Propolis liquid extract and preparation and application thereof
阅读说明：本技术 蜂胶液体提取物及其制剂和用途 (Propolis liquid extract and preparation and application thereof ) 是由 S·拉迪奇 B·拉迪奇 J·苏兰 于 2020-02-12 设计创作，主要内容包括：本发明涉及一种新的标准化蜂胶液体提取物和由所述提取物制备成的药物制剂,及其制备方法和用途。该液体提取物是用PEG200-600(96.5-99.9％w/w)和卵磷脂(0.1-3.5％w/w)的混合物作为提取溶剂提取粗蜂胶制备而成。该提取物的特征在于含有标准化含量的对香豆酸(1),反式阿魏酸(2),咖啡酸(3),以及咖啡酸苯乙酯CAPE(4)。该组合物可作为活性成分用于制备药物,化妆品,兽药,农用化学品或功能性食品。本发明的药物制剂由5-95％w/w的所述蜂胶提取物和制备各种剂型所需的使总量达100％的赋形剂组成。该产品可用于治疗人或动物的疾病,包括炎性疾病,细菌和真菌感染,病毒性疾病,自身免疫性疾病和癌症,还可用于治疗烧伤和促进伤口愈合。(The invention relates to a novel standardized propolis liquid extract, a medicinal preparation prepared from the extract, and a preparation method and application thereof. The liquid extract is prepared by extracting crude propolis with mixture of PEG200-600 (96.5-99.9% w/w) and lecithin (0.1-3.5% w/w) as extraction solvent. The extract is characterized by having standardized contents of p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3), and caffeic acid phenethyl ester CAPE (4). The composition can be used as active ingredient for preparing medicines, cosmetics, veterinary drugs, agrochemicals or functional foods. The pharmaceutical preparation of the present invention consists of 5-95% w/w of said propolis extract and excipients required for the preparation of various dosage forms to bring the total amount to 100%. The product can be used for treating human or animal diseases, including inflammatory diseases, bacterial and fungal infections, viral diseases, autoimmune diseases and cancer, and can be used for treating burn and promoting wound healing.)
1. A liquid propolis extract as a pharmaceutical, cosmetic or agrochemical or food ingredient consisting of:
(A) drying the propolis extract; 0.1-10.0% w/w; and
(B) extracting the solvent; 90.0-99.9% w/w;
the extraction solvent comprises:
(B.l) one or more liquid polyethylene glycol (PEG) 200-600;
96.5-99.9% w/w; and
(b.2) lecithin or hydrolyzed lecithin; 0.1-3.5% w/w;
wherein the liquid propolis extract is standardized as follows:
(i) the mass percentage of the crude propolis and the final extract used as the medicine is as follows: 1:2-1:20 w/w; and
(ii) the mass content of propolis active substances, wherein the propolis active substances comprise p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 3, 4-dihydroxy-trans-cinnamic acid phenethyl ester (4):
wherein the mass composition of at least two of the four active substances is as follows:
(i) p-coumaric acid (1); 100-;
(ii) trans-ferulic acid (2); 75-800 ug/mL;
(iii) caffeic acid (3); 25-300 ug/mL; and
(iv)3, 4-dihydroxy-trans-cinnamic acid phenethyl ester (4, CAPE); 40-400 ug/mL.
2. The liquid propolis extract as a pharmaceutical, cosmetic or agrochemical or food ingredient according to claim 1, wherein the liquid polyethylene glycol (PEG) is selected from: polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 600, or mixtures of these.
3. The liquid propolis extract as a pharmaceutical, cosmetic or agrochemical or food ingredient according to claim 2, wherein the liquid polyethylene glycol (PEG) is selected from: polyethylene glycol 200, polyethylene glycol 400, or mixtures of these.
4. Liquid propolis extract as pharmaceutical, cosmetic or agrochemical or food ingredient according to any of claims 1 to 3, wherein lecithin or hydrolysed lecithin is characterized by a hydrophilic-lipophilic balance factor (HLB) comprised between 2 and 12 and is selected from the following ingredients: soybean lecithin (Glycine max L); sunflower lecithin (Helianthus annus L); rapeseed lecithin (Brassica napus L); rape lecithin (Brassica rapa L); egg chicken lecithin (Gallus galllus domesticl); a de-oiled product of said lecithin; hydrogenated lecithin of said source; hydrolyzed lecithin of the above lecithin; enzymatically modified derivatives of the above lecithins; or mixtures of these substances.
5. A liquid propolis extract as claimed in claim 4 for use as a pharmaceutical, cosmetic or agrochemical or food ingredient wherein the lecithin is selected from the group consisting of: natural lecithin, deoiled, hydrogenated, hydrolyzed or enzymatically modified lecithin, lecithin from soybean, sunflower, rapeseed or canola; or mixtures of these substances.
6. The liquid propolis extract as a pharmaceutical, cosmetic or agrochemical or food ingredient according to any one of claims 1 to 5, wherein the extraction solvent comprises:
(i) polyethylene glycol (PEG)200, polyethylene glycol 300, polyethylene glycol 400, or mixtures thereof; 97-99% w/w; and
(ii) natural lecithin, de-oiled lecithin or hydrolyzed lecithin, from soybean, sunflower, rapeseed or canola; or mixtures of these; 1-3% w/w.
7. Liquid propolis extract as a pharmaceutical, cosmetic or agrochemical or food ingredient according to any of claims 1 to 3, wherein it is standardized as follows:
(i) the mass percentage of the crude propolis used as the medicine to the final extract is 1:3-1:5 w/w; and
(ii) the mass content of the propolis active substances comprises p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 3, 4-dihydroxy trans-cinnamic acid-2 phenethyl ester (4):
wherein the mass composition of at least two of the four key active substances is as follows:
(v) p-coumaric acid (1); 100-;
(vi) trans-ferulic acid (2); 75-800 ug/mL;
(vii) caffeic acid (3); 25-300 ug/mL; and
(viii)3, 4-dihydroxy-trans-cinnamic acid phenethyl ester (4, CAPE); 40-400 ug/mL.
8. A method for preparing a liquid propolis extract as claimed in any one of claims 1 to 7, comprising the steps of:
(i) cooling the crude propolis at-20 deg.C for at least 1 hr;
(ii) grinding frozen propolis, and sieving with 1-8mm hole;
(iii) extracting crude propolis with an extraction solvent under the following conditions:
(a) the weight ratio of the crude propolis to the extraction solvent is 1:2-1:20 w/w;
(b) the extraction temperature is 10-150 ℃; and
(c) the extraction time is 5 minutes to 72 hours;
(iv) filtering the mixture obtained under the above conditions through a filter having pores of 100pm-5pm to produce an undissolved residue and a liquid propolis extract;
(v) performing quantitative analysis on key active propolis substances 1-4 by high performance liquid chromatography; and
(vi) (iv) standardizing the resulting liquid propolis extract with the exact quantitative composition of the key active substances 1-4 determined in step (v), and diluting with the fresh extraction solvent used in step (iii) to bring the active substances 1-4 to the desired content.
9. A method for preparing a liquid propolis extract according to any one of claims 1 to 7, wherein the extraction step (iii) is carried out under the following conditions:
(a) the weight ratio of the crude propolis to the extraction solvent is 1:3-1:5 w/w; (b) the extraction temperature is 15-70 ℃; and
(c) the extraction time is 1-24 hours.
10. The method for preparing liquid propolis extract according to claims 8 and 9, wherein the high performance liquid chromatography method is used for quantitatively determining key active substances 1-4, and the chromatographic conditions are as follows:
(i) a chromatographic column: ascentis Express; c18; size: 15cm × 3.0 mm; the diameter of the particles in the column was 2.7 μm;
(ii) the mobile phase A is 0.1 percent aqueous solution of formic acid, and the mobile phase B is methanol; elution gradient 0min, 80% a, 20% B; 3min, 70% a, 30% B; 60 minutes, 20% a, 80% B; 90 minutes, 20% a, 80% B; 100min, 70% A, 30% B; 105min, 80% A, 20% B;
(iii) the column temperature is 30 ℃;
(iv) the flow rate is 0.25 ml/min;
(v) analysis time: 110 minutes;
(vi) wavelength on UV-VIS detector: 370nm for detection and 290nm for integration;
(vii) the sample injection volume is 10 pl;
(viii) pressure: 210 + 290 bars.
11. Use of a standardized liquid propolis extract as claimed in any one of claims 1 to 7 as an active ingredient or excipient and for the manufacture of a medicament, a pharmaceutical device or a pharmaceutical product of a therapeutic agent.
12. Use of a standardized liquid propolis extract according to any one of claims 1 to 7 as an active ingredient or excipient and for the production of cosmetics.
13. Use of standardized liquid propolis extract according to any of claims 1 to 7 as active ingredient in food products and for the production of functional food products, food supplements and food products for special nutritional purposes.
14. Use of a standardized liquid propolis extract according to any one of claims 1 to 7 as an active pharmaceutical ingredient or excipient for the manufacture of a veterinary product; animal feed; an animal feed additive; or veterinary drugs.
15. Use of a standardized liquid propolis extract according to any one of claims 1 to 7 as agrochemical active ingredient or excipient for the production of agrochemical products, wherein said agrochemical products are selected from: fungicides, bactericides, viricides, insecticides, nematicides and plant enhancers.
16. A pharmaceutical composition comprising:
(i) the liquid propolis extract of any one of claims 1 to 7; 5-95% w/w; and the number of the first and second groups,
(ii) one or more pharmaceutically acceptable excipients required to prepare a dosage form selected from: solutions, suspensions, gels, creams, ointments, oral or nasal sprays; up to 100% w/w of the final composition; wherein
Said composition is characterized in that the quantitative content of two of at least four key active propolis substances is within the following values:
(i) p-coumaric acid (1); 100-;
(ii) trans-ferulic acid (2); 75-800 ug/mL;
(iii) caffeic acid (3); 25-300 ug/mL; and
(iv)3, 4-dihydroxy-trans-cinnamic acid phenethyl ester (4, CAPE); 40-400 ug/mL.
17. The pharmaceutical composition of claim 16, wherein the pharmaceutical excipient is selected from the group consisting of: diluents, wetting agents, preservatives, chelating agents, antioxidants, thickeners, emollients, emulsifiers, surface tension agents and pH adjusting agents.
18. A process for the preparation of a pharmaceutical composition according to claims 16 and 17, wherein it comprises the following steps:
(i) adding a standardised liquid propolis extract according to any of claims 1-7 to a diluent and homogenising it;
(ii) adding one or more other excipients; and homogenizing them;
wherein steps (i) and (ii) are carried out at a temperature of 10-100 ℃, preferably at a temperature of 20-60 ℃, within 1-5 minutes; further, the conditions for preparing the dosage form are as follows:
(iii.a) a solution or a solution for spraying; filtering the final solution, if necessary, sterile filtering;
(iii.b) a gel or suspension; adding a thickening agent, and homogenizing;
(iii.b) a cream; the preparation method of the oil phase comprises the following steps: (iii) mixing emollient and emulsifier and homogenizing at 50-80 deg.C, adding the solution of step (ii) within 1-15 minutes, heating to 50-80 deg.C, emulsifying with high shear or high pressure homogenizer at 50-80 deg.C, preferably 55-65 deg.C, for 1-30 minutes, and homogenizing at 65-20 deg.C for 10-120 minutes; or
(iii.b) an ointment; (iii) mixing the solution of step (ii) with a previously homogenized mixture of emollient and emulsifier over a period of 5-30 minutes at 50-70 ℃, followed by homogenization at 70-20 ℃ over a period of 10-120 minutes.
19. Use of a pharmaceutical composition according to claims 16 and 17 for the treatment of human and animal diseases, in particular selected from: inflammatory diseases; bacterial infection; fungal infections; viral diseases; autoimmune diseases; functional gastrointestinal disorders; for regeneration of mucosa, burn treatment, wound healing; and cancer-related diseases.
20. The use of a pharmaceutical composition according to claim 19, wherein the pharmaceutical composition is for the treatment of an inflammatory disease selected from the group consisting of: gingivitis, periodontitis, laryngitis, gastritis, colitis, hemorrhoids, dermatitis, otitis externa, sinusitis, rhinitis, vaginitis and mastitis.
21. The use of a pharmaceutical composition according to claim 19, wherein the pharmaceutical composition is for the treatment of bacterial infections caused by bacteria in the group of:
(i) gram-positive bacteria: staphylococcus species: staphylococcus aureus, methicillin-resistant staphylococcus aureus (MRSA), methicillin-sensitive staphylococcus aureus (MSSA), staphylococcus intermedius, staphylococcus pseudointermedius, coagulase-negative staphylococcus: staphylococcus epidermidis, staphylococcus saprophyticus, staphylococcus suis; streptococcus genus: streptococcus uberis, Streptococcus bovis, Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus canis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus oralis, Streptococcus thermophilus; the genus Streptococcus; corynebacterium: corynebacterium bovis; steinernema pyogenes; nocardia; b, bacillus subtilis; bacillus cereus; enterococcus: enterococcus faecium, enterococcus faecalis; vancomycin-resistant enterococci (enterococcus casseliflavus); and the number of the first and second groups,
(ii) gram-negative bacteria: e.coli; acinetobacter baumannii; pseudomonas aeruginosa; haemophilus influenzae; salmonella choleraesuis; yersinia enterocolitica; enterobacter: enterobacter cloacae; klebsiella bacteria: klebsiella pneumoniae, klebsiella oxytoca; shigella flexneri; burkholderia cepacia; proteus mirabilis; proteus vulgaris; actinobacillus actinomycetemcomitans; actinomycetes in Israel; bacteroides fragilis; helicobacter pylori; e.coli; campylobacter jejuni; porphyromonas gulila; porphyromonas salivarius; porphyromonas dentata; prevotella intermedia; treponema; paenibacillus sp.
