-phenylethyl 3,4-dihydroxy-trans-cinnamate <4; CAPE>; 100-400 μg/mL. 8. The procedure for obtaining a liquid extract of propolis according to any of the previous claims 1-7, characterized by the fact that it includes the following steps: (i) cooling of raw propolis at –20 °C for a minimum of 1 h; (ii) grinding of cooled propolis while passing through a sieve with pores of 1-8 mm; (iii) extraction of raw propolis with an extraction solvent under the following conditions: (a) mass ratio of raw propolis and extraction solvent = 1:2-1:20 m/m; (b) extraction temperature of 10-150 °C; and (c) extraction time from 5 minutes to 72 h; (iv) filtration of the obtained mixture through a series of filters with a pore size of 100 μm to 5 μm, with the formation of an undissolved residue and a liquid extract of propolis; (v) quantitative analysis of the content of the key active ingredients of propolis 1-4 by means of high-performance liquid chromatography <HPLC>; and (vi) standardization of the obtained liquid extract of propolis, in which the exact quantitative composition of the key active ingredients 1-4 was determined in step (v), through dilution with fresh extraction solvent that was also used in step (iii), to the level of the desired content of active substances 1- 4. 9. The procedure for obtaining a liquid extract of propolis according to claim 8, characterized by the fact that, as in the extraction step (iii), it is carried out under the following conditions: (a) mass ratio of raw propolis and extraction solvent = 1:3-1:5 m/m; (b) extraction temperature of 15-70 °C; and (c) extraction time of 1-24 h. 10. The procedure for obtaining a liquid extract of propolis according to claims 8 and 9, characterized by the fact that for the quantitative determination of the key active ingredients 1-4, the method of high-performance liquid chromatography <HPLC> is used as follows: (i) chromatographic column: Ascentis express; C18; dimensions: 15 cm x 3.0 mm; diameter of particles in the column: 2.7 μm; (ii) mobile phase: A= 0.1% aqueous solution of formic acid, B= methanol; gradient: 0 min, 80% A, 20% B; 3 min, 70% A, 30% B; 60 min, 20% A, 80% B; 90 min, 20% A, 80% B; 100 min, 70% A, 30% B; 105 min, 80% A, 20% B; (iii) column temperature: 30 °C; (iv) flow rate: 0.25 mL/min; (v) analysis time: 110 min; (vi) wavelength on the UV-VIS detector: for detection: 370 nm, for integration 290 nm; (vii) injection volume: 10 μL; (viii) pressure: 210-290 bar. 11. The use of a standardized liquid propolis extract according to claims 1-7, characterized by the fact that it is used as an active pharmaceutical ingredient or auxiliary substance for the production of pharmaceutical products selected from the group consisting of: drugs, medical products or auxiliary medicinal agents. 12. Use of a standardized liquid extract of propolis according to claims 1-7, characterized in that it is used as an active cosmetic ingredient or auxiliary substance for the production of cosmetic products. 13. The use of a standardized liquid propolis extract according to claims 1-7, characterized by the fact that it is used as a food ingredient for the production of functional food products, food supplements or food for special dietary needs. 14. The use of a standardized liquid propolis extract according to claims 1-7, characterized in that it is used as an active pharmaceutical ingredient or auxiliary substance for the production of veterinary products selected from the group consisting of: veterinary medical products; feed for animals; nutritional supplements for animals; or auxiliary medicinal products for use in veterinary medicine. 15. The use of a standardized liquid propolis extract according to claims 1-7, characterized by the fact that it is used as an active agrochemical ingredient or auxiliary substance for the production of agrochemical products selected from the group consisting of: fungicides, bactericides, virucides, insecticides, nematocides and plant enhancers. 16. Pharmaceutical formulation, characterized in that it consists of: (I) a liquid extract of propolis according to any one of claims 1-7; from 5-95% m/m; and, (II) one or more pharmaceutical excipients necessary for the formation of the final dosage form selected from the group consisting of: solution, suspension, gel, cream, ointment, spray for oral or nasal administration; up to 100% m/m of the finished formulation; where said formulation is characterized by quantitative content for at least two of the four key active substances of propolis within the following values: (i) p-coumaric acids <1>; 10-1,300 μg/g; (ii) trans-ferulic acid <2>; 10-800 μg/g; (iii) caffeic acid <3>; 5-300 μg/g; and, (iv) 2-phenylethyl 3,4-dihydroxy-trans-cinnamate <4; CAPE>; 5-400 μg/g. 17. Pharmaceutical formulation according to claim 16, characterized in that the pharmaceutical excipient is selected from the group consisting of: fillers, humectants, preservatives, chelating agents, antioxidants, thickeners, emollients, emulsifiers, tonicity agents and pH control agents. 