WO2013163714A1 - Green propolis pharmaceutical formulation for use in human odontology - Google Patents

Green propolis pharmaceutical formulation for use in human odontology Download PDF

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Publication number
WO2013163714A1
WO2013163714A1 PCT/BR2013/000151 BR2013000151W WO2013163714A1 WO 2013163714 A1 WO2013163714 A1 WO 2013163714A1 BR 2013000151 W BR2013000151 W BR 2013000151W WO 2013163714 A1 WO2013163714 A1 WO 2013163714A1
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Prior art keywords
propolis
containing green
green propolis
pharmaceutical composition
formulation
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PCT/BR2013/000151
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French (fr)
Portuguese (pt)
Inventor
Esther Margarida ALVES FERREIRA BASTOS
Sílvia LIGÓRIO FIALHO
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Fundação Ezequiel Dias
Fundação de Amparo à Pesquisa do Estado de Minas Gerais
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Publication of WO2013163714A1 publication Critical patent/WO2013163714A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/63—Arthropods
    • A61K35/64—Insects, e.g. bees, wasps or fleas
    • A61K35/644—Beeswax; Propolis; Royal jelly; Honey
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds
    • A61K8/988—Honey; Royal jelly, Propolis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses

Definitions

  • PATENT DESCRIPTION REPORT "PHARMACEUTICAL FORMULATION OF GREEN PROPOLIS FOR HUMAN USE IN DENTISTRY"
  • the present invention relates to the process of producing a green propolis pharmaceutical formulation derived from Baccharis dracunculifolia produced by Apis mellifera bees, with antimicrobial properties for the treatment of oral mucositis and peri-implantitis.
  • Said formulation comprises a colloidal dispersion of the glycolic propolis extract composed of nonionic surfactant and viscous agent capable of reducing the hydrophobia of the extract and its tendency to precipitate formation in aqueous medium.
  • the formulation is applicable to buccal administration.
  • Pliny refers to Propolis as a "medicine” capable of reducing swelling and relieving pain (Gonzalez AG, Mendez RB. Colmena products - Good practices for collection, storage and conservation. Lab de Reference for Investigations and Apicultural Health. Sancti Spiritus, Cuba).
  • propolis is derived from the Greek "pro” meaning defense and “polis” meaning city, ie “city defense of bees”. It is a natural resin produced by bees from secretions and exudates from various plants. These constituents are biotransformed, added with wax and then deposited in the hives where they play an important role in sealing cracks, immobilizing invasive agents and their role in protecting against the proliferation of microorganisms (Ghisalberti EL. Propolis: a review. Bee world, v. 60, pp.59-84, 1979).
  • propolis appeared in 1908 and described its chemical properties and composition. In 1968 came the abstract in Chemical Abstracts R patent using propolis (Romanian to produce bath lotions). The first Brazilian publication on propolis, in 1984, presented a comparative study of the effect of propolis and antibiotics on the inhibition of Staphylococcus aureus (Pereira AS, FRMS Seixas, Neto FRA Propolis: 100 years of research and their future perspectives. Quim. Nova, v. 25, pp. 321-326, 2002).
  • propolis is composed of a mixture of bioactive substances: 30% waxes, 55% resins and balms, 10% essential oils, 5% pollen and other compounds.
  • bioactive substances 30% waxes, 55% resins and balms, 10% essential oils, 5% pollen and other compounds.
  • Many natural products are effective in combating free radicals that cause cell oxidation, but so far none of them have been shown to be more effective than green propolis.
  • terpenes major aromatic acids (3-prenyl-4-hydroxycinnamic and 6-propenoic-2,2-dimethyl-2H-1-benzopyran), caffeic acid (potent hepatotoxic activity), fatty acids, amino acids, vitamins (A, B1, B2, B6, C, E, PP), minerals (Mn, Cu, Ca, Al, Si, V, Ni, Zn, Cr) , high molecular weight compounds, carbohydrates and Artepelin C (Pereira AS, Seixas FRMS, Neto FRA. Propolis: 100 years of research and your future prospects. Quim Nova, v. 25, p. 321-326, 2002).
  • propolis has received great attention from the scientific class. It has already been described for its antimicrobial, antimycotic properties (Brumfitt, Hamilton-Miller JMT, Franklin I. Antibiotics of natural products. Propolis Microbes, v. 62, p. 19-22, 1990; Aga H, Shibuya T, Sugimoto T, Kurimoto M, Naka i S. Isolation and identification of antimicrobial compounds in Brazilian propolis. Biosci Biotech Biochem, v.
  • Propolis has also been successfully tested to combat protozoa by inhibiting the proliferation of Toxoplasma perenniali and Trichomonas vaginalis (Starzyk J, Scheller S, Szaflarski J, Moskwa M, Stojko A. Biological properties and clinical application of propolis II: activity of ethanol extract of propolis Arzneim Forsch, v. 27, pp. 1198-1199, 1997) and in the clinical treatment of giardiase (Mirayes C, Hollands I, Castaneda C. Therapeutic trials with the propolis-based product Propolisin in human giardiasis Acta Gastroent Latinoam, v. 18, pp. 195-202, 1988).
  • Higashi & Castro Higashi KO, Castro SLD. Propolis extracts are effective against Trypanosoma cruzi and have an impact on its interaction with host cells. J Ethnopharmacol, v. 43, p. 149-155, 1994) demonstrated that the extract ethanolic propolis was effective against Trypanossoma cruzi infection. Therefore, propolis cannot be relegated to a category of therapeutic fad, given that its virtues have been recognized for centuries, being reported in numerous works that demonstrate different types of biological activity and applications in various therapies.
  • the oral cavity is naturally colonized by several species of microorganisms, which when organized and structured constitute the oral microbiota (Seabra EJG, Lima IPC, Barbosa SV, Lima KC.
  • saliva contains about 10 bacteria / mL and this number reaches 10 11 in dental plaques, with quantitative and qualitative fluctuation (Trabulsi LR, Alterthum, F. Eds. Microbiota or normal flora of the human body.
  • Microbiology Sao Paulo: Atheneu , 4th ed, pp.
  • Streptococcus The predominant genus in the oral cavity is Streptococcus, being represented by the species: S. mutans, S. sanguis and S. salivarius, the latter being isolated mainly from the tongue, while the other species occur in association with the teeth (Moretti PE. Microbiota bacterial activity: oral microbiota Microorganisms Project, Section 1 - Bacteriology, Part 5 Microbial Associations, available at www: //. fam.br/microrganismos/bacteriologia_microbiota_human a_oral.htm. Accessed on 01.03.08).
  • Streptococcus mutans can colonize the surface of teeth and initiate dental plaque formation by being able to synthesize extracellular polysaccharides (such as insoluble glycans) from sucrose using the glycosyl transferase enzyme (Gibbons RJ, Houte J. Bacterial adherence in oral). microbial ecology Annu Rev Microbiol, v. 29, p.19-44, 1975). This adhesion polysaccharide allows other bacteria to adhere to this microenvironment, initiating the formation of microbial biofilms (Moretti PE. Human bacterial microbiota: oral microbiota. Project Microorganisms, Section 1- Bacteriology, Part 5-Microbial Associations, available at www: //. family / microorganisms / microbiology_microbiota_hum ana_oral.htm. Accessed on 01.03.08).
  • extracellular polysaccharides such as insoluble glycans
  • This adhesion polysaccharide allows other bacteria to
  • Cardiovascular infections bacterial endocarditis of oral origin. Pathogens and pophylaxis. J Clin Periodont, v.17, 494-496, 1990).
  • propolis A very effective antiseptic would be propolis.
  • propolis has already been described for its surface anesthetic action (Paintz M, Metzener J. Zur juanasthetician rial von propolis und Effhatsstofen. Pharmazie, v. 34, p.839 - 841, 1979) as a pre-oral mouthwash.
  • Shrock Son O Carvalho ACP. Application of propolis to dental sockets and skin wounds. J Nihon Uni Sch Dent, v.
  • US Patent 6,803,056 entitled “Methods and Composition for the Treatment of Viral Problems” describes a method and composition for treating lesions associated with viral infections, such as Herpes simplex, by applying an effective amount of a topical composition containing: propolis at a concentration of from 0.5 to 10%, preferably from 1 to 8%; a skin protector at a concentration of 0.5 to 50%, a penetration enhancer at a concentration of 5 to 30%, preferably 5 to 25% and a surfactant at a concentration of 1 to 20% (Dolak TM. Method and composition for treating viral outbreaks (US Patent 6,803,056; 2004). This patent further describes that the formulation exhibits better activity in the treatment of the described lesions, stopping their progression when applied and promoting complete healing, usually between 36 and 48 hours.
  • US Patent 6,153,226 entitled "Therapeutic composition containing a phenolic compound and propolis against viruses, especially herpes viruses” describes a therapeutic composition containing a phenolic compound and propolis against lipids, which contains (i) 100 to 650 parts by weight. of the phenolic component for (ii) one part by weight of propolis (Vachy R, Sauvager F, Amoros M. Therapeutic composition containing a phenol compound and propolis, which is useful against lipid capsid viruses, especially herpes viruses.
  • Patent US 6,153,226 / 2000 Said composition is suitable for the treatment of herpes.
  • US Patent 5,561,116 entitled “Solid Product Containing Propolis Components, Preparation and Use” describes a solid, water-dispersible composition containing propolis components (Nakamura S, Miyake T. Solid product containing propolis components, and preparation and uses thereof. 5,561,116; 1996).
  • Said composition is prepared by extracting the propolis components, which are further mixed with at least one saccharide selected from anhydrous saccharides and cyclodextrins.
  • the composition is suitable for use in different diseases by different application forms.
  • Dispersed systems are those with at least two phases, one of which (dispersed or internal phase) is finely distributed in the other (continuous or external phase) generally called dispersion medium or vehicle.
  • dispersed systems are termed coarse dispersion, colloidal dispersion or molecular dispersion (Morrison ID, Ross S. Colloidal dispersions - suspensions, emulsions, and foams. USA: Wiley-Interscience, 2002, 616 p.).
  • Colloidal dispersions are dispersed systems that are situated between molecular dispersion and coarse dispersion systems. Colloids are molecules or aggregates formed from 10 3 to 10 9 atoms, whose particle diameter is on the order of 10 4 to 10 ⁇ 7 cm, and the particles are distributed in a dispersing medium (Dispersiones colloidales. In: Remington - Pharmacy - 19th edition, AR Gennaro ed; wholesome Aires Pan Medica Editorial, 1998. p 366-425) Therefore, the colloidal dispersions are those with particle size ranging from 1 to 10 nm One property....
  • the characteristic feature of colloidal systems is the Tyndall effect, which occurs when a beam of light passes through the system and is scattered by the presence of suspended colloidal particles, promoting light scattering.
  • the extent of turbidity or opalescence of colloidal scatters depends on the nature, of the size and concentration of colloidal particles present (Russell B, Saville DA, Schowalter W. R. Colloidal dispersions. London: Cambridge university press, 1992, 544 p.).
  • One of the main challenges in the development of colloidal dispersions is to avoid the irreversible aggregation of suspended particles that would lead to internal phase sedimentation.
  • a stable colloidal dispersion has particles that remain suspended in the medium by Brownian mechanism.
  • solutes that modify the dielectric constant and raise the viscosity of the external phase may contribute to the reduction of particle aggregation (González-Caballero F., López-Durán JDG Suspension formulation. Eds Nielloud F., Marti-Masters G. New York: Mareei Dekker, 2000, Chapter 4, pp. 127-190).