22. The use of a pharmaceutical composition according to claim 19, wherein the pharmaceutical composition is for the treatment of a fungal infection caused by a fungus selected from the group consisting of: candida species: candida albicans, Candida ducreyi, Candida glabrata, Candida kruensis, Candida tropicalis, Candida paratyphi; the genus Aspergillus: aspergillus niger, aspergillus versicolor; penicillium pinophilum; a variant paecilomyces; trichoderma viride; chaetomium globosum; marylaria pachydermatis.
23. The use of the pharmaceutical composition of claim 19, wherein the pharmaceutical composition is for treating viral diseases caused by: herpes Simplex Virus (HSV); human papilloma virus (HPB); balr Virus (EBV); cytomegalovirus (CMV); poliovirus; influenza a and b viruses; a retrovirus; vaccinia virus; common cold virus: rhinovirus, picornavirus, human parainfluenza virus (HPIV), Human Metapneumovirus (HMPV), coronavirus, adenovirus, Human Respiratory Syncytial Virus (HRSV), enterovirus.
24. The use of a pharmaceutical composition according to claim 19, wherein the pharmaceutical composition is for the treatment of the following autoimmune diseases: psoriasis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, celiac disease, and multiple sclerosis.
25. The use of the pharmaceutical composition of claim 19, wherein the pharmaceutical composition is for treating the following cancer-related diseases: skin and mucosal cancer, gastrointestinal tumors, colorectal cancer.
26. Use of a pharmaceutical composition according to claim 19, characterized in that it is used for the treatment of mastitis in animals.
27. Use of a pharmaceutical composition according to claim 19, characterized in that it is used for the treatment of a functional gastrointestinal disorder selected from the group consisting of: disorders of the esophagus, stomach, duodenum, small intestine and colon, centrally mediated gastrointestinal pain, disorders of the gallbladder and sphincter ODDI, disorders of the anorectum, gastrointestinal disorders specific for children and adolescents.
Technical Field
The present invention relates to a new liquid standardized propolis extract, a process for its preparation, as well as new pharmaceutical compositions based on said extract and uses thereof.
Background
Research has shown that propolis is a mixture of beeswax and a large number of natural organic compounds, and different extraction methods produce different active substances and thus different extracts. By using different selective chemical solvents (ES) for extraction, a certain class of organic compounds can be selectively extracted, and by combining with a suitable analytical method, a consistent and standardized propolis extract composition can be theoretically obtained.
The technical problems solved by the invention include:
(i) a non-alcoholic liquid propolis extract characterized by high content and standardized content of phenolic propolis acids, p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 2-phenethyl ester caffeic acid, 3, 4-dihydroxy-trans-cinnamic acid phenethyl ester (4; CAPE), wherein several pharmacologically active substances known to be beneficial to human and animal organisms are;
(ii) a process for the preparation thereof;
(iii) for the production of medicaments, cosmetics, veterinary drugs, agrochemicals or foodstuffs with known standardised contents of propolis active substances 1-4; and in particular to the fact that,
(iv) a pharmaceutical composition based on the above propolis extract is effective in treating inflammatory and infectious diseases, as well as diseases and conditions in which the antioxidant, anti-inflammatory and immunomodulatory activity of specific propolis active substances and their etiology of pathogenesis are important, as described in some references in the prior art.
The alcohol-free extract and other stable propolis compounds with negligible beeswax content, having a high and standardized content of highly bioactive propolis components, is the basis for the development and production of high-value drugs, veterinary drugs, agrochemicals or functional foods or food supplements. In contrast, in order to achieve the above objects, the standard propolis extract prepared using ethanol, glycerin or 1, 2-propanediol has problems such as low content of phenolic acid in which the content of propolis active is unknown or valuable and insufficient content of flavonoid propolis components.
The basis of the invention is as follows:
(i) using a specific Extraction Solvent (ES) preparation to extract crude propolis with chemoselectivity;
(ii) a method for quantitatively measuring propolis active ingredients of key active substances 1-4;
(iii) standardizing the prepared liquid propolis extract by dilution with a certain amount of the same extraction solvent until the level of the active substances 1-4 according to the invention is reached; wherein the extraction solvent consists of two or more food or pharmaceutically acceptable substances and further comprises a liquid standardized propolis extract in the final product. The same extraction solvent has the function of a carrier or diluent; and the number of the first and second groups,
(iv) pharmaceutical compositions based on said standardized propolis extract of the invention prove to be effective agents for the treatment of inflammatory and infectious diseases. Due to its broad spectrum of pharmacological activity, it is useful in the treatment of various diseases and disorders, such as: inflammatory diseases, bacterial and fungal infections, viral diseases, autoimmune diseases, and also for mucosal regeneration, treatment of burns and wounds, and for cancer-related diseases.
Prior Art
Propolis is a natural product harvested by bees and, for bees, is used to close small openings on the hive that are not needed. Propolis contains beeswax as a main ingredient and various organic compounds in a large amount. Many of the above compounds exhibit significant beneficial pharmacological effects; see, for example, reference 1
1)S.Castaldo,F.Capasso:Propolis,and old remedy used in modern medicine,Fitoterapia 73(2002)S1-6.
In addition to the above active substances 1-4, propolis also contains a series of other natural compounds, for example, as for the acidic component, it also contains trans-cinnamic acid (5), and flavonoid series rich in chrysin (6), coniferol (7), galangin (8), apigenin (9), and kaempferol (10):
the traditional method is to extract crude propolis with ethanol or a mixture of ethanol and water, thereby obtaining a propolis tincture. Such liquid propolis extracts are characterized by the following disadvantages:
(i) the use of a relatively aggressive solvent (ethanol);
(ii) alcohol-containing products are not suitable for children, pregnant and lactating women, and certain patients; and the number of the first and second groups,
(iii) relatively high levels of beeswax which cause depolymerization of beeswax by mixing with water during manufacture of pharmaceuticals and other products, wherein such an extract is used as the active ingredient.
In addition to ethanol, glycerol, water, mixtures of glycerol and water, and other organic solvents are used as extraction solvents.
In this way, Tsukada and co-workers disclose the extraction of propolis using glycerol as the extraction solvent in a ratio of 1:2w/w, at 90-160 ℃ followed by filtration. Such glycerol extracts are water-soluble and suitable for the production of pharmaceutical products as Active Pharmaceutical Ingredients (API); see reference 2
2)JPH05957A；T.Tsukada,W W.Kameda,M.Ide:Production of water-soluble propolis pharmaceutical preparation；Ogawa Koryo KK,Santapuron KK(JP).
Related art also describes propolis aqueous extracts. Extracting with water as solvent at 30-50 deg.C for 6-8 min, and filtering to obtain liquid propolis extract. The latter can be further processed into microcapsule by spray drying to obtain solid extract; see reference 3
3)CN103783349A；Z.Wang,S.Shao,H.Ma,S.Wang,L.Wang,C.Zhang,X.Ma:Process for preparing honeycomb polyphenol extractive microcapsule by adopting spray；University Jiangsu(CN).
There are also prior art extractions using surfactants as extraction solvents, including lecithin. For example, Paradkar and colleagues describe a method of extracting propolis at 40-90 ℃ for 2-24 hours using an aqueous polysorbate solution to obtain a liquid propolis extract; see reference 4:
4)WO2011092511A1；A.Paradkar,R.Dhumal,A.Kelly,S.Gilda:Propolis and process for the treatment thereof and end products formed therefrom；Natures Lab Ltd(GB).
sosnowski discloses a method for extracting propolis with various organic solvents including 1, 2-propanediol, polyethylene glycol (PEG) and mixtures of these solvents with water; see reference 5
5)US4382886；Z.M.Sosnowski:Method for extracting propolis and water soluble dry propolis powder.
Chun and colleagues disclose a pharmaceutical composition, wherein the pharmaceutical composition is based on a liquid propolis extract using 1, 3-butylene glycol as an extraction solvent, which is converted into a pharmaceutical dosage form comprising nanoparticles having the propolis extract by using hydrogenated lecithin and other emulsifiers; see reference 6:
6)KR20130134800A；Y.J.Chun,S.B.Shim,X.Ke:Composition of nano-vesicle containing propolis and manufacturing method of it；University Chungwoon IACF(KR).
although this document does not use lecithin to facilitate the extraction of the propolis active with 1, 3-butanediol, it clearly suggests the possibility of using it as a surfactant, which may eventually improve the extraction and emulsification of certain fat active propolis ingredients in more polar solvents.
Regarding the analysis of propolis, there are a number of analytical methods in the prior art for quantitatively determining propolis active substances in a complex propolis extract containing a large amount of components. For example, an analysis method similar to that employed in the present invention is given in the study of the chinese authors Cui-Ping and their co-workers. They describe a method for quantitative determination of 12 different flavonoids and 8 phenolic propolis acids by High Performance Liquid Chromatography (HPLC). The method is particularly capable of determining p-coumaric acid (1), trans-ferulic acid (2) and caffeic acid (3), which are mentioned as qualitative propolis markers; see reference 7:
7)Z.Cui-ping,H.Shuai,W.Wen-ting,P.Shun,S.Xiao-ge,L.Ya-jing,H.Fu-liang:Development of high-performance liquid chromatographic for quality and authenticity control of Chinese propolis,J.Food Sci.79(2014)C1315-C1322.
propolis and propolis extracts exhibit a series of very valuable and beneficial pharmacological effects on human, animal and plant health due to the presence of a certain amount of active substances 1-10. There are a large number of scientific and patent documents in the prior art, which describe their broad benefits, the most important being as follows: anti-inflammatory; an antioxidant; immune regulation; a liver-protecting agent; antimicrobial agents, including antibacterial, antiviral, antifungal, and antiprotozoal agents; and against cancer; see, e.g., references 8-12:
8)E.L.Ghisalberti:Propolis:A Review,Bee World 60(1979)59-84.
9)A.Banskota,Y.Tezuka,S.Kadota:Recent Progress in Pharmacological Research of Propolis,Phytother.Res.15(2001)561-571.
10)G.A.Burdock:Review of the Biological Properties and Toxicity of Bee Propolis(Propolis),Food Chem.Toxicol.36(1998)347-363.
11)J.M.Sforcin:Propolis and the immune system:a review,J.Ethnopharmacol.113(2007)1-14.
12)J.W.Dobrowolski,S.B.Vohora,K.Sharma,S.A.Shah,S.A.H.Naqvi,P.C.Dandiya:Antibacterial,antifungal,antiamoebic,antiinflammatory and antipyretic studies on propolis bee products,J.Ethnopharmacol.35(1991)77-82.
575/5000
in addition to propolis extracts, the beneficial pharmacological effects of certain isolated (pure) propolis active substances are described in the prior art, 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)3, 4-dihydroxy-trans-cinnamic acid phenethyl ester (4; CAPE); see references 19 and 20; it exhibits significant immunomodulatory, anti-inflammatory and antimicrobial activity:
13)T.F.Bachiega,C.L.Orsatti,A.C.Pagliarone,J.M.Sforcin:The Effects of Propolis and its Isolated Compounds on Cytokine Production by Murine Macrophages,Phyto
14)K.Pei,J.Ou,J.Huang,S.Ou:p-Coumaric acid and its conjugates:dietary sources,pharmacokinetic properties and biological activities,J.Sci.Food Agric.96(2016)2956-2962.
15)N.Kumar,V.Pruthi:Potential applications of ferulic acid from natural sources,Biotechnol.Rep.4(2014)86-93.
16)C.Shi,X.Zhang,Y.Sun,M.Yang,K.Song,Z.Zheng,Y.Chen,X.Liu,Z.Jia,R.Dong,L.Cui,X.Xia:Antimicrobial activity of ferulic acid against Cronobacter sakazakii and possible mechanism of action,Foodborne Pathog.Dis.13(2016)196-204.
17)H.G.Choi,P.T.Tran,J.H.Lee,B.S.Min,J.A.Kim:Anti-inflammatory activity of caffeic acid derivatives isolated from the roots of Salvia miltiorrhiza Bunge,Arch.Pharm.Res.(2018)64-70.
18)V.N.Lima,C.D.Oliveira-Tintino,E.S.Santos,L.P.Morais,S.R.Tintino,T.S.Freitas,Y.S.Geraldo,R.L.Pereira,R.P.Cruz,I.R.Menezes,H.D.Coutinho:Antimicrobial and enhancement of the antibiotic activity by phenolic compounds:Gallic acid,caffeic acid and pyrogallol,Microb.Pathog.99(2016)56-61.
19)K.Frenkel,H.Wei,R.Bhimani,J.Ye,J.A.Zadunaisky,M.-T.Huang,T.Ferraro,A.H.Conney,D.Grunberger:Inhibition of Tumor Promoter-mediatedProcesses in mouse Skin and Bovine Lens by Caffeic Acid Phenethyl Ester,Cancer Res.53(1993)1255-1261.
