BR102012010441A2 - PHARMACEUTICAL FORMULATION OF GREEN PROPOLIS FOR HUMAN USE IN DENTISTRY - Google Patents

Abstract

PHARMACEUTICAL FORMULATION OF GREEN PROPOLIS FOR HUMAN USE IN DENTISTRY, The present invention concerns the process of producing a pharmaceutical formulation based on green propolis with antimicrobial properties capable of treating different dental diseases, such as mucositis and oral peri-implantitis. Said formulation comprises a colloidal dispersion of the glycolic extract of propolis composed of non-ionic surfactant and viscous agent capable of reducing the hydrophobia of the extract and its tendency to form precipitates in aqueous medium. The formulation is applicable to oral administration.

Description

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-based pharmaceutical formulation, originated from dracun Muslim Baccharis produced by. Apis meliifera bees, with non-imitative properties for the treatment of oral mucositis and peri-implantitis.

Said formulation comprises a colloidal dispersion; glycolic propolis extract. compound, ionic, surfactant is / viscous agent capable of reducing the hydrophilicity of the

■ extract and its tendency to precipitate formation; middle. watery. The wording applies to:. buccal administration.

'Practically' / all ancient civilizations ^ · with their

millenary therapies knew and used the products of the

s,: v v. · ■

bees: as valuable resources in medicine. The stories . of the medicines of the Chinese, Tibetan, Egyptian and 'civilizations

- Greco - Romana contain in their writings hundreds of accounts where these products come in (honey, propolis, bee larvae and sometimes the bees themselves) to cure or prevent disease. Sacred Eiblia texts, from the da Civilization ..Hebrew, refer to Propolis as' i The Gilead Balm used to heal wounds. In ancient Egypt (1700 BC) it was used to embalm the dead. The Greeks, among the. which Hippocrates adopted it as an internal and external healer. Roman historian Pliny refers to Propolis as a 'medicine' capable of reducing 'swelling' and relieving pain {GonzaIez AG, Mendez RB. 'The favorite ones. de la colniena ■ Good practice · for su. recoleccion, alrnãcenamiento y '. conservation Lab. 'Of 2 / 2.3 Reference for Investigations e. Salud Apicola.,. Sancti Spiritus, Cuba).

, The term propolis is derived from the Greek "pro" meaning defense and "polis" meaning · city, ie

-'defense of the city of bees '.' It is a 'natural resin, produced by the bees from the secretions and exudates of various plants. These plants. These constituents are bioformed, waxed and then deposited in the hives where · Have an important role in sealing cracks, 10 immobilizing agents, and invaders in addition to their role in protecting.

against the .proliferation of. microorganisms (Ghisalberti EL. ·

/ '·' · ■. '

Propolis: a review. Bee world, v.60, p.59-84, 1979).

In Africa of. South, in the war of the late nineteenth century and World War II, as well as, in the former Soviet Union, propolis - was widely used because of them. - your

■ healing properties, with applications including in the treatment of tuberculosis, observing regression of lung problems (Brooks R, Vick J. A. Studios / above, venom of abe jas. -ΧΧΧΙΊ .. International Congress of

20. Apimondia apiculture. Brazil. Ed. Apimondia, Rumania,

y V N '

pp. 506-51.5, 1989; Gonzalez AG. El. poison of bees · News and perspectives. Ed. Paplo de la Torriente. La Habana, Cuba-, 1998 '; Gonzalez-AG, Mndez'1 RB. The products -of

Ia beehive- Practical buenas for your collection,

. l. ‘

It's a conservation and conservation. Reference Lab for Research in Health and Health Apicola. Sancti Spiritus, Cuba, 200 4) -. .

. . The first scientific work on propolis appeared in 1908 and described its chemical properties and composition. In · 1968 Chemical Abstracts appeared the summary of the 1st patent using propolis (Romanian, to produce 3/2 3 'bath lotions). The first Brazilian publication on propolis in 1984 presented a co-comparative study of the effect of propolis and antibiotics on inhibition of Staphylococcus. alir & us (Pereira AS, Seixas' FRMS, Neto ·, FRA.