  • Colloidal dispersion systems can be classified into colloid dispersion colloid and molecular association colloids (colloidal Dispersiones In: Remington - Pharmacy - 19th edition, AR Gennaro ed; wholesome Aires. Pan Medica Editorial, 1998. 366 p.. - 425).
  • a colloidal association colloid type dispersion has been developed.
  • Association colloids also called micellar colloids, comprise colloidal dispersed systems capable of forming micelles.
  • dispersed molecular substances such as surfactants
  • concentration a certain concentration
  • CMC Generally, hydrophobic or poorly soluble substances are found inside the micelle.
  • Surfactants play another important role in the formulation of colloidal dispersions. They are used as wetting agents, generally with the aim of increasing the miscibility of hydrophobic particles present in an aqueous vehicle (Muller RH, Bohm B, Benita S. Emulsions and nanosuspensions for the formulation of poorly soluble drugs. London: CRC Press. 1998 , 396 p.). These substances tend to accumulate at the interfaces due to their amphiphilic nature, reducing the surface tension of the present phases. The surfactant molecules orient themselves at the liquid-solid interface, so that the hydrocarbon chain is in contact with the solid particle surface, while the polar group is oriented towards the aqueous phase.
  • water-soluble nonionic surfactants are polyoxyethylated, with each molecule consisting of a lipophilic and a polyethylene glycol hydrophilic chain.
  • the surfactant adsorbs at the interface between a hydrophobic solid and water, with its hydrocarbon residue adhered to the solid surface and the water-facing polyethylene glycol residue. In this way, the particle surface is surrounded by a thin layer of. hydrated polyoxyethylene chains.
  • the hydrophilic layer forms a spherical barrier that prevents contact between the particles and hence aggregation (Russell WB, Saville DA, Schowalter W. Colloidal dispersions.
  • Poloxamers are copolymers composed of a polyoxypropylene hydrophobic central chain and two polyoxyethylene hydrophilic chains. Poloxamer is a nonionic surfactant and has the function of emulsifying, solubilizing and wetting agent (Ro and RC, Sheskey PJ, Weller PJ, Ro and R. Handbook of pharmaceutical excipients. 2003. 4 th ed., Washington: APhA Publications, 776p.). It is suitable for the production of aqueous liquid preparations containing hydrophobic substances.
  • Sorbitol is a hexahydric alcohol for mannose and mannitol isomer (Rowe RC, Sheskey PJ, Weller PJ, Rowe R. Handbook of pharmaceutical excipients. 2003.
  • the raw green propolis had its origin confirmed by microscopic laboratory analysis.
  • the extracts were obtained by maceration, centrifugation and sedimentation, for the concentration of phenolic and flavonoid compounds, active components of propolis (Table 1).
  • Antimicrobial activity tests were performed on the green propolis glycol extracts used (Table 1) against microorganisms of clinical interest in the oral cavity: Candida albicans ATCC 36802, Lactobacillus casei ATCC 7469, Porphyromonas gingivalis ATCC 49917, Prevotela melaninogenica ATCC 25845, Staphylococcus aureus ATCC 25923 and Streptococcus mutans ATCC 25175, all from the Reference Laboratory of Microorganisms of the National Institute for Health Quality Control, INCQS / FIOCRUZ, kept viable at 4 ° C in specific solid medium, with periodic subcultures every 20 days. .
  • the positive control was 0.12% (w / v) chlorhexidine hydrochloride salt, a concentration usually employed in clinical practice.
  • the concentrations of glycolic extracts evaluated were: 3, 4, 5 and 7% (v / v). It was evidenced that the concentration of 5% (v / v) of the Glycolic extracts were active against all evaluated microorganisms, inhibiting in 100% their development, similar to that observed for the positive control. This concentration, therefore, was that employed in preparing the formulation of said invention.
  • Example 4 pH measurement of colloidal dispersion containing glycol propolis extract
  • Example 5 Measurement of particle size of colloidal dispersion containing propolis glycolic extract
  • the particle size distribution of the colloidal dispersion was performed by dynamic light scattering at 90 ° optics at 25 ° C using the Zetasizer Model 3000HS (Malvern Instruments Ltd, UK). The result found, 136.5 nm, represents the average particle size. The particle size distribution had a polydispersion equivalent to 0.24.
  • Example 6 Measurement of the electrical charge of the particles of colloidal dispersion containing propolis glycolic extract
  • Colloidal particles are constantly under the influence of attractive forces, so repulsive forces prevent their aggregation (González-Caballero F., López-Durán JDG Suspension formulation. In: Pharmaceutical emulsions and suspensions. Eds. Nielloud F., Marti-Masters G. New York: Mareei Dekker, 2000, chap. 4, pp. 127-190). In most polar liquids, these forces come from particle charges. Potential Zeta is a measure of the intensity of the charge present and represents a clear indication of the stability of a dispersion, so that a value close to zero would indicate colloidal instability and may cause particle aggregation.
  • the viscosity of the developed dispersion was measured using the rotational viscosimeter set at a speed of 60rpm. Measurements were made in triplicate at a constant temperature of 25 ° C. The viscosity value obtained was 5.5 ⁇ 0.4 Cp.
  • Example 8 Measurement of the refractive index of colloidal dispersion containing propolis glycolic extract
  • the refractive index was determined using the refractometer without prior treatment of the formulation. Measurements were made in triplicate at a constant temperature of 25 ° C. The obtained result was 1,366 ⁇ 0,654. This value is close to the water refractive index, thus showing that the system is transparent.
  • Example 9 Evaluation of the stability of the colloidal dispersion containing glycol propolis extract
  • Colloidal dispersions containing propolis glycolic extract prepared for approximately twelve months and stored at 4 ° C, 25 ° C and 40 ° C showed no signs of loss of formulation stability such as: deposit formation, lump formation , crystal growth and phase separation.
  • Antimicrobial activity tests of colloidal dispersion containing green propolis glycolic extract were performed according to the methodology previously described in Example 2. This test was also used as control positive 0.12% (w / v) chlorhexidine digluconate oral solution.
  • FIG. 1 Evaluation of the present invention against Candida albicans yeast (A, B, C) and aerobic bacteria Staphylococcus aureus (D, E, F).
  • a and D Test plates containing formulation of the present invention,
  • B and E microorganism growth control,
  • C and F positive control: 0.12% chlorhexidine di luconate solution.
  • FIG. 3 Evaluation of the present invention against Lactobacillus casei (A, B, C) and Streptococcus mutans (D, E, F).
  • a and D Test plates containing formulation of the present invention,
  • B and E microorganism growth control,
  • C and F positive control: 0.12% chlorhexidine digluconate solution.
  • Porphyromonas gingivalis A, B, C
  • Prevotella melaninogenica D, E, F
  • a and D Test plates containing formulation of the present invention
  • B and E microorganism growth control
  • C and F positive control: 0.12% chlorhexidine digluconate solution.
  • the present invention demonstrated similar antimicrobial activity to that observed for chlorhexidine digluconate oral solution, currently the product of choice in dental offices.
  • the results presented show the therapeutic potential of the present invention as an easily formulated and low cost agent, in substitution for the product currently employed.

Abstract

The present invention relates to the process for preparing a pharmaceutical formulation based on green propolis with antimicrobial properties, which can treat various odontological diseases, such as mucositis and buccal peri-implantitis Said formulation comprises a colloidal dispersion of glycolic propolis extract, composed of a non-ionic surfactant and a thickening agent capable of reducing the hydrophobicity of the extract and the tendency thereof to form precipitates in an aqueous medium. The formulation can be used via buccal administration.

Description

RELATÓRIO DESCRITIVO DA PATENTE DE INVENÇÃO: "FORMULAÇÃO FARMACÊUTICA DE PRÓPOLIS VERDE PARA USO HUMANO EM ODONTOLOGIA"  PATENT DESCRIPTION REPORT: "PHARMACEUTICAL FORMULATION OF GREEN PROPOLIS FOR HUMAN USE IN DENTISTRY"
A presente invenção diz respeito ao processo de produção de formulação farmacêutica a base de própolis verde originada de Baccharis dracunculifolia produzida por abelhas Apis mellifera, com propriedades antimicrobianas para o tratamento de mucosite e periimplantite bucal.  The present invention relates to the process of producing a green propolis pharmaceutical formulation derived from Baccharis dracunculifolia produced by Apis mellifera bees, with antimicrobial properties for the treatment of oral mucositis and peri-implantitis.
A dita formulação compreende uma dispersão coloidal do extrato de própolis glicólico composta por tensoativo não- iônico e agente viscosante capaz de reduzir a hidrofobia do extrato e de sua tendência para formação de precipitados em meio aquoso. A formulação é aplicável à administração bucal .  Said formulation comprises a colloidal dispersion of the glycolic propolis extract composed of nonionic surfactant and viscous agent capable of reducing the hydrophobia of the extract and its tendency to precipitate formation in aqueous medium. The formulation is applicable to buccal administration.
Praticamente todas as civilizações antigas com as suas terapias milenares conheceram e utilizaram os produtos das abelhas como valiosos recursos na medicina. As histórias das medicinas das civilizações Chinesa, Tibetana, Egípcia e Greco - Romana contêm em seus escritos centenas de relatos onde entram esses produtos (mel, própolis, larvas de abelhas e às vezes as próprias abelhas) para curar ou prevenir enfermidades. Textos da Bíblia Sagrada, oriunda da Civilização Hebraica, referem-se à Própolis como "0 Bálsamo de Gileade", utilizada para curar feridas. No antigo Egito (1700 A.C.) era utilizada para embalsamar os mortos. Os Gregos, entre os quais Hipócrates, a adotaram como cicatrizante interno e externo. Plínio, historiador romano, refere-se à Própolis como "medicamento" capaz de reduzir inchaços e aliviar dores (Gonzalez AG, Mendez RB. Los productos de la colmena- Buenas práticas para su recoleccion, almacenamiento y conservacion . Lab. de Referencia para Investigaciones e Salud Apícola. Sancti Spiritus, Cuba) . Virtually all ancient civilizations with their millenary therapies have known and used bee products as valuable resources in medicine. The medicine histories of the Chinese, Tibetan, Egyptian, and Greco - Roman civilizations contain in their writings hundreds of accounts of these products (honey, propolis, bee larvae, and sometimes the bees themselves) to cure or prevent disease. Holy Bible texts, derived from the Hebrew Civilization, refer to Propolis as "0 Balm of Gilead", used to heal wounds. In ancient Egypt (1700 BC) it was used to embalm the dead. The Greeks, including Hippocrates, adopted it as an inner and outer healer. Pliny, Roman historian, refers to Propolis as a "medicine" capable of reducing swelling and relieving pain (Gonzalez AG, Mendez RB. Colmena products - Good practices for collection, storage and conservation. Lab de Reference for Investigations and Apicultural Health. Sancti Spiritus, Cuba).
O termo própolis é derivado do grego "pro" que significa defesa e "polis" que significa cidade, ou seja, "defesa da cidade das abelhas". É uma resina natural produzida pelas abelhas a partir de secreções e exsudatos de várias plantas. Estes constituintes são biotransformados , acrescidos de cera e então depositados nas colméias, onde têm papel importante para vedar frestas, imobilizar agentes invasores além de seu papel na proteção contra a proliferação de microrganismos (Ghisalberti EL. Propolis: a review. Bee world, v.60, p.59-84, 1979).  The term propolis is derived from the Greek "pro" meaning defense and "polis" meaning city, ie "city defense of bees". It is a natural resin produced by bees from secretions and exudates from various plants. These constituents are biotransformed, added with wax and then deposited in the hives where they play an important role in sealing cracks, immobilizing invasive agents and their role in protecting against the proliferation of microorganisms (Ghisalberti EL. Propolis: a review. Bee world, v. 60, pp.59-84, 1979).
