WO2018023182A1 - Red propolis extract-loaded nanospheres, method for obtaining nanospheres, dermocosmetic compositions containing same, and uses - Google Patents

Red propolis extract-loaded nanospheres, method for obtaining nanospheres, dermocosmetic compositions containing same, and uses Download PDF

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Publication number
WO2018023182A1
WO2018023182A1 PCT/BR2017/050088 BR2017050088W WO2018023182A1 WO 2018023182 A1 WO2018023182 A1 WO 2018023182A1 BR 2017050088 W BR2017050088 W BR 2017050088W WO 2018023182 A1 WO2018023182 A1 WO 2018023182A1
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Prior art keywords
red propolis
propolis extract
extract
red
loaded
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PCT/BR2017/050088
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French (fr)
Portuguese (pt)
Inventor
Ticiano Gomes DO NASCIMENTO
Mário Calheiros DE LIMA
Lais Farias AZEVEDO
Priscilla Da Fonseca SILVA
Irinaldo Diniz Basílio JÚNIOR
Maria Cristina Delgado DA SILVA
Josealdo TONHOLO
Camila Braga DORNELAS
Eduardo Jorge Da Silva FONSECA
Círia Vieira BARBOSA
Ana Rúbia Batista RIBEIRO
Cantídio Francisco De Lima FILHO
Isabel Cristina Celerino De Moraes PORTO
Marianna Porfírio BRANDÃO
Louisianny Guerra DA ROCHA
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Universidade Federal De Alagoas
Apícula Fernão Velho
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Publication of WO2018023182A1 publication Critical patent/WO2018023182A1/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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention pertains to the field of natural public health utility products in aesthetics and beauty, more specifically those of the active ingredient loaded nanoparticle systems of the biologically active red propolis extract, intended for dermal administration and in the mucous membranes.
  • Red propolis extract (NPV) loaded nanospheres processes for obtaining NPV, and dermocosmetic compositions containing NPV and uses are proposed.
  • NPV production is linked to the use of standardized red propolis hydroalcoholic extract, active ingredient of NPV, and matrix system of organic pharmaceutical polymers with encapsulating, dispersing and stabilizing function of these suspended nanospheres, which will be nanoprecipitated and lyophilized to obtain nanospheres.
  • NPEPV-containing dermocosmetic compositions have application in dermocosmetic and cosmeceutical sectors, in aesthetic treatment, beauty, in reparative surgeries, due to their antioxidant, anti-inflammatory, anticancer and healing activity, as well as their leishmanicidal action.
  • Flavonoids, phenolic acids and terpenes are the main substances found and used to track quality and, in some cases, to demonstrate the authenticity of propolis in some geographical regions (Volpi, N.; Bergonzini, G., Journal of Pharmaceutical and Biomedical Analysis, V. 42, p. 354-361, 2006).
  • Flavonoids and phenolic acids most commonly used as markers include: quercetin, canferol, naringenin, chrysin, pinocembrine, galangin (flavonoids); gallic acid, caffeic acid, p-coumaric acid, ferulic acid, cinnamic acids and derivatives (phenolic acid), clerodanes (diterpenoids) (Rosalen, PL; Castro, M.L; Cury, JA, Qu ⁇ mica Nova, V. 30, N 07, 1512-1517, 2007); (Yao, L.; Jiang, Y .; Singanusong, R., Datta, N.; Raymont, K., Food Chemistry, V. 86, p.169-177, 2004); (Weston, RJ, Mitchell, KR, Allen, K.L. Food Research International, V. 37, pp. 166-174, 2004).
  • propolis and propolis-containing products such as extracts, creams, ointments, tablets, capsules, or powders (H. Menezes, M Bacci Jr., SD Oliveira, FC Pagnocca, Antibacterial properties of propolis and products. containingpropolis from Brazil, Apidologie, V. 28, p. 71-76, 1997).
  • red propolis As far as red propolis is concerned, it was classified as the 13th subtype of Brazilian propolis found in the mangroves, lagoons, rivers and beaches of northeastern Brazil between the states of Sergipe, Alagoas, Pernambuco and Para ⁇ ba by the researcher (Andreas Daugsch, Cleber S. Moraes, Patricia Fort and Yong K. Park, Brazilian Red Propolis — Chemical Composition and Botanical Origin, eCAM 2008; 5 (4) 435-141 doi: 10.1093 / ecam / nem057).