Propplis: 100 years of research and future perspectives. Quim Nova, v. ‘25, p. 321-326., 2002) '. ■

! The chemical constitution of propolis' is composed of a

mixture of bio bioactive substances: 30% 'of, 55%' of

resins and balms, 10% 'essential oils, 5% pollen and

10- other compounds · Currently there are more than 300 compounds

isolated, and many of them "have already been registered for

. medicinal,. Many natural products are efficient in

combating free radicals that cause the oxidation of

cells, but even now, none of them proved more effective

than · green propolis. ;

Among the various isolated compounds, we can cite the

cinnamic acid, phenolic compounds, is. f lavofiói-des

(Ghisalberti EL. Propolis:> the Rev.iew. Bee World, v. 60, p. 59-

84, 1979; BankOva V, - Popov S, Marekov NL. High performance

* i <'1 liquid chromatographic analysis of 1 flavonoids from

propolis. J. Chromatography,. v. 242, p. 135-143, 198.7;

Arvouet-Grand A. Extrait 'of Propolis: II. Etude of

cicatrisation dé. chez Ie lapin, et chez Ie rat. J

• Pharm - · Belg -, ----- ν ·. · 48, pp. 1'71-178, "19 93"), "terpenes, acids

25 'main aromatics' (3-.prenyl' 4-hydrixicinami, co e ·, the 6-

(2,2-dimethyl-2H-1-benzopyran) · derivatives of

■: caffeic acid (potent - a: hepatotoxic activity), acids

fatty acids, amino acids, vitamins (A, BI, B2, B6, C, E, PP}, '

minerals (Mn, Cu, Ca, Al, Si, V, Ni, Zn, Cr),

high molecular weight, carbohydrates and Artepélin 'C (Pereira

AS, Complaints FRMS / Grandson FPA. Propolis: 100 years of research and future prospects. Ouim ,, New, v. 25, p. 321-326,

. 2002). '

As it is used in the treatment of various diseases in folk medicine since: a. . Antiquity (Hausen. .BM, "Wollwnweber E, Senff H, Post B. Propolis allergy I1. Orri, properties, usage and literature review. Contact Dermatitis, v. 17, p. 163-170, 1987) propiolis has received great attention from the scientific class and has been described for its antimicrobial, antimicotic properties (Brumfitt W, Hamiiton-Miller JMT, Franklin 'I. Antibiotic' ectivity of natural 'products. v. 62, pp. 19-22, 1990; Aga H, Shibuya T. Sugimoto T, Kurimoto M, Nakajima S., Isolation and Identification of Antimicrobial Compounds in Brazilian Propolis Biosci Biotech Biochem, v. 58, p. 945-946, 1994), antioxidants (Scheller S, Wilczoks T, Imielski S, Krol W, Gabrys J, Saani J. Free radical scavenging by ethanol extract of propolis. Int J Radiat Biol., V. 57 , 4 61-4 65, .1990), antithymoral {Scheller S, Szaflarski J, Tustanowski J, Nôlewajka, E., Stojko A. Biological properties and clinical application of propolis s: some .physic-chemical properties of propolis. Ar? Neim Forsch, v. 27, p. 889-890,

''. '■ 1, ‘■' -V-

1989) Regenerative, healing (Arvouet-Gran, A., E-Propolis E-Xteait: II. Cicatrisation Etude of Plaies. Chez Lapin et Chez 1 Erat. J Pharm Belg, v.48, p. 171- 178, 1993), antivirals (Esanu V, Prahoveanu E, Crisau I, Cioca A. The effect of aqueous propolis extract of rutin and frutin-quercetin mixtures on experimental ifluenza 'λ, virus: infection in mice Virologie, v. 32, pp. 213-215, · 1981) and immunomodulatory (Ivanovska ND., DiMov VB, Bankova VS., Popov SS. Imniu η οηιο latory action of propolis IV: - influence of water soluble derivative on complement activity, in vivo. (J Ethnopharmacql (v. 47, p. 145-147, 1995)). Propolis has also been successfully tested in the fight against protozoa by inhibiting a. proliferation. de · Toxpplasma \ gandii and Trichomon.as_. vaginalis ■. (Starzyk J., Scheller, S, Szaflarski - J. Moskwa. M, Stojko. A. Biological properties and clinical application of propolis II: studies on the antiprotozoan "activity of ethanol extract of propolis." Arzneim Forsch, v. 27, pp. 11-98-1199, 1997.) and in the clinical treatment .10 of giardiaSe (Clirayes C, Hollandes L., Castaneda C. Therapeutic trials with the propolis-based product Propolisin in human giardiasis., Acta Gastroent Latinoam ,

(18, p. 195-202, 1988). In addition, Higashi & Castro

■ (Higa-shi KO, Castro, SLD., Propolis extracts, are effective '15 against Trypanosoma-cross and have an impacc, on its interaction with host cells. J Ethnopharmacol, v, 43, p.

'’*'

149-155, 1994) demonstrated that the etaholic extract of propolis was effective against Trypanosome infection.

■ crossed. Thus / propolis cannot be relegated to a category of therapeutic fad, given that its virtues have been 'recognized for centuries', being reported in numerous works demonstrating different types of biological activity and applications are various therapies. .