Na África do Sul, na guerra do fim do século XIX e na 2a Guerra Mundial, assim como na antiga União Soviética, a própolis foi amplamente utilizada devido às suas propriedades cicatrizantes, com aplicações inclusive no tratamento de tuberculose, observando-se a regressão dos problemas pulmonares (Brooks R, Vick J. A. Estúdios sobre el veneno de abejas. XXXII Congresso Internacional de Apicultura de Apimondia. Brasil. Ed. Apimondia, Rumania, pp. 506-515, 1989; Gonzalez AG. El veneno de abelas. Actualidade y perspectivas. Ed. Paplo de la Torriente. La Habana, Cuba, 1998; Gonzalez AG, Mndez RB. Los productos de la colmena- Buenas práticas para su recoleccion, almacenamiento y conservacion . Lab. de Referencia para Investigaciones e Salud Apícola. Sancti Spiritus, Cuba, 2004) . In South Africa, the war in the late nineteenth century and the second World War, as well as the former Soviet Union, propolis has been widely used because of its healing properties, with applications including the treatment of tuberculosis, observing the regression of lung problems (Brooks R, Vick JA Bee venom studios. XXXII International Apiculture Congress of Apimondia. Brazil. Ed. Apimondia, Rumania, pp. 506-515, 1989; Gonzalez AG. Bee venom. Perspectives Ed. Paplo de la Torriente La Habana, Cuba, 1998; Gonzalez AG, Mndez RB The products of beehive- Good practices for collection, storage and conservation Reference Laboratory for Beekeeping Research and Sancti Spiritus , Cuba, 2004).
O primeiro trabalho científico sobre a própolis surgiu em 1908 e descrevia suas propriedades químicas e composição. Em 1968 surgiu no Chemical Abstracts o resumo da Ia patente utilizando a própolis (Romena, para produzir loções de banho) . A primeira publicação brasileira sobre a própolis, em 1984, apresentou um estudo comparativo do efeito da própolis e de antibióticos na inibição de Staphylococcus aureus (Pereira AS, Seixas FRMS, Neto FRA. Própolis: 100 anos de pesquisa e suas perspectivas futuras. Quim. Nova, v. 25, p. 321-326, 2002) . The first scientific work on propolis appeared in 1908 and described its chemical properties and composition. In 1968 came the abstract in Chemical Abstracts R patent using propolis (Romanian to produce bath lotions). The first Brazilian publication on propolis, in 1984, presented a comparative study of the effect of propolis and antibiotics on the inhibition of Staphylococcus aureus (Pereira AS, FRMS Seixas, Neto FRA Propolis: 100 years of research and their future perspectives. Quim. Nova, v. 25, pp. 321-326, 2002).
A constituição química da própolis é composta por uma mistura de substâncias bioativas: 30% de ceras, 55% de resinas e bálsamos, 10% de óleos essenciais, 5% de pólen e outros compostos. Atualmente, há mais de 300 compostos isolados, e muitos deles já foram registrados para uso medicinal. Muitos produtos naturais são eficientes no combate aos radicais livres que causam a oxidação das células, mas até agora, nenhum deles se mostrou mais eficaz do que a própolis verde.  The chemical composition of propolis is composed of a mixture of bioactive substances: 30% waxes, 55% resins and balms, 10% essential oils, 5% pollen and other compounds. There are currently over 300 isolated compounds, and many of them have already been registered for medicinal use. Many natural products are effective in combating free radicals that cause cell oxidation, but so far none of them have been shown to be more effective than green propolis.
Dentre os vários compostos isolados, podemos citar o ácido cinâmico, compostos fenólicos e flavonóides (Ghisalberti EL. Própolis: a review. Bee world, v.60, p.59- 84, 1979; Bankova V, Popov S, Marekov NL. High performance liquid chromatographic analysis of flavonóides from própolis. J. Chromatography, v. 242, p. 135-143, 1987; Arvouet-Grand A. Extrait de própolis: II. Etude de cicatrisation de plaies chez le lapin et chez le rat . J Pharm Belg, v.48, p. 171-178, 1993), terpenos, ácidos aromáticos principais (3-prenil-4-hidrixicinâmico e o 6- propenóico-2, 2 -dimetil-2H-l-benzopirano) , derivados do ácido caféico (potente atividade hepatotóxica) , ácidos graxos, aminoácidos, vitaminas (A, Bl, B2, B6, C, E, PP ) , minerais (Mn, Cu, Ca, Al, Si, V, Ni, Zn, Cr) , compostos de alta massa molecular, carboidratos e Artepelin C (Pereira AS, Seixas FRMS, Neto FRA. Própolis: 100 anos de pesquisa e suas perspectivas futuras. Quim. Nova, v. 25, p. 321-326, 2002) . Among the various isolated compounds we can mention cinnamic acid, phenolic compounds and flavonoids (Ghisalberti EL. Propolis: a review. Bee world, v.60, p.59-84, 1979; Bankova V, Popov S, Marekov NL. High performance liquid chromatographic analysis of flavonoids from propolis J. Chromatography, v. 242, pp 135-143, 1987; Arvouet-Grand A. Extrait of propolis: II Plaque healing et al chez le lapin et chez le rat. Pharm Belg, v.48, pp. 171-178, 1993), terpenes, major aromatic acids (3-prenyl-4-hydroxycinnamic and 6-propenoic-2,2-dimethyl-2H-1-benzopyran), caffeic acid (potent hepatotoxic activity), fatty acids, amino acids, vitamins (A, B1, B2, B6, C, E, PP), minerals (Mn, Cu, Ca, Al, Si, V, Ni, Zn, Cr) , high molecular weight compounds, carbohydrates and Artepelin C (Pereira AS, Seixas FRMS, Neto FRA. Propolis: 100 years of research and your future prospects. Quim Nova, v. 25, p. 321-326, 2002).
Por ser utilizada no tratamento de várias doenças na medicina popular desde a antiguidade (Hausen BM, oll nweber E, Senff H, Post B. Propolis allergy I. Origin, properties, usage and literature review. Contact Dermatitis, v. 17, p. 163-170, 1987), a propolis vem recebendo grande atenção por parte da classe cientifica. Já foi descrita por suas propriedades antimicrobianas , antimicóticas (Brumfitt , Hamilton-Miller JMT, Franklin I. Antibiotic ectivity of natural products. Propolis Micróbios, v. 62, p. 19-22, 1990; Aga H, Shibuya T, Sugimoto T, Kurimoto M, Naka ima S. Isolation and identification of antimicrobial compounds in brazilian propolis. Biosci Biotech Biochem, v. 58, p. 945-946, 1994), antioxidantes (Scheller S, Wilczoks T, Imielski S, Krol , Gabrys J, Saani J. Free radical scavenging by etanol extract of propolis. Int J Radiat Biol, v. 57, p. 461-465, 1990), antitumorais (Scheller S, Szaflarski J, Tustanowski J, Nolewajka E, Stoj ko A. Biological propoerties and clinicai application of propolis: some physic-chemical properties of propolis. Arzneim Forsch, v. 27, p. 889-890, 1989) regenerativas, cicatrizantes (Arvouet-Grand A. Extrait de propolis: II. Etude de cicatrisation de plaies chez le lapin et chez 1 erat. J Pharm Belg, v.48, p. 171- 178, 1993), antivirais (Esanu V, Prahoveanu E, Crisau I, Cioca A. The effect of aqueous propolis extract of rutin ando f rutin-quercetin mixtures on experimental influenza vírus infection in mice. Virologie, v. 32, p. 213-215, 1981) e imunomodulatórias (Ivanovska ND, Dimov VB, Bankova VS, Popov SS. Immunomodulatory action of propolis IV: influence of water soluble derivative on complemente activity in vivo. J Ethnopharmacol , v. 47, p. 145-147, 1995) . A própolis também foi testada com sucesso no combate a protozoários, inibindo a proliferação de Toxoplasma gandii e Trichomonas vaginalis (Starzyk J, Scheller S, Szaflarski J, Moskwa M, Stojko A. Biological propoerties and clinicai application of propolis II: studies on the antiprotozoan activity of etanol extract of propolis. Arzneim Forsch, v. 27, p. 1198-1199, 1997) e no tratamento clinico de giardiase (Mirayes C, Hollands I, Castaneda C. Therapeutic trials with the propolis-based product Propolisina in human giardiasis. Acta Gastroent Latinoam, v. 18, p. 195-202, 1988). Além disso, Higashi & Castro (Higashi KO, Castro SLD. Propolis extracts are effective against Trypanosoma cruzi and have an impact on its interaction with host cells. J Ethnopharmacol, v. 43, p. 149-155, 1994) demonstraram que o extrato etanólico de própolis foi efetivo contra a infecção por Trypanossoma cruzi. Assim sendo, não se pode relegar a própolis a uma categoria de modismo terapêutico, tendo em vista que suas virtudes são reconhecidas há séculos, sendo relatadas em inúmeros trabalhos que demonstram diferentes tipos de atividade biológica e aplicações em diversas terapias. As it has been used to treat various diseases in folk medicine since ancient times (Hausen BM ,ollweber E, Senff H, Post B. Propolis allergy I. Origin, properties, usage and literature review. Contact Dermatitis, v. 17, p. 163-170, 1987), propolis has received great attention from the scientific class. It has already been described for its antimicrobial, antimycotic properties (Brumfitt, Hamilton-Miller JMT, Franklin I. Antibiotics of natural products. Propolis Microbes, v. 62, p. 19-22, 1990; Aga H, Shibuya T, Sugimoto T, Kurimoto M, Naka i S. Isolation and identification of antimicrobial compounds in Brazilian propolis. Biosci Biotech Biochem, v. 58, pp. 945-946, 1994), antioxidants (Scheller S, Wilczoks T, Imielski S, Krol, Gabrys J, Saani J. Free radical scavenging by ethanol extract of propolis Int J Radiat Biol, v. 57, pp. 461-465, 1990), antitumor (Scheller S, Szaflarski J, Tustanowski J, Nolewajka E, Stojko A. Biological propoerties and clinical application of propolis: some physicochemical properties of propolis Arzneim Forsch, v. 27, pp. 889-890, 1989) regenerative, healing (Arvouet-Grand A. Extrait of propolis: II. le lapin et chez 1 erat J Pharm Belg, v.48, pp. 171-178, 1993), antivirals (Esanu V, Prahoveanu E., Cr. isau I, Cioca A. The effect of aqueous propolis extract from rutin and frutin-quercetin mixtures on experimental influenza virus infection in mice. Virologie, v. 32, p. 213-215, 1981) and immunomodulatory (Ivanovska ND, Dimov VB, Bankova VS, Popov SS. Immunomodulatory action of propolis IV: influence of water soluble derivative on complement activity in vivo. J Ethnopharmacol, v. 47, p. 145-147, 1995). Propolis has also been successfully tested to combat protozoa by inhibiting the proliferation of Toxoplasma gandii and Trichomonas vaginalis (Starzyk J, Scheller S, Szaflarski J, Moskwa M, Stojko A. Biological properties and clinical application of propolis II: activity of ethanol extract of propolis Arzneim Forsch, v. 27, pp. 1198-1199, 1997) and in the clinical treatment of giardiase (Mirayes C, Hollands I, Castaneda C. Therapeutic trials with the propolis-based product Propolisin in human giardiasis Acta Gastroent Latinoam, v. 18, pp. 195-202, 1988). In addition, Higashi & Castro (Higashi KO, Castro SLD. Propolis extracts are effective against Trypanosoma cruzi and have an impact on its interaction with host cells. J Ethnopharmacol, v. 43, p. 149-155, 1994) demonstrated that the extract ethanolic propolis was effective against Trypanossoma cruzi infection. Therefore, propolis cannot be relegated to a category of therapeutic fad, given that its virtues have been recognized for centuries, being reported in numerous works that demonstrate different types of biological activity and applications in various therapies.