  • the main botanical origin of red propolis is the Dalbergia ecastophyllum plant, locally known as the Howler's Tail, which has a red resinous exudate released from its sap.
  • flavonoid subclasses have also been identified, including new types of flavonoids from northeastern red propolis that we can mention: rutin, quercetin, luteoline, pinocembrin and biochanin A, formononetin, daidzein, liquiritigenin, pinobanksin, dalbergin (neoflavonoids) (Andreas Daugsch) , Cleber S. Moraes, Patricia Fort and Yong K. Park, Brazilian Red Propolis - Chemical Composition and Botanical Origin, eCAM, Evidence-based complementary and alternative medicine, 2008; 5 (4) 435-441 doi: 10.1093 / ecam / nem057 ).
  • red propolis With a view to the discovery of molecules and their therapeutic targets for the cure of diseases, red propolis has been subjected to several detailed studies on its biological activities. Research into the identification and structural elucidation of new substances continues to be developed in the identification of new substances present in red propolis extract. A new chalcone, 3, 4, 2 ' , 3 ' tetrahydroxchalcona and a new C-glycosylated flavonoid, narigenin-8-C-hexoside, as well as (3S) -vestitol and (3S) -7-O-methylvestitol have been identified.
  • Xanthochimol and formononetin enriched red propolis extracts have been showing in vitro antitumor activity against different cell lines such as melanoma using B16F10 cell line, ATCC CRL6475 (Maria Novak, Martha Silveira and Costa Silva, Maria Academic Marcucci, Alexandra Christine Helena Frankland Sawaya, Begoha Giménez-Cassina Lopez, Maria Angela Henriques Zanella Fortes, Ricardo Rodrigues Giorgi, Kamila Tamie Marumo, Rosangela Felipe Rodrigues, Durvanei Augusto Maria Antitumoural activity of Brazilian red propolis fraction enriched with xanthochymol and formononetin vivo study, Journal of Functional Foods, 11, (2014), 91-102).
  • Red propolis ethanolic extract has been showing activity against cancer cells of the glioblastoma (SF-295), ovary (Ovcar-8) and colon (HCT-1 16) strains (Izabel Academic Gomes de Mendonça, Isabel Chris Celerino Porto, Ticiano Gomes do Nascimento, Naiana Soares de Souza, Jose Marcos dos Santos Oliveira, Rodolfo Elleson dos Santos Arruda, Kristiana Cerqueira Mousinho, Aldenir Feitosa dos Santos, Irinaldo Diniz Basilio Junior, Abhishek Parolia, Francisco Stefendio Barreto, Brazilian red propolis: phytochemical screening, antioxidant activity and effect against cancer cells, BMC Complementary and Alternative Medicine (2015) 15: 357.DOI 10.1 186 / s12906-015-0888-9).
  • African Journal of Microbiology Research. 4,556-560. leishmanicidal activity against tropic Leishmania with IC50 of 32 ⁇ g / mL.
  • Comparative study of Brazilian green propolis extracts and Bulgarian propolis extract by Machado et al. (2007) showed activity against leishmanicides 4 different species of Leishmania chagasi (amazonensis braziliensis and Major).
  • Brazilian green propolis extract showed IC50 close to 49 ⁇ g / mL against braziliensis chagasi and Major species
  • Bulgarian propolis extract showed leishmanicidal activity for amazonensis, chagasi and Major species with IC50 between 2.8 ⁇ g / mL and 41 ⁇ g / mL.
  • the skin acts as a large organ that covers a surface of over 20,000 cm 2 with varying properties and functions, as it protects the body from microorganism invasion, protects against UV rays, produces melanin and immunoglobulins, and controls body temperature, blood pressure as well as having sensory function.
  • the skin has several histological layers, being divided into stratum corneum, epidermis, dermis and subcutaneous adipose tissue.
  • stratum corneum and hair follicles and glands are considered the first barriers that limit the entry speed of the active substances that make up a cosmetic cream (Leon Lachman, Herbert A. Lieberman, Joseph L Kanig, Theory and Practice in Industry Pharmaceuticals, Vol. II, Chapter 18, pages 907-953, 2001).
  • the choice of active extract encapsulating agents and excipients used in cosmetology with multifunctional properties is extremely important, ie excipients with various functions such as: skin permeation facilitators, photoprotective action, specific cosmetics, lipophilic / hydrophilic system stabilizers, viscosity and sensory control, as well as being biocompatible and biodegradable.