20)A.Russo,R.Longo,A.Vanella:Antioxidant activity of propolis:role of caffeic acid phenethyl ester and galangin,Fitother.73(2002)S21-S29.
furthermore, the propolis active substance can be used as fungicide, bactericide, viricide, insecticide, nematicide, and can be used for protecting plants. Propolis, due to its strong antioxidant activity, strengthens plants and enhances their resistance to abiotic stress and helps them to resist infection; see references 21-25:
21)C.A.Guginski-Piva,I.dos Santos,A.W.Júnior,D.W.Heck,M.F.Flores,K.Pazolini:Propolis for the control of powdery mildew and the induction ofphytoalexins in cucumber,IDESIA(Chile)33(2015)39-47；
22)Z.Ararso,G.Legesse:Insecticidal action of honeybees propolis extractagainst larvae of lesser wax moth,Agric.Biol.J.North Am.(2015)doi:10.5251/abjna.2016.7.6.302.306.
23)L.Kumar,M.K.Mahatma,K.A.Kalariya,S.K.Bishi,A.Mann:PlantPhenolics:Important Bio-Weapon against Pathogens and Insect Herbivores,Popular Kheti 2(2014)149-152；
24)E.M.Noweer,M.G.Dawood:Efficiency of propolis extract on faba beanplants and its role against nematode infection,Commun.Agric.Appl.Biol.Sci.74(2009)593-603；
25)K.Kulbat:The role of phenolic compounds in plant resistance,Biotechnol.Food Sci.80(2016)97-108.
as known from recent studies, the use of a specific Extraction Solvent (ES) based on liquid polyethylene glycol having a low (0.1-3.5% m/m) lecithin content as a chemoselectivity enhancer for propolis extraction has not been described in the prior art. The specific Extraction Solvent (ES) adopted by the invention can enhance the chemical selectivity of the crude propolis extract, thereby obtaining the corresponding liquid extract, wherein the content of the corresponding active substances 1-4 is obviously improved.
Furthermore, the use of the specific, standardized liquid propolis extract mentioned in the present invention as Active Pharmaceutical Ingredient (API) in pharmaceutical compositions produces unexpected improvements in a range of different indications for its use; as described in the detailed description of the invention.
Disclosure of Invention
The crude propolis extracted by the specific Extraction Solvent (ES) has unexpected efficacy and chemical selectivity. The latter consists of a mixture of liquid polyethylene glycol (PEG) (e.g. PEG400) and lecithin or hydrolysed lecithin in a ratio of 96.5-99.9: 0.1-3.5% w/w.
The Extraction Solvent (ES) effectively and chemoselectively extracts active substances in propolis, such as p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 3, 4-dihydroxy trans-cinnamic acid 2-phenylethyl ester (4; CAPE). The active substances 1-4 in the prepared primary extract can be quantitatively determined using suitable High Performance Liquid Chromatography (HPLC) analytical methods. The latter is further standardized by using the same dilution of ES in the extraction step until the desired content of active substances 1-4 according to the invention is reached. In this way, a liquid propolis extract according to the invention is obtained containing known and standardized concentrations of the key active substances 1-4. The extract can be further used as Active Pharmaceutical Ingredient (API), Active Cosmetic Ingredient (ACI), or as a food ingredient for the manufacture of functional foods and food supplements.
The composition of the present invention is based on said liquid propolis extract, which contains key active substances p-coumaric acid (1; 10-1,300. mu.g/mL), trans-ferulic acid (2; 10-800. mu.g/mL), caffeic acid (3; 5-300. mu.g/mL) and 2-phenylethyl 3, 4-dihydroxy trans-cinnamate (4; 5-400. mu.g/mL), is an effective agent for the treatment of inflammatory diseases, bacterial infections, fungal infections, viral diseases, autoimmune diseases, functional gastrointestinal diseases, mucosal regeneration, treatment of burns and wounds and treatment of cancer diseases.
Drawings
FIG. 1 shows typical HPLC chromatograms of propolis key components 1-4 and concomitant components 5-10.
FIGS. 2.1-2.4 show the quantitative composition results of propolis active substances 1-4 in liquid propolis extracts extracted with ethanol (96%) and mixtures of ethanol with various kinds and concentrations of lecithin.
FIGS. 2.5-2.10 show the quantitative composition results of concomitant substances 5-10 in liquid propolis extracts obtained by extraction with ethanol (96%) and mixtures of ethanol with various kinds and concentrations of lecithin.
FIGS. 3.1 to 3.4 show the quantitative composition results of propolis active substances 1 to 4 in liquid propolis extracts obtained by extraction with polyethylene glycol 400 and mixtures of polyethylene glycol 400 with various kinds and concentrations of lecithin.
FIGS. 3.5 to 3.10 show the quantitative composition results of concomitant substances 5 to 10 in liquid propolis extracts obtained by extraction with polyethylene glycol 400 and mixtures of polyethylene glycol 400 with various kinds and concentrations of lecithin.
Figure 4 shows a block diagram of a method for the production of a standardized liquid propolis extract according to the invention.
Figure 5 shows a typical HPLC chromatogram for quantitative analysis of a pharmaceutical formulation of the invention, example 11.
Abbreviations
ES-extraction solvent
SL-soy lecithin (Glycine max. L.)
RL-rapeseed lecithin (Brassica napus L.)
HRL-hydrolyzed Soybean 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-Rosevir park memorial park
TTC-triphenyltetrazolium chloride, redox indicator
PBS-phosphate buffer
CFU-colony forming units; number of viable microorganisms capable of forming colonies
XTT-XTT sodium salt; 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazole-5-carboxyanilide inner salt, redox indicator
MRSA-methicillin-resistant Staphylococcus aureus
MSSA-methicillin-resistant sensitive Staphylococcus aureus
CLSI-American society for clinical laboratory standardization
EUCAST-European Committee for antibiotic susceptibility testing
API-active pharmaceutical ingredient/substance
DER-weight ratio of drug to extract; measurement of extract Strength expressed as the weight ratio of starting drug to Final extract
SCC-somatic cell count
Mam. -intramammary (application)
ATCC-american type culture collection; non-profit organization for collection, storage and distribution of standard reference microorganisms
CNS-coagulase negative staphylococci
Detailed Description
The present invention relates to a novel propolis liquid extract, a preparation method and use thereof, wherein the content of key active substances of the propolis liquid extract is standardized, said key active substances being selected from the group consisting of: p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 3, 4-dihydroxy-trans-cinnamic acid phenethyl ester (4).
According to the present invention, the liquid propolis extract as a pharmaceutical, cosmetic or agrochemical or food ingredient is composed of:
(A) drying the propolis extract; 0.1-10.0% w/w; and
(B) extracting the solvent; 90.0-99.9% w/w;
wherein the extraction solvent consists of:
(B.1) one or more liquid polyethylene glycols (PEG) 200-600; 96.5-99.9% w/w; and
(b.2) lecithin or hydrolyzed lecithin; 0.1-3.5% w/w;
the propolis liquid extract is standardized by the following steps:
(I) the quantitative weight ratio of crude propolis to final extract (DER) as a drug was:
1:2-1:20 w/w; and
(II) a quantitative content of propolis active substances selected from the group consisting of p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3), and 3, 4-dihydroxy-trans-cinnamic acid phenethyl ester (4), wherein two of at least four of the key active substances are quantitatively composed as follows:
(i) p-coumaric acid (1); 100-;
(ii) trans-ferulic acid (2); 75-800 mug/mL;
(iv) caffeic acid (3); 25-300 mug/mL; and
(v)3, 4-dihydroxy-trans-cinnamic acid phenethyl ester (4; CAPE); 40-400. mu.g/mL.
In a preferred embodiment of the present invention, the liquid polyethylene glycol (PEG) as a component of the Extraction Solvent (ES) is selected from: polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 600 or mixtures of these.
Specifically, the liquid polyethylene glycol (PEG) as a component of the Extraction Solvent (ES) is selected from: polyethylene glycol 200, polyethylene glycol 400 or mixtures of these.
Further, the lecithin or hydrolyzed lecithin is selected from the group consisting of: a product characterized by a hydrophilic-lipophilic balance (HLB) factor of 2-12 and selected from the group consisting of: soy lecithin (SL, from Glycine max. l.); sunflower lecithin (SUL; from sunflower (Helianthus annuus L.)); rapeseed lecithin (RL; from Brassica napus L.); canola lecithin (Brassica rapa L.); lecithin from chicken (Gallus Gallus domesticus L.) egg; a de-oiled product of said lecithin; hydrogenated lecithin from the source; hydrolyzed lecithin from the source; an enzyme-modified derivative of said lecithin; or mixtures of these substances.
Specifically, natural lecithins including deoiled, hydrogenated, hydrolyzed, or enzyme-modified lecithins from soybean (Glycine max.l.), sunflower (Helianthus annuus L.), rapeseed (Brassica napus L.), or canola (Brassica rapa L.), can be used as the lecithin in the present invention; or mixtures of these substances.
The term "enzymatically modified lecithin" includes hydrolyzed lecithin obtained by enzymatically hydrolyzing one higher fatty acid moiety, wherein monoglyceride of a higher fatty acid is produced, with the remaining phosphate and choline groups in the molecule. This results in a significant increase in the HLB factor of this hydrolyzed lecithin.
Preferably, according to the present invention, the following mixture may be used as an extraction solvent for preparing the liquid propolis extract:
(i) polyethylene glycol (PEG)200, polyethylene glycol 300, polyethylene glycol 400, or mixtures thereof; 97-99% w/w; and
(ii) natural lecithin, defatted lecithin 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; 1-3% w/w;
in a preferred embodiment of the invention, the liquid propolis extract according to the invention is standardized in the following respects:
(I) the mass percentage of the crude propolis and the final extract (DER) as the medicine is as follows:
1:3-1:5 w/w; and
(II) a 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-dihydroxycinnamate (4), wherein two of at least four of the key active substances correspond to the following quantitative contents:
(i) p-coumaric acid (1); 500-1,300. mu.g/mL;
(ii) trans-ferulic acid (2); 300-;
(iii) caffeic acid (3); 100-300 μ g/mL; and
(iv)3, 4-dihydroxy-trans-cinnamic acid phenethyl ester (4; CAPE); 100-.
Analysis of active substances in propolis liquid extract
In order to develop the novel standardized liquid propolis extract of the present invention, the present application establishes a suitable analytical method for the quantitative determination of:
(i) a key active propolis substance selected from the group consisting of: p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 3, 4-dihydroxy-trans-cinnamic acid phenethyl ester (4); and
(ii) a concomitant active selected from: trans-cinnamic acid (5), chrysin (6), coniferyl (7), galangin (8), apigenin (9) and kaempferol (10), which are important.
In order to initially study the analysis method, a liquid propolis extract model was prepared. Propolis extract model was prepared using ethanol (96%) and polyethylene glycol. Prepared by standard extraction methods, soaking at room temperature for 24 hours. The undissolved residue was then separated by filtration and the clear filtrate was used for further studies as a liquid propolis extract model. Polyethylene glycol 400(PEG400) was used as the polyethylene glycol as the model liquid. Examples 1 (96% ethanol) and 2(PEG400) describe methods for preparing liquid propolis extract models in the classical extraction solvents ethanol (96%) and polyethylene glycol 400.
The present invention establishes a suitable analytical method based on High Performance Liquid Chromatography (HPLC) by which a successful separation of all 10 of said compounds 1-10 is achieved. This method is described precisely in example 3.
A typical HPLC chromatogram obtained by the present analytical method is shown in fig. 1. Retention times (t) for Compounds 1-10R) Given in table 1.
Table 1 determination of the retention of propolis active substances 1-10 according to the HPLC method of the present inventiona
No.
Propolis ActivitySubstance(s)
Retention time (t)R)[min]
1
P-coumaric acid (1)
14.13
2
Trans-ferulic acid (2)
15.31
3
Caffeic acid (3)
10.57
4
2-phenethyl 3, 4-dihydroxy-trans-cinnamate (4)
48.62
5
Trans cinnamic acid (5)
29.76
6
Chrysin (6)
44.68
7
Pinocembrin (7)
47.39
8
Galangin (8)
49.94
9
Apigenin (9)
37.72
10
Kaempferol (10)
36.87
aA chromatographic column: ascentis express; c18; the size is 15cm multiplied by 3.0 mm; the particle diameter in the column was 2.7 μm; mobile phase, A is 0.1% formic acid water solution, B is methanol; elution gradient: 0min, 80% a, 20% B; 3min, 70% a, 30% B; 60 minutes, 20% a, 80% B; 90 minutes, 20% a, 80% B; 100min, 70% a, 30% B; 105 minutes, 80% a, 20% B; the column temperature is 30 ℃; the flow rate is 0.25 mL/min; analysis time: 110 min; wavelength on UV-VIS detector: 370nm for detection and 290nm for integration; injection volume is 10 μ L; pressure: 210 + 290 bars.
Research on influence of lecithin on extraction effect of propolis active substances 1-4
In the course of the subsequent studies of this study, the effect of different Extraction Solvents (ES) and different kinds (SL, RL, HRL) of lecithin and lecithin concentration (1-30% w/w) on the extraction efficacy of propolis active substances 1-4 was studied; see table 2.
