Liquid propolis extract, its preparation and uses

Abstract

The present invention discloses a novel standardized liquid propolis extract and pharmaceutical formulations based on the extract, as well as their production and use. The liquid extract is produced by extraction of crude propolis with an extraction solvent based on a mixture of PEG 200-600 (96.5-99.9% w / w) and lecithin (0.1-3.5% w / w). The extract is characterized by a standardized content of p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3), and CAPE (4). It is used as an active ingredient in the manufacture of pharmaceuticals, cosmetics, veterinary medicines, agrochemicals, or functional foods. The pharmaceutical formulations of the present invention consist of 5-95% w / w propolis extract and up to 100% excipients necessary for the preparation of various dosage forms. They are used for the treatment of human and animal diseases and conditions, such as inflammatory diseases, bacterial and fungal infections, viral, autoimmune, and cancer diseases, as well as for the treatment of burns and wound healing.

Description

[Technical field]

[0001] The present invention relates to a novel liquid standardized propolis extract, a process for its preparation into novel pharmaceutical compositions based on said extract, and its uses.

[0002] technical challenges Due to the fact that propolis is a mixture of beeswax and numerous natural organic compounds, different extraction processes result in extracts of active substances with very different compositions. Using chemically selective extractants (ES), which have the ability to selectively extract primarily certain groups of organic compounds, together with the use of appropriate analytical methods, it is theoretically possible to achieve a consistent and standardized propolis extract composition.

[0003] The technical problems solved by the present invention include: (i) A non-alcoholic liquid propolis extract characterized by a high standardized content of the phenolic propolis acids, para-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3), and the 2-phenylethyl ester of caffeic acid, 2-phenethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE), which are known for several beneficial pharmacological activities on both human and animal organisms; [ka] (ii) its generation process; (iii) use for the manufacture of a pharmaceutical, cosmetic, veterinary, pesticide, or food product having a known standardized content of propolis active substances 1 to 4; and in particular (iv) Pharmaceutical compositions based on the aforementioned propolis extracts which are effective in the treatment of inflammatory and infectious diseases and diseases and conditions in which, as suggested by certain state-of-the-art references, the antioxidant, anti-inflammatory and immunomodulatory activity of certain propolis active substances plays an important role in the pathogenesis of their occurrence.

[0004] Alcohol-free extracts with negligible beeswax content and other ballast propolis compounds with high standardized content of highly bioactive propolis components are the basis for the development and production of highly valuable pharmaceutical, veterinary products, agrochemicals or functional foods or food supplements. In contrast, the use of standardized propolis extracts based on ethanol, glycerol or 1,2-propylene glycol for this purpose is accompanied by various difficulties such as unknown content of the above-mentioned propolis active substances or low quantitative composition of valuable phenolic acids and CAPE relative to flavonoid propolis components.

[0005] The present invention relates to (i) the use of specific extraction solvent (ES) formulations that aid in the chemoselective extraction process of crude propolis; (ii) A method for quantifying active propolis components, including major active substances 1-4; (iii) based on standardization of the liquid propolis extract thus prepared by dilution with a certain amount of the same extraction solvent according to the present invention to the desired level of active substances 1-4, the extraction solvent being composed of two or more food and pharma-ceutically acceptable substances, and in the final product, consisting of a liquid standardized propolis extract. This same extraction solvent has the role of a carrier or diluent, and the present invention also provides: (iv) Based on a pharmaceutical composition based on the standardized propolis extract according to the present invention, which has been proven to be an effective agent in the treatment of inflammatory and infectious diseases. Due to its wide range of pharmacological activity, it can be used in the treatment of various diseases and conditions, such as inflammatory diseases, bacterial and fungal infections, viral diseases, autoimmune diseases, mucosal regeneration, treatment of burns and wounds, and cancer diseases. [Background technology]

[0006] prior art Propolis is a natural product harvested by bees, who act as a glue to close small undesirable openings in the honeycomb. Propolis contains beeswax as its main component and a large number of different organic compounds, many of which have shown significant beneficial pharmacological effects, see for example Reference 1:

[0007] 1) Non-Patent Document 1

[0008] In addition to the above-mentioned active substances 1-4, propolis contains a whole series of other natural compounds, such as trans-cinnamic acid (5) among its acids, and a series of flavonoids, including relatively abundant amounts of chrysin (6), pinocembrin (7), galangin (8), apigenin (9) and kaempferol (10). [ka]

[0009] Traditionally, crude propolis is extracted with ethanol or a mixture of ethanol and water to obtain the so-called tinctures of propolis. Such liquid propolis extracts are characterized by several disadvantages: (i) the presence of a relatively aggressive solvent (ethanol); (ii) alcohol-based products are not suitable for children, pregnant or breastfeeding women and certain patients; and (iii) A relatively high content of beeswax, which causes its degradation through a mixed phase with water during the manufacture of medicines and other products in which such extracts are used as active ingredients.

[0010] Besides ethanol, glycerol, water, mixtures of glycerol and water, as well as other organic solvents have been used as extraction solvents.

[0011] Thus, Tsukada et al. disclosed the use of glycerol as an extraction solvent at a propolis:extraction solvent ratio of 1:2 w / w and 90-160°C followed by filtration. Such glycerol extracts are water-soluble and suitable for the manufacture of pharmaceuticals as active pharmaceutical ingredients (APIs). See Reference 2:

[0012] 2) Patent Document 1

[0013] Aqueous propolis extracts have also been described in the prior art. Extraction with water as ES can be carried out at a temperature of 30-50°C for 6-8 minutes, which gives directly a liquid propolis extract after filtration. The latter can finally be further processed into a solid extract via microencapsulation by spray drying techniques onto suitable carriers such as maltodextrin and gum arabic. See Reference 3:

[0014] 3) Patent Document 2

[0015] The use of surfactants as components of extraction solvents (ES) containing lecithin is generally known in the art. For example, Paradkar et al. described a method for extracting propolis using an aqueous polysorbate solution at 40-90°C for 2-24 hours to obtain a liquid propolis extract. See Reference 4:

[0016] 4) Patent Document 3

[0017] Sosnowski disclosed methods for propolis extraction using various organic solvents including 1,2-propylene glycol, polyethylene glycol (PEG) and mixtures of these solvents with water. See Reference 5:

[0018] 5) Patent Document 4

[0019] Chun et al., inter alia, disclosed a pharmaceutical composition based on a liquid propolis extract in 1,3-butylene glycol as an extraction solvent, which is converted into a pharmaceutical dosage form containing nanoparticles containing the propolis extract by using hydrogenated lecithin together with other emulsifiers. See Reference 6:

[0020] 6) Patent Document 5

[0021] Despite the fact that this document does not use lecithin to enhance the extraction of propolis active substances with 1,3-butylene glycol, it certainly suggests its possible use as a surfactant, which could ultimately improve the extraction and emulsification in more polar solvents of certain lipoactive propolis components.

[0022] Concerning propolis analysis, there are numerous analytical methods in the prior art for the quantification of propolis active substances in complex propolis extracts containing a large number of components. As an example, an analytical method similar to the one used in the present invention is shown in the work of Chinese authors Cui-Ping et al. They described a method for the quantification of 12 different flavonoids and 8 phenolic propolis acids via high performance liquid chromatography (HPLC). This method allows, among others, the determination of p-coumaric acid (1), trans-ferulic acid (2) and caffeic acid (3), which are referred to as qualitative propolis markers. See Reference 7:

[0023] 7) Non-patent literature 2

[0024] Due to their content of active substances 1-10 etc., propolis and propolis extracts exhibit a series of highly valuable beneficial pharmacological effects on human, animal and plant health. The prior art contains a large number of scientific and patent literature supporting a wide range of beneficial effects, the most important of which are anti-inflammatory; antioxidant; immunomodulatory; hepatoprotective; antimicrobial, including antibacterial, antiviral, antifungal and antiprotozoal; and anticancer. See for example references 8-12:

[0025] 8) Non-patent literature 3 9) Non-patent literature 4 10) Non-patent literature 5 11) Non-patent document 6 12) Non-Patent Document 7

[0026] In addition to propolis extracts, a whole range of beneficial pharmacological effects have been described in the prior art for certain isolated (pure) propolis active substances, such as, 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); and (iv) 2-Phenylethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE) (see References 19 and 20), which exhibits, inter alia, immunomodulatory, anti-inflammatory and antibacterial activity.

[0027] 13) Non-patent document 8 14) Non-Patent Document 9 15) Non-Patent Document 10 16) Non-Patent Document 11 17) Non-Patent Document 12 18) Non-Patent Document 13 19) Non-Patent Document 14 20) Non-Patent Document 15

[0028] Furthermore, propolis active substances are fungicides, bactericides, virucides, insecticides and nematicides and are used in plant protection.

[0029] Due to its strong antioxidant activity, propolis strengthens plants, increases their resistance to abiotic stresses, and helps them fight infections. See References 21-25: 21) Non-patent literature 16; 22) Non-Patent Document 17 23) Non-patent literature 18; 24) Non-Patent Document 19; 25) Non-Patent Document 20

Prior Art Documents

Patent Documents

[0030]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0031]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Outdoor Tools 14

Outdoor Tools 15

Outdoor Content 16

Outdoor Track 17

[0032] To the best of our knowledge, the use of specific liquid polyethylene glycol-based extraction solvents (ES) containing low (0.1-3.5% m / m) lecithin content as an enhancer of propolis extraction chemoselectivity has not been described in the prior art. The application of such specific ES from the present invention allows for an improvement of chemoselectivity in crude propolis extraction, resulting in corresponding liquid extracts with significantly enhanced contents of the respective active substances 1-4.

[0033] Also, the use of said particular standardized liquid propolis extract as an active pharmaceutical ingredient (API) in the pharmaceutical composition according to the present invention provides unexpected improvements in a range of different indications for its use, as described in the detailed description of the invention. [Means for solving the problem]

[0034] The present invention is based on the unexpected efficacy and chemoselectivity of crude propolis extraction with a specific extraction solvent (ES), which consists of a mixture of liquid polyethylene glycol (PEG), e.g., PEG 400, and lecithin or hydrolyzed lecithin in a ratio of 96.5-99.9:0.1-3.5% w / w.

[0035] The extraction solvent (ES) effectively and chemoselectively extracts the propolis active substances p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 2-phenethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE). The use of an appropriate analytical method based on high performance liquid chromatography (HPLC) allows the quantification of the active substances 1-4 in the primary extract so prepared. The latter is further standardized by dilution with the same ES used in the extraction step to the desired content of active substances 1-4 according to the invention. In this way, a liquid propolis extract is obtained according to the invention with a known standardized concentration of the main active substances 1-4. This can be further used as an active pharmaceutical ingredient (API), an active cosmetic ingredient (ACI) or as a food ingredient for producing functional foods and food supplements.