  • Cosmetic excipients are essential in a dermocosmetic composition as they have characteristics that ensure stability to the composition, facilitate administration, promote the release of active substances from the dermal matrix thus promoting the bioavailability of active substances in different layers of the skin and consequently the appropriate pharmacological action. without toxicity, as well as consumer acceptability from a sensory and aesthetic point of view.
  • excipients that will adequately release the drug or active substances from a dermocosmetic matrix and among them are the excipients: emulgents, emollients, humectants, water absorbent bases, hydrophilic bases. , waxes, hydrocarbons, oilseeds, alcohols, fatty acids, esters, polyols, insoluble powder, essences, preservatives, antiseptics.
  • Some excipients used in cosmetic compositions may alter the internal structure of the stratum corneum by altering the barrier function and increasing the permeation rate of active substances by the stratum corneum.
  • cosmetic excipients may generate negative influences for dermocosmetic composition.
  • Cosmetic compositions due to the presence of lipid excipients, or the presence of lipid active substances, are subject to self-oxidation reactions with subsequent decomposition, which is a drawback for these formulations, resulting in aroma and appearance. unpleasant and rancid reactions. Therefore, the choice of excipients should be well defined to avoid problems of drug-excipient or excipient-excipient incompatibility, reducing the stability of the active substance (s) in the composition as well as toxicity problems.
  • Some active substances may be highly reactive in a dermocosmetic composition and produce various chemical reactions reducing the stability of these semi-solid compositions. Commonly anticipated chemical reactions include hydrolysis reactions of esters, amides, imides, lactone; oxidation reactions due to the presence of high oxygen concentration in the composition and self-oxidation; isomerization reactions; Photodegradation reactions initiated by the action of light causing the potency of the active substances to decrease (Katsunori Yoshida, Tomoko Sekine, Fumiaki Matsuzaki, Toshio Yanaki * , and Michihiro Yamaguchi. Stability of Vitamin A in Oil-ln-Water-In-Oil-Type Multiple Emulsions, JAOCS, Vol. 76, No.
  • propolis extracts have in their composition several active substances of different phytochemical classes, namely: flavonoids, phenolic acids, terpenes, clerodanes, xanthones, benzophenones among others, which may in contact with different dermocosmetic excipients promote chemical reactions of the propolis. More diverse reducing power, stability, and result in unpleasant looking products and also result in toxic effects. In this sense, it is necessary to develop micro or nanoencapsulation techniques of active substances of natural origin.
  • Micro and nanoencapsulation techniques are used by pharmaceutical, food and cosmetic industries in order to solve technological bottlenecks among them: protecting the active substance (s) against extrinsic agents (moisture, light, oxygen) that may cause hydrolysis, photodegradation, oxidation reactions; prevent loss of volatile substances; increase the shelf life of the product; promote controlled release of active substances; as well as improving specific delivery characteristics of active substances in target tissues.
  • extrinsic agents moisture, light, oxygen
  • Nanoparticles in general, are obtained by various preparation techniques which among them we can mention the techniques of nanoemulsion formation, and among them we can mention: double emulsification method, emulsification-coacervation method, polymer coating method , layer-by-layer coating method, emulsification-diffusion method, and nanoprecipitation method (Mora-Huertas, CE Fessi, H. Elaissari, A. Polymer-based nanocapsules for drug delivery. International Journal of Pharmaceutics 385 (2010) 1 13-142 ).
  • Nanoparticle drying techniques such as solvent evaporation by spin-evaporator, spray-drying, lyophilization and others.
  • One method of choice for obtaining widely used microencapsulated and nanoencapsulated is the freeze drying method. Freeze drying is widely used in drying substances thermolabile, as a material containing protein substances, microorganisms such as lactic bacteria used in the production of yeast, edible fungi, among others. It is also employed as a method of encapsulating temperature sensitive and high pressure active materials. Nanoparticle drying has the advantages of extending shelf life of active substances, prevents degradation of substances in aqueous and suspended media, facilitates redispersion process and reincorporation into semi-solid systems.
  • KR1020030063053 produces granules from a mixture of 10 to 30% propolis, 40 to 70% lactose, 0.1 to 1.0% vitamin and 10 to 30% dehydrated honey by lyophilization.