TABLE 2 Extraction Solvent (ES) test for extracting active substances 1-4 from propolis
The contents of key active substances 1-4 and accompanying active substances 5-10 of the thus prepared liquid propolis extract were quantitatively analyzed. The results are given in tables 3-6.
In the case of using an Extraction Solvent (ES) based on 96% ethanol (EtOH) and a mixture of EtOH with different species (SL, RL, HRL) and concentrations (1-30% w/w in the ES composition), the primary liquid propolis extract contained, as the main active ingredients, p-coumaric acid (1; about 800-; see table 3.
Table 3 quantitative composition of active substances 1-4 in liquid propolis extract obtained with ethanol as Extraction Solvent (ES).
Wherein the concentration of auxiliary active substance 5-10 is at the following level: trans-cinnamic acid (5; about 40-65. mu.g/mL), chrysin (6; about 500-; see table 4.
In the case of using an Extraction Solvent (ES) based on polyethylene glycol (PEG400) and mixtures of PEG400 with various kinds (SL, RL, HRL) and concentrations (1-10% w/w in the ES composition) of lecithin, the primary liquid extract also contained p-coumaric acid (1; about 750-; see table 5.
Table 4 quantitative composition of concomitant active substances 5-10 in liquid propolis extract obtained with ethanol as Extraction Solvent (ES).
Table 5 quantitative composition of active substances 1-4 in liquid propolis extract obtained with polyethylene glycol 400 as Extraction Solvent (ES).
In this case, the concentrations of the auxiliary active substances 5 to 10 are at the following levels: trans-cinnamic acid (5; about 30-70. mu.g/mL), chrysin (6; about 400-; see table 6.
Table 6 quantitative composition of active substance 5-10 in liquid propolis extract obtained with polyethylene glycol 400 as Extraction Solvent (ES).
From the results it can be seen that the combination of lecithin (SL, RL, HRL) with 96% ethanol or polyethylene glycol 400(PEG400) as tested contributes more to the selective extraction of the chemically active substances 1-4 than any pure solvent when the composition Extraction Solvent (ES) is used at lower concentrations of 1-3% w/w.
The unexpected effect of the combination of PEG400 and lecithin (soybean lecithin, rapeseed lecithin, hydrolyzed rapeseed lecithin) is reflected in the fact that: although PEG400 was used as a pure solvent, the ability to extract the active substances 1-4 was significantly weaker than 96% ethanol (table 3, row 1, column 2); when used in combination with lecithin (soy lecithin, rapeseed lecithin, hydrolyzed rapeseed lecithin) at a concentration of 1-3% w/w, compounds 1-4 can be extracted significantly more efficiently than a similar combination of 96% ethanol and the same lecithin. For example, while the concentration of p-coumaric acid (1) in the extract obtained with 100% PEG400 as the extraction solvent was in the range of 750. mu.g/mL (Table 5, line 1, column 2) and the concentration of p-coumaric acid (1) in the extract obtained with 96% EtOH as the extraction solvent was in the range of 916. mu.g/mL (Table 3, line 1, column 2), the concentration of p-coumaric acid (1) in the extract obtained with PEG400 was in the range of 1.112. mu.g/mL (Table 5, line 2, column 2) relative to the level of 820. mu.g/mL (Table 3, line 2, column 2) using 3% w/w soybean lecithin and 96% ethanol as the extraction solvent.
From this exemplary example, the completely unexpected effect achieved is evident in an extraction solvent of a combination of polyethylene glycol and lecithin at a concentration of 1-3% w/w. This can be inferred by those skilled in the art as an acceptable range for achieving an optimal weight percent lecithin in the ES composition of 0.1-3.5% w/w.
When a higher weight percentage of lecithin is used in the Extraction Solvent (ES) composition, the beneficial effect of lecithin on propolis extraction chemoselectivity is lost compared to similar systems based on ethanol. For example, by using lecithin in ES at a concentration of 4% RL, 7.7% HRL or 10% SL, lower concentrations of key actives 1-4 were obtained compared to using a similar ethanol-based ES system. Typical results are shown in tables 7 and 8 for the two most abundant actives, p-coumaric acid (1) and trans-ferulic acid (2).
Table 7 quantitative composition of p-coumaric acid (1) in liquid propolis extracts obtained with different extraction solvents of the present invention.
In table 7, the unexpected results typical of the present invention can be seen in row 8, column 3. Wherein a 50% increase in p-coumaric acid (1) concentration was observed when using the extraction solvent PEG400+ RL (98.9:1.1, w/w) compared to a similar EtOH group Es (EtOH + RL 98.9:1.1, w/w).
Table 8 shows the quantitative composition of trans-ferulic acid (2) in liquid propolis extracts obtained with different extraction solvents of the invention.
As another typical example of unexpected results, in line 12 of Table 8, the data in column 3 records that when the Extraction Solvent (ES) PEG400+ HRL (97.8:2.2, w/w) is used, the trans-ferulic acid (2) concentration increases by 53.5% compared to a similar 96% ethanol-based ES.
Method for producing standardized liquid propolis extract according to the present invention
The preparation method of the liquid propolis extract according to the present invention comprises the steps of:
(i) cooling the crude propolis at-20 deg.C for at least 1 hr;
(ii) grinding frozen propolis, and sieving with 1-8mm hole;
(iii) extracting crude propolis with an extraction solvent under the following conditions:
(a) the weight ratio of the crude propolis to the extraction solvent is 1:2-1:20 w/w;
(b) the extraction temperature is 10-150 ℃; and
(c) the extraction time is 5 minutes to 72 hours;
(iv) filtering the mixture thus obtained through a series of filters having pores ranging from 100pm to 5pm, producing an undissolved residue and a liquid propolis extract;
(v) performing quantitative analysis on key active propolis substances 1-4 by High Performance Liquid Chromatography (HPLC); and
(vi) (iv) standardizing the liquid propolis extract thus obtained, having a precisely quantified composition of the key active substances 1-4 determined in step (v), by dilution with the fresh extraction solvent used in step (iii), until the desired content of active substances 1-4 is reached.
In a preferred embodiment for carrying out the method for preparing a liquid propolis extract according to the present invention, the extraction step (iii) is carried out under the following conditions:
(a) the weight ratio of the crude propolis to the extraction solvent is 1:3-1:5 w/w;
(b) the extraction temperature is 15-70 ℃; and
(c) the extraction time is 1-24 hours.
Furthermore, the quantitative determination steps of the key active substances 1 to 4 and the concomitant monitoring of the auxiliary active substances 5 to 10, the analytical high performance liquid chromatography method (HPLC) developed for this purpose were carried out as follows:
(i) a chromatographic column: ascentis Express; c18; the size is 15cm multiplied by 3.0 mm; the diameter of the particles in the column was 2.7 pm;
(ii) mobile phase, A is 0.1% formic acid water solution, B is methanol; elution 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) the column temperature is 30 ℃;
(iv) the flow rate is 0.25 ml/min;
(v) analysis time: 110 min;
(vi) wavelength on UV-VIS detector: 370nm for detection and 290nm for integration;
(vii) injection volume 10 pl;
(viii) pressure: 210 + 290 bars.
Examples 4-9 disclose experimental methods for preparing the liquid propolis extract of the present invention. In example 9, an optimized form of the preparation process of the standardized liquid strength extract according to the invention is described, consisting of the parameters drug-extract ratio (DER) weight percentage 1: and 2, are shown.
Example 3 describes an analytical method for the quantitative determination of key active substances 1 to 4 and auxiliary active substances 5 to 10.
The present invention standardizes the determination of antimicrobial efficacy of liquid extracts. Minimal inhibition of model pathogenic microorganisms Determination of concentration (MIC)
The antimicrobial efficacy of the propolis extract was measured at the molecular medical college, university of Rudjer Boskovic, located in captain, crohnia. According to the present invention, the Minimum Inhibitory Concentration (MIC) of the standardized liquid propolis extract is determined by the instructions of CLSI (american clinical laboratory standards institute) and EUCAST (european antibiotic susceptibility test committee). Antimicrobial susceptibility testing) as described in references 26-29. The product of example 9 was used.
26)M.Balouiri,M.Sadiki,S.K.Ibnsouda:Methods for in vitro evaluating antimicrobial activity:A review,J.Pharm.Anal.6(2016)71-79.
27)CLSI,Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically,Approved Standard,9th ed.,CLSI document M07-A9.Clinical and Laboratory Standards Institute,950West 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,Suite 1400,Wayne,Pennsylvania 19087-1898,USA,2002.
29)CLSI,Methods for Determining Bactericidal Activity of Antimicrobial Agents.Approved Guideline,CLSI document M26-A.Clinical and Laboratory Standards Institute,950West Valley Road,Suite 2500,Wayne,Pennsylvania 19087,USA,1998.
The following ATCC strains of model pathogenic microorganisms (M) were tested for antimicrobial efficacy under in vitro conditions:
(i) staphylococcus aureus ATCC 29293 (M1);
(ii) methicillin-resistant staphylococcus aureus; MRSA (MFBF Collection; M2);
(iii) methicillin-sensitive staphylococcus aureus; MSSA (MFBF set; M3);
(iv) enterococcus faecalis ATCC 9212 (M4);
(v) enterococcus faecalis VRE (MFBF collection) (M5);
(vi) e.coli ATCC 10536 (M6);
(vii) acinetobacter baumannii ATCC 43498 (M7);
(viii) pseudomonas aeruginosa ATCC 9027 (M8); and
(ix) candida albicans ATCC 90028 (M9).
A series of microdilution procedures were performed to determine the Minimum Inhibitory Concentration (MIC) of the extract. MIC values were determined as propolis extract concentrations at which an 80% reduction in bacterial or fungal counts occurred (MIC 80); the minimum inhibitory concentrations (MIC80) shown in table 9 are shown as dilutions (%) of the corresponding liquid extracts in a given solvent. The starting liquid propolis extract, i.e. the product of example 9, was obtained at a ratio of 1:2 of crude propolis to final extract (DER). If the dilution of the liquid extract is greater, i.e. the MIC values of the active substances 1-10 are lower, the antibacterial effect of the test extract obtained is higher.
A detailed description of the experimental procedure for MIC value determination is disclosed in example 10 and the results are listed in table 9.
Table 9 results of minimum inhibitory concentration (MIC;%) assay of diluted propolis extract of the product of example 9, compared to liquid extract obtained using 96% ethanol (example 1) or polyethylene glycol 400 (example 2) as an extraction solvent.
In tables 10-15, 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 are given; these are obtained by using the following method:
(i) 96% ethanol; the product of example 1 was used as a reaction product,
(ii) polyethylene glycol 400(PEG 400); the product of example 2; and the number of the first and second groups,
(iii) a mixture of PEG400 (97% w/w) and soya lecithin (3% w/w); the product of example 9; alternatively, the mass concentration of the respective active substance at which each specific liquid extract reaches the MIC value is 1-10. From DER to 1:2, when the MIC is reached, it is separated with a diluent.
Table 10 mass concentration (γ) of propolis active substance 1-4 in unit of [ μ g/ml ] in an effective dilution of propolis liquid extract obtained with 96% ethanol; product of example 1
Table 11 mass concentration (γ) of propolis active substance 5-10 in unit of [ μ g/ml ] in an effective dilution of propolis liquid extract obtained with 96% ethanol; the product of example 1.
Table 12 shows the mass concentration (γ) of propolis active substances 1 to 4 in [ μ g/ml ] in an effective dilution of a liquid propolis extract obtained with polyethylene glycol 400(PEG 400); the product of example 1.
Table 13 shows the mass concentration (γ) of propolis active substance 5-10 in [ μ g/ml ] in an effective dilution of propolis liquid extract obtained with polyethylene glycol 400(PEG 400); the product of example 2.
Table 14 shows the mass concentration (. gamma.) of propolis active substances 1 to 4 in [ μ g/ml ] in the liquid propolis extract obtained by effectively diluting a mixture of polyethylene glycol 400(PEG 400; 97% w/w) and soybean lecithin (3% w/w); the product of example 9.
Table 15 shows the mass concentration (. gamma.) of propolis active substances in the unit of [ μ g/ml ] of 5 to 10 in the liquid propolis extract obtained by effectively diluting a mixture of polyethylene glycol 400(PEG 400; 97% w/w) and soybean lecithin (3% w/w); the product of example 9.
The total antimicrobial effect (MIC) of each tested liquid propolis extract was achieved by the synergistic effect of several active substances 1-10. Interestingly and totally unexpectedly, mixtures of low concentrations of active substances 1-10 are more effective than mixtures of high concentrations of certain (pure) active substances 1-10.
The formulations of the present invention are most effective against gram-positive microorganisms such as staphylococci. However, as shown in Table 9, it was also effective against some gram-negative bacteria such as E.coli and Vibrio baumannii, and the fungus Candida albicans, at higher concentrations.
Application of standardized liquid propolis extractThe propolis extract as a pharmaceutical, cosmetic, agrochemical or food ingredient according to the present invention contains standardized concentrations of highly bioactive substances:
(i) p-coumaric acid (1); 100-;
(ii) trans-ferulic acid (2); 75-800 mug/ml;
(iii) caffeic acid (3); 25-300 mug/ml; and
(iv)3, 4-dihydroxy-trans-cinnamic acid phenethyl ester (4; CAPE); 40-400. mu.g/ml.