[0036] The composition of the present invention based on a liquid propolis extract containing the main 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-phenethyl 3,4-dihydroxy-trans-cinnamate (4; 5-400 μg / mL) is an effective agent for the treatment of inflammatory diseases, bacterial infections, fungal infections, viral diseases, autoimmune diseases, functional gastrointestinal disorders, mucosal regeneration, treatment of burns and wounds, and treatment of cancer diseases. [Brief description of the drawings]

[0037] [Figure 1] FIG. 1 shows typical HPLC chromatograms of major propolis components 1-4 and associated components 5-10. [Figure 2-1] Figure 2.1 shows the results of the quantitative composition of propolis active substance 1 in liquid propolis extracts obtained by extraction with ethanol (96%) and with mixtures of ethanol and different types and concentrations of lecithin. Figure 2.2 shows the results of the quantitative composition of propolis active substance 2 in liquid propolis extracts obtained by extraction with ethanol (96%) and with mixtures of ethanol and different types and concentrations of lecithin. Figure 2.3 shows the results of the quantitative composition of propolis active substance 3 in liquid propolis extracts obtained by extraction with ethanol (96%) and with mixtures of ethanol and different types and concentrations of lecithin. Figure 2.4 shows the results of the quantitative composition of propolis active substance 4 in liquid propolis extracts obtained by extraction with ethanol (96%) and with mixtures of ethanol and different types and concentrations of lecithin. [Figure 2-2]Figure 2.5 shows the results of the quantitative composition of the accompanying substance 5 in the liquid propolis extract obtained by extraction with ethanol (96%) and with mixtures of ethanol and different types and concentrations of lecithin. Figure 2.6 shows the results of the quantitative composition of the accompanying substance 6 in the liquid propolis extract obtained by extraction with ethanol (96%) and with mixtures of ethanol and different types and concentrations of lecithin. Figure 2.7 shows the results of the quantitative composition of the accompanying substance 7 in the liquid propolis extract obtained by extraction with ethanol (96%) and with mixtures of ethanol and different types and concentrations of lecithin. Figure 2.8 shows the results of the quantitative composition of the accompanying substance 8 in the liquid propolis extract obtained by extraction with ethanol (96%) and with mixtures of ethanol and different types and concentrations of lecithin. Figure 2.9 shows the results of the quantitative composition of the accompanying substance 9 in the liquid propolis extract obtained by extraction with ethanol (96%) and with mixtures of ethanol and different types and concentrations of lecithin. Figure 2.10 shows the results of the quantitative composition of accompanying substances 10 in liquid propolis extracts obtained by extraction with ethanol (96%) and mixtures of ethanol with different types and concentrations of lecithin. [Figure 3-1]Figure 3.1 shows the results of the quantitative composition of propolis active substance 1 in liquid propolis extracts obtained by extraction with polyethylene glycol 400 and mixtures of polyethylene glycol 400 and different types and concentrations of lecithin. Figure 3.2 shows the results of the quantitative composition of propolis active substance 2 in liquid propolis extracts obtained by extraction with polyethylene glycol 400 and mixtures of polyethylene glycol 400 and different types and concentrations of lecithin. Figure 3.3 shows the results of the quantitative composition of propolis active substance 3 in liquid propolis extracts obtained by extraction with polyethylene glycol 400 and mixtures of polyethylene glycol 400 and different types and concentrations of lecithin. Figure 3.4 shows the results of the quantitative composition of propolis active substance 4 in liquid propolis extracts obtained by extraction with polyethylene glycol 400 and mixtures of polyethylene glycol 400 and different types and concentrations of lecithin. [Figure 3-2]Figure 3.5 shows the results of the quantitative composition of the accompanying substance 5 in the liquid propolis extract obtained by extraction with polyethylene glycol 400 and with mixtures of polyethylene glycol 400 and different types and concentrations of lecithin. Figure 3.6 shows the results of the quantitative composition of the accompanying substance 6 in the liquid propolis extract obtained by extraction with polyethylene glycol 400 and with mixtures of polyethylene glycol 400 and different types and concentrations of lecithin. Figure 3.7 shows the results of the quantitative composition of the accompanying substance 7 in the liquid propolis extract obtained by extraction with polyethylene glycol 400 and with mixtures of polyethylene glycol 400 and different types and concentrations of lecithin. Figure 3.8 shows the results of the quantitative composition of the accompanying substance 8 in the liquid propolis extract obtained by extraction with polyethylene glycol 400 and with mixtures of polyethylene glycol 400 and different types and concentrations of lecithin. Figure 3.9 shows the results of the quantitative composition of accompanying substances 9 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 3.10 shows the results of the quantitative composition of accompanying substances 10 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 4] FIG. 1 shows a block scheme of a method for producing a standardized liquid propolis extract according to the present invention. [Diagram 5] FIG. 1 shows a typical HPLC chromatogram of the quantitative analysis of the product of Example 11, a pharmaceutical formulation of the present invention.

[0038] Abbreviation ES-extraction solvent SL-Soy Lecithin (Glycine max.L.) RL-rapeseed lecithin (Brassica napus L.) HRL - Hydrolyzed Soy Lecithin SUL-Deoiled 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-2fi-tetrazolium chloride, redox indicator PBS-phosphate buffered saline CFU- colony forming units; the number of viable microorganisms capable of forming colonies XTT - XTT sodium salt; 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2fi-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 Extractable Weight Ratio; a measure of extractable strength expressed by the weight ratio of starting drug and final extractable SCC-Somatic cell count i.mam.-Intramammary (application) ATCC-American Type Culture Collection; a non-profit organization that collects, preserves, and distributes standard reference microorganisms CNS-coagulase-negative staphylococci DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Detailed Description of the Invention The present invention relates to a novel liquid propolis extract standardized for the content of the main active substances selected from the group consisting of p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and caffeic acid 2-phenylethyl ester, 2-phenethyl 3,4-dihydroxy-trans-cinnamate (4), its preparation method and its use. [ka]

[0040] The liquid propolis extract according to the present invention as a pharmaceutical, cosmetic or agricultural chemical ingredient or food ingredient has the following properties: (A) 0.1 to 10.0% w / w of dried propolis extract; and (B) Extraction solvent of 90.0 to 99.9% w / w The extraction solvent is (B.1) one or more liquid polyethylene glycols (PEG) 200-600, 96.5-99.9% w / w; and (B.2) 0.1 to 3.5% w / w of lecithin or hydrolyzed lecithin The liquid propolis extract comprises: (I) A quantitative weight ratio of crude propolis and final extract as drug (DER) of 1:2 to 1:20 w / w; and (II) Quantitative content of propolis active substances selected from the group consisting of p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 2-phenylethyl 3,4-dihydroxycinnamate (4). The quantitative composition of at least two of the four main active substances mentioned above is (i) 100–1,300 μg / mL p -coumaric acid ( 1 ); (ii) 75–800 μg / mL trans -ferulic acid (2); (iii) 25–300 μg / mL of caffeic acid (3); and (iv) 40-400 μg / mL 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE) It is.

[0041] In a preferred embodiment of the present invention, the liquid polyethylene glycol (PEG) as a component of the extraction solvent (ES) is selected from the group consisting of polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 600, or a mixture of these substances.

[0042] In particular, the liquid polyethylene glycol (PEG) as a component of the extraction solvent (ES) is selected from the group consisting of polyethylene glycol 200, polyethylene glycol 400, or a mixture of these substances.

[0043] Further, the lecithin or hydrolyzed lecithin is selected from the group consisting of products characterized by a hydrophilic-lipophilic balance (HLB) factor of 2 to 12 and is selected from the group consisting of soybean 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; deoiled products of lecithin; hydrogenated lecithin from a source; hydrolyzed lecithin from a source; enzymatically modified derivatives of lecithin; or mixtures of these substances.

[0044] Specifically, the lecithin used in the present invention may be natural lecithin derived from soybean (Glycine max. L.), sunflower (Helianthus annuus L.), rapeseed (Brassica napus L.) or canola (Brassica rapa L.), deoiled lecithin, hydrogenated lecithin, hydrolyzed lecithin or enzyme-modified lecithin, or a mixture of these substances.

[0045] The term "enzyme-modified lecithin" includes hydrolyzed lecithin obtained by enzyme-catalyzed hydrolysis of one higher fatty acid moiety with the production of glycerol monoesters of higher fatty acids with remaining phosphate and choline groups in the molecule, which significantly increases the HLB factor of such hydrolyzed lecithin.

[0046] Preferably, as extraction solvent (ES) for the preparation of the liquid propolis extract according to the invention, (i) 97-99% w / w of polyethylene glycol (PEG) 200, polyethylene glycol 300, polyethylene glycol 400, or mixtures thereof; and (ii) 1 to 3% w / w of natural, deoiled or hydrolyzed lecithin derived from soybean (Glycine max. L.), sunflower (Helianthus annuus L.), rapeseed (Brassica napus L.) or canola (Brassica rapa L.); or mixtures of these substances. A mixture of may be used.

[0047] In a preferred embodiment of the present invention, the liquid propolis extract according to the present invention comprises: (I) Quantitative weight ratio of crude propolis as drug to final extract (DER) in the ratio of 1:3 to 1:5 w / w; and (II) Quantitative composition of propolis active substances selected from the group consisting of p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 2-phenylethyl 3,4-dihydroxy-cinnamate (4). Standardized for at least two of the four main active substances listed above with a quantitative content of: (i) 500–1,300 μg / mL p-coumaric acid (1); (ii) 300–800 μg / mL trans -ferulic acid (2); (iii) 100–300 μg / mL of caffeic acid (3); and (iv) 100-400 μg / mL 2-phenylethyl 3,4-dihydroxy-cinnamate (4; CAPE) Corresponds to.

[0048] Analysis of propolis active substances in liquid extracts For the development of the novel standardized liquid propolis extract according to the present invention, (i) a major active propolis substance selected from the group consisting of p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3), and 2-phenylethyl 3,4-dihydroxy-cinnamate (4) described above; and (ii) an accompanying active substance selected from the group consisting of trans-cinnamic acid (5), chrysin (6), pinocembrin (7), galangin (8), apigenin (9) and kaempferol (10); The development of suitable analytical methods for the quantification of was essential.

[0049] [ka]

[0050] For the initial study of the aforementioned analytical methods, it was necessary to prepare model liquid propolis extracts. Model propolis extracts in ethanol (96%) and polyethylene glycol were used. These were prepared by standard extraction methods by maceration for 24 hours at room temperature. The undissolved residue was then separated by filtration, and the clear filtrate was used as a model liquid propolis extract for further studies. Polyethylene glycol 400 (PEG400) was used as a model liquid polyethylene glycol. The procedures for preparing model liquid propolis extracts in the classical extraction solvents ethanol (96%) and polyethylene glycol 400 are described in Example 1 (96% ethanol) and Example 2 (PEG400).

[0051] A suitable analytical method based on high performance liquid chromatography (HPLC) was developed, by which successful separation of all ten aforementioned compounds 1-10 was achieved. This method is precisely described in Example 3.

[0052] A typical HPLC chromatogram obtained by this analytical method is shown in Figure 1. The retention times (t R ) are shown in Table 1.

[0053] [Table 1]

[0054] Study of the effect of lecithin on the effectiveness of the extraction of propolis active substances 1-4 In continuing this research, the effect of different extraction solvents (ES) containing various types (SL, RL, HRL) of lecithin and concentrations (1-30% w / w) on the effectiveness of propolis active substances 1-4 extraction was studied, see Table 2.

[0055] [Table 2]

[0056] The liquid propolis extract thus prepared was subjected to quantitative analysis for the contents of the main active substances 1-4 and the associated active substances 5-10. The results are shown in Tables 3-6.

[0057] When using extraction solvents (ES) based on 96% ethanol (EtOH) and mixtures of EtOH with different types (SL, RL, HRL) and concentrations (1-30% w / w in the ES composition), the primary liquid propolis extract contains p-coumaric acid (1; ca. 800-1250 μg / mL), trans-ferulic acid (2; ca. 485-770 μg / mL), caffeic acid (3; ca. 185-290 μg / mL) and 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE; ca. 215-320 μg / mL) as main active ingredients. See Table 3.

[0058] [Table 3]

[0059] Here, the concentrations of the auxiliary active substances 5-10 were at the following levels: 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.

[0060] When using extraction solvents (ES) based on polyethylene glycol (PEG400) and mixtures of PEG400 with lecithin of various types (SL, RL, HRL) and concentrations (1-10% w / w in the ES composition), the primary liquid extract also contains p-coumaric acid (1; approx. 750-1300 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; approx. 190-370 μg / mL). See Table 5.

[0061] [Table 4]

[0062] [Table 5]

[0063] In this case, the concentrations of the auxiliary active substances 5-10 were at the following levels: 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.

[0064] [Table 6]

[0065] As can be seen from the results, all tested lecithins (SL, RL, HRL) in combination with any of the pure solvents, 96% ethanol or polyethylene glycol 400 (PEG400), when used at lower concentrations of 1-3% w / w in the extraction solvent (ES) composition, contribute significantly to an increase in the extraction chemoselectivity of actives 1-4 compared to the pure solvents.