  • Chinese document CN171 1992 obtains propolis microcapsules using in the high cost excipient composition beta-cyclodextrin followed by lyophilization process.
  • JP2006028174 produces propolis granules using a 50% amino acid mixture (propolis extract: amino acid mixture).
  • Propolis microencapsulates containing a modified starch (starch octenyl succinate) and gum arabic were obtained by Spray-Dryer technique (Silva F.
  • propolis microencapsulates for various purposes is described in the prior art.
  • CN102920652 prepares propolis microencapsulates with other biopolymers: chitosan, hydroxypropyl betacyclodextrin and sodium glycerophosphate for treatment of periodontal disorders.
  • CN102772596 uses multimixture of plant extract, propolis and 5% chitin to obtain preparations using the spray-dryer technique for obesity treatment.
  • CN102641256 obtains microencapsulates of propolis with urea, gelatin, paraffin, and toxic solvent (hexane) for preparation of microencapsulates with large particle size between 70 to 150 ⁇ .
  • CN102429141 uses gelatin / pectin and propolis system for preparation of slow release microcapsules using lyophilization.
  • microcapsules are formed by a nucleus active ingredient, coated with a coating based for example on ethylcellulose, polyvinylpyrrolidone, castor oil and magnesium stearate.
  • European patent EP 548356 claims a rapidly disintegrating multiparticulate tablet comprising an active substance in the form of microcrystals or microgranules provided with a coating.
  • WO 2014/166994 A1 develops a mixed active ingredient release system and comprises a nano-micro particulate system which has active ingredients within the nanoparticle as well as within the microparticle for use in oral compositions.
  • a polymeric propolis nanoparticle formulation (nanofood propolis) was developed using cross-linked micellar aggregates of N-isopropylacrylamide (NIPAAm) with N-vinyl-2-pyrrolidone (VP) and poly (ethylene glycol) monoacrylate (PEG-A) (Kim, Dong-Myung, Lee, Gee-Dong, Aum, Seung-Hyun, Kim, Ho-Jun, Biological & Pharmaceutical Bulletin, (2008) 31, 1704-1710).
  • NIPAAm N-isopropylacrylamide
  • VP N-vinyl-2-pyrrolidone
  • PEG-A poly (ethylene glycol) monoacrylate
  • US 20130295181 and EP2633862 of the same patent family develops polyvinyl alcohol (PVA) propolis nanocapsules, submicrometic particle size ( ⁇ 1000nm) ranging from 220nm to 438nm with different applicability and different areas: agronomic, pharmaceutical and cosmetics, etc.
  • PVA polyvinyl alcohol
  • WO / 2011/001 181 and CA2765920 utilize propolis extract to obtain a solid carrier matrix of active substances, specifically developing a propolis-containing solid film.
  • WO2015 / 022471 A1 uses coating nanocapsules for active oleophilic substances present in UVA / UVB sunscreens.
  • KR20100078349 discloses propolis nano emulsion preparations using ⁇ -cyclodextrin excipient.
  • WO 2015/003155 A1 shows applicability of biocompatible nanofibers containing chitosan, honey, acetic acid, gelatin, collagen, alginates, BSA and other crosslinked nanometric network forming agents and natural or synthetic active substances against various pathologies such as bacteria, hypertension artery, wound healing, cancer.
  • PCT / US2013 / 075714 deals with compositions containing anhydrous powder containing liquid submicron particulate system ( ⁇ 1000nm) used for the preparation of emulsions, suspensions, solutions and powders for cosmetic or pharmaceutical purposes.
  • This system consists of a hydrophilic core containing a hydrophobic coating that will protect all active ingredients in its core.
  • Chinese Patent Document CN103520069 utilizes honey and propolis in nail beauty products and compositions including gel-like compositions, creams and adhesives. Soft gelatin capsules have been developed in the patent document to maintain beauty and youth as well as regulate immunity, cholesterol and senescence.
  • Chinese Patent Document CN1031 160399 develops soap with natural extracts, propolis and base ingredients for soap making for multifunctional treatment of cleaning, bacterial removal, allergy reduction and antiseptic.
  • Chinese patent CN101461846 develops a gel containing plant extract and herpes zoster propolis.
  • Japanese Patent Document JP2008247830 utilizes propolis extract and amaranth extract in cosmetic composition to inhibit skin blemishes, pigmentation, age spots.