Oral Cavity and the Use of Propolis The oral cavity is naturally colonized by several

species of microorganisms, which when organized and structured constitute the oral mycrobiotic. (S.eabra EJG, Lima IPC, Barbosa SV, Lima KC. In vitro antimicrobial activity of calcium hydroxide and tergenol based compounds in 30. different 'concentrations' over 'oral bacteria Acta Cirurggica Brasileira, v. 20, - p. 18,' 2005). It is estimated that the saliva contains about 108 bacteria / ml and this number reaches 1011 n, the dermal plates. having quantitative and qualitative fluctuation (Trabulsi LR, Alter.thum, F .. Eds., Microbiot. or normal flora of the human body. Microbiology,

• 5 St. Paul: Atheneu, 4a; ed, p .; 10, -109, 2005). ■■ High humidity, relatively 'constant' temperature (34 to 36 ° C) and continuous nutrient availability make this place a very microenvironment. conducive to the establishment of approximately 500. bacterial groups' .. Among groups 10 plus. representative are the genera Lactobacillus, Actinobacillus, Perphyromonas, Prévotella, Staphylococcus and Streptococcus {-Mqretti PE. Human Bacterial Mycrobiotic: ", Mycrobiotic. oral. Project Microorganisms, 'Section 1- Bacteriology, Part 5-Microbial Associations, available 15 ertiwww: //. fan. br / microorganisms / bacteriology_microbibta_h'a ana__oral.htm. Accessed Mar. 01, 2008), 'In addition to Candida yeast species (Assis EM. Comparison of the efficacy of green propolis in relation to' miconazole 'in the treatment of chronic atrophic candidiasis in' de users').

removable full denture. Specialization Monograph,

- · ´ · f 't i: ^

Ont. Pontifical Catholic University of Minas Gerais, 38 p., 2006).

The predominant gender J in the oral cavity is; Streptococcus, represented by the species: 'S.

. 25 mutans, S. sanguis and S ~. , salj-varius, the latter being isolated mainly from the tongue, while the 'other species' occur in association with the teeth (Moretti PE. Human bacterial myrobiotic: oral mycrobiotic. Project Microorganisms, Section 1 - Bacteriology, Part 5 30 Associations / Microbials, available at: www. / Amf / br / microorganismds / bacteria_microbiota_human ■ a_or-al.htm (Accessed OL.03.08). . 1 .

The 'Streptococcus mutans' species can colonize the 'superferitious' teeth and start. dental plaque formation by being; capable of synthesizing 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, pp. 19-44, 1975). This adhesion polysaccharide allows the adhesion of other bacteria to this microenvironment, initiating the formation of microbial biofilms (Morettí, PE., Human bacterial microbiota: oral mycrobiot. Project Microorganisms, Section 1- Bacteriology, Pairt; Microbial Associations, 'available at www. // fam.br/micro;i:organisms/bacteriology_microbiota_hum ana__oral.htm .Access on 03.01.08) ..

Studies have shown the similarity between the flora around the implants and the surrounding natural teeth (LekhoIm U. The condition of the soft tissues at 20 tcoth, and. Fixture abutments supporting fixed, bridges. J. Clin-Periodont, v. 13, p. 558-56.2, 1985; Mombelli A, Lang NP. Microbial aspects of implant dentistry. Périodontol-, v. 4, -p. 74-80, -,

■ 1994), indicating that therapeutic procedures, except for some specific, minor care, become common to both. Thus, it is known that periodontal disease is a 'treatable' infection. and, . therefore, amenable to control and prevention when developed around natural teeth or implants (Bauman GR, Plaque-induced inflammation. 30. around implants. Int J · Oral & 'Max Impl., v. 7, p. 330- 33.7,

. ' 1992). Dental procedures, especially those that cause harm to soft tissues, can spread microorganisms to vital organs such as the heart, brain, and liver (Slots J, Taubman MAi 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 updaite. Oral Surg, v. ■ 57 ,. p..606 - 615, .1984), was. It has been shown that 92% of the infectious endocarditis cases analyzed originated from. due to the 'dissemination of' microorganisms' of the oral cavity, being the main factor. de virulence, dextran production (Nord CE, Heimdahl A. Cardiovascular infections: .. bacterial endocarditis 'of. oral origin * Pathogens an.d. pophyl.ax.is .J Clin Periodont, v. 17, p.' 4 94 - 49.6, 1990). ■ Some researchers share the view that a reduction in the number of oral microorganisms by the use of. antiseptic- 'before ·. ■ any procedure .. invasive. decreases the likelihood of bacteremia during dental treatment (MacFarlane. TW, Ferguson MM, Mulgrew CJ. Pos -; '’*

extraction bacteraemia: role ', of antisep.tics and antibiotics'. Br Dent J, v. 156, p.179 - 191, 1984).