[0066] The unexpected effect of the combination of PEG400 and lecithin (SL, RL, HRL) becomes evident in the fact that PEG400, even as a pure solvent, extracts active substances 1-4 significantly weaker than 96% ethanol (Table 3; row 1, column 2). When used in combination with lecithin (SL, RL, HRL) at concentrations of 1-3% w / w, it extracts compounds 1-4 significantly more effectively compared to similar combinations of 96% ethanol with the same lecithin. For example, the achieved p-coumaric acid (1) concentrations in extracts obtained using 100% PEG400 as the extraction solvent were in the range of 750 μg / mL (Table 5; row 1, column 2) and 916 μg / mL using 96% EtOH as the ES (Table 3; row 1, column 2), while the use of 3% w / w SL in PEG400 resulted in a concentration of 1.112 μg / mL (Table 3; row 2, column 2) compared to a level of 820 μg / mL for the use of an ES based on 96% ethanol containing 3% w / w SL (Table 5; row 2, column 2).

[0067] From this typical example, one can clearly see the totally unexpected effect of the combination of polyethylene glycol and lecithin at a concentration of 1-3% w / w in the extraction solvent (ES), which can be extrapolated by one skilled in the art to an acceptable range of an optimal lecithin weight percentage of 0.1-3.5% w / w in the ES composition.

[0068] Using a higher weight percentage of lecithin in the extraction solvent (ES) composition eliminates the beneficial effect of lecithin on the chemoselectivity of propolis extraction compared to a similar system based on ethanol. For example, by using lecithin at a concentration of 4% RL, 7.7% HRL or 10% SL in ES, lower concentrations of the main actives 1-4 were recorded compared to the use of a similar ethanol-based ES system. Typical results for the two most abundant actives, p-coumaric acid (1) and trans-ferulic acid (2), are shown in Tables 7 and 8.

[0069] [Table 7]

[0070] In Table 7, row 8, column 3, a typical unexpected result of the present invention can be seen, where a 50% increase in p-coumaric acid (1) concentration was observed when using the extraction solvent (ES), PEG400+RL (98.9:1.1, w / w), compared to a similar EtOH-based ES (EtOH+RL=98.9:1.1, w / w).

[0071] [Table 8]

[0072] As a further representative example of the unexpected results, data from row 12, column 3 of Table 8 are presented herein where a 53.5% increase in trans-ferulic acid (2) concentration was recorded using the extraction solvent (ES) PEG400+HRL (97.8:2.2, w / w) compared to a similar 96% ethanol-based ES.

[0073] Method for producing standardized liquid propolis extract according to the present invention

[0074] The method for producing a liquid propolis extract according to the present invention comprises the steps of: (i) chilling the crude propolis at −20° C. for at least 1 hour; (ii) crushing the cooled propolis by sieving through 1-8 mm pores; (iii) Under the following conditions: (a) weight ratio of crude propolis and extraction solvent = 1:2~1:20 w / w; (b) an extraction temperature of 10 to 150°C; and (c) Extraction time from 5 minutes to 72 hours extracting the crude propolis with an extraction solvent; (iv) The mixture thus obtained is mixed with 100 μ m~5 μ filtering the propolis through a series of filters having pores of 0.1 mm to produce an undissolved residue and a liquid propolis extract; (v) quantitatively analyzing the main active propolis substances 1-4 by high performance liquid chromatography (HPLC); (vi) standardizing the liquid propolis extract thus obtained, the exact quantitative composition of the main active substances 1-4 being determined in step (v) by diluting it with fresh extraction solvent used in step (iii) to the desired content of active substances 1-4. Includes.

[0075] In a preferred embodiment of the realization of the method for preparing a liquid propolis extract according to the invention, the extraction step (iii) is carried out under the following conditions: (a) weight ratio of crude propolis and extraction solvent = 1:3~1:5 w / w; (b) an extraction temperature of 15 to 70°C; and (c) Extraction time of 1 to 24 hours It is held.

[0076] Also, an analytical high performance liquid chromatography method (HPLC) developed for this purpose for the quantitative steps of the main active substances 1-4, and for the concomitant monitoring of the auxiliary active substances 5-10, was carried out under the following conditions: (i) Chromatography column: Ascentis express; C18; dimensions: 15 cm × 3.0 mm; particle size in the column: 2.7 μm; (ii) Mobile phase: A = 0.1% formic acid in water, 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: 0.25mL / min; (v) Analysis time: 110 minutes; (vi) UV-VIS detector wavelengths: for detection: 370 nm, for integration: 290 nm; (vii) Injection volume: 10μL; (viii) Pressure: 210-290 bar Used in.

[0077] Experimental procedures for preparing a liquid propolis extract according to the invention are disclosed in Examples 4 to 9. In Example 9, an optimized version of the preparation process of a liquid extract of standardized strength, represented by the parameter Drug-to-Extract Ratio (DER) weight percentage of 1:2 according to the invention, is described.

[0078] Analytical methods for the quantification of primary actives 1-4 and co-actives 5-10 are described in Example 3.

[0079] Determination of the antibacterial effect of the standardized liquid extract according to the invention. Determination of the minimum inhibitory concentration (MIC) against model pathogenic microorganisms

[0080] The antibacterial effect of the propolis extract was measured at the Department of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia. The minimum inhibitory concentration (MIC) of the standardized liquid propolis extract according to the invention was determined according to the instructions of the CLSI (Clinical and Laboratory Standards Institute) and EUCAST (European Committee on Antimicrobial Susceptibility Testing) methods, as described in references 26-29. The product of Example 9 was used.

[0081] 26) M.Balouiri, M.Sadiki, SKIbnsouda: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,940West Valley Road,Suite1400,Wayne,Pennsylvania19087-1898,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

[0082] The antibacterial effects were tested under in vitro conditions against ATCC strains of the following model pathogenic microorganisms (M): (i)Staphylococcus aureus ATCC29293(M1); (ii) Methicillin-resistant Staphylococcus aureus; MRSA (MFBF collection; M2); (iii) Methicillin-susceptible Staphylococcus aureus; MSSA (MFBF collection; M3); (iv)Enterococcus faecalis ATCC9212(M4); (v) Enterococcus faecalis VRE (MFBF collection) (M5); (vi) Escherichia coli ATCC10536(M6); (vii) Acinetobacter baumanii ATCC43498(M7); (viii) Pseudomonas aeruginosa ATCC9027(M8); and (ix) Candida albicans ATCC 90028 (M9).

[0083] A serial microdilution procedure was performed to determine the minimum inhibitory concentration (MIC) of the extracts. The MIC value was determined as the propolis extract concentration at which an 80% reduction in the bacterial or fungal count occurred (MIC80). The minimum inhibitory concentrations (MIC80) presented in Table 9 are given in the form of a dilution (%) of the corresponding liquid extract in a given solvent. The starting liquid propolis extract was obtained with a crude propolis to final extract ratio (DER) of 1:2. This is the product of Example 9. If the dilution of the liquid extract is greater, i.e. the concentrations of active substances 1 to 10 are lower relative to the MIC, the obtained antibacterial effect of the tested extract is higher.

[0084] A detailed description of the experimental procedures for MIC determination is disclosed in Example 10 and the results are presented in Table 9.

[0085] [Table 9]

[0086] Tables 10-15 show the mass concentrations of active substances 1-10 in the effective dilutions of the propolis extract in each of the three tested extraction solvent systems. (i) 96% ethanol; the product of Example 1; (ii) polyethylene glycol 400 (PEG400); the product of Example 2; and (iii) Mixture of PEG400 (97% w / w) and soy lecithin (3% w / w); product of Example 9 obtained using Alternatively, the mass concentration of the corresponding active substance 1–10 at which each particular liquid extract reached its MIC is shown, which was determined from the primary liquid extract with a DER of 1:2 divided by the dilution at which the MIC was achieved.

[0087] [Table 10]

[0088] [Table 11]

[0089] [Table 12]

[0090] [Table 13]

[0091] [Table 14]

[0092] [Table 15]

[0093] The overall antibacterial efficacy (MIC) of each of the tested liquid propolis extracts is achieved by the synergistic effect of several active substances 1 to 10. What is interesting and totally unexpected is that mixtures of low concentrations of active substances 1 to 10 are more effective than mixtures of certain (pure) active substances 1 to 10 at much higher concentrations.

[0094] The formulations of the present invention are most effective against gram-positive microorganisms such as Staphylococcus spp., but at higher concentrations are also effective against some gram-negative bacteria: E. coli and A. baumanii, and the fungus C. albicans, as can be seen from Table 9.

[0095] Use of the standardized liquid propolis extract according to the present invention

[0096] The propolis extract according to the invention as a pharmaceutical, cosmetic or agrochemical ingredient or as a food ingredient contains highly bioactive substances in standardized concentrations: (i) 100–1,300 μg / mL p -coumaric acid ( 1 ); (ii) 75–800 μg / mL trans -ferulic acid (2); (iii) 25–300 μg / mL of caffeic acid (3); and (iv) 40-400 μg / mL 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE) Includes.

[0097] The standardized content of the main active substances 1 to 4 makes the liquid propolis extract according to the invention an active pharmaceutical ingredient (API), an active cosmetic ingredient (ACI) for human and animal use or a functional ingredient of food or animal feed, which is characterized by the following beneficial pharmacological effects: (i) anti-inflammatory (see references 1, 8, 9, 11, 12, 13, 17); (ii) antioxidant properties (see references 8, 9, 14, 15, 20); (iii) immunomodulatory (see references 1, 8, 11, 13, 15, 17, 18, 19, 20); (iv) Hepatoprotective (see Reference 9): (v) antibacterial, including antibacterial, antiviral, antifungal, and antiprotozoal (see References 9, 10, 12, 15, 16, and 18); (vi) antitumor properties (see References 9, 10, 11, 14, 19); and (vii) Anticancer properties (see References 11 and 14).

[0098] Other beneficial effects of the liquid propolis extract according to the invention are its fungicidal, bactericidal, virucidal, insecticidal and nematicidal effects in plant protection. Due to its strong antioxidant activity, the liquid propolis extract according to the invention indirectly strengthens plants and their resistance to abiotic stress factors and helps to combat infectious diseases. For this reason, it is used as a plant strengthening agent. See references 21-25.

[0099] The liquid propolis extract according to the present invention comprises: (i) an active pharmaceutical ingredient or excipient for the manufacture of a pharmaceutical product selected from the group consisting of a drug, a medical device, or a therapeutic agent; (ii) Active cosmetic ingredients or excipients for the manufacture of cosmetic products; (iii) Food ingredients for the production of functional foods, food supplements or foods for special nutritional purposes; (iv) an active pharmaceutical ingredient or excipient for the manufacture of a veterinary product selected from the group consisting of a veterinary pharmaceutical product, an animal feed, an animal feed supplement, or a therapeutic agent for veterinary use; or (v) Active pesticide ingredients or excipients for producing pesticide products selected from the group consisting of fungicides, bactericides, virucides, insecticides, nematicides and plant strengthening agents, in particular those particularly suitable for ecological agriculture. Used as.

[0100] Pharmaceutical compositions based on standardized liquid propolis extracts according to the present invention

[0101] Furthermore, the present invention discloses a pharmaceutical composition based on a standardized liquid propolis extract as an active pharmaceutical ingredient (API). (I) 5 to 95% w / w of a liquid propolis extract according to the present invention; and (II) up to 100% w / w of the final composition, one or more pharmaceutical excipients necessary for preparing the final dosage form selected from the group consisting of solutions, suspensions, gels, creams, ointments, sprays for oral or nasal application It consists of: The aforementioned composition contains four main active propolis substances within the following value ranges: (i) 10–1,300 μg / g p-coumaric acid (1); (ii) 10–800 μg / g trans -ferulic acid (2); (iii) 5 to 300 μg / g caffeic acid (3); and (iv) 5-400 μg / g 2-phenylethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE) It is characterized by a quantitative content of at least two of the following:

[0102] Thereby, the pharmaceutical excipients (auxiliary substances) are selected from the group consisting of diluents, humectants, preservatives, chelating agents, antioxidants, thickeners, emollients, emulsifiers, tonicity agents, and pH control agents.