Due to the standardisation of the content of key active substances 1-4, the liquid propolis extract according to the invention represents an Active Pharmaceutical Ingredient (API), an Active Cosmetic Ingredient (ACI) or a functional ingredient for food or animal feed for humans and animals, characterized by the following beneficial pharmacological effects:
(i) anti-inflammatory; see literature references 1, 8, 9, 11, 12, 13, 17;
(ii) an antioxidant; see literature references 8, 9, 14, 15, 20;
(iii) an immunomodulator; see literature references 1, 8, 11, 13, 15, 17, 18, 19, 20;
(iv) protecting liver; see literature reference 9;
(v) an antibacterial agent; including antibacterial, antiviral, antifungal and antiprotozoal; see literature references 9, 10, 12, 15, 16, 18;
(vi) anti-tumor; see literature references 9, 10, 11, 14, 19; and
(vii) resisting cancer; see literature references 11, 14.
Other valuable effects of the liquid propolis extract of the present invention also result from fungicidal, bactericidal, virucidal, insecticidal and nematicidal effects in plant protection. The liquid propolis extracts of the present invention indirectly enhance the resistance of plants and their to abiotic stress factors and help them to resist infection due to the strong antioxidant activity. Therefore, it can be used as a plant enhancer; see the literature reference ILLS. The liquid propolis extract according to the present invention can also be used as:
(i) an active pharmaceutical ingredient or excipient for the manufacture of a pharmaceutical product selected from: a drug, a pharmaceutical device or a therapeutic agent;
(ii) active cosmetic ingredients or excipients for the production of cosmetics;
(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 use in the manufacture of a veterinary product derived from: a veterinary pharmaceutical product; animal feed; an animal feed additive; or veterinary drugs; or
(v) An active agrochemical or excipient for the production of an agrochemical product derived from: fungicides, bactericides, viricides, insecticides, nematicides and plant enhancers; is particularly suitable for ecological agriculture.
Pharmaceutical composition based on said standardized liquid propolis extract according to the invention
Furthermore, the present invention discloses a pharmaceutical composition based on said standardized liquid propolis extract as Active Pharmaceutical Ingredient (API). The pharmaceutical composition according to the invention comprises:
(i) a liquid propolis extract according to the present invention; 5-95% w/w; and the number of the first and second groups,
(ii) one or more pharmaceutically acceptable excipients required for formulation of the final dosage form, derived from: solutions, suspensions, gels, creams, ointments, oral or nasal sprays; up to 100% w/w of the final composition;
wherein said composition is characterized by a quantitative content of at least two of the four key active propolis substances within the following values:
(i) p-coumaric acid (1); 10-1300 mu g/g;
(ii) trans-ferulic acid (2); 10-800 mug/g;
(iii) caffeic acid (3); 5-300 mug/g; and the number of the first and second groups,
(iv)3, 4-dihydroxy-trans-cinnamic acid phenethyl ester (4; CAPE); 5-400 mu g/g.
Thus, the pharmaceutically acceptable excipients (adjuvants) are selected from diluents, wetting agents, preservatives, chelating agents, antioxidants, thickeners, emollients, emulsifiers, tonicity agents and pH control agents.
The diluent is a pharmaceutically acceptable liquid derived from: purifying the water; ethanol; 1, 2-propanediol; liquid polyethylene glycol (PEG), such as PEG200, PEG400 or PEG 600; or mixtures of these substances.
The humectant is derived from glycerin, sorbitol, 1, 2-propylene glycol or a mixture of these materials.
The preservative comes from: methyl p-hydroxybenzoate, ethyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate, butyl 4-hydroxybenzoate; 4-chloro-cresol; triclosan; benzyl alcohol; 2-phenoxyethanol; benzoic acid and its salts such as sodium benzoate; sorbic acid or a salt thereof such as potassium sorbate; dehydroacetic acid (3-acetyl-2-hydroxy-6-methyl-4 a-pyran-4-one); chlorhexidine and its salts such as chlorhexidine digluconate; quaternary ammonium salts such as benzalkonium chloride or cetylammonium bromide; or mixtures of these substances.
The chelating agent is derived from: sodium or potassium salts of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA); a soluble citrate such as trisodium citrate dihydrate (Na3C6H5O 7.2H 2O), a typical chelating agent is disodium ethylenediaminetetraacetic acid dihydrate (Na2 edta.2h2O).
The antioxidant is derived from: alpha-tocopherol and esters thereof such as alpha-tocopherol succinate; ascorbic acid and its salts such as sodium ascorbate; 2, 6-di-tert-butyl-4-methylphenol (BHT); tert-butyl methyl ether (BHA); or mixtures of these substances.
The thickening agent is derived from: 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 polyvinyl pyrrolidone (PVP); various gums such as acacia, xanthan gum, tragacanth gum; alginic acid and salts thereof such as sodium alginate; metal salts of higher fatty acids, such as aluminum monostearate, aluminum distearate, aluminum tristearate; or mixtures of these substances.
The emollient is derived from: vaseline; mineral oil; vegetable oils such as almond oil, sunflower oil 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; a silicone oil; higher fatty acids such as oleic acid or stearic acid; higher aliphatic alcohols such as cetyl alcohol; or mixtures of these substances.
The emulsifier is derived from: lanolin; ethoxylated lanolin; lanolin alcohol; ethoxylated lanolin alcohols; lecithin; hydrolyzing lecithin; mono-and diesters of glycerol and higher fatty acids such as glycerol monostearate; sorbitol esters of higher fatty acids such as sorbitol monostearate; ethoxylated higher aliphatic alcohols such as polyoxyethylene (23) lauryl ether or polyoxyethylene (2) oleate, wherein the number 23 or 2 represents the number of ethylene oxide units; ethoxylated sorbitan esters such as esters of 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. The tonicity agent is primarily used in pharmaceutical dosage forms for administration to mucous membranes, such as the nasal mucosa, and is selected from the group consisting of sodium chloride (NaCl), glycerin, 1, 2-propanediol, or mixtures of these substances.
pH control agents include pharmaceutically acceptable acids and bases for lowering or increasing pH and buffer systems derived from: hydrochloric acid (HCl), sulfuric acid (H)2SO4), phosphoric acid (H)3PO4), citric acid, acetic acid, sodium hydroxide (NaOH), potassium hydroxide (KOH), ammonium hydroxide (NH)4OH), sodium dihydrogen phosphate (NaH)2PO4), sodium hydrogen phosphate (Na)2HPO4) Sodium dihydrogen citrate (N)aH2C6H5O7), sodium hydrogen citrate (Na)2HC6H5O7) Sodium citrate (Na)3C6H5O7) Or mixtures of these substances.
Preparation of the pharmaceutical composition according to the inventionThe pharmaceutical composition of the present invention is prepared by a process comprising the steps of:
(i) adding the standardized liquid propolis extract of the present invention to a diluent and homogenizing it;
(ii) adding one or more other excipients; and their homogenization;
wherein steps (i) and (ii) are carried out at a temperature of 10-100 ℃, preferably at a temperature of 20-60 ℃, within 1-5 minutes; next, in the case of formulation preparation:
(iii.a) a solution or a solution for spraying; performing filtration of the final solution, including sterile filtration if necessary;
(iii.b) a gel or suspension; adding a thickening agent, and homogenizing;
(iii.c) a cream; (iii) by mixing emollient and emulsifier and homogenising at a temperature of 50-80 ℃ for 1-15 minutes, then adding the solution of step (ii) heated to 50-80 ℃, followed by emulsification at a temperature of 50-80 ℃, preferably 55-65 ℃ for 1-30 minutes with a high shear or high pressure homogeniser followed by homogenisation at a temperature of 65-20 ℃ for 10-120 minutes; or
(III. d) ointments; (iii) mixing the solution of step (ii) with a mixture of the emollient and the final emulsifier previously melted at a temperature of 50-70 ℃ during 5-30 minutes, followed by homogenization at a temperature of 70-20 ℃ during 10-120 minutes.
The steps for preparing a pharmaceutical composition from the present invention may also include various alternative conventional technical steps for producing the dosage form, which are well known to those skilled in the art.
Examples 11-16 describe representative examples of the manufacture of pharmaceutical compositions according to the present invention.
As a specific example, the final dosage form of the solution disclosed in example 11 for intramammary administration is described herein. In this case, the primary standardized liquid propolis extract, the preparation of which is described in example 9; in this case, a mixture of polyethylene glycol 400 (97% w/w) and soybean lecithin (3% w/w) was used, which served as a diluent therein. The intramammary solutions obtained in the above manner lead to the given ranges of quantitative contents of the key active substances 1 to 4, see table 16.
Table 1 results of quantitative analysis of active substances 1 to 10 by High Performance Liquid Chromatography (HPLC) of the pharmaceutical composition of the present invention
A typical HPLC chromatogram for quantitative analysis of the composition of the invention is shown in fig. 5.
Research on pharmacological action of the pharmaceutical composition
The selected pharmacological effects of the compositions from the invention in dosages from solutions were studied in clinical studies, and the product from example 11, useful for the following treatments:
(i) cow mastitis, mastitis;
(ii) mastitis of goats; and the number of the first and second groups,
(iii) the wound of the horse heals.
Study on therapeutic effect of cow mastitis
The study of the treatment of mastitis in cows was carried out in 5 farms with cows of the holstein breed. A total of 86 cows (339 udder portions) were enrolled in the study, with one udder portion serving as a statistical unit. Animals were freely placed in thick bedding and fed standard premix to cows without antibiotic addition. The study was approved by the veterinary association. Including healthy animals and udder portions with no clinical symptoms of mastitis with Somatic Cell Counts (SCC) below 200,000/mL, and infected udder portions with SCC above 200,000/mL. Randomized cross-clinical studies of safety and efficacy were conducted by intramammary (i.mam.) application of the compositions of the present invention. The composition of the invention in example 11 was administered three times in all four udder portions of cattle: the early milking session, the late milking session and the day after the early milking. A detailed procedure is described in example 17, while table 17 shows the bacteriological cure of each pathogen identified in a certain number of breast portions prior to the first i.mam. application.
Table 17 is a test of the effectiveness of the bacterial treatment/cure of cow mastitis after i.mam. application of the formulation of example 11 of the present invention to cows
The three (two-day) dosing regimen of the composition of example 11 provided a 100% cure rate within only 7 days.
Compared with the traditional Chinese medicine, the antibacterial cure rate of the cephalosporin antibiotics ceftiofur reaches 66%, but after 8 days of daily i.mam application, only 54% of cases are cured after 5 days; see literature reference 30. For subclinical mastitis caused by streptococcus rubberis, 58.1% of cases can be healed by treating two days with pirlimycin, and 68.8% and 80% of cases can be healed by treating 5 and 8 days; see literature reference 30:
30)S.P.Oliver,B.E.Gillespie,S.J.Headrick,H.Moorehead,P.Lunn,H.H.Dowlen,D.J.Johnson,K.C.Lamar,S.T.Chester,W.M.Moseley:Efficacy of extended Ceftiofur intramammary therapy for treatment of subclinical mastitis in lactating dairy cows.J.Dairy Sci.87(2004)2393-2400.
alternatively, in the treatment of bovine mastitis, compositions of the present invention in the form of suspensions for intramammary administration may also be used successfully, as described in example 12.
Study on treatment of goat mastitis
The study of goat mastitis treatment was performed on 25 goats in the alpine goat farm of OPG Matijasec, of Sigetec Ludbreski, cropland. All goats diagnosed subclinical mastitis in the left and right galactomas. Goats whose goat milk was positive by microbiological examination were divided into two groups: one group administered the composition of the invention of example 11 and the other group administered an intramammary suspension of amoxicillin and clavulanic acid (Klavuxil r) (geneva, crohn's); all three dosing regimens were used. The tolerance and bacterial immobilization of both sets of breasts were monitored in parallel after i.mam. application using the composition of example 11. Three (two days) administrations of the formulation of the invention provided bacterial treatment in 75% of the infected breast and 85% after 14 days. While intramammary antibiotic administration enables bacterial treatment in 73.3% of cases. This proves that the former is more effective than the latter. The results of the study show that treatment of mastitis in goats with the composition of example 11 is timely to provide bacterial treatment without the use of antibiotics.
The detailed procedure of the study is described in example 18, while the results of the bacterial treatment are given in table 18.
Table 18 shows the formulations of example 11 of the invention in combination with antibiotics (fixed combination of amoxicillin and clavulanic acid)aThe mixture of (a) is applied to a goat for testing the effectiveness of bacterial treatment/cure of goat mastitis after i.mam
Comparing the activity of amoxicillin as a broad spectrum antibiotic in i.mam applications, which is suitable for treating mastitis caused by pathogens found in milk samples and the composition of example 11, the latter proved to have a stronger activity. At 7 days after amoxicillin administration, only 60% of the galactans were cured, whereas the results for the composition of example 11 were 75%. The total percent of healed galacta with antibiotic was 73.3% and the total percent of test composition was 85% 14 days after the first administration.
From the results, it can be seen that the anti-inflammatory and antimicrobial activity of the composition of the present invention has a high efficacy in treating mastitis. The curative effect is higher than that of the classical amoxicillin and clavulanic acid fixed combination as broad-spectrum antibiotics.
Alternatively, in the treatment of goat mastitis, compositions of the present invention in the form of suspensions for intramammary administration as described in example 12 may also be used successfully.