A very effective antiseptic would be propolis. There are reports of use of this product in oral cavity by. civ-üi-z-actions-s-ant-tg-as (.Castaldro ^ -Sv Capasso · F. Propolis, An Old Remedy Used in Modern Medicine. Herbal Medicine, v. 73, p.

Sl - S6, ‘2002). In dentistry, propolis has been described by its surface anesthetic action {Paintz MJ, Metzener ■ J. Zur lokalanasthetischen wirkung von propolis und einigen ‘inhatsstofen. Pha.rmazie-, v. .34, p.839 -. 841, 1979) as a pre-surgical mouthwash (Magro Filho O, Carvalho ACP. Application of dental sockets and skin wounds. J Nihon Uni Sch Dent, v. 32, pp. 4-13, 1990) ), daily mouthwash (Ikenò K, Ikeno P,, ■, ■ ■ ■■ ■ ■ /

Miyazawa C. Effects of propolis on dental caries in rats. Caries Res, - v. 25, p. 347 - 351, 1991): in 5 'ulcer treatments; recurrent oral lesions ('Silveira ■ MG. Preliminary study on the effects of prophylaxis on treatment of chronic, chronic and ulcers')

Mouthpieces. Cuban Rev of Stomat, v. 25, p. 36 44, 1988) and 1. ■ ■. '' 'Vv-

oral thrush (Garcia CL, Garguera EG. Efectos dei propolan

in the treatment of oral thrush. Rev Cuba Med Thousand, v. '

, 22, 42-45 / 1993). Antimicrobial activity on '

some ralis bacteria too, 'has already been' demonstrated,., so

as inhibition of the enzyme glycosyl transferase, responsible for

by plaque formation. : ·, /. '

15 'Some ... patents related to.

use of propolis in pharmaceutical formulations. ■ ■

US patent 6,803,056. entitled ■ "Methods. and

composition for 'treatment of viral problems' describes.

a method and composition for the treatment of injuries

associated with viral infections such as Herpes slmplex,

by applying an effective amount of a composition

containing: y propolis extract at concentration: 0.5-

at 10%, preferably from 1 to 8%; a skin protector on

• concentration of 0.5 to 50%, a penetration enhancer in the

'25. concentration of 5 to 30%, preferably 5. at .25% and one.

■ surfactant at a concentration of 1 · 20% (Dolak TM. Method and composition for treating viral outbreaks. Patent

■ US 6, 803,056; '2004)., This patent further describes that the formulation'. has better activity in the treatment of ■

- described lesions, stopping their * progression when applied 'and promoting complete healing, usually between 36 and .48 hours., -.

U.S. Patent No. 6, 153, 226 entitled. "Therapeutic composition, containing a virus-efficient, phenolic compound and propolis .5 compound, especially herpes viruses" describes a therapeutic composition containing a phenolic compound and a lipid-virus-efficient propolis, which contains (i.e. ) 100 to 650 parts by weight of the phenolic component for '(ii). one part by weight of propolis (Vachy R, 10 Sauvager1F, Amoros M. Therapè.utic composition containing a

■ phenol eompound and propolis, which is useful against. Iipid capsid. viruses, especially herpes ■ viruses. US Patent 6, 153, 22.6; , 2000). Said composition is suitable for treating herpes.

- 15 US Patent 5,561,116 entitled "Solid Product

containing propolis components, preparation and use "describes a solid, dispersible, ■ water

■ propolis components (Nakamura S, Miyake T. Solid, Productivity Containing propolis components, and preparation and 20 uses thereof. Patent 5,561,116; 1996). Said composition is prepared by extracting the propolis components, which are further mixed together with at least one saccharide selected from anhydrous and cioiodèxtine saccharides. The composition is suitable for use in 25 different diseases by different application forms.

At present, we find commercially available formulations containing propolis for use in dentistry. ■ These formulations are present in the form of oral solution or extract, concentrated. Generally, however, alcohol 30 is present in the composition of these formulations which can cause mucosal irritation. · and non-adherence of the patient to treatment. , ■ -. ■. · ■ · ■■ ’J \

Gs .. dispersed systems are those that present at least. two phases, one of which (dispersed phase. or internal 5) is finely distributed in the other (continuous or external phase) generally referred to as dispersion means - or vehicle. Depending on the particle size, the particles. Dispersed systems are, called coarse dispersion, colloidal dispersion or molecular dispersion (Morrison. I D,