[0103] The diluent is a pharma- ceutically acceptable liquid selected from the group consisting of purified water, ethanol, 1,2-propylene glycol, liquid polyethylene glycol (PEG) such as PEG200, PEG400 or PEG600, or mixtures of these substances.

[0104] The humectant is selected from the group consisting of glycerol, sorbitol, 1,2-propylene glycol, or mixtures of these substances.

[0105] 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.

[0106] The chelating agent is selected from the group consisting of sodium or potassium salts of ethylenediaminotetraacetic acid (EDTA), diethylenetriaminopentaacetic acid (DTPA), nitrilotriacetic acid (NTA), soluble citrate salts such as trisodium citrate dihydrate (Na3C6H5O7·2H2O), or mixtures of these substances. An exemplary chelating agent is disodium edetate dihydrate (Na2EDTA·2H2O).

[0107] 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 a mixture of these substances.

[0108] The thickening agent is selected from the group consisting of cellulose gums such as hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), methylcellulose (MC), sodium carboxymethylcellulose (NaCMC); synthetic polymers such as polyvinyl alcohol (PVA), polyacrylic acid (PAA) and its copolymers, polyvinylpyrrolidone (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.

[0109] The emollient is selected from the group consisting of petrolatum; mineral oil; vegetable oil, such as almond, sunflower or olive oil; medium chain triglycerides; natural or synthetic esters of higher fatty acids and monohydric alcohols, such as isopropyl myristate or jojoba oil; waxes, such as beeswax; silicone oils; higher fatty acids, such as oleic acid or stearic acid; higher fatty alcohols, such as cetyl alcohol; or mixtures of these substances.

[0110] The emulsifier is selected from the group consisting of lanolin; ethoxylated lanolin; lanolin alcohol; ethoxylated lanolin alcohol; lecithin; hydrolyzed lecithin; mono- and diesters of glycerol and higher fatty acids, such as glycerol monostearate; sorbitan esters of higher fatty acids, such as sorbitan monostearate; ethoxylated higher fatty alcohols, such as polyoxyethylene(23) lauryl ether or polyoxyethylene(2) oleate (the numbers 23 or 2 represent the number of ethylene oxide units); esters of ethoxylated sorbitan esters, such as polysorbate 60; water-soluble soaps, such as sodium stearate; water-soluble sulfates of higher fatty alcohols, such as sodium lauryl sulfate; water-soluble phosphates of fatty alcohols, such as potassium cetyl phosphate; or mixtures of these substances.

[0111] Isotonicity agents are primarily used in pharmaceutical dosage forms applied to mucous membranes, such as the nasal mucosa, and are selected from the group consisting of sodium chloride (NaCl), glycerol, 1,2-propylene glycol or mixtures of these substances.

[0112] The pH control agent includes pharma- ceutically acceptable acids and bases for lowering or raising the pH value, as well as buffer systems, which are selected 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.

[0113] Preparation of pharmaceutical compositions according to the invention

[0114] The pharmaceutical composition according to the present invention comprises (i) adding the standardized liquid propolis extract according to the present invention to a diluent and homogenizing them; (ii) adding one or more other excipients and homogenizing them; Includes; Steps (i) and (ii) are carried out at a temperature between 10 and 100°C, preferably between 20 and 60°C, for a period of 1 to 5 minutes; Next, (iii.a) in the case of preparing a solution or spray solution dosage form, performing filtration of the final solution, including sterile filtration as necessary; (iii.b) in the case of preparing a gel or suspension dosage form, adding a thickening agent and homogenizing it; (iii.c) in case of preparation of a cream formulation, preparation of a fatty phase by mixing an emollient and an emulsifier and homogenizing at a temperature of 50-80°C for 1-15 minutes, then adding the solution of step (ii) and heating to 50-80°C, followed by emulsification using a high shear or high pressure homogenizer at a temperature of 50-80°C, preferably 55-65°C, for 1-30 minutes, followed by homogenization at a temperature of 65-20°C for 10-120 minutes; or (iii.d) for preparation of an ointment dosage form, mixing the solution of step (ii) with a pre-melted mixture of emollients and finally emulsifiers at a temperature of 50-70°C for 5-30 minutes, followed by homogenization at a temperature of 70-20°C for 10-120 minutes. It is prepared by a method comprising:

[0115] The procedures for preparing the pharmaceutical compositions of the present invention may also include various alternative general technical procedures for producing the aforementioned dosage forms, as known to those skilled in the pharmaceutical art.

[0116] Representative examples of the preparation of pharmaceutical compositions according to the present invention are described in Examples 11-16.

[0117] As a specific example, here we outline the final dosage form of a solution for intramammary application, disclosed in Example 11. In this case, the primary standardized liquid propolis extract, the preparation of which is described in Example 9, is simply diluted with an extraction solvent according to the invention, in this case a mixture of polyethylene glycol 400 (97% w / w) and soy lecithin (3% w / w), which here is the function of the diluent. The solution for intramammary application thus obtained gives a quantitative content of the main active substances 1 to 4 within the abovementioned limits. See Table 16.

[0118] [Table 16]

[0119] A typical HPLC chromatogram from a quantitative analysis of this composition is shown in FIG.

[0120] Study of the pharmacological effects of the pharmaceutical composition of the present invention

[0121] Selected pharmacological effects of the compositions of the invention at doses from the solution of the product of Example 11 are shown in Table 1. (i) Bovine mastitis, or mastitis; (ii) mastitis in goats; and (iii) Wound healing in horses It has been studied in clinical trials in the treatment of

[0122] Research into the treatment of bovine mastitis

[0123] A study of bovine mastitis treatment was carried out on five farms with Holstein dairy cows. A total of 86 cows or 339 quarters were included in the study, with the udder quarter used as the statistical unit. The animals were kept free on deep bedding and fed a standard premix for dairy cows without added antibiotics. The study was approved by the Veterinary Ethics Committee. Healthy animals and quarters without clinical signs of mastitis with a somatic cell count (SCC) of less than 200,000 / mL, as well as infected quarters with an SCC of more than 200,000 / mL, were included. A randomized crossover clinical trial of safety and efficacy was carried out with intramammary (i.mam.) application of the composition of the invention in the form of a solution under topographical conditions. The composition of the invention of Example 11 was applied three times to all four quarters of the cow's udder during the morning milking, during the evening milking and after the next morning milking. Detailed procedures are described in Example 17, but Table 17 presents the bacteriological cure of each pathogen identified in a given number of quarters prior to the first i.mam. application.

[0124] [Table 17]

[0125] A three-times (two-day) dosing regimen of the composition from Example 11 resulted in bacteriological cure in 100% of cases within only seven days.

[0126] In comparison, the cephalosporin antibiotic ceftiofur achieves a bacteriological cure of 66%, but only after 8 days of daily i.mam. application, while after 5 days cure occurs in only 54% of cases. See Reference 30. In subclinical mastitis caused by S. uberis, 2 days of treatment with pirlimycin resulted in cure in 58.1% of cases, whereas 5 and 8 days of treatment resulted in cure in 68.8 and 80% of cases. See Reference 30.

[0127] 30)SPOliver,BEGillespie,SJHeadrick,H.Moorehead,P.Lunn,HHDowlen,DJJohnson,KCLamar,STChester,WMMoseley:Efficacy of extended Ceftiofur intramammary therapy for treatment of subclinical mastitis in lactating dairy cows.J.Dairy Sci.87(2004)2393-2400

[0128] Alternatively, in the treatment of bovine mastitis, compositions of the invention in the form of a suspension for intramammary application, as described in Example 12, can also be used successfully.

[0129] Research into the treatment of mastitis in goats

[0130] A study of caprine mastitis treatment was carried out on 25 goats at the alpine goat farm at OPG Matijasec, Sigetec Ludbreski, Croatia. All goats were diagnosed with subclinical mastitis in the left and right udder halves. Goats whose milk tested positive to microbiological investigations were divided into two groups. One group was applied with the composition of the invention from Example 11, the other with an intramammary suspension of amoxicillin and clavulanic acid (Clavxil®; Genera, Croatia). All with a three-application regimen. Tolerance and bacteriological cure of the halves were monitored in parallel after i.mam. application of the composition from Example 11. Three applications (2 days) of the formulation of the invention resulted in 75% of infected halves and 85% bacteriological cure after 14 days. This proved to be more effective than intramammary antibiotic administration, which allowed bacteriological cure in 73.3% of cases. The results of the study showed that treatment of mastitis in goats with the composition of Example 11 could result in bacteriological cure in time without the use of antibiotics.

[0131] Detailed procedures of the study are described in Example 18, and the bacteriological cure results are shown in Table 18.

[0132] [Table 18]

[0133] Comparing the activity of amoxicillin as a broad spectrum antibiotic suitable for the treatment of mastitis caused by pathogens found in milk samples and the i.mam. application of the composition from Example 11, the stronger activity of the latter was demonstrated. After 7 days from the application of amoxicillin, only 60% of the halves were cured, whereas the result for the composition of Example 11 was 75%. After 14 days from the first application, the overall percentage of bacteriologically cured halves with the antibiotic application was 73.3%, whereas for the test composition it was 85%.

[0134] From the above results, it can be seen that the anti-inflammatory and antibacterial activity of the composition of the present invention is highly effective in treating mastitis, the effectiveness being higher than that of classical treatments such as those with a fixed combination of amoxicillin and clavulanic acid as broad spectrum antibiotics.

[0135] Alternatively, in the treatment of mastitis in goats, compositions of the invention in the form of a suspension for intramammary application, as described in Example 12, can also be used successfully.

[0136] It can be concluded that the composition of the present invention, in addition to other properties, is characterized by antibacterial activity against a range of bacteria and fungi, and the antibacterial effect is higher in vivo than under in vitro conditions, compared to classical antibiotics. It is not only an antibacterial agent, but also an anti-inflammatory and immunomodulatory agent.

[0137] The immunomodulatory effects of propolis are closely linked to its antioxidant effects, and oxidative stress is an integral part of the pathogenesis of mastitis. See, for example, reference 31:

[0138] 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

[0139] A review of wound healing cases in horses

[0140] The case of wound healing of the front leg of a female horse is described. This is a deep wound in the ergot area of ​​the front leg, which occurred when the rear leg grips the front leg during landing. Before starting the administration of the composition from Example 11, the wound was treated conservatively with different preparations without success.

[0141] The wound was then washed with physiological solution, dried and treated with the composition from Example 11 once a day for 5 days. Improvement such as epithelialization was observed after 48 hours, and complete healing of the wound occurred after 96 hours. The mare limped heavily for several days, but no signs of lameness were observed thereafter. Recovery was complete. A detailed description of this case is provided in Example 19.

[0142] Although detailed clinical studies are required for more precise conclusions, it is clear to those skilled in the art that this composition effectively acts as a wound healing agent.It is known from the literature that anti-inflammatory activity, antioxidant activity and epithelialization activity are important for the wound healing process, so that stimulation of collagen synthesis is part of the range of pharmacological effects, as in the composition of the present invention, along with the proven antibacterial activity.For comparison, please refer to reference 32:

[0143] 32) S. Marinotti, E. Ranzato: Propolis: a new frontier for wound healing, Burns Trauma (2015) 3:9, doi 10.1186 / s41038-015-0010-z.

[0144] Uses of the pharmaceutical composition according to the present invention

[0145] The pharmaceutical compositions herein are used for the treatment of human and animal diseases and conditions from the group of inflammatory diseases, bacterial infections, fungal infections, viral diseases, autoimmune diseases, functional gastrointestinal disorders, mucosal regeneration, burn treatment and wound healing, and cancer diseases.

[0146] Inflammatory diseases and conditions included are gingivitis, periodontitis, laryngitis, gastritis, colitis, haemorrhoidal disease, dermatitis, ear inflammation externally, sinusitis, rhinitis, vaginitis and mastitis.