  • CN101 181 198 utilizes kojic acid, fibroin, propolis and other ingredients in the preparation of a nourishing beauty cream for removing skin blemishes.
  • Patent utilizes honey, pearl powder, propolis, glycerin to produce a multifunctional massage and beauty cream reducing inflammatory skin processes such as acne.
  • JP2006016373 with multifunctional property of weight reduction, hair reduction and beauty treatment utilizes propolis, royal jelly, plant extracts, and collagen.
  • KR1020030093890 uses propolis extract and green tea extract and other plant extracts to produce an antibacterial, antiseptic, multi-functional soap, atopic dermatitis and odorant action soap.
  • Japanese Patent JP2003055138 uses honey and propolis extract from Brazilian rosemary to produce a type of plaster with antibacterial and antifungal action, as well as for beauty treatment.
  • Chinese patent CN1244386 uses propolis and perilla extract to produce particles between 0.01 to 10 ⁇ , in compositions used in the treatment and prevention of caries, periodontal inflammation, antibacterial agent.
  • nanoencapsulated In the nanocapsulation process of drugs and active substances some polymers with nanoencapsulant properties have been used in compositions of said nanospheres containing a solid core or nanocapsules containing an oily or liquid core. A combination of nanoencapsulant polymers has also been used to form so-called solid polymeric matrices (nanospheres) or solid-walled liquid core nanocapsules. The term "nanoencapsulated” can also be used more broadly without defining the type of polymeric nanoparticle (nanosphere or nanocapsule).
  • polymeric systems or also called polymeric matrices has been found in the scientific literature and can combine one or more of the following polymers: Span, polyvinyl alcohol, polyethylene glycol, polyoxypropylene, Tween, pluronic, polyoxyethylene, polycaprolactone, ceramide, acid polylactic / polyglycolic, alpha-tocopherol polyethylene glycol succinate, trimethyl chitosan chloride, among others (M. Wulff-Perez, A. Torcello-Gomez, MJ. Galvez-Ruiz, A.
  • polymeric coating matrices guarantees advantages to the formulator among them, stability to the nanoparticulate matrix system, avoids degradation problems or hydrolysis reactions, photodegradation, among others, and also ensures standardized process of release of active substances.
  • polymeric matrices in the process of micro or nanoencapsulation of drugs with pharmacological activity against cancer (tamoxifen, paclitaxel), using plant extract, propolis extract as well as phytopharmaceuticals (rutin) (Juliana S. Almeida , Fernanda Lima, Simoni Da Ros, Luis OS Bulh ⁇ es, Leandro M.
  • US20130295181 and EP2633862 develops nanocapsules of polyvinyl alcohol (PVA) -based propolis ranging in size from 220nm to 438nm with different applicability including agronomic, pharmaceutical and cosmetic areas.
  • PVA polyvinyl alcohol
  • the invention proposed herein seeks to fill a technological gap in the application of red propolis as an active element of modified release dermocosmetic compositions for the treatment and prevention of diseases by proposing a modified release form of nanoparticles.
  • propolis extract containing polymeric compounds NPEPV
  • polymeric nanoparticles containing modified release red propolis extract, processes for obtaining NPEPV, dermocosmetic compositions with NPEPV, preparation processes for dermocosmetic compositions with NPEPV and uses of these antioxidant, anti-inflammatory, cytostatic dermocosmetic compositions are proposed. / cytotoxic, wound healing in the postoperative period, decubitus wounds, beauty and beauty treatments, hygiene and cleanliness.
  • red propolis is a determining feature in innovation, in terms of new health technologies with the use of propolis and opotherapics, since red propolis has a characteristic composition, which will play a primary role in the activity of NPEPV and other dermocosmetic compositions for disease prevention and treatment, aesthetics and beauty.
  • the red propolis-containing polymeric nanoparticles and dermocosmetic compositions proposed in the present invention utilize encapsulating, stabilizing and dispersing compounds of the core in the intermediate matrix which will promote controlled release as well as avoid oxidative processes between phenolic compounds present in red propolis with other cosmetic excipients present in the external matrix, as well as a polymultifunctional external oil / aqueous matrix with accessory activities that contain biocompatible, biodegradable, non-toxic and stable cosmetic excipients with rheological characteristic for cosmetic semisolids / modified release cosmeceuticals.
  • the composition disclosed herein can be used to solve semi-industrial and industrial technical problems in the dermocosmetic areas, as well as some therapeutic applications.