It can be concluded that the compositions of the invention are characterized by, among other properties, antibacterial activity against a range of bacteria and fungi, wherein the antibacterial efficacy is higher in vivo than under in vitro conditions compared to classical antibiotics. It is not only an antimicrobial agent, but also an anti-inflammatory and immunomodulatory agent.
The immunoregulation propolis effect is closely related to the antioxidation effect of the immunoregulation propolis, and the oxidative stress is a component of mastitis pathogenesis; see, for example, reference 31:
31)O.Atakisi,H.Oral,E.Atakisi,O.Merhan,S.Metin Pancarci,A.Ozcan,S.Marasli,B.Polat,A.Colak,S.Kaya:Subclinical mastitis causes alterations in nitric oxide,total oxidant and antioxidant capacity in cow milk,Res.Vet.Sci.89(2010)10-13.
study of wound healing status in horses
The situation is a wound healing situation describing the mare's foreleg, which is a deep wound created in the ergot area of the foreleg when the hind leg scratches the foreleg during landing. The wound was treated with different formulations conservatively, unsuccessfully, before the start of administration of the composition of example 11.
The wound was then washed with physiological solution, dried and treated once daily with the composition of example 11 over a period of 5 days. An improvement similar to epithelial neogenesis was seen after 48 hours, and the wound was completely healed after 96 hours. Mares flex vigorously over several days, after which time no lameness was observed and treatment was complete. A detailed description of this is described in example 19.
Although detailed clinical studies must be conducted for more accurate conclusions, it is obvious to those skilled in the art that the composition of the present invention effectively functions as a wound healing agent. Since it is known from the literature that anti-inflammatory, antioxidant and epithelializing activities are crucial for the wound healing process, the stimulation of collagen synthesis, as well as the compositions of the present invention, together with the demonstrated antimicrobial activity, is part of the pharmacological spectrum of action; for comparison, see reference 32:
32)S.Marinotti,E.Ranzato:Propolis:a new frontier for wound healing,Burns Trauma(2015)3:9,doi 10.1186/s41038-015-0010-z.
application of the pharmaceutical composition
The pharmaceutical compositions of the invention are useful for treating diseases and conditions in humans and animals, comprising: inflammation; bacterial infection; fungal infections; viral diseases; autoimmune diseases; functional gastrointestinal disorders; for mucosal regeneration, burn treatment and wound healing; and cancer diseases.
Inflammation and its disorders include: gingivitis, periodontitis, laryngitis, gastritis, colitis, hemorrhoids, dermatitis, otitis externa, sinusitis, rhinitis, vaginitis and mastitis.
The pharmaceutical composition of the invention is used for treating bacterial infections caused by bacteria in the following group:
(i) gram-positive bacteria: staphylococcus genus: staphylococcus aureus, MRSA (methicillin-resistant staphylococcus aureus), MSSA (methicillin-sensitive staphylococcus aureus), staphylococcus intermedius, staphylococcus pseudointermedius; coagulase-negative staphylococci: staphylococcus epidermidis, staphylococcus putrefaction, staphylococcus suis; streptococcus genus: staphylococci, Streptococcus bovis, Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus canis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus oralis, Streptococcus thermophilus; the genus peptostreptococcus; corynebacterium: corynebacterium bovis; steviosin pyogenes; nocardia; b, bacillus subtilis; bacillus cereus; enterococcus: enterococcus faecium, enterococcus faecalis; vancomycin Resistant Enterococci (VRE): enterococcus cassavae; and the number of the first and second groups,
(ii) gram-negative bacteria: e.coli; acinetobacter baumannii; pseudomonas aeruginosa; haemophilus influenzae; salmonella choleraesuis; yersinia enterocolitica; enterobacter (enterobacter cloacae); klebsiella bacteria: klebsiella pneumoniae, klebsiella oxytoca; shigella flexneri; burkholderia cepacia; proteus mirabilis; a proteus bacterium; an actinomycete aggregate; an ether actinomycete; bacteroides fragilis; helicobacter pylori; e.coli; campylobacter jejuni; porphyromonas gulila; porphyromonas salivarius; porphyromonas dentata; prevotella intermedia; treponema; paenibacillus sp.
Furthermore, the pharmaceutical composition of the invention is useful for the treatment of fungal infections caused by fungi, such as: candida species: candida albicans, Candida ducreyi, Candida glabrata, Candida kruensis, Candida tropicalis, Candida paratyphi; the genus Aspergillus: aspergillus niger, aspergillus versicolor; penicillium pinophilum; a variant paecilomyces; trichoderma viride; chaetomium globosum; and malassezia pachydermatis.
Furthermore, the compositions of the invention are useful for the treatment of viral diseases caused by viruses, such as: herpes Simplex Virus (HSV); human Papilloma Virus (HPV); EB virus (EBV); cytomegalovirus (CMV); poliovirus; influenza a and b viruses; a retrovirus; vaccinia virus; common cold virus, rhinovirus, picornavirus, human parainfluenza virus (HPIV), Human Metapneumovirus (HMPV), coronavirus, adenovirus, Human Respiratory Syncytial Virus (HRSV), enterovirus.
Alternatively, the pharmaceutical composition according to the invention is used for the treatment of autoimmune diseases, such as: psoriasis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, celiac disease, and multiple sclerosis.
In addition, the pharmaceutical compositions of the invention are useful for treating cancer diseases, such as: skin and mucosal cancer, gastrointestinal tumors, colorectal cancer.
The pharmaceutical composition of the invention is used for treating the following functional gastrointestinal diseases: esophageal, gastric, duodenal, small intestinal and colonic diseases, centrally mediated gastrointestinal pain, gall bladder and sphincter of ODDI diseases, anorectal diseases, children and adolescent specific gastrointestinal diseases. In particular, the compositions of the present invention are useful for treating mastitis in animals.
Examples
General remarks
Crude propolis of poplar type from Hedera was used as primary propolis. Ethanol (96%) and polyethylene glycols 200,400 and 600, as well as powdered Soybean Lecithin (SL) were purchased from Fagron Cratia (HR). Rapeseed Lecithin (RL) and Hydrolyzed Rapeseed Lecithin (HRL) were purchased from Pfannenschmidt (DE). Deoiled sunflower lecithin (SUL) was purchased from Barentz (NL). Aluminum distearate was purchased from Sigma-Aldrich (US). Other starting materials were purchased from local suppliers.
Samples of chemically pure compounds for analytical purposes: p-coumaric acid (1; 98% or more HPLC), trans-ferulic acid (2; 99%), caffeic acid (3; 98% or more HPLC), 3, 4-dihydroxy-trans-cinnamic acid 2-phenylethyl ester (CAPE; 4; 97% or more HPLC), trans-cinnamic acid (5; 96.5% or more, DC), chrysin (6; 98% or more HPLC, strobilurin (7; 95% or more TLC), galangin (8; 95% or more HPLC), apigenin (9; 99% or more HPLC) and kaempferol (10; 90% or more HPLC) as quantitative analytical standards for determining their quantitative content in liquid propolis extracts, which are available from Sigma-Aldrich (US).
The term "room temperature" refers to a temperature interval of 20-25 ℃. The abbreviation "min" means minutes. The yield (percentage of theoretical yield) is expressed as the weight (% w/w) of the isolated liquid propolis extract relative to the mass of the starting extraction solvent (ethanol, PEG, ethanol + lecithin, PEG + lecithin).
The quantitative content of the active substance 1-10 in the liquid propolis extract is expressed in units of micrograms per milliliter [ μ g/ml ] in terms of mass concentration (γ), and its quantitative composition is expressed in micrograms per gram of the final product (dosage form) [ μ g/g ] in the pharmaceutical composition of the present invention: .
Example 1 preparation of liquid propolis extract with 96% ethanol as extraction solvent
Pretreatment of propolis before extraction: will be provided withA sample of crude propolis (poplar; 1kg) was frozen in a freezer at-20 ℃ for at least 1 hour. The sample was then ground in a grinder.
Extracting with 96% ethanol: ethanol (96%; 70.00g) was added to ground propolis (30.00 g). The obtained mixture was left at room temperature for 72 hours while being periodically stirred. Then, the mixture was filtered through a filter paper (black band) to obtain 60.00g (85.7%) of liquid propolis extract as a dark brown solution with a slightly strong propolis odor.
To test the Minimum Inhibitory Concentration (MIC) of the alcohol propolis extract described in example 10, the ratio of drug to extract (DER) was 1:2 repeat the same steps.
Example 2 preparation of liquid propolis extract using polyethylene glycol 400 as extraction solvent
Propolis (30.00g) pre-treated and ground as described in example 1 was mixed with polyethylene glycol 400(PEG 400; 70.00 g). The obtained mixture was left at room temperature for 72 hours while being periodically stirred. The mixture was then filtered through filter paper (black tape) yielding 55.00g (78.6%) of liquid propolis extract in the form of a dark brown viscous liquid with a slightly strong smell similar to propolis.
To test the Minimum Inhibitory Concentration (MIC) of the polyethylene glycol propolis extract described in example 10, the ratio of drug to extract (DER) was 1:2 repeat the same steps.
Example 3 an HPLC analytical method for quantitatively determining active substances 1-10 in liquid propolis extract
Quantitative analysis was performed by High Performance Liquid Chromatography (HPLC), which was specifically developed for monitoring key active substances 1-4 and accompanying active ingredients 5-10 from propolis.
By mixing with ethanol: 75 parts of water: 25 to 100 μ g/ml to prepare a sample of a commercially available standard of active substance 1-10 for analysis.
Prior to analysis, a sample of the liquid propolis extract (100pL) of the present invention was extracted with ethanol: 75 parts of water: 25, V/V (900pL) was diluted in a 1:10w/w (ten fold dilution ratio) mixture.
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) a chromatographic column: ascentis Express; c18; the size is 15cm multiplied by 3.0 mm; the diameter of the particles in the column was 2.7 pm;
(ii) mobile phase, A is 0.1% formic acid water solution, B is methanol; elution 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) the column temperature is 30 ℃;
(iv) the flow rate is 0.25 ml/min;
(v) analysis time: 110 min;
(vi) wavelength on UV-VIS detector: 370nm for detection and 290nm for integration;
(vii) injection volume 10 pl;
(viii) pressure: 210 + 290 bars.
Under the above conditions, the key active substances 1 to 4 and the accompanying ingredients 5 to 10 have the following retention times (tR):
(i)TR[ P-coumaric acid (1)]＝14.13min；TR[ Trans-ferulic acid (2)]＝15.31min；
TR[ caffeic acid (3)]＝10.57min；TR[3, 4-dihydroxy-trans-cinnamic acid phenethyl ester;
CAPE(4)]＝48.62min；
(ii)TR[ trans-cinnamic acid (5)]＝29.76min；TR[ Chrysin (6)]＝44.68min；
TR[ coniferyl element (7)]＝47.39min；TR[ galangin (8)]＝49.94min；
TR[ apigenin (9)]＝37.72min；TR[ Kaempferol (10)]＝36.87min。
The retention times of the key propolis actives 1-4 and the accompanying actives 5-10 are listed in table 1.
Example 4 preparation of a target Using an extraction solvent based on a mixture of ethanol and lecithinStandardized liquid propolis extract
Ground propolis, pretreated as described in example 1, 30.00g of this propolis was mixed with the extraction solvent of the following composition (experiment EL-8):
e1: 96% ethanol (67.00 g; 95.7%) and soya lecithin (SL; 3.00 g; 4.3%);
e2: 96% ethanol (60.00 g; 85.7%) and soya lecithin (SL; 10.00 g; 14.3%);
e3: 96% ethanol (50.00 g; 74%) and soya lecithin (SL; 20.00 g; 28.6%);
e4: 96% ethanol (40.00 g; 57.1%) and soya lecithin (SL; 30.00 g; 42.9%);
e5 96% ethanol (67.00 g; 98.9%) and rapeseed lecithin (RL; 3.00g 25% -preparation; 0.75g lecithin 1.1%); theoretical yield E5 ═ 67g ethanol +0.75g lecithin ═ 67.75 g;
e6 96% ethanol (60.00 g; 96.0%) and rapeseed lecithin (RL; 10.00g 25% -preparation; 2.50g lecithin; 4.0%); theoretical yield E6 ═ 60g ethanol +2.50g lecithin ═ 62.50 g;
e7 96% ethanol (67.00 g; 97.8%) and hydrolysed rapeseed lecithin (HRL; 3.00g 50% -preparation; 50g hydrolysed rapeseed lecithin; 2.2%); theoretical yield E7 ═ 67g ethanol +1.50g lecithin ═ 68.50 g;
e8 96% ethanol (60.00 g; 92.3%) and hydrolysed rapeseed lecithin (HRL; 10.00g 50% -preparation; 5.00g lecithin; 7.7%); theoretical yield E8 ═ 60g ethanol +5.00g lecithin ═ 65.00 g.
The obtained mixture was stirred at room temperature for 1 hour, and left to stand at room temperature for 72 hours while being periodically stirred. Then, the mixture was filtered through filter paper (black band) to give 50-60g (71.0-85.7%) of liquid propolis extract as dark brown viscous liquid with a slightly strong smell like propolis.