[0147] The pharmaceutical composition of the present invention comprises: (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; Enterococcus faecium, Enterococcus faecalis; Vancomycin-resistant Enterococcus (VRE): Enterococcus casseliflavus; and (ii) Gram-negative bacteria: Escherichia coli; Acinetobacter baumanii; Pseudomonas aeruginosa; Haemophilus influenzae; Salmonella choleraesuis; 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 It is used in the treatment of bacterial infections caused by bacteria from the group.

[0148] Further, the pharmaceutical composition is used for the treatment of fungal infections caused by fungi such as 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; and Malassezia pachydermatis.

[0149] The compositions of the present invention are also used to treat viral diseases caused by viruses such as herpes simplex virus (HSV); human papillomavirus (HPV); Epstein-Barr virus (EBV); cytomegalovirus (CMV); poliovirus; influenza A and B viruses; retroviruses; vaccinia virus; common cold viruses: rhinovirus, picornavirus, human parainfluenza virus (HPIV), human metapneumovirus (HMPV), coronavirus, adenovirus, human respiratory syncytial virus (HRSV), enterovirus, and the like.

[0150] Alternatively, the pharmaceutical compositions according to the invention are used to treat autoimmune diseases such as psoriasis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, celiac disease and multiple sclerosis.

[0151] Furthermore, the pharmaceutical composition of the present invention is used to treat cancer diseases such as skin and mucosal cancer, gastrointestinal tumors, colorectal cancer, and the like.

[0152] The pharmaceutical compositions of the present invention are used to treat functional gastrointestinal disorders such as disorders of the esophagus, stomach, duodenum, small intestine and colon, central gastrointestinal pain, gallbladder and sphincter of Oddi disorders, anorectal disorders, and pediatric and adolescent specific gastrointestinal disorders.

[0153] Specifically, the compositions of the present invention are used to treat mastitis in animals. EXAMPLES

[0154] Overview

[0155] As primary propolis, crude propolis of poplar type from Hedera Ltd was used. Ethanol (96%) and polyethylene glycol 200, 400 and 600, as well as powdered soy lecithin (SL) were purchased from Fagron Croatia Ltd (HR). Rapeseed lecithin (RL) and hydrolyzed rapeseed lecithin (HRL) were purchased from Pfannenschmidt (DE). Deoiled sunflower lecithin (SUL) was purchased from Barentz (NL). Aluminium distearate was purchased from Sigma-Aldrich (US). All other starting materials were purchased from local suppliers. Samples of chemically pure compounds of analytical interest: p-coumaric acid (1; ≥ 98% HPLC), trans-ferulic acid (2; 99%), caffeic acid (3; ≥ 98% HPLC), 2-phenethyl 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 to determine their quantitative content in the liquid propolis extracts, were purchased from Sigma-Aldrich (USA).

[0156] The term room temperature refers to the temperature interval 20-25 °C. The abbreviation "min" stands for minutes. Yields (% from theoretical yield) are expressed as the weight (% w / w) percentage of isolated liquid propolis extract relative to the mass of the starting extraction solvent (ethanol, PEG, ethanol + lecithin, PEG + lecithin).

[0157] The quantitative content of active substances 1-10 in the liquid propolis extract is expressed as mass concentration (γ) in micrograms per milliliter [μg / mL], whereas their quantitative composition in the pharmaceutical composition of the present invention is expressed in micrograms per gram of final product (dosage form) [μg / g].

[0158] Example 1: Preparation of liquid propolis extract by using 96% ethanol as the extraction solvent

[0159] Pretreatment of propolis before extraction: A sample of crude propolis (poplar type; 1 kg) was cooled in a refrigerator at -20°C for a minimum of 1 hour. The sample was then ground in a mill.

[0160] Extraction with 96% ethanol: Ethanol (96%; 70.00 g) was added to ground propolis (30.00 g). The mixture thus obtained was left at room temperature for 72 hours with periodic stirring. The mixture was then filtered through a filter paper (black ribbon) to obtain 60.00 g (85.7%) of liquid propolis extract in the form of a deep brown solution with a slightly strong propolis odor.

[0161] For the purpose of testing the minimum inhibitory concentration (MIC) of the alcoholic propolis extract described in Example 10, the same procedure was repeated with a drug to extract (DER) ratio of 1:2.

[0162] Example 2: Preparation of liquid propolis extract by using polyethylene glycol 400 as the extraction solvent

[0163] Ground propolis (30.00 g), the pretreatment of which is described in Example 1, was mixed with polyethylene glycol 400 (PEG 400; 70.00 g). The mixture thus obtained was left at room temperature for 72 hours with periodic stirring. The mixture was then filtered through a filter paper (black ribbon). This yielded 55.00 g (78.6%) of liquid propolis extract in the form of a deep brown viscous liquid with a slightly strong odor similar to propolis.

[0164] For the purpose of testing the minimum inhibitory concentration (MIC) of the polyethylene glycol propolis extract described in Example 10, the same procedure was repeated with a drug to extract (DER) ratio of 1:2.

[0165] Example 3: HPLC analysis method for quantifying active substances 1-10 in liquid propolis extract

[0166] Quantitative analysis by high performance liquid chromatography (HPLC) was performed with a method developed specifically to monitor the main active substances 1–4 and the associated active components 5–10 from propolis.

[0167] Prepare samples of commercial standards of active substances 1 to 10 for analysis by diluting with an ethanol:water mixture, 75:25, V / V, to a concentration of 100 µg / mL.

[0168] Samples (100 μL) of liquid propolis extract according to the invention were diluted in a 1:10 w / w ratio (10-fold dilution) with an ethanol:water mixture, 75:25, V / V (900 μL) prior to analysis.

[0169] The analysis was carried out on a Shimadzu LC201CHT instrument equipped with an autosampler, pump, degasser, column oven and UV-VIS detector under the following conditions: (i) Chromatography column: Ascentis express; C18; dimensions: 15 cm × 3.0 mm; particle size in the column: 2.7 μm; (ii) Mobile phase: A = 0.1% formic acid in water, 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: 0.25mL / min; (v) Analysis time: 110min; (vi) UV-VIS detector wavelengths: for detection: 370 nm, for integration: 290 nm; (vii) Injection volume: 10μL; (viii) Pressure: 210-290 bar.

[0170] Under the aforementioned conditions, the main active substances 1-4 and the accompanying components 5-10 have the following retention times (t R ) (i)t R [p-Coumaric acid (1)] = 14.13 min;t R [trans-ferulic acid (2)] = 15.31 min;t R [Caffeic acid (3)] = 10.57 min;t R [2-phenethyl 3,4-dihydroxy-trans-cinnamate; CAPE(4)] = 48.62 min; (ii) t R [trans-cinnamic acid (5)] = 29.76 min;t R [Chrysin(6)] = 44.68 min;t R [Pinocembrin(7)]=47.39min;t R [galangin(8)]=49.94min;t R [Apigenin(9)]=37.72min;t R [Kaempferol(10)]=36.87min.

[0171] The retention times of the major propolis active substances 1-4 and the associated active ingredients 5-10 are shown in Table 1.

[0172] Example 4: Preparation of a standardized liquid propolis extract by using an extraction solvent based on a mixture of ethanol and lecithin

[0173] Crushed propolis (30.00 g), pretreated as described in Example 1, was mixed with an extraction solvent of the following composition (Experiments E1-8): E1: 96% ethanol (67.00 g; 95.7%) and soy lecithin (SL; 3.00 g; 4.3%); E2: 96% ethanol (60.00 g; 85.7%) and soy lecithin (SL; 10.00 g; 14.3%); E3: 96% ethanol (50.00 g; 71.4%) and soy lecithin (SL; 20.00 g; 28.6%); E4: 96% ethanol (40.00 g; 57.1%) and soy lecithin (SL; 30.00 g; 42.9%); E5: 96% ethanol (67.00 g; 98.9%) and rapeseed lecithin (RL; 3.00 g 25%-prepared; 0.75 g lecithin; 1.1%); theoretical yield in E5 = 67 g ethanol + 0.75 g lecithin = 67.75 g; E6: 96% ethanol (60.00 g; 96.0%) and rapeseed lecithin (RL; 10.00 g 25%-prepared; 2.50 g lecithin; 4.0%); theoretical yield in E6 = 60 g ethanol + 2.50 g lecithin = 62.50 g; E7: 96% ethanol (67.00 g; 97.8%) and hydrolyzed rapeseed lecithin (HRL; 3.00 g 50%-prepared; 1.50 g hydrolyzed rapeseed lecithin; 2.2%); theoretical yield in E7 = 67 g ethanol + 1.50 g lecithin = 68.50 g; E8: 96% ethanol (60.00 g; 92.3%) and hydrolyzed rapeseed lecithin (HRL; 10.00 g 50%-preparation; 5.00 g lecithin; 7.7%); theoretical yield in E8 = 60 g ethanol + 5.00 g lecithin = 65.00 g.

[0174] The mixture thus obtained was stirred for 1 hour at room temperature and left for 72 hours at room temperature with periodic stirring. The mixture was then filtered through a filter paper (black ribbon). This resulted in 50-60 g (71.0-85.7%) of liquid propolis extract in the form of a deep brown viscous liquid with a slightly strong odor similar to propolis.

[0175] The primary liquid propolis extract thus prepared was subjected to quantitative analysis of the contents of the main active substances 1-4 and the accompanying components 5-10 according to the analytical method described in Example 3. The results are shown in Tables 3 and 4.

[0176] The primary liquid propolis extracts so prepared in the extraction solvents (ES) described in experiments E1-E8 with known quantitative contents of active substances 1-4 were standardized by diluting with the same ES used in the extraction step to the desired level of quantitative composition of active substances 1-4 according to the invention.

[0177] Example 5: Preparation of a standardized liquid propolis extract by using an extraction solvent based on a mixture of polyethylene glycol 400 and lecithin

[0178] Ground propolis (30.00 g), pretreated as described in Example 1, was mixed with extraction solvents of the following compositions (Experiments E1-E8): E1: polyethylene glycol 400 (PEG400; 67.00 g; 97%) and soy lecithin (SL; 3.00 g; 3%); E2: polyethylene glycol 400 (PEG400; 60.00 g; 90%) and soy lecithin (SL; 10.00 g; 10%); E3: Polyethylene glycol 400 (PEG400; 67.00 g; 98,9%) and rapeseed lecithin (RL; 3.00 g 25%-prepared; 0.75 g lecithin; 1.1%); theoretical yield of E3 = 67 g PEG400 + 0.75 g lecithin = 67.75 g; E4: Polyethylene glycol 400 (PEG400; 60.00 g; 96.0%) and rapeseed lecithin (RL; 10.00 g 25%-prepared; 2.50 g lecithin; 4.0%); theoretical yield in E4 = 60 g PEG400 + 2.50 g lecithin = 62.50 g; E5: Polyethylene glycol 400 (PEG400; 67.00 g; 97,8%) and hydrolyzed rapeseed lecithin (HRL; 3.00 g 50%-prepared; 1.50 g hydrolyzed lecithin; 2.2%); theoretical yield with E5 = 67 g PEG400 + 1.50 g lecithin = 68.50 g; E6: Polyethylene glycol 400 (PEG400; 60.00 g; 92.3%) and hydrolyzed rapeseed lecithin (HRL; 10.00 g 50%-preparation; 5.00 g lecithin; 7.7%); theoretical yield with E6 = 60 g PEG400 + 5.00 g lecithin = 65.00 g.

[0179] The mixture thus obtained was stirred for 1 hour at room temperature and left for 72 hours at room temperature with periodic stirring. The mixture was then filtered through a filter paper (black ribbon). This resulted in 45-55 g (69.0-78.6%) of liquid propolis extract in the form of a deep brown viscous liquid with a slightly strong odor similar to propolis.

[0180] The primary liquid propolis extract thus prepared was subjected to quantitative analysis of the contents of the main active substances 1-4 and the accompanying components 5-10 according to the analytical method described in Example 3. The results are shown in Tables 5 and 6.