Cavidade Oral e o Uso da Própolis  Oral Cavity and the Use of Propolis
A cavidade oral é naturalmente colonizada por várias espécies de microrganismos, que quando organizados e estruturados constituem a microbiota oral (Seabra EJG, Lima IPC, Barbosa SV, Lima KC. Atividade antimicrobiana in vitro de compostos a base de hidróxido de cálcio e tergenol em diferentes concentrações sobre bactérias orais. Acta Cirúrgica Brasileira, v. 20, p. 12 - 18, 2005) . Calcula-se que a saliva contenha cerca de 10 bactérias/mL e que este número atinja IO11 nas placas dentais, havendo flutuação quantitativa e qualitativa (Trabulsi LR, Alterthum, F. Eds . Microbiota ou flora normal do corpo humano. Microbiologia, São Paulo: Atheneu, 4a ed, p. 101-109, 2005) . A alta umidade, temperatura relativamente constante (34 a 36 °C) e disponibilidade continua de nutrientes fazem deste local um microambiente muito propicio ao estabelecimento de aproximadamente 500 grupos bacterianos. Entre os grupos mais representativos estão os géneros Lactobacillus, Actinobacillus, Porphyromonas, Prevotella, Staphylococcus e Streptococcus (Moretti PE. Microbiota bacteriana humana: microbiota oral. Projeto Microrganismos, Seção 1- Bacteriologia, Parte 5-Associações Microbianas, disponível emwww : // . fam. br/microrganismos/bacteriologia_microbiota_hum ana_oral.htm. Acesso em 01.03.08), além das espécies de leveduras do género Cândida (Assis EM. Comparação da eficácia da própolis verde em relação ao miconazol no tratamento de candidíase atrófica crónica - em usuários de prótese total removível. Monografia de Especialização, Pontifícia Universidade Católica de Minas Gerais, 38 p., 2006) . The oral cavity is naturally colonized by several species of microorganisms, which when organized and structured constitute the oral microbiota (Seabra EJG, Lima IPC, Barbosa SV, Lima KC. In vitro antimicrobial activity of calcium hydroxide and tergenol compounds in different concentrations on oral bacteria Acta Cirurggica Brasileira, v. 20, pp. 12 - 18, 2005). It is calculated saliva contains about 10 bacteria / mL and this number reaches 10 11 in dental plaques, with quantitative and qualitative fluctuation (Trabulsi LR, Alterthum, F. Eds. Microbiota or normal flora of the human body. Microbiology, Sao Paulo: Atheneu , 4th ed, pp. 101-109, 2005). The high humidity, relatively constant temperature (34 to 36 ° C) and continuous nutrient availability make this place a very favorable microenvironment for the establishment of approximately 500 bacterial groups. Among the most representative groups are the genera Lactobacillus, Actinobacillus, Porphyromonas, Prevotella, Staphylococcus and Streptococcus (Moretti PE. Human bacterial microbiota: oral microbiota. Project Microorganisms, Section 1- Bacteriology, Part 5-Microbial Associations, available at www: //. family / microorganisms / bacteriology_microbiota_hum ana_oral.htm. Accessed on 01.03.08), besides Candida yeast species (Assis EM. Comparison of the efficacy of green propolis in relation to miconazole in the treatment of chronic atrophic candidiasis - in users of total removable prosthesis (Specialization Monograph, Pontifical Catholic University of Minas Gerais, 38 pp., 2006).
O género predominante na cavidade oral é o Streptococcus, sendo representado pelas espécies: S. mutans, S. sanguis e S. salivarius, sendo esta última isolada principalmente da língua, enquanto as demais espécies ocorrem em associação com os dentes (Moretti PE. Microbiota bacteriana humana: microbiota oral. Projeto Microrganismos, Seção 1 - Bacteriologia, Parte 5 Associações Microbianas, disponível em www: // . fam.br/microrganismos/bacteriologia_microbiota_human a_oral.htm. Acesso em 01.03.08). The predominant genus in the oral cavity is Streptococcus, being represented by the species: S. mutans, S. sanguis and S. salivarius, the latter being isolated mainly from the tongue, while the other species occur in association with the teeth (Moretti PE. Microbiota bacterial activity: oral microbiota Microorganisms Project, Section 1 - Bacteriology, Part 5 Microbial Associations, available at www: //. fam.br/microrganismos/bacteriologia_microbiota_human a_oral.htm. Accessed on 01.03.08).
A espécie Streptococcus mutans pode colonizar a superfície dos dentes e iniciar a formação da placa dentária por ser capaz de sintetizar polissacarídeos extracelulares (como glicanas insolúveis) a partir de sacarose, utilizando a enzima glicosil transferase (Gibbons RJ, Houte J. Bacterial adherence in oral microbial ecology. Annu Rev Microbiol, v. 29, p.19-44, 1975). Este polissacarídeo de aderência permite a adesão de outras bactérias neste microambiente, iniciando a formação de biofilmes microbianos (Moretti PE. Microbiota bacteriana humana: microbiota oral. Projeto Microrganismos, Seção 1- Bacteriologia, Parte 5-Associações Microbianas, disponível emwww : // . fam. br/microrganismos/bacteriologia_microbiota_hum ana_oral.htm. Acesso em 01.03.08).  Streptococcus mutans can colonize the surface of teeth and initiate dental plaque formation by being able to synthesize extracellular polysaccharides (such as insoluble glycans) from sucrose using the glycosyl transferase enzyme (Gibbons RJ, Houte J. Bacterial adherence in oral). microbial ecology Annu Rev Microbiol, v. 29, p.19-44, 1975). This adhesion polysaccharide allows other bacteria to adhere to this microenvironment, initiating the formation of microbial biofilms (Moretti PE. Human bacterial microbiota: oral microbiota. Project Microorganisms, Section 1- Bacteriology, Part 5-Microbial Associations, available at www: //. family / microorganisms / microbiology_microbiota_hum ana_oral.htm. Accessed on 01.03.08).
Estudos têm demonstrado a semelhança entre a flora ao redor dos implantes e a existente ao redor de dentes naturais (Lekholm U. The condition of the soft tissues at tooth and fixture abutments supporting fixed bridges. A microbiological and histological study. J Clin Periodont, v. 13, p. 558-562, 1985; Mombelli A, Lang NP. Microbial aspects of implant dentistry. Periodontol, v. 4, p. 74-80, 1994), indicando que procedimentos terapêuticos, salvo alguns pequenos cuidados específicos, tornam-se comuns aos dois. Assim, sabe-se que a doença periodontal é uma infecção tratável e, portanto, passível de controle e prevenção, quando desenvolvida ao redor de dentes naturais ou implantes (Bauman GR. Plaque-induced inflammation around implants. Int J Oral & Max Impl, v. 7, p. 330-337, 1992) . Os procedimentos odontológicos, principalmente os que causam injúrias aos tecidos moles, podem disseminar microrganismos para órgãos vitais, como coração, cérebro e fígado (Slots J, Taubman MA. Contemporary oral microbiology and immunology. St Louis: Mosby Year Book, 1992). Em estudo desenvolvido por Jasper & Little (Jasper MT, Little JW. Infective endocarditis : a review and update. Oral Surg, v. 57, p.606 - 615, 1984), foi demonstrado que 92% dos casos analisados de endocardite infecciosa teriam sido originados em decorrência da disseminação de microrganismos da cavidade oral, sendo o principal fator de virulência a produção de dextrano (Nord CE, Heimdahl A. Cardiovascular infections: bacterial endocarditis of oral origin. Pathogens and pophylaxis. J Clin Periodont, v.17, p. 494 - 496, 1990) . Alguns pesquisadores partilham da opinião de que a redução do número de microrganismos orais pelo uso de antissépticos antes de qualquer procedimento invasivo diminui a probabilidade de bacteriemia durante tratamento odontológico (MacFarlane TW, Ferguson MM, Mulgrew CJ. Pos - extraction bacteraemia: role of antiseptics and antibiotics. Br Dent J, v. 156, p.179 - 191, 1984). Studies have shown the similarity between the flora around the implants and that surrounding natural teeth (Lekholm U. The condition of the soft tissues at the tooth and the fixation abutments supporting fixed bridges. A Clinical and microbiological study. J Clin Periodont, v 13, pp. 558-562, 1985; Mombelli A, Lang NP Microbial Aspects of Implant Dentistry (Periodontol, v. 4, pp. 74-80, 1994), indicating that therapeutic procedures, except for some minor specific care, render common to both. Thus, periodontal disease is known to be a treatable infection and therefore controllable and preventable when developed around natural teeth or implants (Bauman GR. Plaque-induced inflammation around implants. Int J Oral & Max Impl, v. 7, pp. 330-337, 1992). Dental procedures, especially those that cause soft tissue injuries, can spread microorganisms to vital organs such as the heart, brain and liver (Slots J, Taubman MA. Contemporary oral microbiology and immunology. St Louis: Mosby Year Book, 1992). In a study by Jasper & Little (Jasper MT, Little JW. Infective endocarditis: a review and update. Oral Surg, v. 57, p.606 - 615, 1984), it was shown that 92% of the analyzed cases of infectious endocarditis would have originated from the spread of microorganisms from the oral cavity, the major virulence factor being dextran production (Nord CE, Heimdahl A. Cardiovascular infections: bacterial endocarditis of oral origin. Pathogens and pophylaxis. J Clin Periodont, v.17, 494-496, 1990). Some researchers share the view that reducing the number of oral microorganisms by using antiseptics before any invasive procedure decreases the likelihood of bacteremia during dental treatment (MacFarlane TW, Ferguson MM, Mulgrew CJ. Post - extraction bacteremia: role of antiseptics and antibiotics, Br Dent J, v. 156, p.179 - 191, 1984).
Um antisséptico bastante eficaz seria a própolis. Há relatos de utilização deste produto na cavidade oral por civilizações antigas (Castaldo S, Capasso F. Propolis, an old remedy used in modern medicine. Fitoterapia, v. 73, p. SI - S6, 2002). Na odontologia, a própolis já foi descrita por sua ação anestésica de superfície (Paintz M, Metzener J. Zur lokalanasthetischen wirkung von propolis und einigen inhatsstofen . Pharmazie, v. 34, p.839 - 841, 1979), como enxaguatório bucal pré-cirúrgico (Magro Filho O, Carvalho ACP. Application of propolis to dental sockets and skin wounds. J Nihon Uni Sch Dent, v. 32, p.4 - 13, 1990), enxaguatório bucal de uso diário (Ikeno K, Ikeno P, Miyazawa C. Effects of propolis on dental caries in rats. Caries Res, v. 25, p.347 - 351, 1991), em tratamentos de úlceras bucais recorrentes (Silveira MG. Estúdio preliminary sobre los efectos dei propolan en el tratamiento de la gingivitis crónica y de las ulceras bucales. Rev Cubana de Estomat, v. 25, p. 36 - 44, 1988) e aftas bucais (Garcia CL, Garguera EG. Efectos dei propolan en el tratamiento de aftas bucales. Rev Cuba Med Mil, v. 22, p.42 - 45, 1993). A atividade antimicrobiana sobre algumas bactérias orais também já foi demonstrada, assim como a inibição da enzima glicosil transferase, responsável pela formação de placa. A very effective antiseptic would be propolis. There are reports of use of this product in the oral cavity by ancient civilizations (Castaldo S, Capasso F. Propolis, an old remedy used in modern medicine. Phytotherapy, v. 73, p. SI - S6, 2002). In dentistry, propolis has already been described for its surface anesthetic action (Paintz M, Metzener J. Zur lokalanasthetischen wirkung von propolis und einigen inhatsstofen. Pharmazie, v. 34, p.839 - 841, 1979) as a pre-oral mouthwash. (Slim Son O, Carvalho ACP. Application of propolis to dental sockets and skin wounds. J Nihon Uni Sch Dent, v. 32, p.4 - 13, 1990), daily mouthwash (Ikeno K, Ikeno P, Miyazawa C. Effects of propolis on dental caries in rats. Caries Res, v. 25, p.347 - 351, 1991) , in treatments for recurrent mouth ulcers (Silveira MG. Preliminary studio on the effects of prophylaxis on the treatment of chronic gingivitis and oral ulcers. Rev Cubana de Estomat, v. 25, p. 36 - 44, 1988) and thrush (Garcia CL, Garguera EG. Effects of propolis on the treatment of oral thrush. Rev Cuba Med Mil, v. 22, p.42 - 45, 1993). Antimicrobial activity on some oral bacteria has also been demonstrated, as has inhibition of the enzyme glycosyl transferase, which is responsible for plaque formation.