  • the present invention features nanobeads loaded with red propolis extract (NPV), composed of standardized extract of red propolis from the red propolis raw material, and three nanopolymer matrix systems composed of coating and dispersing / stabilizing polymers.
  • NDV red propolis extract
  • These nanoencapsulant systems are composed of binary or ternary systems, with biocompatible, biodegradable and modified release properties promoting the active principle, protect dermocosmetic compositions from oxidative attack of phenolic substances present in the active extract of red propolis, besides presenting leishmanicidal activity.
  • the invention also relates to the process of obtaining polymeric nanoparticle suspensions which are nanoencapsulated in a modified release system by the nanoprecipitation technique followed by centrifugation and lyophilization to produce solid state nanospheres loaded with red propolis extract. Additionally, it is presented in this embodiment of the invention in terms of semisolid compositions containing the NPV, preferably in the form of nonionic cream, nonionic cream gel, nonionic gel cream, nonionic lotions, and may also prepare cationic creams.
  • the disclosed composition comprises: i) a core containing red propolis active substance (s) combined with a polymeric nanoclay matrix that may modify or delay the release of the active substance (s) contained in the red propolis extract.
  • the invention addresses the process of preparing the dermocosmetic composition from preparing suspensions of red propolis-containing polymeric nanoparticles, as well as from solid state polymeric nanoparticles containing red propolis extract.
  • the attached Figure 1 shows NPV Electron Scanning Photomicroscopy - NCEU20% (A and B), NCEU 12% (C and D) and NCPCL30% (E and F) using colloidal silicon dioxide cryoprotectants (A, B and E) and sodium starch glycolate (C, D and F). Photomicrographs with magnification between 3000 times (5 ⁇ scale) and magnification up to 12000 times (1 ⁇ scale) showing nanoscale particles.
  • NEU NEU
  • NPCL NPCL
  • C NEUPCL
  • the attached Figure 3 shows NPV FTIR-ATR spectra.
  • Red propolis extract, placebo composition and 30% NEU composition A and B.
  • Red propolis extract, placebo composition and NPCL composition 30% C and D.
  • Red propolis extract, placebo composition and composition 30% NEUPCL E and F).
  • the attached Figure 4 shows the chromatograms (UPLC-DAD) of red propolis extract (A), Placebo NEU (B), NEU30% composition (C), NPCL 30% composition (D) and NEUPCLC 30% composition ( E) which identifies flavonoids / isoflavonoid present in red propolis extract and compositions and absence of flavonoids in placebo.
  • ULC-DAD chromatograms
  • FIG. 5 shows a standardized IC50 determination plot in an in vitro leishmanicidal assay against Leishmania (V.) braziliensis (A). Schematic illustration of some hypothetical biochemical mechanisms of cellular inhibition of Leishmania (V.) braziliensis by flavonoids present in the red propolis extract from Alagoas as well as in nanospheres loaded with red propolis extract (B).
  • the attached Figure 6 shows photos of non-ionic creams containing NPCL30% loaded with red propolis extract to obtain final concentration in the cosmetic cream of 2.0% (A), 1.5% (B), 1.0% (C) and 0.5% (D) of the red propolis extract.
  • Solid state NPV nanospheres were either directly incorporated into the nonionic cream or using polymeric nanoparticle suspensions (B).
  • the nanoparticles (solid or in suspension) presented herein have differentiated flavonoid, isoflavonoid, chalcone and phenolic acid concentrations due to the specificity of red propolis.
  • the nanoparticles in question have an important feature of protecting the semi-solid matrix system (cosmetic cream) from the pro-oxidant action of flavonoids and phenolic compounds present in red propolis extract, since cosmetic creams prepared directly with red propolis extract are darkened. (brown color) within 10 days, due to oxidizing reactions of phenolic compounds against the cosmetic excipients present in this matrix dermal release system.
  • the proposed polymeric coating has a second important functionality, modified release of active compounds, delaying the release of flavonoids / isoflavonoids, for a longer action, and maintenance of active principles longer with biological activity in the layers. It is important in treatments against skin inflammation, cancer, leishmanicidal action, wound healing in leishmaniasis, besides promoting anti-aging action due to its antioxidant properties.
  • red propolis extract loaded nanospheres presented in this invention may also be applied to the production of red propolis capsules.