10 Ros.s S. Golloidal dispersions - suspensions, .emulsion.s,. and. foams. USA: Wiley-Interscience, 2002, 616 p.).

The dispersions, solid, are systems. Dispersions ■ that are situated between the systems of molecular dispersion. and the coarse dispersion. They are called colloid as / 1 ^! 'molecules' or aggregates; formed by 10 3 to 10 9 atoms, whose. diameter. The particle is of the order of 10 -4 'to 10 -7 cm, the particles being distributed in a dispersing medium (Dispersiones colloidales., In: Remington - Pharmacy - 19a ,,,' edition; AR Genríaro, Buenos Aires: Editorial Medical 20 (Panamericana, 1998, pp. 366-425). Therefore, solid dispersions are those which have particles of varying size. between -I and 10 nm. A characteristic optical property of the coloidal systems is the Tyndall effect, which occurs when a beam. of light passes through the system and is dispersed by the presence of suspended solid particles. promoting light scattering. The extent of turbidity or palescence of the solid dispersions depends on the nature, size and color of the solid particles present (Ruissel WB, Saville DA, Schowalter M.R.30 Colloidals dispersions. · London: Cambridge university press, 1992, 544. p. One of the major challenges in the development of solid dispersions is to prevent the irreversible aggregation of suspended particles,. which would lead to sedimentation of the internal phase. A stable colloidal dispersion has '5 particles' which 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, as 'os'. polyols, 'can. contribute to the reduction of particle aggregation (González-Caballero F., López-Durán JDG 10 Suspension formulation. In: · Pharrtiaceutical emul.s-ions and suspensions. Eds. Nielloud F., Marti-Mèstres G. 'New' York: Marcel Dekker, 2000, chap. 4, pp. 127-190). - ■ *,

The 'systems' give colloidal dispersion. can be classified into dispersion colloids, association colloids & molecular colloids (Dispersiones eoloidales. In:.-Remington - Pharmacy - ·, 19th edition; ed. AR Gennaro; Buenos Aires: Editprial Medica Panaméricana, 1,99,8 (pp. 366-425). In the present invention, a colloidal colloid type dispersion of association type has been developed. The colloids of. Association, also known as micolar colloids, comprise the solid dispersed systems capable of forming micelles. In this case, dispersed molecular substances, such as surfactants, are capable of improving miscibility. without producing changes in structure. Chemistry of these, by the formation of molecular aggregates called micelles, originated in a surfactant solution above a certain concentration, known as the Critical Mircellar Concentration (CMC). . Generally, hydrophobic or poorly water-soluble substances are found within the micelle (González-Caballero F., López-Durán J. D. G. Suspendion 13/23-formulation. In: Pharmaceutical Eraulsions and Suspensions.

Eds. Nielloud F., 'Marti-Masters G. New York: Marcel Dekker,

2000, chap. 4, p. 127-19.0).

Surfactants play another important role in

formulation of solid dispersions. They are used

as wetting agents, generally for the purpose of

increase the sci.ability of hydrophobic particles,

present in an aqueous vehicle (Muller RH, Bohm Bf-Benita

S. Emulsions and nanosuspensions for th.e formulation. of

. ' poorly soluble. drugs. London: CRC Press. 1998, 396. p.

'-These substances' tend to accumulate at interfaces / '- - ■ ■ J' · .. ·· due to their amphiphilic nature, reducing stress

surface of the present phases. The molecules of t.ensive

oriented at the liquid-solid interface, so that the

1-5 hydrocarbon chain is in contact with

surface of the solid particle while the polar group

orientates to the aqueous phase. Ά most non-surfactants 1

Water soluble ionic compounds are polyoxyethylated, each molecule consisting of a lipophilic hydrocarbon chain and a polyethylene glycol hydrophilic. The surfactant adsorbs at the interface between a hydrophobic solid and water, with its hydrocarbon residue adhered to the solid surface and the polyethylene glycol residue returned to water. Thus, the surface of the particle is surrounded by a thin layer of hydrated polyoxyethylene chains. The hydrophilic layer forms one. ■ steric barrier that prevents contact between particles, and ■ consequently aggregation. (Russell WB, Saville DA, Schowalter WR. Colloidal dispersions. London: Cambridge University Press, 1992, 544 p.}. To date, the state of the art has not clearly described a system that allows the form of oral administration of extract of propolis without the formation of

precipitated. An alternative to this formulation is to

· \ -; in the use of dispersions. The present invention is based on the

Use of a colloidal dispersion compatible with oral administration in mammals, containing extract of 1 propolis. This formulation is applicable for use as a mouthwash in the treatment of different dental diseases. For the composition of the system poloxamer and sorbitol are used as pharmaceutical adjuvants.