[0181] The primary liquid propolis extracts so prepared in the extraction solvents (ES) described in experiments E1-E6 with known quantitative contents of active substances 1-4 were standardized by diluting with the same ES used in the extraction step to the desired level of quantitative composition of active substances 1-4 according to the invention.

[0182] Example 6: Preparation of a standardized liquid propolis extract by using an extraction solvent based on a mixture of polyethylene glycol 200 and soy lecithin

[0183] The ground propolis (30.00 g), the pretreatment of which is described in Example 1, was mixed with a mixture of the extraction solvent, polyethylene glycol 200 (149.85 g; 99.9%) and soy lecithin (SL; 0.15 g; 0.1%). The mixture thus obtained was stirred for 1 hour at room temperature and left for 48 hours at room temperature with periodic stirring. The mixture was then filtered through a filter paper (black ribbon). This resulted in 137.00 g (91.3%) of liquid propolis extract in the form of a deep brown viscous liquid with a slightly strong odor similar to propolis.

[0184] Then, quantitative HPLC analysis was performed according to the method described in Example 3 to obtain the quantitative contents of the main active substances 1-4. The filtrate was diluted with the same extraction solvent, a mixture of polyethylene glycol 200 and soy lecithin (SL), 99.9:0.1 w / w, to the total level of active substances 1-4 content according to the specification of the standardized liquid extract of the present invention.

[0185] Example 7: Preparation of a standardized liquid propolis extract by using an extraction solvent based on a mixture of polyethylene glycol 600 and deoiled sunflower lecithin

[0186] The ground propolis (30.00 g), the pretreatment of which is described in Example 1, was mixed with a mixture of the extraction solvent, polyethylene glycol 600 (PEG600; 297.00 g; 99%) and deoiled sunflower lecithin (SUL; 3.00 g; 1.0%). The mixture thus obtained was heated to 70°C and stirred vigorously for 3 hours. The mixture was then cooled to room temperature and filtered through a filter paper (black ribbon). This resulted in 261.00 g (87.0%) of liquid propolis extract in the form of a deep brown viscous liquid with a slightly strong odor similar to propolis.

[0187] Then, quantitative HPLC analysis was performed according to the method described in Example 3 to determine therefrom the quantitative contents of the main active substances 1-4. The filtrate was diluted with the same extraction solvent, a mixture of polyethylene glycol 600 and deoiled sunflower lecithin (SUL), 99:1 w / w, to the total content level of active substances 1-4, according to the specifications of the standardized liquid extract of the present invention.

[0188] Example 8: Preparation of a standardized liquid propolis extract by using an extraction solvent based on a mixture of polyethylene glycol 200, polyethylene glycol 600 and hydrolyzed rapeseed lecithin

[0189] The ground propolis (30.00 g), the pretreatment of which is described in Example 1, was mixed with a mixture of the extraction solvent, polyethylene glycol 200 (PEG200; 150.00 g; 50%), polyethylene glycol 600 (139.50 g; 46.5%) and hydrolyzed rapeseed lecithin (HRL; 10.50 g; 3.5%). The mixture thus obtained was heated at 100°C with vigorous stirring for 3 hours. The mixture was then cooled to room temperature and filtered through filter paper (black ribbon). This resulted in 245.00 g (81.7%) of liquid propolis extract in the form of a deep brown viscous liquid with a slightly strong odor similar to propolis.

[0190] Then, quantitative HPLC analysis was performed according to the method described in Example 3 to determine the quantitative contents of the main active substances 1-4 therefrom. 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 (w / w / w), to the total content level of active substances 1-4 according to the specifications of the standardized liquid extract of the present invention.

[0191] Example 9: Preparation of standardized liquid propolis extract using polyethylene glycol 400 and soy lecithin mixture

[0192] Ground propolis (30.00 g), pretreated as described in Example 1, was mixed with a mixture of extraction solvent, polyethylene glycol 400 (PEG400; 87.30 g; 97% m / m) and soy lecithin (SL; 2.70 g; 3% w / w). The mixture thus obtained was left to macerate for 72 hours with periodic stirring. It was then filtered through a filter paper (800 pores / cm 2 The mixture was filtered through a sieve. This yielded 50-55 g of a deep brown viscous liquid with a strong odor similar to propolis. The product thus obtained was diluted with the same extraction solvent to a mass of 60.00 g. In this way, a final extract with a drug-to-extract (DER) ratio of 1:2, or 60 g of extract as mentioned above was obtained from 30 g of starting propolis.

[0193] The liquid propolis extract thus prepared is used for the purpose of preparing the pharmaceutical composition of the present invention. (I) subjecting to quantitative HPLC analysis according to the method described in Example 3 to determine active substances 1-4, and then with respect to the actual mass concentrations of active substances 1-4 in such prepared extracts; (II) Pure (fresh) solvent mixture: polyethylene glycol 400 (97% w / w) and soy lecithin (3% w / w) with active substances 1-4: (i) 10–1,300 μg / mL p -coumaric acid ( 1 ); (ii) 10–800 μg / mL trans -ferulic acid (2); (iii) 5–300 μg / mL of caffeic acid (3); and (iv) 5–400 μg / mL of 2-phenethyl 3,4-dihydroxy-trans-cinnamate (4; CAPE); The mass concentration of was standardized by diluting it by a factor X / 100 (X=weight percentage %, w / w, of said standardized liquid propolis extract in the formulation of the pharmaceutical composition).

[0194] Example 10: Determination of the antibacterial effect of the standardized liquid extract according to the invention. Determination of the minimum inhibitory concentration (MIC) against model pathogenic microorganisms

[0195] The minimum inhibitory concentration (MIC) of the standardized liquid propolis extract according to the invention was determined using the product of Example 9 in comparison with similar liquid propolis extracts obtained with 96% ethanol (product from Example 1) or PEG400 (product from Example 2) according to the instructions of the CLSI and EUCAST methods, see references 26-29.

[0196] The antibacterial effect was tested under in vitro conditions against ATCC strains of the following model pathogenic microorganisms (M): Staphylococcus aureus ATCC29293 (M1); methicillin-resistant Staphylococcus aureus; MRSA (MFBF collection; M2); methicillin-susceptible Staphylococcus aureus; MSSA (MFBF collection; M3); Enterococcus faecalis ATCC9212 (M4); Enterococcus faecalis VRE (MFBF collection) (M5); Escherichia coli ATCC10536 (M6); Acinetobacter baumanii ATCC43498 (M7); Pseudomonas aeruginosa ATCC9027 (M8); and Candida albicans ATCC 90028 (M9).

[0197] A serial microdilution procedure was performed to determine the minimum inhibitory concentration (MIC) of the extracts. Cell suspensions were prepared from parent cultures in PBS buffer (pH 7.4) and these were adjusted to 0.5 McFarland units by turbidimetry. Tests were performed in serial dilutions in 96-well microtiter plates ranging from 100 to 0.7125 μg / mL by adding 100 μL of a solution of propolis extract dissolved in Mueller-Hinton broth. After seeding with 100 μL of each bacterial culture adjusted to 105 cfu / mL, the plates were incubated at 37 °C for 24 h. The MIC was determined by adding 10 μL of a 0.5 mg / mL solution of 2,3,5-triphenyl-2H-tetrazolium chloride (TTC; a redox indicator) per single well and the absorbance was determined spectrophotometrically at a wavelength of 490 nm after 4 h of incubation at 30 °C.

[0198] The MIC value was determined as the concentration of propolis extract that caused 80% reduction of bacteria (MIC80).

[0199] For fungal species, MIC values ​​were determined in RPMI medium with additional glucose according to the same scheme as for bacteria: after incubation (48 h, 37 °C, aerobic conditions, dark), XTT (a redox indicator) was added in combination with menadione and the absorbance was determined spectrophotometrically at a wavelength of 540 nm.

[0200] MIC values ​​were determined as the concentration of propolis extract that caused 80% reduction of bacteria or fungi (MIC80).

[0201] Negative controls contained medium and solvent only (no added microorganisms and propolis) and positive controls were exposed to antibiotics or antifungals.

[0202] The antibacterial activity of propolis solutions was determined in vitro by measuring the minimum inhibitory concentration (MIC80) and is given in Table 9 as a percentage of the liquid extract diluted in a given solvent. The starting liquid propolis extract was obtained with a drug to extract (DER) weight ratio of 1:2. This is the product from Example 9. The greater the dilution of the liquid extract, i.e. the lower the concentration of active substances 1-10 relative to the MIC, the higher the resulting antibacterial effect of the tested extract.

[0203] The results are shown in Table 9.

[0204] Tables 10-15 show the calculated mass concentration (γ) in [μg / mL] of each specific active substance 1-10 (from each of the three tested liquid propolis extracts) at which each specific extract achieved its MIC. The following extraction solvents (ES) were used to obtain the extracts: 96% ethanol (product from Example 1), polyethylene glycol 400 (PEG400; product from Example 2), and a mixture of PEG400 (97% w / w) and soy lecithin (3% w / w) (product from Example 9). These are the values ​​determined from the primary liquid extract with a DER of 1:2, divided by the dilution (factor) at which the corresponding MIC was achieved.

[0205] Example 11: Preparation of a composition according to the invention in the form of a solution for intramammary application containing at least 150 μg / g of active substances 1 to 4

[0206] Formulation (for 100 g solution): (1) 50.00 g (50.00% w / w) of a liquid propolis extract according to the invention, the product of Example 9, standardized with a minimum of 300 μg / mL of active substances 1 to 4. (2) 50.00 g (20.00% w / w) of polyethylene glycol 400

[0207] Preparation: Components (1) and (2) were mixed and homogenized by stirring at room temperature for 5 minutes. The solution was filtered through filter paper and filled into intramammary syringes in portions of 4-8 g each. Solution composition: total concentration of active substances 1-4 of at least 150 μg / g. The results of the quantitative HPLC analysis are shown in Table 16, and the corresponding typical HPLC chromatogram is shown in FIG.

[0208] Example 12: Preparation of a composition according to the invention in the form of a suspension for intramammary application containing at least 100 μg / g of active substances 1 to 4

[0209] Formulation (for 100g suspension): (1) 77.00 g (77.00% w / w) of liquid propolis extract according to the invention, the product of Example 9, standardized with a minimum of 150 μg / mL of active substances 1 to 4. (2) 20.00 g (20.00% w / w) of polyethylene glycol 4000 (3) 3.00 g (3.00% w / w) of aluminum distearate

[0210] Preparation: Polyethylene glycol (2) was melted at 60°C, aluminum distearate (3) was added and homogenized at this temperature for 15 minutes. The mixture was then cooled with stirring and propolis extract (1) was added at 40-45°C. The mixture was further cooled to room temperature with stirring and added and homogenized at room temperature for 15 minutes. A pale yellow viscous suspension was obtained, which was further filled into intramammary syringes in portions of 4-8 g each.

[0211] Suspension composition: total concentration of active substances 1-4 of at least 100 μg / g

[0212] Example 13: Preparation of a composition according to the invention in the form of a gel containing at least 250 μg / g of active substances 1 to 4

[0213] Formulation (for 100g gel): (1) 30.00 g (30.00% w / w) of a liquid propolis extract according to the invention, the product of Example 9, standardized with a minimum of 850 μg / mL of active substances 1 to 4. (2) 2.00 g (2.00% w / w) of 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) A suitable amount of sodium hydroxide (20% solution) (8) 100% purified water

[0214] Preparation: 30 g of the liquid propolis extract according to the present invention was added with (2) and homogenized by stirring at room temperature for 20 minutes. Then, 50 g of purified water (8) was added and homogenized by stirring at room temperature for 5 minutes. After that, ingredients (3-6) were added and dissolved by stirring for 10 minutes. Then, the pH value was adjusted to 5.5-6 with (7). The remaining amount of purified water was added to the gel thus obtained up to a total mass of 100 g and homogenized by stirring at room temperature for 15 minutes, and finally the product was degassed.

[0215] Gel composition: total concentration of active substances 1-4 of minimum 250 μg / g.