São encontradas algumas patentes relacionadas à utilização da propolis em formulações farmacêuticas.  Some patents related to the use of propolis in pharmaceutical formulations are found.
A patente US 6,803,056 intitulada "Métodos e composição para o tratamento de problemas virais" descreve um método e uma composição para o tratamento de lesões associadas a infecções virais, tais como Herpes simplex, pela aplicação de uma quantidade efetiva de uma composição tópica contendo: extrato de propolis na concentração de 0,5 a 10%, preferencialmente de 1 a 8%; um protetor cutâneo na concentração de 0,5 a 50%, um promotor de penetração na concentração de 5 a 30%, preferencialmente de 5 a 25% e um agente tensoativo na concentração de 1 a 20% (Dolak TM. Method and composition for treating virai outbreaks. Patent US 6,803,056; 2004). Esta patente descreve, ainda, que a formulação apresenta melhor atividade no tratamento das lesões descritas, interrompendo a sua progressão quando aplicada e promovendo cicatrização completa, geralmente entre 36 e 48 horas. US Patent 6,803,056 entitled "Methods and Composition for the Treatment of Viral Problems" describes a method and composition for treating lesions associated with viral infections, such as Herpes simplex, by applying an effective amount of a topical composition containing: propolis at a concentration of from 0.5 to 10%, preferably from 1 to 8%; a skin protector at a concentration of 0.5 to 50%, a penetration enhancer at a concentration of 5 to 30%, preferably 5 to 25% and a surfactant at a concentration of 1 to 20% (Dolak TM. Method and composition for treating viral outbreaks (US Patent 6,803,056; 2004). This patent further describes that the formulation exhibits better activity in the treatment of the described lesions, stopping their progression when applied and promoting complete healing, usually between 36 and 48 hours.
A patente US 6,153,226 intitulada "Composição terapêutica contendo um composto fenólico e própolis eficiente contra virus, especialmente os virus da herpes" descreve uma composição terapêutica contendo um composto fenólico e própolis eficiente contra virus lipidicos, qual contem (i) 100 a 650 partes por peso do componente fenólico para (ii) uma parte por peso de própolis (Vachy R, Sauvager F, Amoros M. Therapeutic composition containing a phenol compound and própolis, which is useful against lipid capsid viruses, especially herpes viruses. Patent US 6,153,226/ 2000). A referida composição é adequada para o tratamento de herpes.  US Patent 6,153,226 entitled "Therapeutic composition containing a phenolic compound and propolis against viruses, especially herpes viruses" describes a therapeutic composition containing a phenolic compound and propolis against lipids, which contains (i) 100 to 650 parts by weight. of the phenolic component for (ii) one part by weight of propolis (Vachy R, Sauvager F, Amoros M. Therapeutic composition containing a phenol compound and propolis, which is useful against lipid capsid viruses, especially herpes viruses. Patent US 6,153,226 / 2000) . Said composition is suitable for the treatment of herpes.
A patente US 5,561,116 intitulada "Produto sólido contendo componentes da própolis, preparo e uso" descreve uma composição sólida, dispersivel em água contendo componentes da própolis (Nakamura S, Miyake T. Solid product containing própolis components, and preparation and uses thereof. Patent US 5,561,116; 1996). A referida composição é preparada pela extração dos componentes da própolis, os quais são posteriormente misturados com pelo menos um sacarideo selecionado de sacarideos anidros e ciclodextrinas . A composição é adequada para utilização em diferentes doenças por diferentes formas de aplicação.  US Patent 5,561,116 entitled "Solid Product Containing Propolis Components, Preparation and Use" describes a solid, water-dispersible composition containing propolis components (Nakamura S, Miyake T. Solid product containing propolis components, and preparation and uses thereof. 5,561,116; 1996). Said composition is prepared by extracting the propolis components, which are further mixed with at least one saccharide selected from anhydrous saccharides and cyclodextrins. The composition is suitable for use in different diseases by different application forms.
Atualmente, encontramos disponíveis no mercado algumas formulações contendo própolis para uso em odontologia. Estas formulações estão presentes na forma de solução oral ou extrato concentrado. No entanto, geralmente, o álcool está presente na composição destas formulações, o que pode causar irritação para a mucosa oral e não adesão do paciente ao tratamento. We currently find some formulations containing propolis available for use in dentistry. These formulations are present in the form of oral solution or concentrated extract. However, alcohol is generally present in the composition of these formulations, which may cause irritation to the oral mucosa and nonadherence to treatment.
Os sistemas dispersos são aqueles que apresentam pelo menos duas fases, sendo que uma delas (fase dispersa ou interna) encontra-se finamente distribuída na outra (fase contínua ou externa) geralmente denominada meio de dispersão ou veículo. Dependendo do tamanho das partículas, os sistemas dispersos são denominados dispersão grosseira, dispersão coloidal ou dispersão molecular (Morrison I D, Ross S. Colloidal dispersions - suspensions, emulsions, and foams. USA: Wiley-Interscience, 2002, 616 p.).  Dispersed systems are those with at least two phases, one of which (dispersed or internal phase) is finely distributed in the other (continuous or external phase) generally called dispersion medium or vehicle. Depending on particle size, dispersed systems are termed coarse dispersion, colloidal dispersion or molecular dispersion (Morrison ID, Ross S. Colloidal dispersions - suspensions, emulsions, and foams. USA: Wiley-Interscience, 2002, 616 p.).
As dispersões coloidais são sistemas dispersos que se encontram situados entre os sistemas de dispersão molecular e os de dispersão grosseira. São denominados colóides as moléculas ou agregados formados por IO3 a IO9 átomos, cujo diâmetro de partícula é da ordem de IO"4 a 10~7 cm, sendo que as partículas estão distribuídas em um meio dispersante (Dispersiones coloidales . In: Remington - Farmácia - 19a edição; ed. A R Gennaro; Buenos Aires: Editorial Medica Panamericana, 1998. p. 366-425). Portanto, as dispersões coloidais são aquelas que apresentam partículas de tamanho que varia entre 1 e 10 nm. Uma propriedade óptica característica dos sistemas coloidais é o efeito Tyndall, que ocorre quando um feixe de luz atravessa o sistema e é dispersado pela presença de partículas coloidais suspensas, promovendo a dispersão da luz. A extensão da turbidez ou da opalescência das dispersões coloidais depende da natureza, do tamanho e da concentração de partículas coloidais presentes (Russel B, Saville D A, Schowalter W R. Colloidal dispersions. London: Cambridge university press, 1992, 544 p. ) . Um dos principais desafios no desenvolvimento de dispersões coloidais é evitar a agregação irreversível das partículas suspensas, que levariam a sedimentação da fase interna. Uma dispersão coloidal estável apresenta partículas que permanecem suspensas no meio por mecanismo Browniano. A adição de solutos que modificam a constante dielétrica e elevam a viscosidade da fase externa, como os polióis, pode contribuir para a redução da agregação das partículas (González-Caballero F. , López-Durán J. D. G. Suspension formulation. In: Pharmaceutical emulsions and suspensions. Eds . Nielloud F., Marti-Mestres G. New York: Mareei Dekker, 2000, cap. 4, p. 127-190) . Colloidal dispersions are dispersed systems that are situated between molecular dispersion and coarse dispersion systems. Colloids are molecules or aggregates formed from 10 3 to 10 9 atoms, whose particle diameter is on the order of 10 4 to 10 ~ 7 cm, and the particles are distributed in a dispersing medium (Dispersiones colloidales. In: Remington - Pharmacy - 19th edition, AR Gennaro ed; Buenos Aires Pan Medica Editorial, 1998. p 366-425) Therefore, the colloidal dispersions are those with particle size ranging from 1 to 10 nm One property.... The characteristic feature of colloidal systems is the Tyndall effect, which occurs when a beam of light passes through the system and is scattered by the presence of suspended colloidal particles, promoting light scattering. The extent of turbidity or opalescence of colloidal scatters depends on the nature, of the size and concentration of colloidal particles present (Russell B, Saville DA, Schowalter W. R. Colloidal dispersions. London: Cambridge university press, 1992, 544 p.). One of the main challenges in the development of colloidal dispersions is to avoid the irreversible aggregation of suspended particles that would lead to internal phase sedimentation. A stable colloidal dispersion has particles that remain suspended in the medium by Brownian mechanism. The addition of solutes that modify the dielectric constant and raise the viscosity of the external phase, such as polyols, may contribute to the reduction of particle aggregation (González-Caballero F., López-Durán JDG Suspension formulation. Eds Nielloud F., Marti-Masters G. New York: Mareei Dekker, 2000, Chapter 4, pp. 127-190).
Os sistemas de dispersão coloidal podem ser classificados em colóides de dispersão, colóides de associação e colóides moleculares (Dispersiones coloidales. In: Remington - Farmácia - 19a edição; ed. A R Gennaro; Buenos Aires: Editorial Medica Panamericana, 1998. p. 366- 425) . Na presente invenção, foi desenvolvida uma dispersão coloidal do tipo colóides de associação. Os colóides de associação, também denominados colóides micelares, compreendem os sistemas dispersos coloidais capazes de formar micelas. Neste caso, substâncias moleculares dispersas, como os tensoativos, são capazes de melhorar a miscibilidade de substâncias hidrofóbicas sem produzir alterações na estrutura química destas, pela formação de agregados moleculares denominados micelas, originados em uma solução de tensoativos acima de determinada concentração, conhecida como concentração micelar críticaColloidal dispersion systems can be classified into colloid dispersion colloid and molecular association colloids (colloidal Dispersiones In: Remington - Pharmacy - 19th edition, AR Gennaro ed; Buenos Aires. Pan Medica Editorial, 1998. 366 p.. - 425). In the present invention, a colloidal association colloid type dispersion has been developed. Association colloids, also called micellar colloids, comprise colloidal dispersed systems capable of forming micelles. In this case, dispersed molecular substances, such as surfactants, are capable of improving the miscibility of hydrophobic substances without producing changes in their chemical structure by the formation of molecular aggregates called micelles, originated in a surfactant solution above a certain concentration, known as concentration. critical micellar
(CMC) . Geralmente, substâncias hidrofóbicas ou pouco solúveis em água, encontram-se no interior da micela(CMC). Generally, hydrophobic or poorly soluble substances are found inside the micelle.