Poloxamers are copolymers composed of a

hydrophobic power plant. polyoxypropylene and two chains,

hydrophilic. polyoxyethylene. Poloxamer is a surfactant

not ionic and has the function of agent, emulsifier,

- ■ solubilizing ■ and wetting (Rowe -RC, Sheskey PJ, - Weller -PJ,

Rowe R. Handbok of 'pharmaceutical' excipients. 2003. 4th

ed. ', .Washington: APhA Publications, 776p.). It is appropriate 'pa'ra

the production of liquid, aqueous preparations containing · '1 ■. ■; '

hydrophobic substances. Its function: is reached by the

process Where surfactant molecules are adsorbed by

particles forming a film around them,. and

decreasing the: surface tension between ^, substance

dispersed— (in this case, the propolis extract particles) '.

.25 ■ is the liquid phase. ('Water).' This way, the decrease.

or even preventing the formation of lumps of

particles in the liquid and the 'fluctuation phenomenon,

difficult to wet (very hydrophobic) substances in

presence of water group together by lining clusters containing

30. gases that attach to the surface and float the

,. agglomerate in the suspension liquid. Sorbitol is a hexahydric alcohol referring to the mannose and isomer of the raariital (Rowe RC, Sheskey. PJ, Weller PJ., Rowe R. Handbook, pharmaçeutical excipiènts. 2003. 4th ed., WasKingtori: APhA * Publications, .7 7 6p ·.; 'Sweetman' 'SC; Martindale - the complete., Drug reference. 2002. 32 ed. London.:'Pharmaceutical Press .. 2483: p.)., Is relatively inert and compatible with. The. most ■ '' excipients; does not darken or decompose at 'high temperatures' It is used in ■ liquid preparations for use. oral, in 10 'injectable formulations, in suspensions and emulsions for internal and external use. Its main function in dispersed systems is that of an agent; viscous without, however, promoting. significant changes in behavior

-I 1 '

of the final product. 1 ' ,

The present invention is further described by the

■ following examples:

Example 1 - Obtaining Green Propolis

In the present invention was used as base the extract

■ glycolic of ..propolis green, originating in the species. vegetable 20 Baccharis dracunculifolia, popular - field rosemary, obtained commercially from beekeepers in the state of Minas Gerais. Green propolis is intensively produced by 'African bees. of the species 'Apis, mellifera, in / different regions of the Sado - (- cerrado savannas, South of Minas,' Zôna da 25,. resin supply species.

For the production of extracts, green propolis in brutb state had its origin confirmed. .. through analysis, microscopic laboratories. Extracts have been obtained by: maceration; centrifugation and; sedimentation, for the concentration of phenolic and flavonoid compounds,

Stivos' components of propolis (Table 1).

Table 1 - Detail of propolis extracts. green

used_; ., - ·, · '.: S - ■ · ■ ■. - ■

■ ..... "" iJ ... ..... ■: ·. \. Physical-Sample Analysis, Chemical Description. (%) · at. Phenolic Flavonoid '‘-' os 'des · ■ 156, Glycolic Extract *', PO 13/07 4, 5,, 1.28, 164 Glycolic Extract, POa 13/07, 3.7. 1.4. Glycemic Extract, Lot 16/08 6.85 · 1.44; 176 Glycolic Extract., Lot 19/08; 7.38 3.22 Benchmark values for · ', ^, 0, 5% S: green propolis · 0.25% /' ■ '. ■ Example I '' 1 '. Evaluation of antimicrobial activity of glycolic extracts of propolis; used

Antimicrobial activity tests were performed on the green propolis glycolic extracts used. (Table 11) against microorganisms: of clinical 'interest' in the oral cavity, namely: Candida albicans, ATCC 36802,

, Lactobacillus casei ATCC 7469, Porphyromonas gingivalis. ATCC 49917, Prevotela melani ngenica ATCC 2,584 5., Staphylocoocus aureus, ATCC 259,2,3 and Streptococcus mutans 15 ATCC 25175, all from the National Laboratory of Microorganisms' Quality Control Reference in Health, INCQS / FIOCRUZ, kept viable at 4 ° C in specific solid medium, with periodic subcultures every 20 days.

■ In order to compare the effectiveness of '' extracts. propolis-

green, was used as positive control 'the salt

■ /

chlorhexidine hydrochloride, a./ 0; 12% (w / vh, concentration · usually used in clinical practice. Concentrations of glycolic extracts evaluated were: 3, 4, 5 and 7%

25, (v / v) .. It was evidenced. what; the concentration of 5%. (, v / v) of the glycolic extracts was 'active' against all evaluated microorganisms. inhibiting by 100% their development, similar to that observed --for positive control. This finding, therefore, was the one undertaken in the preparation of the formulation of said invention. ,

Example 3 - Preparation of Colloidal Dispersion Formulation Containing Propolis Extract

. For the preparation of the mouthwash: buccal thorium were employed,. only extracts showing concentration of compounds;