The primary liquid propolis extract thus prepared was subjected to quantitative analysis of the contents of key active substances 1 to 4 and accompanying components 5 to 10 according to the analytical method described in example 3. The results are shown in tables 3 and 4.
Primary liquid propolis extract, prepared in Extraction Solvent (ES) as described in experiments E1-E8, with an active substance content of 1-4, was standardized by dilution with the same ES used in the extraction step, up to the desired level of quantitative composition of active substances 1-4 according to the invention.
Example 5 preparation of standardized liquid propolis Using an extraction solvent based on a mixture of polyethylene glycol 400 and lecithin Extract of plant
Ground propolis, pretreated as described in example 1, 30.00g of this propolis were mixed with the extraction solvents of the following composition (experiments E1-E8):
EL: polyethylene glycol 400(PEG 400; 67.00 g; 97%) and soya lecithin (SL; 3.00 g; 3%);
e2: polyethylene glycol 400(PEG 400; 60.00 g; 90%) and soya lecithin (SL; 10.00 g; 10%);
e3 polyethylene glycol 400(PEG 400; 67.00 g; 98.9%) and rapeseed lecithin (RL; 3.00g 25% -preparation; 0.75g lecithin; 1.1%); theoretical yield E3 ═ 67g PEG400+ 0.75g lecithin ═ 67.75 g;
e4 polyethylene glycol 400(PEG 400; 60.00 g; 96.0%) and rapeseed lecithin (RL; 10.00g 25% -preparation; 2.50g lecithin; 4.0%); theoretical yield E4 ═ 60g PEG400+ 2.50g lecithin ═ 62.50 g;
e5 polyethylene glycol 400(PEG 400; 67.00 g; 97.8%) and hydrolysed rapeseed lecithin (HRL; 3.00g 50% -formulation; 50g hydrolysed lecithin; 2.2%); theoretical yield E5 ═ 67g PEG400+ 50g lecithin ═ 68.50 g;
e6 polyethylene glycol 400(PEG 400; 60.00 g; 92.3%) and hydrolysed rapeseed lecithin (HRL; 10.00g 50% -formulation; 5.00g lecithin; 7.7%); theoretical yield E6 ═ 60g PEG400+ 5.00g lecithin ═ 65.00 g.
The obtained mixture was stirred at room temperature for 1 hour, and left to stand at room temperature for 72 hours while being periodically stirred. The mixture was then filtered through filter paper (black band). Yielding 45-55g (69.0-78.6%) of liquid propolis extract in the form of a dark brown viscous liquid with a slightly rich smell similar to propolis.
The primary liquid propolis extract thus prepared was subjected to quantitative analysis of the contents of key active substances 1 to 4 and accompanying components 5 to 10 according to the analytical method described in example 3. The results are shown in tables 5 and 6.
This primary liquid propolis extract, prepared in the Extraction Solvent (ES) described in experiments E1-E6, was diluted with the same ES used in the extraction step to standardize the known quantitative content of active substances 1-4 up to the desired level of the quantitative composition of active substances 1-4 according to the invention.
Example 6 preparation of a standardized liquid Using an extraction solvent based on a mixture of polyethylene glycol 200 and soya lecithin Propolis extract
Propolis was ground and pretreated as described in example 1, and 30.00g of this propolis was mixed with an extraction solvent, polyethylene glycol 200(149.85 g; 99.9%) and soybean lecithin (SL; 0.15 g; 0.1%). The obtained mixture was stirred at room temperature for 1 hour, and left to stand at room temperature for 48 hours while being periodically stirred. The mixture was then filtered through filter paper (black band). 137.00g (93%) of liquid propolis extract in the form of a dark brown viscous liquid with a slightly strong smell similar to propolis was obtained.
Quantitative HPLC analysis was then performed according to the method described in example 3 to obtain quantitative contents of key active substances 1-4. The filtrate was diluted with the same extraction solvent, wherein the mixing ratio of the mixture of polyethylene glycol 200 and Soybean Lecithin (SL) was 99.9:0.1w/w, and the highest level of active substance 1-4 could be obtained according to the specification of standardized liquid extracts of the present invention.
Example 7 preparation of standards Using an extraction solvent based on a mixture of polyethylene glycol 600 and deoiled sunflower lecithin Liquefied propolis extract
Propolis was ground, pretreated as described in example 1, and 30.00g of this propolis was mixed with a mixture of an extraction solvent, polyethylene glycol 600(PEG 600; 297, 00 g; 99%) and deoiled sunflower lecithin (SUL; 3.00 g; 0%). The resulting mixture was heated to 70 ℃ and stirred vigorously for 3 hours. The mixture was then cooled to room temperature and filtered through filter paper (black band). 2600g (87.0%) of liquid propolis extract in the form of a dark brown viscous liquid with a slightly strong smell similar to propolis were obtained.
Then, quantitative HPLC analysis was performed according to the method described in example 3, thereby determining the quantitative content of the key active substances 1 to 4. The filtrate was diluted with the same extraction solvent, i.e. a mixture of polyethylene glycol 600 and deoiled sunflower lecithin (SUL) in a ratio of 99:1w/w, and the highest level of active substance 1-4 could be obtained according to the specification of the standardized liquid extract of the present invention.
Example 8 extraction with a mixture based on polyethylene glycol 200, polyethylene glycol 600 and hydrolysed rapeseed lecithin Preparing standardized liquid propolis extract from solvent
Propolis was ground, pretreated as described in example 1, and 30.00g of this propolis was mixed with a mixture of an extraction solvent, polyethylene glycol 200(PEG 200; 150.00 g; 50%), polyethylene glycol 600(139.50 g; 46.5%) and hydrolyzed rapeseed lecithin (HRL; 10.50 g; 3.5%). The mixture obtained is heated at 100 ℃ for 3 hours with vigorous stirring. The mixture was then cooled to room temperature and filtered through filter paper (black band). 245.00g (81.7%) of liquid propolis extract in the form of a dark brown viscous liquid with a slightly strong smell similar to propolis were obtained.
Then, quantitative HPLC analysis was performed according to the method described in example 3, thereby determining the quantitative content of the key active substances 1 to 4. The filtrate was diluted with the same extraction solvent and a mixture of polyethylene glycol 200, polyethylene glycol 600 and Hydrolysed Rapeseed Lecithin (HRL), 50:46.5:3.5, w/w/w, according to the specification of the standardized liquid extract of the present invention, the highest levels of active substance 1-4 could be obtained.
Example 9 preparation of standardized liquid propolis extract Using mixture of polyethylene glycol 400 and Soybean lecithin
Propolis was ground, pretreated as described in example 1, and 30.00g of this propolis was mixed with a mixture of an extraction solvent, polyethylene glycol 400(PEG 400; 87.30 g; 97% m/m) and soybean lecithin (SL; 2.70 g; 3% w/w). The resulting mixture was left to stand for 72 hours under periodic stirring for impregnation. The mixture was then passed through filter paper (800 pores/cm 2) to give 50-55g of a dark brown viscous liquid with a strong smell similar to propolis. The resulting product was diluted with the same extraction solvent until a mass of 60.00g was reached. In this way, a drug to extract (DER) ratio of 1:2, or said 60g extract is obtained from 30g of starting propolis.
For the pharmaceutical composition formulation of the present invention, the liquid propolis extract prepared is:
(i) quantitative HPLC analysis was carried out according to the method described in example 3 to determine the actual mass concentration of active substances 1-4 in the extract prepared therefrom in the manner described previously,
(ii) pure (fresh) solvent mixture dilutions were normalized: polyethylene glycol 400 (97% w/w) and soya lecithin (3% w/w); until a mass concentration of active substances 1-4 is reached:
(i) p-coumaric acid (1); 10-1300 mu g/ml;
(ii) trans-ferulic acid (2); 10-800 mug/ml;
(iii) caffeic acid (3); 5-300 mug/ml; and the number of the first and second groups,
(iv)3, 4-dihydroxy-trans-cinnamic acid phenethyl ester (4; CAPE); 5-400 mug/ml;
dividing by a factor X/100, wherein X is the weight percent%, w/w, of the standardized liquid propolis extract in the pharmaceutical composition formulation.
Example 10 determination of antimicrobial efficacy of standardized liquid extracts of the invention against model pathogenic microorganisms Determination of the Minimum Inhibitory Concentration (MIC)
The Minimum Inhibitory Concentration (MIC) of the standardized liquid propolis extract of the present invention was determined using the product from example 9, as compared to a similar liquid propolis extract obtained with 96% ethanol (product from example 1) or PEG400 (product from example 2), according to the instructions of the american clinical laboratory standards institute and european antibiotic susceptibility test committee methods. See literature references 26-29.
The following ATCC strains of model pathogenic microorganisms (M) were tested for antimicrobial efficacy under in vitro conditions: staphylococcus aureus ATCC 29293 (M1); methicillin-resistant staphylococcus aureus; MRSA (MFBF Collection; M2); methicillin-sensitive staphylococcus aureus; MSSA (MFBF Collection; M3); enterococcus faecalis ATCC 9212 (M4); enterococcus faecalis (MFBF Collection) (M5); e.coli ATCC 10536 (M6); acinetobacter baumannii ATCC 43498 (M7); pseudomonas aeruginosa ATCC 9027 (M8); and candida albicans ATCC 90028 (M9).
Serial microdilution procedures were performed to determine the Minimum Inhibitory Concentration (MIC) of the extract. Cell suspensions were prepared from parental cultures in PBS buffer (ph7.4) and adjusted to 0.5McFarland units by the turbidity method. The assay was performed in 96-well microtiter plates with serial dilutions ranging from 100-0.7125 μ g/ml, adding 100pl of propolis extract solution dissolved in Milletton broth. 100pl of each bacterial culture was adjusted to 105CFU/mL after incubation. The plates were then incubated at 37 ℃ for 24 hours. MIC was determined by adding 10pl of a 0.5mg/ml solution of 2,3, 5-triphenyl-2H-tetrazolium chloride (TTC; redox indicator) to each individual well and, after 4 hours of incubation at 30 ℃, absorbance was determined spectrophotometrically at a wavelength of 490 nm.
MIC values were determined as propolis extract concentration at which 80% bacterial reduction occurred (MIC 80);
for fungal species, MIC values were determined in RPMI medium supplemented with glucose by the same protocol as for bacteria. After incubation (48h, 37 ℃, aerobic conditions, in the dark), XTT (redox indicator) was added in combination with menadione and the absorbance was determined spectrophotometrically at a wavelength of 540 nm.
MIC values were determined as propolis extract concentration at which 80% bacterial or fungal reduction occurred (MIC 80);
the negative control contained only medium and solvent (no added microorganisms and propolis), while the positive control was exposed to the effects of antibiotics or antifungal agents.
The minimum inhibitory concentration (MIC80) was measured by measuring the antimicrobial activity of the propolis solution in vitro, and is shown in table 9 as a dilution (%) of the liquid extract in a given solvent. The weight ratio of the medicine to the extract (DER) is 1:2 obtaining the starting liquid propolis extract, i.e. the product of example 9. If the dilution of the liquid extract is greater, i.e. the concentration of the MIC active 1-10 is lower, the antibacterial effect of the resulting test extract is higher.
The results are shown in Table 9.
In tables 10 to 15, calculated values of mass concentration () (expressed in [ μ g/ml ]) of each specific active substance, in which each specific extract reached MIC, are given for 1 to 10. The extract was obtained with the following extraction solvents: 96% ethanol (i.e., the product of example 1), polyethylene glycol 400(PEG 400; i.e., the product of example 2), and a mixture of PEG400 (97% w/w) and soy lecithin (3% w/w) (i.e., the product of example 9). These are derived from DER being 1:2 in the primary liquid extract; extraction to reach the dilution (factor) of the corresponding MIC.
Example 11A hair preparation is prepared with at least 150. mu.g/g of active substances 1 to 4 in the form of a solution for intramammary administration Pharmaceutical composition
Formulation (for 100g solution):
---------------------------------------------------------------------
(1)50.00g (50.00% w/w) of a liquid propolis extract according to the invention, product from example 9, standardized to a minimum of 300 μ g/mL of active substance 1-4
(2)50.00g (20.00% w/w) polyethylene glycol 400
---------------------------------------------------------------------
Preparation:ingredients (1) and (2) were mixed and homogenized at room temperature with stirring for 5 minutes. The solution was filtered through filter paper and filled into intramammary syringes in amounts of 4-8 g.
The composition of the solution is as follows: the active substances 1 to 4 have a minimum total concentration of 150. mu.g/g. The results of the quantitative analysis of the HPLC are shown in Table 16, while the corresponding typical HPLC chromatograms are shown in FIG. 5.
EXAMPLE 12 intramammary administration of compositions of the invention with preparation of active substances 1 to 4 at a concentration of at least 100. mu.g/g With suspensions
Formulation (for 100g suspension):
--------------------------------------------------------------------
(1)77.00g (77.00% w/w) of a liquid propolis extract of the invention, from the product of example 9, standardized to a minimum of 150 μ g/mL of active substance 1-4
(2)20.00g (20.00% w/w) polyethylene glycol 4000
(3)3.00g (3.00% w/w) of aluminum distearate
---------------------------------------------------------------------
Preparation: polyethylene glycol (2) was melted at 60 ℃, aluminum distearate (3) was added and homogenized at this temperature for 15 minutes. Then, the mixture was cooled under stirring, and the propolis extract (1) was added at 40-45 ℃. The mixture was further cooled to room temperature while stirring and homogenized for another 15 minutes at room temperature. A pale yellow viscous suspension was obtained, which was filled into intramammary syringes every 4-8 g.