(González-Caballero F., López-Durán J. D. G. Suspension formulation. In: Pharmaceutical emulsions and suspensions. Eds. Nielloud F. , Marti-Mestres G. New York: Mareei Dekker, 2000, cap. 4, p. 127-190) . (González-Caballero F., López-Durán JDG Suspension formulation. In: Pharmaceutical emulsions and suspensions. Eds. Nielloud F., Marti-Masters G. New York: Mareei Dekker, 2000, ch. 4, p. 127-190).
Os tensoativos apresentam um outro papel importante na formulação de dispersões coloidais. Eles são utilizados como agentes molhantes, geralmente com o objetivo de aumentar a miscibilidade das partículas hidrofóbicas presentes em um veículo aquoso (Muller RH, Bohm B, Benita S. Emulsions and nanosuspensions for the formulation of poorly soluble drugs. London: CRC Press. 1998, 396 p.). Estas substâncias tendem a acumular-se nas interfaces devido a sua natureza anfifílica, reduzindo a tensão superficial das fases presentes. As moléculas de tensoativo se orientam na interface líquido-sólido, de forma que a cadeia hidrocarbônica encontra-se em contato com a superfície da partícula sólida, enquanto o grupo polar se orienta para a fase aquosa. A maioria dos tensoativos não iónicos, solúveis em água, é polioxietilada, sendo que cada molécula é constituída por uma cadeia de hidrocarbonos lipofílica e uma hidrofílica de polietilenoglicol . O tensoativo se adsorve na interface entre um sólido hidrófobo e a água, com seu resíduo de hidrocarbono aderido à superfície sólida e o resíduo polietilenoglicol voltado para a água. Desta forma, a superfície da partícula encontra-se rodeada por uma camada delgada de. cadeias de polioxietileno hidratadas. A camada hidrofílica forma uma barreira esférica que impede o contato entre as partículas e consequentemente, a agregação (Russel W B, Saville D A, Schowalter W R. Colloidal dispersions. London: Cambridge university press, 1992, 544 p.) Até o momento, o estado da arte não descreve claramente um sistema que permita a forma de administração oral de extrato de própolis, sem que ocorra a formação de precipitados. Uma alternativa para esta formulação consiste no emprego das dispersões. A presente invenção baseia-se na utilização de uma dispersão coloidal compatível com a administração bucal em mamíferos, contendo extrato de própolis. Esta formulação é aplicável para utilização como enxaguatório bucal no tratamento de diferentes doenças odontológicas. Para composição do sistema são utilizados, como adjuvantes farmacêuticos, o poloxamer e o sorbitol. Surfactants play another important role in the formulation of colloidal dispersions. They are used as wetting agents, generally with the aim of increasing the miscibility of hydrophobic particles present in an aqueous vehicle (Muller RH, Bohm B, Benita S. Emulsions and nanosuspensions for the formulation of poorly soluble drugs. London: CRC Press. 1998 , 396 p.). These substances tend to accumulate at the interfaces due to their amphiphilic nature, reducing the surface tension of the present phases. The surfactant molecules orient themselves at the liquid-solid interface, so that the hydrocarbon chain is in contact with the solid particle surface, while the polar group is oriented towards the aqueous phase. Most water-soluble nonionic surfactants are polyoxyethylated, with each molecule consisting of a lipophilic and a polyethylene glycol hydrophilic chain. The surfactant adsorbs at the interface between a hydrophobic solid and water, with its hydrocarbon residue adhered to the solid surface and the water-facing polyethylene glycol residue. In this way, the particle surface is surrounded by a thin layer of. hydrated polyoxyethylene chains. The hydrophilic layer forms a spherical barrier that prevents contact between the particles and hence aggregation (Russell WB, Saville DA, Schowalter W. Colloidal dispersions. London: Cambridge university press, 1992, 544 p.) To date, the state of the art does not clearly describe a system that allows the oral administration of propolis extract without precipitate formation. An alternative to this formulation is the use of dispersions. The present invention is based on the use of a colloidal dispersion compatible with buccal administration in mammals containing propolis extract. This formulation is applicable for use as a mouthwash in the treatment of different dental diseases. For the system composition, poloxamer and sorbitol are used as pharmaceutical adjuvants.
Os poloxamers são copolímeros compostos por uma cadeia central hidrofóbica de polioxipropileno e duas cadeias hidrofílicas de polioxietileno . 0 poloxamer é um tensoativo não iônico, e tem função de agente emulsionante, solubilizante e molhante (Ro e RC, Sheskey PJ, Weller PJ, Ro e R. Handbook of pharmaceutical excipients. 2003. 4th ed., Washington:APhA Publications , 776p.). É adequado para a produção de preparações líquidas aquosas contendo substâncias hidrofóbicas. Sua função é alcançada pelo processo onde moléculas do tensoativo são adsorvidas pelas partículas formando uma película ao redor delas e diminuindo a tensão superficial entre a substância dispersada (no caso, as partículas do extrato de própolis) e a fase líquida (água) . Desta forma, ocorrerá diminuição ou até mesmo impedimento da formação de grumos de partículas no líquido e o fenómeno de flutuação, em que substâncias dificilmente molháveis (muito hidrofóbicas) em presença de água se agrupam formando aglomerados que contêm gases que se fixam à superfície e fazem flutuar o aglomerado no líquido de suspensão. O sorbitol é um álcool hexahidrico referente à manose e isômero do manitol (Rowe RC, Sheskey PJ, Weller PJ, Rowe R. Handbook of pharmaceutical excipients. 2003. 4th ed., Washington :APhA Publications , 776p.; Sweetman SC. artindale - the complete drug reference. 2002. 32 ed. London : Pharmaceutical Press. 2483 p.). É relativamente inerte e compatível com a maioria dos excipientes ; não escurece nem decompõe em altas temperaturas. Ele é utilizado em preparações líquidas de uso oral, em formulações injetáveis, em suspensões e emulsões de uso interno e externo. Sua principal função em sistemas dispersos é a de agente viscosante, sem, entretanto, promover significativas alterações no comportamento reológico do produto final. Poloxamers are copolymers composed of a polyoxypropylene hydrophobic central chain and two polyoxyethylene hydrophilic chains. Poloxamer is a nonionic surfactant and has the function of emulsifying, solubilizing and wetting agent (Ro and RC, Sheskey PJ, Weller PJ, Ro and R. Handbook of pharmaceutical excipients. 2003. 4 th ed., Washington: APhA Publications, 776p.). It is suitable for the production of aqueous liquid preparations containing hydrophobic substances. Their function is achieved by the process where surfactant molecules are adsorbed by the particles forming a film around them and decreasing the surface tension between the dispersed substance (in this case the propolis extract particles) and the liquid phase (water). This will reduce or even prevent particle lumps from forming in the liquid and the fluctuation phenomenon, in which difficult-to-water (very hydrophobic) substances in the presence of water will group together to form clusters that contain surface-bound gases and make float the agglomerate in the suspension liquid. Sorbitol is a hexahydric alcohol for mannose and mannitol isomer (Rowe RC, Sheskey PJ, Weller PJ, Rowe R. Handbook of pharmaceutical excipients. 2003. 4 th ed., Washington: APhA Publications, 776p .; Sweetman SC. Artindale - the complete drug reference, 2002. 32 ed. London: Pharmaceutical Press. 2483 p.). It is relatively inert and compatible with most excipients; does not darken or decompose at high temperatures. It is used in oral liquid preparations, injectable formulations, internal and external suspensions and emulsions. Its main function in dispersed systems is viscous agent, without, however, promoting significant changes in the rheological behavior of the final product.
A presente invenção é adicionalmente descrita pelos seguintes exemplos:  The present invention is further described by the following examples:
Exemplo 1 - Obtenção da própolis verde  Example 1 - Obtaining Green Propolis
Na presente invenção foi empregado como base o extrato glicólico de própolis verde, originária da espécie vegetal Baccharis dracunculifolia , popular alecrim do campo, obtida comercialmente de apicultores instalados no Estado de Minas Gerais. A própolis verde é intensamente produzida por abelhas africanas da espécie Apis mellifera, em diferentes regiões do Estado (áreas de cerrado, Sul de Minas, Zona da Mata e em áreas antrópicas), pela abundância da espécie fornecedora de resina.  Glycolic extract of green propolis from the plant species Baccharis dracunculifolia, popular field rosemary, obtained commercially from beekeepers in the state of Minas Gerais, was used as a base. Green propolis is intensively produced by African bees of the species Apis mellifera, in different regions of the state (cerrado areas, southern Minas Gerais, Zona da Mata and in anthropic areas), due to the abundance of the resin supplying species.
Para produção dos extratos, a própolis verde em estado bruto teve sua origem confirmada através de análises laboratoriais microscópicas. Os extratos foram obtidos por meio de maceração, centrifugação e sedimentação, para a concentração dos compostos fenólicos e flavonóides, componentes ativos da própolis (Tabela 1) . For the production of extracts, the raw green propolis had its origin confirmed by microscopic laboratory analysis. The extracts were obtained by maceration, centrifugation and sedimentation, for the concentration of phenolic and flavonoid compounds, active components of propolis (Table 1).
Tabela 1 - Detalhamento dos extratos de própolis verde utilizados  Table 1 - Detailing of green propolis extracts used
Análise Físico- Physical Analysis-
Amostr química Chemical sample
Descrição (%) as Fenólic Flavonói os des  Description (%) as Phenolic Flavonoid des
156 Extrato glicólico®, PO 13/07 4,5 1, 28156 Glycolic Extract ® , PO 13/07 4,5 1, 28
164 Extrato glicólico, POa 13/07, 3,7 1,4 164 Glycolic Extract, POa 13/07, 3.7 1.4
174 Extrato glicólico, lote 16/08 6, 85 1,44 174 Glycolic Extract, Lot 16/08 6, 85 1.44
176 Extrato glicólico, lote 19/08 7, 38 3, 22 176 Glycolic Extract, lot 8/19 7, 38 3, 22
Valores de referência para ≥  Reference values for ≥
> 0,5%  > 0.5%
própolis verde 0,25%  green propolis 0.25%
Exemplo 2 - Avaliação da atividade antimicrobiana dos extratos glicólicos de própolis verde utilizados Example 2 - Evaluation of antimicrobial activity of green propolis glycol extracts used
Foram realizados testes de atividade antimicrobiana dos extratos glicólicos de própolis verde utilizados (Tabela 1) frente a microrganismos de interesse clínico na cavidade oral, sendo estes: Cândida albicans ATCC 36802, Lactobacillus casei ATCC 7469, Porphyromonas gingivalis ATCC 49917, Prevotela melaninogenica ATCC 25845, Staphylococcus aureus ATCC 25923 e Streptococcus mutans ATCC 25175, todas provenientes do Laboratório de Microrganismos de Referência do Instituto Nacional de Controle de Qualidade em Saúde, INCQS/FIOCRUZ , mantidas viáveis a 4°C em meio sólido específico, com subculturas periódicas a cada 20 dias.  Antimicrobial activity tests were performed on the green propolis glycol extracts used (Table 1) against microorganisms of clinical interest in the oral cavity: Candida albicans ATCC 36802, Lactobacillus casei ATCC 7469, Porphyromonas gingivalis ATCC 49917, Prevotela melaninogenica ATCC 25845, Staphylococcus aureus ATCC 25923 and Streptococcus mutans ATCC 25175, all from the Reference Laboratory of Microorganisms of the National Institute for Health Quality Control, INCQS / FIOCRUZ, kept viable at 4 ° C in specific solid medium, with periodic subcultures every 20 days. .