1.0 'phenolics greater than or equal to OiSI (v / v) and flavonoids greater than or equal to 0.25% - (v / v) (Table 1). Glycolic extracts were chosen for the pharmaceutical formulation of the present invention because it is propylene glycol a product with excellent

, · ·, · · \ ■ '/' ·. ■ '■ /. Λ'1 ·

solvency, acting on Solubility is dispersion of the active - contained in the propolis samples. Further, propylene glycol is easy to handle, an important factor in the formulation process of a mouthwash, and of great acceptability by patients because it does not 'cause irritation' to the oral mucosa. //

For the preparation of colloidal dispersion, initially 1-

3% nonionic surfactant (polysorbate 80 or poloxamer F127) was weighed and added to one. vial · containing 7.5-9.5 ml distilled water under constant agitation. Then 20-30% of the sorbitol was weighed and added to the flask under constant stirring until the solution was completely homogeneous. Then 5% of the glycol extract of propolis was added to the solution. previously prepared under çonstahte agitation until: formation of a colloidal dispersion. The volume was completed to 10.0 mL and the dispersion formed was stirred until complete homogenization. Figure 1 'shows a photo of the glycolic extract' of water-diluted propolis and the same propolis extract prepared 'as per the formulation of the present invention. It was observed that the' formulation obtained was homogeneous, 5 'transparent , without the deposition of clumps or particles, contrary to the preparation containing the extract diluted in water.

Figure 1 - Photograph 'showing: A - aspect of the formulation

propolis rinsing system developed (transparent) in

compared to a suspension of propolis extract in water

(milky); B - Bottles in reverse order to show the

\ - deposition of propolis- extract particles at the bottom of the

,. suspension bottle of propolis extract in water (arrow),

- fact not observed in the developed formulation. , -

Example- 4Measurement of -ρΗ da; colloidal dispersion containing -1

propolis glycolic extract

The pH of the developed formulation was measured using

í

the Digimed pHmeter, model DM-20 (Digicron Analytical LTDAf Brazil). The value established for the desired range that did not allow oral mucosal irritation to occur was 6.5-7.5. Values below or above have been corrected to reach this range. . Example 5: Measurement of particle size of the colloidal dispersion containing glycol extract1 of 'propolis'

The size distribution of the. particles of colloidal dispersion was performed by means of dynamic dispersion of the. 5 optical light at 90 ° at 25 ° C / 'Zetásizer Model 300, OHS (Malvern' Instruments Ltd, UK). The result found, 136.5 nm, represents the average particle size. The particle size distribution showed an equivalent polydispersion of 100, 24.. . ^,. . '-

Example 6: Measurement of the electric charge of the particles of the colloidal dispersion containing glycolic propolis extract.

The colloidal particles are constantly,. under the influence of. forces . attractive, 'so' that repulsive forces, avoid their aggravation (González-Caballero .. F., 'López-Durán JD. G. Suspension formulation. In: Pharmaceutical emulsions and suspensions. Eds. Nielloud F., Marti-Masters G. New York: Marcel Dekker, 2000, chap. 4, pp. 127-190) In most polar liquids, these forces come from charges, from particles. : measured, of the intensity of the present charge and represents an indicative, clear, stable stability of a dispersion, so that a value close to zero would indicate colloidal instability and may cause aggregation of, particles.

The Zeta Potential of Colloidal Dispersion Particles

was measured by electrophoretic scattering of light at 25 ° C using the Zetasizer Model 3000HS (Malvern Instrurfients Ltd, UK). The encpntradip result is' -14, 2. mVr ± 3.85 represents the mean ± o-, deviation .30 paclron, and indicates a colloidal dispersion stability. Example 7: Viscosity methyide gives ■ colloidal dispersion containing glycol propolis extract · ■ ■

The viscosity of the developed dispersion was. measured using Si meter 'rotacignal.' adjusted on yelocida.de

■ * ’/ ■ ■, '■' 'v' · '*'; ■ Λ '■■ ■ ■

60rpm. The measurements. were performed in triplicate at a constant temperature of 25 ° C. The obtained viscosity value was 5.5 ± 0.4 Cp.

Example 8: 'Measurement of Refractive Index of, Dispersion

1 colloid containing glycolic extract of propolis.

'Aiming / evaluating system-transparency, the index

Refraction rate was determined using the refractometer without prior treatment of the formulation. Measurements were made in triplicate at a constant temperature of 25 ° C. The result obtained was · 1.366 ± 0.654. This value is

15 'near the water-refractive index,' showing, 'portánto,

■ that the system is transparent.

Example 9: Evaluation of the stability of the glycolic extract-containing colloidal dispersion of propolis.