[0216] Example 14: Preparation of a composition according to the invention in the form of a cream containing at least 100 μg / g of active substances 1 to 4

[0217] Formulation (for 100g cream): (1) 20.00 g (20.00% w / w) of liquid propolis extract according to the invention, the product of Example 9, standardized with a minimum of 500 μg / mL of active substances 1 to 4. (2) 5.00 g (5.00% w / w) of 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) Vaseline, white (7) 10.00 g (10.00% w / w) mineral oil, concentrated (8) 0.20 g (0.20% w / w) of 4-chloro-m-cresol (9) 100% purified water

[0218] Preparation: The oil phase was prepared by melting a mixture of components (2-7) at 65-70°C for 15-20 minutes until a nearly colorless oily liquid was formed. The aqueous phase was prepared by dissolving (8) and (1) in purified water and then heating to 65-70°C with stirring. The aqueous phase was then slowly added to the oil phase with vigorous mixing for 15-20 minutes, preferably using a homogenizer allowing high shear homogenization at 1,000-3,000 revolutions per minute (rpm). The emulsion thus obtained was further stirred vigorously and gradually cooled at a temperature of 65-20°C for 30 minutes.

[0219] Cream composition: total concentration of active substances 1-4 of at least 100 μg / g

[0220] Example 15: Preparation of a composition according to the invention in the form of an ointment containing at least 500 μg / g of active substances 1 to 4

[0221] Formulation (for 100g ointment): (1) 50.00 g (50.00% w / w) of liquid propolis extract according to the invention, the product of Example 9, standardized with a minimum of 1,000 μg / mL of active substances 1 to 4. (2) 10.00 g (10.00% w / w) polyethylene glycol 400 (3) 40.00 g (40.00% w / w) of polyethylene glycol 4000 Preparation: Ingredients (1-3) were carefully mixed, heated to 60°C, homogenized by stirring for 5 minutes, and slowly cooled to room temperature with mixing.

[0222] Ointment composition: total concentration of active substances 1-4 of at least 500 μg / g

[0223] Example 16: Preparation of compositions of the invention in the form of a nasal spray containing at least 50 μg / g of active substances 1 to 4

[0224] Formulation (for 100 g solution for spray): (1) 5.00 g (5.00% w / w) of liquid propolis extract according to the invention, the product of Example 9, standardized with active substances 1-4 at a minimum of 1,000 μg / mL (2) 0.60 g (0.60% w / w) sodium chloride (3) 0.10 g (0.10% w / w) of 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) of sodium edetate (Na2EDTA 2H2O) (8) 100% purified water

[0225] Preparation: Ingredients (2-7) were added to 90 g of purified water (1) and dissolved by stirring at room temperature for 15 minutes. Then, (1) was added and the mixture was homogenized at room temperature for 15 minutes. The solution was then diluted with 0.2 mL of sterilized water. μ The solution was filtered through a .mu filter and filled into suitable sterile bottles equipped with closures and spray pumps for nasal application.

[0226] Solution composition: Total concentration of active substances 1-4 of at least 50 μg / g

[0227] Example 17: Study of the anti-inflammatory activity of the composition of the invention in the form of an intramammary solution of Example 11 in the treatment of mastitis in dairy cows

[0228] A study of mastitis treatment in cows was carried out in five farms of Holstein dairy cows. A total of 86 cows or 339 quarters were included in the study, with the udder quarter used as the statistical unit. The animals were kept free on deep bedding and fed a standard premix for dairy cows without added antibiotics. The study was approved by the Veterinary Ethics Committee. Healthy animals and quarters without clinical signs of mastitis with a somatic cell count (SCC) of less than 200,000 / mL, as well as infected quarters with an SCC of more than 200,000 / mL, were included. A randomized crossover clinical trial of safety and efficacy was carried out with intramammary (i.mam.) application of the composition of the invention in the form of a solution under topographical conditions. The composition of the invention of Example 11 was applied three times to all four quarters of the cow's udder during the morning milking, during the evening milking and after the next morning milking. First, the tolerability of the composition in intramammary application was tested. Changes in cow behavior were monitored, as well as the gross appearance of the udder (edema and redness), milk and udder sensitivity to touch. Changes in somatic cell count (SCC) in milk were monitored from the period before the first i.mam. application of the composition until 7 days after the first application. Milk samples and also quarters were grouped according to whether the SCC was elevated or below 200,000 / mL and whether the samples were positive or negative for bacteriological investigation. See Reference 33:

[0229] 33)European Medicines Agency(1992):Local Tolerance of Intramammary Preparations in Cows.Directive81 / 852 / EEC

[0230] A bacteriological cure efficacy test was then performed with the propolis composition according to the instructions of the European Medicines Agency (EMA), which is the only measure of efficacy of i.mam. preparations in the treatment of subclinical mastitis. This is defined as the absence of previously identified pathogens in milk samples collected within a certain period of time after i.mam. application of a given preparation. See Reference 34:

[0231] 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

[0232] Milk sampling was performed according to standard methods. Microbiological investigations were performed according to standard instructions. See reference 35:

[0233] 35)JWHogan:Laboratory handbook on bovine mastitis.National Mastitis Council(1999)Madison,Wisconsin,SAD

[0234] The results of bacteriological cure of cows from mastitis are shown in Table 17.

[0235] Example 18: Study of the anti-inflammatory activity of the composition of the invention in the form of an intramammary solution of Example 11 in the treatment of mastitis in goats

[0236] A study of mastitis treatment in goats was carried out on 25 goats diagnosed with subclinical mastitis in the left and right udder halves. Goats whose milk was positive for microbiological investigation were divided into two groups. One group was applied with the composition of the invention from Example 11, the other group was applied with an intramammary suspension of amoxicillin and clavulanic acid (Clavxil®; Genera, Croatia), all with a three-application regimen.

[0237] The tolerability and bacteriological cure of the hamsters were monitored in parallel after i.mam. application of the composition from Example 11. The preparations were applied according to a three times (2 days) regimen with the aforementioned antibiotics or the composition from Example 11.

[0238] During the study, the goats were kept in deep bedding in the shed. There were 170 lactating goats, with an average milk production of 500-600 kg per goat during one lactation lasting about 300 days. They were fed, if necessary, with a minimum of 2 kg of hay per goat and about 1 kg of a premix containing about 16% protein per goat. They were divided into two parts during morning and evening milking. A 2% vitamin-mineral premix Ovisan® (Sano Company, Croatia) was added to the premix. Goats whose milk tested positive for bacteria were divided into two groups. One group (number of treated halves, N=20) received the composition from Example 11, the other group (N=15) received an intramammary suspension of amoxicillin and clavulanic acid (Clavxil®; Genera Company, Croatia). All with a three-application regimen.

[0239] During the test of the tolerability of the composition from Example 11, no changes in the behavior of the goats were observed, nor in the gross appearance of the udder (edema and redness) and milk, nor in the udder sensitivity to touch. After the first few milkings of the jets, milk samples from the left and right halves of the udder were taken in pre-marked sterile plastic tubes. Samples were taken before the first application of the formulation from Example 11, 12 hours after the first application, 24 hours after the first application, and 7 days after the first application of the composition. Milk samples were kept at 4°C until the next day and were analyzed in the laboratory for mastitis and raw milk quality at the Croatian Veterinary Institute.

[0240] Testing of bacteriological cure efficacy was performed in accordance with the instructions of the European Medicines Agency (EMA), see Reference 34. Milk sampling was performed using standard methods and microbiological testing was performed according to standard procedures, see Reference 35.

[0241] The results of bacteriological cure of goats from mastitis are shown in Table 18.

[0242] Example 19: Review of successful wound healing in mares' legs by administration of the composition of the invention from Example 11 in the form of a solution

[0243] A wound healing case of a mare's front leg is described. It was a deep wound in the ergot area of ​​the front leg, which was caused when the mare's hind leg gripped the front leg while downhauling. Before starting the administration of the composition from Example 11, the wound was treated conservatively with different preparations without success.

[0244] The wound was formed when the mare overextended her hind legs while she was galloping. The hind hoof and the head of the horse's hoof then injured the knuckle area of ​​the front leg. This resulted in an oval-shaped wound with dimensions of 2 x 4 cm. The mare showed signs of lameness immediately after the injury and was scored as 4 / 5 (American Equine Medical Association). Immediately after the injury, the wound was shaved and washed with normal cold water, then treated with iodine (povidone-iodine, 0.01%) and sprayed with silver nitrate spray. The wound was closed to avoid infectious contamination. No TAT serum was applied, as the mare had already been vaccinated against tetanus. The mare was kept at rest for 2 weeks and the wound was treated daily by mechanical cleaning and kept clean and dry. The wound was treated every 48 hours with iodine and silver nitrate spray. After 5 days, the wound started to heal and was recovering. Treatment of the wound with zinc-vitamin ointment was then started. After two weeks, the mare was again introduced to work. However, as soon as the mare started to gallop, the wound began to bleed. The wound was again disinfected with iodine and a topical cephalosporin-based antibiotic in a formulation for intramammary application was applied (Cobactan®). Together with mechanical wound cleansing, the wound was treated topically with antibiotics for a further 5 days. Further work was then attempted with the mare, but the wound began to bleed again.

[0245] The wound was then washed with physiological solution, dried and treated with the composition of Example 11 once a day for 5 days. Visible improvement by epithelialization could be observed after 48 hours, but complete healing occurred after 96 hours. The mare was lame for the first few days only, but no signs of lameness were observed thereafter. Recovery was complete.

[0246] conclusion

[0247] Experimental results showed that certain mixtures of liquid polyethylene glycol (PEG), such as PEG 400, in combination with lecithin in amounts ranging from 0.1 to 3.5% by weight of the extraction solvent (ES) unexpectedly extracted the active propolis substances p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3), and 2-phenethyl 3,4-dihydroxy-trans-cinnamate (4) more effectively and chemoselectively than pure solvents, such as 96% ethanol, PEG 400, or mixtures of EtOH and the same lecithin.

[0248] The exact quantitative composition of the main active substances 1-4 and the accompanying components 5-10 in the primary liquid propolis extract is determined by the use of a suitable analytical HPLC method developed in the present invention. Such a primary extract is then standardized with the same extraction solvent (ES) used in the extraction step. This results in a liquid propolis extract according to the present invention, containing known standardized concentrations of the main active substances 1-4. The standardized propolis extract thus prepared is used as an active pharmaceutical ingredient (API), active cosmetic ingredient (ACI), or food ingredient for producing functional foods and food supplements.

[0249] The composition of the present invention based on the aforementioned liquid propolis extract containing the main 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-phenethyl 3,4-dihydroxy-trans-cinnamate (4; 5-400 μg / g) is an effective agent for the treatment of inflammatory diseases, bacterial infections, fungal infections, viral diseases, autoimmune diseases, functional gastrointestinal disorders, mucosal regeneration, burn treatment and wound healing, as well as the treatment of cancer diseases. [Industrial Applicability]

[0250] The wide range of practical uses of liquid propolis extracts and pharmaceutical compositions based thereon demonstrate the industrial applicability of the present invention.

Claims

1. (A) 0.1-10.0% w / w of a propolis extract; and (B) 90.0-99.9% w / w of an extraction solvent A liquid propolis extract as a pharmaceutical, cosmetic or agricultural chemical ingredient or a food ingredient, comprising: The extraction solvent is (B.1) one or more liquid polyethylene glycols <PEG> 200-600, from 96.5 to 99.9% w / w; and (B.2) 0.1 to 3.5% w / w of lecithin or hydrolyzed lecithin The liquid propolis extract comprises: (I) A quantitative weight ratio of crude propolis as drug and final extract <DER> of 1:2 to 1:20 w / w; and (II) p-Coumaric acid <1>, trans-ferulic acid <2>, caffeic acid <3> and 2-phenylethyl 3,4-dihydroxycinnamate <4>: 【Chemistry 1】 Quantitative content of propolis active substances selected from the group consisting of: The quantitative composition of at least two of the four main active substances is: (i) 100 to 1,300 μg / mL of p-coumaric acid <1>; (ii) 75 to 800 μg / mL of trans-ferulic acid <2>; (iii) 25 to 300 μg / mL of caffeic acid <3>; and (iv) 40 to 400 μg / mL of 2-phenylethyl 3,4-dihydroxy-trans-cinnamate <4; CAPE> Liquid propolis extract.