A fim de comparar a eficácia dos extratos de própolis verde, foi empregado como controle positivo o sal cloridrato de clorexidina a 0,12% (p/v), concentração usualmente empregada na prática clínica. As concentrações dos extratos glicólicos avaliadas foram: 3, 4, 5 e 7 % (v/v) . Foi evidenciado que a concentração de 5% (v/v) dos extratos glicólicos foi ativa frente a todos os microrganismos avaliados, inibindo em 100% o desenvolvimento destes, semelhante ao observado para o controle positivo. Esta concentração, portanto, foi a empregada no preparo da formulação da dita invenção. In order to compare the efficacy of green propolis extracts, the positive control was 0.12% (w / v) chlorhexidine hydrochloride salt, a concentration usually employed in clinical practice. The concentrations of glycolic extracts evaluated were: 3, 4, 5 and 7% (v / v). It was evidenced that the concentration of 5% (v / v) of the Glycolic extracts were active against all evaluated microorganisms, inhibiting in 100% their development, similar to that observed for the positive control. This concentration, therefore, was that employed in preparing the formulation of said invention.
Exemplo 3 - Preparo da formulação de dispersão coloidal contendo extrato de própolis Example 3 - Preparation of Colloidal Dispersion Formulation Containing Propolis Extract
Para o preparo do enxaguatório bucal foram empregados apenas extratos que apresentaram concentração de compostos fenólicos maior ou igual a 0,5% (v/v) e flavonóides maior ou igual a 0,25% (v/v) (Tabela 1). Os extratos glicólicos foram escolhidos para a formulação farmacêutica da' presente invenção por ser o propilenoglicol um produto com excelente solvência, atuando na solubilidade e dispersão dos ativos contidos nas amostras de própolis. Ainda, o propilenoglicol é de fácil manipulação, fator importante no processo de formulação de um enxaguatório bucal, e de grande aceitabilidade pelos pacientes, por não causar irritação à mucosa oral.  For the mouthwash preparation, only extracts with phenolic compounds concentration greater than or equal to 0.5% (v / v) and flavonoids greater than or equal to 0.25% (v / v) were used (Table 1). Glycolic extracts were chosen for the pharmaceutical formulation of the present invention because propylene glycol is a product with excellent solvency, acting on the solubility and dispersion of the actives contained in the propolis samples. Moreover, propylene glycol is easy to handle, an important factor in the process of formulating a mouthwash, and of great acceptance by patients, as it does not cause oral mucosa irritation.
Para o preparo da dispersão coloidal, inicialmente, 1- 3% de tensoativo do tipo não iônico (polissorbato 80 ou poloxamer F127) foram pesados e adicionados a um frasco contendo 7,5-9,5 ml de água destilada, sob agitação constante. Em seguida, 20-30% de sorbitol foi pesado e adicionado ao mesmo frasco, sob agitação constante, até a solução se encontrar completamente homogénea. Em seguida, 5% do extrato glicólico de própolis foi adicionado sobre a solução anteriormente preparada, sob agitação constante até formação de uma dispersão coloidal. O volume foi completado para 10,0 mL e a dispersão formada foi agitada até completa homogeneização . A Figura 1 mostra uma foto do extrato glicólico de propolis diluído em água e do mesmo extrato de propolis preparado conforme a formulação da presente invenção. Foi observado que a formulação obtida se apresentou homogénea, transparente, sem o depósito de grumos ou partículas, contrário da preparação que continha o extrato diluído em água . To prepare the colloidal dispersion, initially 1-3% nonionic surfactant (polysorbate 80 or poloxamer F127) were weighed and added to a flask containing 7.5-9.5 ml distilled water under constant stirring. Then 20-30% sorbitol was weighed and added to the same flask under constant stirring until the solution was completely homogeneous. Then 5% of the glycol propolis extract was added over the previously prepared solution under constant stirring until a colloidal dispersion formed. The volume was completed to 10.0 mL and the dispersion formed was stirred until complete homogenization. Figure 1 shows a photo of the water-diluted glycolic propolis extract and the same propolis extract prepared according to the formulation of the present invention. It was observed that the formulation obtained was homogeneous, transparent, without the deposition of lumps or particles, contrary to the preparation containing the extract diluted in water.
Figure imgf000019_0001
fato não observado na formulação desenvolvida.
Figure imgf000019_0001
fact not observed in the developed formulation.
Exemplo 4: Medida do pH da dispersão coloidal contendo extrato glicólico de propolis Example 4: pH measurement of colloidal dispersion containing glycol propolis extract
O pH da formulação desenvolvida foi medido utilizando o pHmetro Digimed, modelo DM-20 (Digicron Analítica LTDA, Brasil) . O valor estabelecido para a faixa desejada que não possibilitasse a ocorrência de irritação na mucosa oral foi de 6,5-7,5. Valores abaixo ou acima foram corrigidos para atingir esta faixa. Exemplo 5: Medida do tamanho das partículas da dispersão coloidal contendo extrato glicólico de própolis The pH of the developed formulation was measured using the Digimed pHmeter, model DM-20 (Digicron Analytical LTDA, Brazil). The value established for the desired range that did not allow the occurrence of oral mucosa irritation was 6.5-7.5. Values below or above have been corrected to reach this range. Example 5: Measurement of particle size of colloidal dispersion containing propolis glycolic extract
A distribuição do tamanho das partículas da dispersão coloidal foi realizada por meio de espalhamento dinâmico da luz com óptica a 90°, na temperatura de 25°C, utilizando o equipamento Zetasizer modelo 3000HS (Malvern Instruments Ltd, UK) . 0 resultado encontrado, de 136,5 nm, representa a média do tamanho das partículas. A distribuição do tamanho das partículas apresentou uma pol-idispersão equivalente a 0,24.  The particle size distribution of the colloidal dispersion was performed by dynamic light scattering at 90 ° optics at 25 ° C using the Zetasizer Model 3000HS (Malvern Instruments Ltd, UK). The result found, 136.5 nm, represents the average particle size. The particle size distribution had a polydispersion equivalent to 0.24.
Exemplo 6: Medida da carga elétrica das partículas da dispersão coloidal contendo extrato glicólico de própolis Example 6: Measurement of the electrical charge of the particles of colloidal dispersion containing propolis glycolic extract
As partículas coloidais estão constantemente sob a influência de forças atrativas, de modo que forças repulsivas evitam sua agregação (González-Caballero F., López-Durán J. D. G. Suspension formulation. In: Pharmaceutical emulsions and suspensions. Eds . Nielloud F. , Marti-Mestres G. New York: Mareei Dekker, 2000, cap. 4, p. 127-190) . Na maioria dos líquidos polares, estas forças são provenientes das cargas das partículas. O potential Zeta é uma medida da intensidade da carga presente e representa um indicativo claro de estabilidade de uma dispersão, de forma que um valor próximo de zero indicaria instabilidade coloidal, podendo causar agregação das partículas. Colloidal particles are constantly under the influence of attractive forces, so repulsive forces prevent their aggregation (González-Caballero F., López-Durán JDG Suspension formulation. In: Pharmaceutical emulsions and suspensions. Eds. Nielloud F., Marti-Masters G. New York: Mareei Dekker, 2000, chap. 4, pp. 127-190). In most polar liquids, these forces come from particle charges. Potential Zeta is a measure of the intensity of the charge present and represents a clear indication of the stability of a dispersion, so that a value close to zero would indicate colloidal instability and may cause particle aggregation.
O potencial Zeta das partículas da dispersão coloidal foi medido por meio de espalhamento eletroforético da luz, na temperatura de 25°C, utilizando o equipamento Zetasizer modelo 3000HS (Malvern Instruments Ltd, UK) . O resultado encontrado de -14,2 mV ± 3,85 representa a média ± o desvio padrão, e indica uma estabilidade da dispersão coloidal. Exemplo 7: Medida da viscosidade da dispersão coloidal contendo extrato glicolico de propolis The Zeta potential of the colloidal scattering particles was measured by electrophoretic light scattering at 25 ° C using the Zetasizer Model 3000HS (Malvern Instruments Ltd, UK). The result found of -14.2 mV ± 3.85 represents the mean ± standard deviation, and indicates a stability of colloidal dispersion. Example 7: Measurement of viscosity of colloidal dispersion containing glycol propolis extract
A viscosidade da dispersão desenvolvida foi medida utilizando o viscosimetro rotacional ajustado na velocidade de 60rpm. As medidas foram realizadas em triplicata em temperatura constante de 25°C. O valor de viscosidade obtido foi de 5,5 ± 0,4 Cp.  The viscosity of the developed dispersion was measured using the rotational viscosimeter set at a speed of 60rpm. Measurements were made in triplicate at a constant temperature of 25 ° C. The viscosity value obtained was 5.5 ± 0.4 Cp.
Exemplo 8: Medida do índice de refração da dispersão coloidal contendo extrato glicólico de propolis  Example 8: Measurement of the refractive index of colloidal dispersion containing propolis glycolic extract
Visando avaliar a transparência do sistema, o índice de refração foi determinado utilizando o refratômetro sem necessidade de tratamento prévio da formulação. As medidas foram realizadas em triplicata em temperatura constante de 25°C. O resultado obtido foi de 1,366 ± 0,654. Este valor é próximo do índice de refração da água, mostrando, portanto, que o sistema é transparente.  In order to evaluate the transparency of the system, the refractive index was determined using the refractometer without prior treatment of the formulation. Measurements were made in triplicate at a constant temperature of 25 ° C. The obtained result was 1,366 ± 0,654. This value is close to the water refractive index, thus showing that the system is transparent.
Exemplo 9: Avaliação da estabilidade da dispersão coloidal contendo extrato glicólico de propolis  Example 9: Evaluation of the stability of the colloidal dispersion containing glycol propolis extract
Dispersões coloidais contendo o extrato glicólico de propolis, preparadas há aproximadamente doze meses e armazenadas às temperaturas de 4°C, 25°C e 40°C não mostraram sinais de perda de estabilidade da formulação, tais como: formação de depósitos, formação de grumos, crescimento de cristais e separação de fases.  Colloidal dispersions containing propolis glycolic extract prepared for approximately twelve months and stored at 4 ° C, 25 ° C and 40 ° C showed no signs of loss of formulation stability such as: deposit formation, lump formation , crystal growth and phase separation.
Exemplo 10: Avaliação da atividade antimicrobiana da dispersão coloidal contendo extrato glicolicos de propolisExample 10: Evaluation of Antimicrobial Activity of Colloidal Dispersion Containing Propolis Glycol Extracts
Foram realizados testes de atividade antimicrobiana da dispersão coloidal contendo extrato glicólico de propolis verde conforme metodologia anteriormente descrita no Exemplo 2. Neste teste também foi empregado como controle positivo solução oral de digluconato de clorexidina a 0,12% (p/v) . Antimicrobial activity tests of colloidal dispersion containing green propolis glycolic extract were performed according to the methodology previously described in Example 2. This test was also used as control positive 0.12% (w / v) chlorhexidine digluconate oral solution.