Dispersions: solid containing glycolic extract of

'' '' '' L,

propolis, prepared there. approximately twelve months and stored at temperatures of 4 ° C, 25 ° C. at 40 ° C showed no signs of loss of formulation stability, such as: deposit formation, lump formation, crystal growth and phase separation.

Example 10: Evaluation of aVitimicrobial Activity of Glycolic Containing Extract Containing Glycolic Extract of Propolis

■; 'Antimicrobial activity tests of colloidal dispersion containing green propolis glycolic extract were performed according to the methodology previously described in

J 'J-

Example 2. In this test was also employed as a positive control 0> 12% chlorhexidine® digluconate oral solution

, (w / v). v

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) propyl gilcolic extract. This result was similar to that observed - for oral solution,

chlorhexidine digluconate. currently the product of choice in dental offices but which has high adverse effects. To confirm the similarity of the present invention to the current product of choice, the independence test / exact test was performed, shown in. Table 2: / /. ■: '

Table 2. Exact test .conditional, e. odds ratio for comparison between the present invention and the chlorhexidine hydrochloride salt

Exact Conditional Test, Independence, and Controls S '_P-R value. C IC \ PI x C + 12, 00 1,000 0,000 [0'- inf] PI x CC 0,00 0,000 0,000 [0 - 0, 04] 'C + x CC. 14.0.0 0.000 ·. Inf [94.01 - Inf] C +; · Chlorhexidine digiucchate solution (C, .12%) PI: ', present invention' CC: growth control '.

The exact conditional independence test showed 20. that, there is no difference. between the present invention and the oral solution. of chlorhexidine digluconate, 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.

25 Figure. 2. Evaluation, from the pressing injury to the yeast

1 Candida albicans ■ (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 digluconate solution.

Figure 3. Evaluation 'of. present invention against Lactobacillus casei (A, B, C) and Streptocoçcus. mutans, · (D,. EVv. F) .. (A and D) Test plates containing formulation of this

'invention, (B-e E) growth control of the microorganism;

■■■ ■ '' '', -. * ■ ". 23 / 23- (Ce F) control, positivei: digluconate solution of

^ II '.:

0.12% chlorhexidine.

Figure A. ■ Evaluation of the present invention in the face of Porphyromonas gipgivalis (A, B, C) and Prevella melayninogenic (D, E, F). . (A and D) Test plates containing formulation of the present invention, ■ (B and E). growth control of the microorganism, (C and F) positive control: 'digluconate solution of. 0.12% chlorhexidineâ. ·. ·

The results presented demonstrated that the present

Green Propolis, at 5% concentration, is capable of totally inhibiting myGrobial growth of six major, commonly isolated oral cavity microorganisms implicated in cases of mueositis and peri-implantitis-s. demonstrated activity

antimicrobial similar to that observed for the oral solution; Chlorhexidine digluconate base, currently the product of choice in -> dental offices'. The results presented show the therapeutic potential of the present. invention as a low cost, easily formulated agent,

. in place of the product currently employed.

Claims (15)

1. Process for the production of a pharmaceutical formulation containing green propolis, which is not a dispersion, colloidal. A pharmaceutical formulation as described in claim 1 containing green propolis, the propolis being employed as glycolic extract. A pharmaceutical formulation as described in claim 2 containing green propolis, whose glycolic extract has activity against microorganisms of clinical interest in the oral cavity. Pharmaceutical formulation as described in claim 2 containing 'verdant propolis capable of reducing'. hydrophobia of propolis extract and its tendency to precipitate formation in aqueous medium. - A pharmaceutical formulation as described in claim 2 containing propolis. green that can be given by mouth. Pharmaceutical formulation as described in claim 2 containing green propolis which may be applied to the treatment of different dental diseases. A pharmaceutical formulation as described in claim 2 containing green propolis green propolis, wherein such formulation consists of a non-ionic surfactant and a concomitant agent such as sorbitol. A pharmaceutical composition as described in claim 7 containing green propolis having a pH between 6.5-7.5. A pharmaceutical composition as described in claim 7 containing green propolis having particulates less than 200 nm in size, preferably ranging from 100 to 18 µm. ' A pharmaceutical composition as described in claim 7 containing green propolis which has a zeta potential of about -15mV. ., A pharmaceutical composition as described in claim 5 containing propolis which has a viscosity-in. around 5.5 Cp. Pharmaceutical composition as described in claim 7 containing green propolis having a refractive index of around 1.4. A pharmaceutical composition as described in claim 7 containing green propolis which. stable stability at different temperatures over a period of twelve months. A pharmaceutical composition as described in claim 7 containing green propolis which does not induce formation of deposits and lumps, crystal growth and phase separation. '' A pharmaceutical composition as described in claim 7 containing green propolis which exhibits antimicrobial activity to different microorganisms of clinical interest in oral charity.