18. The process of obtaining a pharmaceutical formulation according to claims 16 and 17, characterized in that it includes the following steps: (i) adding the standardized liquid propolis extract according to claims 1-7 to the filler and their homogenization; (ii) addition of one or more other excipients; and their homogenization; wherein steps (i) and (ii) are carried out at a temperature of 10-100 °C, preferably at a temperature of 20-60 °C, for 1-15 minutes; and then, in the case of preparing a dosage form: (iii.a) solutions or spray solutions; filtration of the finished solution is performed, including, if necessary, sterile filtration; (iii.b) gel or suspension; addition of thickener and its homogenization are carried out; (iii.c) creams; the fat phase is prepared by mixing emollients and emulsifiers and their homogenization at a temperature of 50-80 °C for 1-15 minutes, then adding the solution from step (ii) heated to 50-80 °C, and then emulsifying with the use of a homogenizer with a high number of revolutions of the mixing element or high pressure, at a temperature of 50-80 °C, preferably of 55-65 °C, for 1-30 minutes, with subsequent homogenization at temperatures of 65-20 °C, for 10-120 minutes; or at (iii.d) fats; the solution from step (ii) is mixed with a previously melted mixture of emollients and possibly emulsifiers at a temperature of 50-70 °C for 5-30 minutes, with subsequent homogenization at temperatures of 70-20 °C for 10-120 minutes. 19. The use of a pharmaceutical formulation according to claims 16 and 17, characterized in that it is used for the treatment of diseases and conditions in humans and animals, which are selected from the group consisting of: inflammatory diseases; bacterial infections; fungal infections; viral diseases; autoimmune diseases; functional gastrointestinal disorders; for regeneration of mucous membranes, treatment of burns and wound healing; and tumor diseases. 20. The use of a pharmaceutical formulation according to claim 19, characterized in that it is used for the treatment of inflammatory diseases selected from the group consisting of: gingivitis, periodontitis, laryngitis, gastritis, colitis, hemorrhoidal disease, dermatitis, inflammation of the external ear, sinusitis, rhinitis, vaginitis and mastitis . 21. The use of a pharmaceutical formulation according to claim 19, characterized in that it is used for the treatment of bacterial infections caused by bacteria selected from the group consisting of: (i) Gram-positive bacteria: Staphylococcus spp: Staphylococcus aureus, MRSA <methicillin-resistant Staphylococcus aureus>, MSSA <methicillin-susceptible Staphylococcus aureus>, Staphylococcus intermedius, Staphylococcus pseudintermedius; coagulase-negative staphylococci: Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus hyicus; Streptococcus spp: Streptococcus uberis, Streptococcus bovis, Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus canis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus oralis, Streptococcus thermophilus; Peptostreptococcus spp; Corynebacterium spp: Corynebacterium bovis; Trueperella pyogenes; Nocardia spp; Bacillus subtilis; Bacillus cereus; enterococci: Enterococcus faecium, Enterococcus faecalis; vancomycin-resistant enterococci <VRE>: Enterococcus casseliflavus; and, (ii) Gram-negative bacteria: Escherichia coli; Acinetobacter baumannii; Pseudomonas aeruginosa; Haemophilus influenzae; Salmonella enterica; Yersinia enterocolitica; Enterobacter spp <Enterobacter cloacae>; Klebsiella spp: Klebsiella pneumoniae, Klebsiella oxytoca; Shigella flexneri; Burkholderia cepacia; Proteus mirabilis; Proteus vulgaris; Aggregatibacter actinomycetemcomitans; Actinomyces israelii; Bacteroides fragilis; Helicobacter pylori; Campylobacter coli; Campylobacter jejuni; Porphyromonas gulae; Porphyromonas salivosa; Porphyromonas denticanis; Prevotella intermedia; Treponema spp; Bacteroides splanchnicus. 22. The use of a pharmaceutical formulation according to claim 19, characterized in that it is used for the treatment of fungal infections caused by fungi selected from the group consisting of: Candida spp <Candida albicans, Candida dubliniensis, Candida glabrata, Candida kruzei, Candida tropicalis, Candida parapsilosis>; Aspergillus spp. <Aspergillus niger, Aspergillus versicolor>; Penicillium pinophilum; Paecilomyces variotii; Trichoderma virens; Chaetomium globosum; Malassezia pachydermatis. 23. The use of a pharmaceutical formulation according to claim 19, characterized in that it is used for the treatment of viral diseases caused by viruses selected from the group consisting of: Herpes simplex virus <HSV>; Human papilloma virus <HPV>; Epstein-Barr virus <EBV>; Cytomegalovirus <CMV>; poliovirus; influenza viruses A and B; retroviruses; Vaccinia virus; cold viruses: rhinovirus, picornavirus, human parainfluenza virus <HPIV>, human metapneumovirus <HMPV>, coronavirus, adenoviruses, human respiratory syncytial virus <HRSV>, enteroviruses. 24. The use of a pharmaceutical formulation according to claim 19, characterized in that it is used for the treatment of autoimmune diseases selected from the group consisting of: psoriasis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, celiac disease and multiple sclerosis. 25. The use of a pharmaceutical formulation according to claim 19, characterized in that it is used for the treatment of tumor diseases selected from the group consisting of: skin and mucous membrane cancer, tumors of the digestive system, colorectal cancer. 26. Use of the pharmaceutical formulation according to claim 19, characterized in that it is used for the treatment of mastitis in animals. 27. The use of a pharmaceutical formulation according to claim 19, characterized by the fact that it is used for the treatment of functional gastrointestinal disorders consisting of: disorders of the esophagus, stomach, duodenum, small and large intestine, centrally mediated gastrointestinal pain, disorders of the gallbladder and sphincter of Oddi, anorectal disorders, gastrointestinal disorders specific for children and adolescents.