Os resultados mostraram que todos os microrganismos demonstraram susceptibilidade à formulação descrita na presente invenção (Tabela 2; Figuras 2, 3 e 4), a qual contem 5% (v/v) de extrato gilcólico de própolis. Este resultado foi semelhante ao observado para a solução oral digluconato de clorexidina, atualmente o produto de escolha em consultórios odontológicos, mas que apresenta elevados efeitos adversos. Para confirmar a semelhança da presente invenção com o produto de escolha atual, foi realizado o teste exato condicional de independência, demonstrado na Tabela 2:  The results showed that all microorganisms demonstrated susceptibility to the formulation described in the present invention (Table 2; Figures 2, 3 and 4), which contains 5% (v / v) propolis gilcolic extract. This result was similar to that observed for chlorhexidine digluconate oral solution, currently the product of choice in dental offices, but which has high adverse effects. To confirm the similarity of the present invention with the current product of choice, the exact conditional independence test was performed, shown in Table 2:
Tabela 2. Teste exato condicional e razão de chance para comparação entre a presente invenção e o sal cloridrato de clorexidina  Table 2. Exact conditional test and odds ratio for comparison between the present invention and chlorhexidine hydrochloride salt
Tratamentos Teste Exato Condicional de Independência e Controles S P -valor R.C IC  Treatments Exact Conditional Independence Test and Controls S P-value R.C IC
PI x C+ 12, 00 1, 000 0, 000 [ 0 - inf ] PI x C + 12.00 1,000 0.00 [0 - inf]
PI x CC 0, 00 0, 000 0, 000 [ 0 - 0,04 ]PI x CC 0.00 0.00 000 [0 - 0.04]
C+ x CC 14, 00 0, 000 Inf [ 94,01 - Inf ]C + x CC 14.00 0.00 Inf [94.01 - Inf]
: Solução de digluconato de clorexidina (0,12%) PI: prese: invenção CC: controle de crescimento : Chlorhexidine digluconate solution (0.12%) PI: prosthesis: invention CC: growth control
O teste exato condicional de independência evidenciou que não existe diferença significativa entre a presente invenção e a solução oral de digluconato de clorexidina, destacando o potencial terapêutico da presente invenção em relação à solução de uso na odontologia clinica, com um p- valor menor que 0,001. The exact conditional independence test showed that there is no significant difference between the present invention and the chlorhexidine digluconate oral solution, highlighting the therapeutic potential of the present invention in relation to the solution for use in clinical dentistry, with a p-value less than 0.001. .
Figure imgf000023_0001
Figure imgf000023_0001
Figura 2. Avaliação da presente invenção frente à levedura Cândida albicans (A, B,C) e bactéria aeróbica Staphylococcus aureus (D, E, F) . (A e D) Placas-teste contendo formulação da presente invenção, (B e E) controle de crescimento do microrganismo, (C e F) controle positivo: solução de di luconato de clorexidina a 0,12%.  Figure 2. Evaluation of the present invention against Candida albicans yeast (A, B, C) and aerobic bacteria Staphylococcus aureus (D, E, F). (A and D) Test plates containing formulation of the present invention, (B and E) microorganism growth control, (C and F) positive control: 0.12% chlorhexidine di luconate solution.
Figure imgf000023_0002
Figure imgf000023_0002
Figura 3. Avaliação da presente invenção frente a Lactobacillus casei (A, B, C) e a Streptococcus mutans (D, E, F) . (A e D) Placas-teste contendo formulação da presente invenção, (B e E) controle de crescimento do microrganismo, (C e F) controle positivo: solução de digluconato de clorexidina a 0,12%. Figure 3. Evaluation of the present invention against Lactobacillus casei (A, B, C) and Streptococcus mutans (D, E, F). (A and D) Test plates containing formulation of the present invention, (B and E) microorganism growth control, (C and F) positive control: 0.12% chlorhexidine digluconate solution.
frente à
Figure imgf000024_0001
compared to
Figure imgf000024_0001
Porphyromonas gingivalis (A, B, C) e a Prevotella melaninogenica (D, E, F) . (A e D) Placas-teste contendo formulação da presente invenção, (B e E) controle de crescimento do microrganismo, (C e F) controle positivo: solução de digluconato de clorexidina a 0,12%. Porphyromonas gingivalis (A, B, C) and Prevotella melaninogenica (D, E, F). (A and D) Test plates containing formulation of the present invention, (B and E) microorganism growth control, (C and F) positive control: 0.12% chlorhexidine digluconate solution.
Os resultados apresentados demonstraram que a presente invenção a base de própolis verde, à concentração de 5%, é capaz de inibir totalmente o crescimento microbiano de seis importantes microrganismos comumente isolados da cavidade oral, implicados em casos de mucosites e periimplantites .  The results presented demonstrate that the present invention based on green propolis, at a concentration of 5%, is able to totally inhibit the microbial growth of six important microorganisms commonly isolated from the oral cavity, implicated in cases of mucositis and periimplantitis.
A presente invenção demonstrou atividade antimicrobiana semelhante ao observado para a solução oral a base de digluconato de clorexidina, atualmente o produto de escolha em consultórios odontológicos. Os resultados apresentados evidenciam o potencial terapêutico da presente invenção, como um agente de fácil formulação e baixo custo, em substituição ao produto atualmente empregado.  The present invention demonstrated similar antimicrobial activity to that observed for chlorhexidine digluconate oral solution, currently the product of choice in dental offices. The results presented show the therapeutic potential of the present invention as an easily formulated and low cost agent, in substitution for the product currently employed.

Claims

REIVINDICAÇÕES: CLAIMS:
1. Processo de produção de formulação farmacêutica contendo própolis verde, que se apresenta na forma de dispersão coloidal .  1. Production process of pharmaceutical formulation containing green propolis, which is in colloidal dispersion form.
2. Formulação farmacêutica como descrito na reivindicação 1 contendo própolis verde, sendo que a própolis é empregada na forma de extrato glicólico.  A pharmaceutical formulation as described in claim 1 containing green propolis, the propolis being employed as glycolic extract.
3. Formulação farmacêutica como descrito na reivindicação 2 contendo própolis verde, cujo extrato glicólico possui atividade frente a microorganismos de interesse clinico na cavidade oral.  Pharmaceutical formulation as described in claim 2 containing green propolis, whose glycolic extract has activity against microorganisms of clinical interest in the oral cavity.
4. Formulação farmacêutica como descrito na reivindicação 2 contendo própolis verde capaz de reduzir a hidrofobia do extrato de própolis e sua tendência para formação de precipitados em meio aquoso.  A pharmaceutical formulation as described in claim 2 containing green propolis capable of reducing the hydrophobia of propolis extract and its tendency to precipitate formation in aqueous medium.
5. Formulação farmacêutica como descrito na reivindicação 2 contendo própolis verde que pode ser administrada pela via bucal .  Pharmaceutical formulation as described in claim 2 containing green propolis which may be administered via the oral route.
6. Formulação farmacêutica como descrito na reivindicação 2 contendo própolis verde, que pode ser aplicada ao tratamento de diferentes doenças odontológicas.  A pharmaceutical formulation as described in claim 2 containing green propolis which may be applied to the treatment of different dental diseases.
7. Formulação farmacêutica como descrito na reivindicação 2 contendo própolis verde própolis verde, onde tal formulação consiste de um tensoativo não-iônico e um agente viscosante tal como o sorbitol.  A pharmaceutical formulation as described in claim 2 containing green propolis green propolis, wherein such formulation consists of a nonionic surfactant and a viscous agent such as sorbitol.
8. Composição farmacêutica como descrito na reivindicação 7 contendo própolis verde que apresenta pH entre 6,5-7,5. A pharmaceutical composition as described in claim 7 containing green propolis having a pH between 6.5-7.5.
9. Composição farmacêutica como descrito na reivindicação 7 contendo própolis verde que apresenta partículas de tamanho inferior a 200nm, variando preferencialmente de 100 a 180nm. A pharmaceutical composition as described in claim 7 containing green propolis having particles less than 200nm in size, preferably ranging from 100 to 180nm.
10. Composição farmacêutica como descrito na reivindicação 7 contendo propolis verde que apresenta potencial Zeta em torno de -15mV. Pharmaceutical composition as described in claim 7 containing green propolis having Zeta potential around -15mV.
11. Composição farmacêutica como descrito na reivindicação 7 contendo propolis verde que apresenta viscosidade em torno de 5,5 Cp.  A pharmaceutical composition as described in claim 7 containing green propolis having a viscosity around 5.5 Cp.
12. Composição farmacêutica como descrito na reivindicação 7 contendo propolis verde que apresenta índice de refração em torno de 1,4.  A pharmaceutical composition as described in claim 7 containing green propolis having a refractive index of about 1.4.
13. Composição farmacêutica como descrito na reivindicação 7 contendo propolis verde que apresenta estabilidade frente a diferentes temperaturas por um período de doze meses. A pharmaceutical composition as described in claim 7 containing green propolis which exhibits stability at different temperatures over a period of twelve months.
14. Composição farmacêutica como descrito na reivindicação 7 contendo propolis verde que não induz a formação de depósitos e grumos, o crescimento de cristais e a separação de fases. A pharmaceutical composition as described in claim 7 containing green propolis which does not induce deposit and lump formation, crystal growth and phase separation.
15. Composição farmacêutica como descrito na reivindicação 7 contendo propolis verde que apresenta atividade antimicrobiana frente a diferentes microorganismos de interesse clínico na cavidade oral.  Pharmaceutical composition as described in claim 7 containing green propolis which exhibits antimicrobial activity against different microorganisms of clinical interest in the oral cavity.
PCT/BR2013/000151 2012-05-03 2013-05-03 Green propolis pharmaceutical formulation for use in human odontology WO2013163714A1 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9968548B2 (en) 2014-11-07 2018-05-15 Vets Plus, Inc. Dental hard chew supplements containing antimicrobial actives
RU2786136C1 (en) * 2022-02-18 2022-12-19 Сергей Владимирович Сирак Paste for the prevention and treatment of peri-implantitis
ES2850277R1 (en) * 2019-01-10 2023-07-07 Innovacorium Inc Pharmaceutical administration compositions and uses thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0109993A1 (en) * 1982-12-01 1984-06-13 Zenon M. Sosnowski Method for extracting propolis and water soluble dry propolis powder obtained thereby and cosmetic and pharmaceutical preparations containing same
KR20020076979A (en) * 2001-03-28 2002-10-11 진승관 Preparation of water-soluble propolis, employ propylen glycol as solvent
BR0105471A (en) * 2001-09-26 2003-09-23 Maria Cristina Marcucci Ribeir Propolis-based formulations for dental use
BRPI0800363A2 (en) * 2008-03-10 2009-10-27 Univ Sao Paulo process of obtaining standardized extracts, fractions and substances isolated from propolis or baccharis species, standardized extract formulations, fractions and substances isolated from propolis or baccharis species, and the use of standardized extract formulations, fractions and substances isolated from propolis or baccharis species. baccharis species such as sanitizers, antiseptics and / or disinfectants

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0109993A1 (en) * 1982-12-01 1984-06-13 Zenon M. Sosnowski Method for extracting propolis and water soluble dry propolis powder obtained thereby and cosmetic and pharmaceutical preparations containing same
KR20020076979A (en) * 2001-03-28 2002-10-11 진승관 Preparation of water-soluble propolis, employ propylen glycol as solvent
BR0105471A (en) * 2001-09-26 2003-09-23 Maria Cristina Marcucci Ribeir Propolis-based formulations for dental use
BRPI0800363A2 (en) * 2008-03-10 2009-10-27 Univ Sao Paulo process of obtaining standardized extracts, fractions and substances isolated from propolis or baccharis species, standardized extract formulations, fractions and substances isolated from propolis or baccharis species, and the use of standardized extract formulations, fractions and substances isolated from propolis or baccharis species. baccharis species such as sanitizers, antiseptics and / or disinfectants

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9968548B2 (en) 2014-11-07 2018-05-15 Vets Plus, Inc. Dental hard chew supplements containing antimicrobial actives
ES2850277R1 (en) * 2019-01-10 2023-07-07 Innovacorium Inc Pharmaceutical administration compositions and uses thereof
RU2786136C1 (en) * 2022-02-18 2022-12-19 Сергей Владимирович Сирак Paste for the prevention and treatment of peri-implantitis

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