The suspension composition is: active substance 1-4 with minimum total concentration of 100 μ g/g
EXAMPLE 13 preparation of gel formulations containing the composition of the invention with a minimum of 250. mu.g/g of active substance 1 to 4
Formulation (for 100g gel):
---------------------------------------------------------------------
(1)30.00g (30.00% w/w) of a liquid propolis extract according to the invention, from the product of example 9, standardized to a minimum of 850. mu.g/mL of active substance 1-4
(2)2.00g (2.00% w/w) carbomer 940
(3)1.00g (1.00% w/w) Polysorbate 60
(4)0.20g (0.20% w/w) Potassium sorbate
(5)0.30g (0.30% w/w) sodium benzoate
(6)0.30g (0.30% w/w) anhydrous citric acid
(7) Appropriate amount of sodium hydroxide (20% solution)
(8) Adding 100% purified water to 100%
---------------------------------------------------------------------
Preparation: to 30g of the liquid propolis extract of the present invention, (2) is added and homogenized at room temperature for 20 minutes under stirring. Then, 50g of purified water (8) was added, and homogenized for 5 minutes by stirring at room temperature. Thereafter, the components (3-6) were added and dissolved with stirring for 10 minutes. Then, the pH was adjusted to 5.5-6 with (7). To the gel thus obtained, the remaining amount of purified water was added to reach a total mass of 100g, and homogenized by stirring at room temperature for 15 minutes, and finally the product was degassed.
Gel composition: active substance 1-4 with minimum total concentration of 250 μ g/g
Example 14 cream preparation of a composition according to the invention with a minimum of 100. mu.g/g active substance 1 to 4
Formulation (for 100g cream):
---------------------------------------------------------------------
(1)20.00g (20.00% w/w) of a liquid propolis extract according to the invention, from the product of example 9, standardized with a minimum of 500. mu.g/mL of active substance 1-4
(2)5.00g (5.00% w/w) Polysorbate 60
(3)5.00g (5.00% w/w) lanolin, anhydrous
(4)2.00g (2.00% w/w) beeswax, white
(5)8.00g (8.00% w/w) cetyl alcohol
(6)8.00g (8.00% w/w) Vaseline, white
(7)10.00g (10.00% w/w) mineral oil, thick
(8)0.20g (0.20% w/w) 4-chloro-m-cresol
(9) Adding 100% purified water to 100%
---------------------------------------------------------------------
Preparation: the oil phase was prepared by melting the mixture of the components (2-7) at 65-70 ℃ for 15-20 minutes until an almost colorless oily liquid was formed. The aqueous phase was prepared by dissolving (8) and (1) in purified water, followed by heating to 65-70 ℃ with stirring. Then, the aqueous phase is slowly added to the oil phase and mixed well, preferably using a homogenizer capable of high shear homogenization at 1,000-3,000 revolutions per minute (r.p.m.) over a period of 15-20 minutes. The emulsion thus obtained was further stirred vigorously at a temperature of 65 ℃ to 20 ℃ 3 over 30 minutes and gradually cooled.
Cream composition: active substance 1-4 with minimum total concentration of 100 μ g/g
Example 15 preparation of an ointment of a composition according to the invention with a minimum of 500. mu.g/g active substance 1 to 4
Formulation (for 100g ointment):
---------------------------------------------------------------------
(1)50.00g (50.00% w/w) of a liquid propolis extract of the invention, from the product of example 9, standardized to a minimum of 1,000. mu.g/mL of active substance 1-4
(2)10.00g (10.00% w/w) polyethylene glycol 400
(3)40.00g (40.00% w/w) polyethylene glycol 4000
---------------------------------------------------------------------
Preparation: ingredients (1-3) were carefully mixed and heated to 60 ℃, homogenized for 5 minutes by stirring and gradually cooled to room temperature with mixing.
Ointment composition: the minimum total concentration is 1-4 active substances at 500. mu.g/g.
EXAMPLE 16 nasal spray formulations of the compositions of the invention with a minimum of 50. mu.g/g active 1 to 4 were prepared
Formulation (for 100g spray solution):
---------------------------------------------------------------------
(1)5.00g (5.00% w/w) of a liquid propolis extract according to the invention, product from example 9, standardized to a minimum of 1,000. mu.g/mL of active substance 1-4
(2)0.60g (0.60% w/w) sodium chloride
(3)0.10g (0.10% w/w) Polysorbate 60
(4)0.10g (0.10% w/w) potassium sorbate
(5)0.10g (0.10% w/w) sodium benzoate
(6)0.20g (0.20% w/w) citric acid, anhydrous
(7)0.01g (0.01% w/w) sodium ethylenediaminetetraacetate (Na2 EDTA-2H 2O)
(8) Adding 100% purified water to 100%
---------------------------------------------------------------------
Preparation: to 90g of purified water (1), the components (2 to 7) were added and dissolved with stirring at room temperature for 15 minutes. Then, (1) was added and the mixture was homogenized for 15 minutes at room temperature. Thereafter, the solution was filtered through a sterile 0.2 μm filter and filled into suitable sterile bottles equipped with closures and spray pumps for nasal administration.
The composition of the solution is as follows: the minimum total concentration is 50. mu.g/g of active substance 1-4.
EXAMPLE 17 anti-mastitis treatment of cows by a composition of the invention in the form of an intramammary solution of example 11 Investigation of inflammatory Activity
A study of the treatment of cow mastitis was conducted on five holstein cow farms. A total of 86 cows or 339 udder fractions were involved in the study, with the udder fraction as the statistical unit. Animals were kept freely in thick bedding and fed with standard cow premix without antibiotic addition. The study was approved by the veterinary ethics committee. Healthy animals and regions with no clinical symptoms of mastitis and with Somatic Cell Counts (SCC) below 200,000/mL, and infected regions with SCC above 200,000/mL are included. Randomized cross-clinical studies of safety and efficacy were conducted by intramammary (i.mam.) administration of the compositions of the present invention in solution under topographic conditions. The composition of the invention from example 11 was applied three times during the morning milking, during the evening milking and the day after the morning milking at all quarters of the cow udder part. First, the compositions were tested for tolerance in intramammary administration. Changes in cow behavior were monitored, as well as the macroscopic appearance of the udder (edema and redness), milk and sensitivity of the udder to touch. Changes in Somatic Cell Count (SCC) in milk are monitored from the period prior to the first intramammary (i.mam.) administration. The composition is applied the second day of the first application. Milk samples and milk regions were grouped according to whether SCC was high or low 200,000/mL, and whether the samples were positive or negative for bacteriological investigation; see reference 33:
33)European Medicines Agency(1992):Local Tolerance of Intramammary Preparations in Cows.Directive 81/852/EEC.
the propolis composition was then used for efficacy testing of bacterial treatment according to the guidelines of the European Medical Agency (EMA), which is the only measure of efficacy of i.mam. A preparation for treating subclinical mastitis. It is defined as the absence of previously identified pathogens in milk samples collected within some time period after i.mam. Administering a given formulation; see reference 34:
34)European Medicines Agency(2017):Guideline on the conduct of efficacy studies for intramammary products for use in cattle.CVMP.EMA/CVMP/344/1999-Rev.2.
milk sampling was performed using standard methods. Carrying out microbial investigation according to the standard; see reference 35:
35)J.W.Hogan:Laboratory handbook on bovine mastitis.National Mastitis Council(1999)Madison,Wisconsin,SAD.
the bacterial healing results for mastitis in cows are shown in table 17.
EXAMPLE 18 anti-mastitis treatment in goats with a composition according to the invention in the form of an intramammary solution according to example 11 Inflammatory Activity Studies
A study of goat mastitis treatment was performed on 25 goats diagnosed with subclinical mastitis for the left and right galacta. Goats positive for goat milk microbiological survey were divided into two groups: one group to which example 11 of the present invention was appliedThe compound, and the other group was administered amoxicillin and clavulanic acid (cromet)Genus) of the genus; all were dosed using a three-dose regimen.
Tolerance and bacteriological healing of the mammary halves were monitored in parallel after application of composition i.mam of example 11. The antibiotics or compositions from example 11 were administered together by a three-time (two-day) regimen.
During the study, the goats were kept in deep-padded stalls. There were 170 lactating goats and the average lactation yield per goat was 500-600kg during one lactation period lasting about 300 days. They were fed hay as required, a minimum of 2kg per goat, and a premix containing 16% protein, about 1kg per goat; the method is divided into a morning milking part and an evening milking part. Mixing 2% vitamin-mineral premix(Sano Company, Croatia) was added to the premix. Goats with goat milk positive for bacteriological examination were divided into two groups: one group (half of treatment, N ═ 20), administered the composition from example 11; and in another group (N ═ 15), amoxicillin and clavulanic acid (a ═ 15) are administeredAll three administration regimens were used.
During the period of testing for tolerability of the composition from example 11, neither behavioral changes in any goat, nor macroscopic appearance (edema and redness) and changes in milk of the breast, nor sensitivity of the breast to touch were observed. After the first few milkings, milk samples from the left and right galactans were sampled in pre-labeled sterile plastic tubes. Samples were taken before the first administration from the formulation of example 11, 12 hours after the first administration, 24 hours after the first administration and 7 days after the first administration. Milk samples were kept at 4 ℃ until the next day and analyzed for mastitis and raw milk quality in the laboratory of the veterinary institute of Crowdia.
Bacterial treatment efficacy testing was performed according to the guidelines of the European Medicinal Agency (EMA); see reference 34. Taking milk by a standard method, and carrying out microbial examination according to a standard program; see reference 35.
The results of the bacteriological treatment of goat mastitis are shown in table 18.
This example describes the healing of a female horse's foreleg wound. When the mare jumps down, the hind leg presses on the foreleg, creating a deep wound in the ergot area of the foreleg. Conservative treatment of wounds with different formulations was unsuccessful before starting administration of the composition from example 11.
The wound is formed during the mare dance, when the mare jumps, the hind leg is overstretched to the foreleg. The horseshoe shape of the hoof head and hind legs then injures the joint area of the forefoot. This produced oval wounds of 2 x 4cm in size. The horses showed signs of lameness immediately after injury, scored 4/5 (american society of horse practitioners). Immediately after wounding, the wounds were shaved and rinsed with normal cold water, then treated with iodine (povidone iodine, 0.01%) and sprayed with a silver nitrate spray. The wound is closed to avoid infectious contamination. Since the mares had been vaccinated against tetanus, they were not treated with TAT serum. The mares were allowed to stand for two weeks, the wounds were treated daily by mechanical cleaning and kept clean and dry. The wounds were treated with iodine and silver nitrate sprays for 48 hours each. After 5 days, the wound began to heal and became better. Then, the wound treatment with zinc-vitamin ointment was started. After two weeks, the mare was allowed to continue working. However, once the mare begins to dance, the wound begins to bleed. The wound is again disinfected with iodine and a topical antibiotic of cephalosporin in a formulation for intramammary administration is appliedWhile mechanical wound was cleaned, topical treatment with antibiotics was performed for 5 more days. Then, the mare is allowed to continue working and the wound begins bleeding again.
Thereafter, the wound was washed with physiological solution, dried and treated with the composition of example 11 once a day for 5 days. A visible improvement in epithelialization was observed after 48 hours, while the wound was completely healed after 96 hours. Mares lame only in the first few days, but no signs of lameness were observed thereafter. And (5) finishing the mare recovery.
Conclusion
The experimental results show that the specific mixture of liquid polyethylene glycol (PEG) (e.g. PEG400) in combination with 0.1-3.5% by mass of lecithin of the Extraction Solvent (ES) effectively and chemoselectively extracts the active substances p-coumaric acid (1), trans-ferulic acid (2), caffeic acid (3) and 2-phenylethyl 3, 4-dihydroxy-trans-cinnamate (4) in propolis in an unexpected manner compared to pure solvents (e.g. 96% ethanol, PEG400 or a mixture of EtOH and the same lecithin).
By using the proper HPLC analysis method established by the invention, the key active substances 1-4 and the accompanying components 5-10 in the propolis primary liquid extract are accurately and quantitatively determined. Such primary extract is then standardized with the same Extraction Solvent (ES) as in the extraction step. Thus obtaining the liquid propolis extract of the present invention, which has known and standardized concentrations of key active substances 1-4. The standardized propolis extract thus prepared is used as an Active Pharmaceutical Ingredient (API), an Active Cosmetic Ingredient (ACI), or a food ingredient for manufacturing functional foods and food supplements.
The composition of the present invention, which is obtained based on the liquid propolis extract, contains key active substances p-coumaric acid (1; 10-1,300. mu.g/g), trans-ferulic acid (2; 10-800. mu.g/g), caffeic acid (3; 5-300. mu.g/g) and 2-phenylethyl 3, 4-dihydroxy-trans-cinnamate (4; 5-400. mu.g/g), 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 wound healing and treatment of cancer diseases.
INDUSTRIAL APPLICABILITY
The industrial applicability of the present invention is evident due to the wide practical application of liquid propolis extracts and pharmaceutical compositions based thereon.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：通过纳米乳化配制的大麻油粉末、其产生方法和用途