2. 2. The liquid propolis extract as a pharmaceutical, cosmetic or agrochemical ingredient or food ingredient according to claim 1, wherein 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 these substances.

3. 3. The liquid propolis extract as a pharmaceutical, cosmetic or agrochemical ingredient, or food ingredient, according to claim 2, wherein the liquid polyethylene glycol <<PEG>> is selected from the group consisting of polyethylene glycol 200, polyethylene glycol 400, or a mixture of these substances.

4. 4. The liquid propolis extract as pharmaceutical, cosmetic or agrochemical ingredient or food ingredient according to claims 1 to 3, wherein the lecithin or hydrolyzed lecithin is characterized by a hydrophilic-lipophilic balance <HLB> factor between 2 and 12 and is selected from the group consisting of soy lecithin <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, deoiled products of said lecithins, hydrogenated lecithins from said sources, hydrolyzed lecithins from said sources, enzymatically modified derivatives of said lecithins or mixtures of these substances.

5. 5. The liquid propolis extract as a pharmaceutical, cosmetic or agrochemical ingredient or food ingredient according to claim 4, wherein the lecithin is selected from the group consisting of natural lecithin from soybean (Glycine max L), sunflower (Helianthus annuus L), rapeseed (Brassica napus L) or canola (Brassica rapa L), deoiled, hydrogenated, hydrolyzed or enzymatically modified lecithin, or a mixture of these substances.

6. The extraction solvent is (i) 97-99% w / w of polyethylene glycol <PEG> 200, polyethylene glycol 300, polyethylene glycol 400 or mixtures thereof; and (ii) 1 to 3% w / w of natural, deoleinized or hydrolyzed lecithin from soybean (Glycine max L), sunflower (Helianthus annuus L), rapeseed (Brassica napus L) or canola (Brassica rapa L); or mixtures of these substances. A liquid propolis extract as a pharmaceutical, cosmetic or agricultural chemical ingredient or a food ingredient according to claims 1 to 5.

7. (I) Quantitative weight ratio of crude propolis as drug to final extract in the ratio of 1:3 to 1:5 w / w <DER> and (II) Quantitative composition of propolis active substances selected from the group consisting of p-coumaric acid <1>, trans-ferulic acid <2>, caffeic acid <3> and 2-phenylethyl 3,4-dihydroxy-cinnamate <4> and at least two of the four main active substances have the following quantitative content: (i) 500 to 1,300 μg / mL of p-coumaric acid <1>; (ii) 300 to 800 μg / mL of trans-ferulic acid <2>; (iii) 100 to 300 μg / mL of caffeic acid <3>; and (iv) 100-400 μg / mL of 2-phenylethyl 3,4-dihydroxy-cinnamate <4; CAPE> 4. A liquid propolis extract as a pharmaceutical, cosmetic or agricultural ingredient or food ingredient according to claims 1 to 3, which corresponds to

8. A method for producing a liquid propolis extract according to any one of claims 1 to 7, comprising the steps of: (i) chilling the crude propolis at −20° C. for a minimum of 1 hour; (ii) crushing the cooled propolis by sieving through 1-8 mm pores; (iii) under the following conditions: (a) weight ratio of crude propolis and extraction solvent=1:2 to 1:20 w / w; (b) an extraction temperature of 10 to 150° C.; and (c) Extraction time from 5 minutes to 72 hours Extracting the crude propolis with an extraction solvent; (iv) filtering the mixture thus obtained through a series of filters having pores between 100 μm and 5 μm to produce an undissolved residue and a liquid propolis extract; (v) quantitatively analyzing the main active propolis substances 1-4 by high performance liquid chromatography (HPLC); (vi) standardizing the liquid propolis extract thus obtained, the exact quantitative composition of the main active substances 1-4 being determined in step (v), by diluting with fresh extraction solvent used in step (iii) to the desired content of active substances 1-4; The method includes:

9. The extraction step (iii) is carried out under the following conditions: (a) weight ratio of crude propolis and extraction solvent=1:3-1:5 w / w; (b) an extraction temperature of 15 to 70° C.; and (c) Extraction time of 1 to 24 hours 9. The method for producing a liquid propolis extract according to claim 8, wherein the method is carried out in a step S10.

10. The high performance liquid chromatography (HPLC) method was carried out under the following conditions: (i) Chromatography column: Ascentis express; C18; Dimensions: 15 cm x 3.0 mm; particle size in column: 2.7 μm; (ii) Mobile phase: A = 0.1% formic acid in water, B = methanol; Gradient: 0min, 80%A, 20%B; 3min, 70%A, 30%B; 60min, 20%A, 80%B; 90min, 20%A, 80%B; 100min, 70%A, 30%B; 105min, 80%A, 20%B; (iii) Column temperature: 30° C.; (iv) Flow rate: 0.25mL / min; (v) Analysis time: 110min; (vi) UV-VIS detector wavelengths: detection: 370 nm, integration: 290 nm; (vii) Injection volume: 10 μL; (viii) Pressure: 210-290 bar 10. The method for producing a liquid propolis extract according to claims 8 and 9, which is used for quantifying the main active substances 1 to 4 in

11. 8. Use of the standardized liquid propolis extract according to claims 1 to 7, wherein the liquid propolis extract is used as an active pharmaceutical ingredient or excipient for the manufacture of a pharmaceutical product selected from the group consisting of a drug, a medical device or a therapeutic agent.

12. Use of the standardized liquid propolis extract according to claims 1 to 7, wherein said liquid propolis extract is used as an active cosmetic ingredient or excipient for producing a cosmetic product.

13. Use of the standardized liquid propolis extract according to claims 1 to 7, wherein said liquid propolis extract is used as a food ingredient for producing functional foods, food supplements and foods for special nutritional purposes.

14. 8. Use of the standardized liquid propolis extract according to claims 1 to 7, wherein said liquid propolis extract is used as an active pharmaceutical ingredient or excipient for the manufacture of a veterinary product selected from the group consisting of a veterinary medicinal product, an animal feed, an animal feed supplement, or a therapeutic agent for veterinary use.

15. Use of the standardized liquid propolis extract according to claims 1 to 7, wherein said liquid propolis extract is used as an active pesticide ingredient or excipient for the manufacture of an agrochemical product selected from the group consisting of fungicides, bactericides, virucides, insecticides, nematicides and plant strengthening agents.

16. (I) 5-95% w / w of a liquid propolis extract according to any one of claims 1 to 7; and (II) one or more pharmaceutical excipients necessary to prepare the final dosage form selected from the group consisting of solutions, suspensions, gels, creams, ointments, sprays for oral or nasal application, up to 100% w / w of the final composition; A pharmaceutical composition comprising: The composition contains four major active propolis substances within the following ranges: (i) 10 to 1,300 μg / g of p-coumaric acid <1>; (ii) 10 to 800 μg / g trans-ferulic acid <2>; (iii) 5 to 300 μg / g of caffeic acid <3>; and (iv) 5 to 400 μg / g of 2-phenylethyl 3,4-dihydroxy-trans-cinnamate <4; CAPE> A pharmaceutical composition characterized by a quantitative content of at least two of the following:

17. 17. The pharmaceutical composition of claim 16, wherein the pharmaceutical excipient is selected from the group comprising diluents, humectants, preservatives, chelating agents, antioxidants, thickening agents, emollients, emulsifiers, tonicity agents, and pH control agents.

18. A method for producing the pharmaceutical composition of claims 16 and 17, comprising the steps of: (i) adding the standardized liquid propolis extract according to claims 1 to 7 to a diluent and homogenizing them; (ii) adding one or more other excipients and homogenizing them; Including; Steps (i) and (ii) are carried out at a temperature of 10 to 100°C, preferably at a temperature of 20 to 60°C, for 1 to 5 minutes; Next, (iii.a) in the case of preparing a solution or spray solution formulation, carrying out filtration of the final solution, including sterile filtration if necessary; (iii.b) in case of preparing a gel or suspension dosage form, adding a thickening agent and homogenizing it; (iii.c) in case of cream formulation preparation, preparation of the fatty phase by mixing the emollients and emulsifiers and homogenizing at a temperature of 50-80°C for 1-15 minutes, then adding the solution of step (ii) and heating to 50-80°C, followed by emulsification using a high shear or high pressure homogenizer at a temperature of 50-80°C, preferably 55-65°C for 1-30 minutes, followed by homogenization at a temperature of 65-20°C for 10-120 minutes; or (iii.d) in case of preparing an ointment dosage form, mixing the solution of step (ii) with a pre-melted mixture of emollients and finally emulsifiers at a temperature of 50-70° C. for 5-30 minutes, followed by homogenization at a temperature of 70-20° C. for 10-120 minutes. The method includes:

19. 18. A pharmaceutical composition according to claim 16 or 17 for the treatment of human and animal diseases and conditions from the group of inflammatory diseases, bacterial infections, fungal infections, viral diseases, autoimmune diseases, functional gastrointestinal disorders, mucosal regeneration, burn treatment and wound healing, and cancer diseases.

20. 20. A pharmaceutical composition according to claim 19 for the treatment of inflammatory diseases from the group: gingivitis, periodontitis, laryngitis, gastritis, colitis, hemorrhoidal diseases, dermatitis, ear inflammation externally, sinusitis, rhinitis, vaginitis and mastitis.

21. The following groups of bacteria: (i) Gram-positive bacteria: Staphylococcus spp: Staphylococcus aureus, MRSA <methicillin-resistant Staphylococcus aureus>, MSSA <methicillin-susceptible Staphylococcus aureus>, Staphylococcus intermedius, Staphylococcus pseudointermedius; Coagulase-negative Staphylococcus: Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus Streptococcus 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 baumanii; Pseudomonas aeruginosa; Haemophilus influenzae; Salmonella choleraesuis; 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 20. The pharmaceutical composition according to claim 19 for the treatment of a bacterial infection caused by Bacillus splanchnicus.

22. Candida spp: Candida albicans, Candida dubliniensis, Candida glabrata, Candida cruzei, Candida tropicalis, Candida parapsilosis; Aspergillus spp: Aspergillus niger, Aspergillus versicolor; Penicillium pinophilum; Paecilomyces variotii; Trichoderma virens; Chaetomium globosum; Malassezia 20. The pharmaceutical composition according to claim 19 for the treatment of fungal infections caused by fungi from the group of P. pachydermatis.

23. 20. The pharmaceutical composition according to claim 19, for the treatment of viral diseases caused by viruses from the group of herpes simplex virus (HSV); human papilloma virus (HPV); Epstein-Barr virus (EBV); cytomegalovirus (CMV); poliovirus; influenza A and B viruses; retroviruses; vaccinia virus; common cold viruses: rhinoviruses, picornaviruses, human parainfluenza virus (HPIV), human metapneumovirus (HMPV), coronaviruses, adenoviruses, human respiratory syncytial virus (HRSV), enteroviruses.

24. 20. The pharmaceutical composition according to claim 19 for the treatment of autoimmune diseases from the group: psoriasis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, celiac disease and multiple sclerosis.

25. 20. The pharmaceutical composition according to claim 19 for the treatment of cancer diseases from the group: cancer of the skin and mucous membranes, gastrointestinal tumors, colorectal cancer.

26. 20. A pharmaceutical composition according to claim 19 for the treatment of mastitis in animals.

27. 20. The pharmaceutical composition according to claim 19, for the treatment of functional gastrointestinal disorders selected from the group consisting of disorders of the esophagus, stomach, duodenum, small intestine and colon, central gastrointestinal pain, disorders of the gallbladder and sphincter of Oddi, anorectal disorders, child and adolescent specific gastrointestinal disorders.

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