  • NPV in addition to the extract with the active principles of interest, obtained from red propolis, have in their composition pharmaceutical excipients with different functionalities: active compound (extract of red propolis), polymeric coating matrix with nanoencapsulant function. , emulsifier, dispersant, stabilizer and antioxidant.
  • NEU polymeric nanoparticle in different percentages of red propolis extract is presented as a composition of: standardized red propolis extract from the red propolis raw material in a proportion of 5 to 80%, preferably between 10 and 55%; 50 to 70% encapsulating agent and 15 to 30% dispersing / stabilizing agent.
  • an NPCL polymeric nanoparticle in different percentages of red propolis extract has the following composition: standardized hydroalcoholic extract of red propolis from the red propolis raw material in a ratio of 10 to 80%. preferably between 15 and 55%; 40 to 90% encapsulating agent and 1 to 20% dispersing / stabilizing agent.
  • a NEUPCL polymeric nanoparticle in different percentages of red propolis extract has the following composition: standardized hydroalcoholic extract of red propolis from the red propolis raw material in a proportion of 10 to 95%. preferably between 15 and 50%; encapsulating agents in a ratio of 30 to 80% and a dispersing / stabilizing agent in a ratio of 1 to 20%.
  • the polymeric matrix system is characterized by being a binary system that promotes nanoparticle nanoencapsulation, in addition to stabilization and dispersion after aqueous nanoprecipitation, composed of Eudragit® E100, also known as poly (butyl-co (2-dimethylaminoethyl methacrylate) (methyl methacrylate) (PM: 47,000) and pluronic F-108 triblock copolymer (PM: 14000)
  • the dispersing / stabilizing system can also be characterized by being composed of Eudragit®E100 (PM: 47,000) and pluronic F-68 triblock copolymer ( PM: 14,000).
  • the polymeric nanospheres loaded with red propolis extract consisting of biocompatible and biodegradable excipients, as well as conferring modified release to the nanoencapsulated, also reduces attack.
  • red propolis extract consisting of biocompatible and biodegradable excipients, as well as conferring modified release to the nanoencapsulated, also reduces attack.
  • the polymeric matrix system is characterized by being a ternary system that promotes nanoparticle nanoencapsulation as well as stabilization and dispersion after nanoprecipitation.
  • the dispersing / stabilizing system in aqueous medium consisting of poly-s-caprolactone or PCL, Eudragit® E100, also known as poly (butyl-co (2-dimethylaminoethyl) methacrylate (methyl methacrylate) (PM: 47,000) and pluronic F-108 triblock copolymer (PM: 14000)
  • the dispersing / stabilizing system in NEUPCL20%, NEUPCL25%, NEUPCL30% and NEUPCL40%, can also be characterized by being composed of poly-s-caprolactone (PM: 10,000), Eudragit ®E100 (PM: 47,000) and pluronic F-68 triblock copolymer (PM: 14,000).
  • the polymeric matrix system is characterized by being a binary system that promotes nanoparticle nanoencapsulation, in addition to stabilization and dispersion after nanoprecipitation in aqueous medium composed of poly-s-caprolactone or PCL (PM: 10,000) and pluronic F-108 triblock copolymer (PM: 14,000).
  • the dispersing / stabilizing system in this embodiment of the invention in NPCL20%, NPCL25%, NPCL30%, NPCL40% and NPCL50%, can also be characterized by being composed of poly-s-caprolactone (PM: 10,000) and pluronic F-68 triblock copolymer. (PM: 14,000).
  • PM poly-s-caprolactone
  • PM: 14,000 pluronic F-68 triblock copolymer.
  • Preparations of the polymeric nanoparticles containing red propolis extract are obtained by superconcentrated and standardized red propolis hydroalcoholic extract with residual solvent percentage from 0.01 to 10%, preferably from 0.01 to 3% using the process. maceration and ethyl alcohol at 75 to 85 Q GL for a time period between 48 to 72 hours of extraction and extract concentration in rotary evaporator using a temperature of 45 ° C and a pressure of 650 immHg.
  • red propolis extract A dark red solid mass, with a yield between 50 and 70%, will be obtained with a solvent percentage between 0.01 and 35%, being called red propolis extract.
  • the extract should have a solvent percentage of 0.01 to 3%.
  • NPV steps include: 1) Weighing of organic phase components including standardized red propolis extract, poly-s-caprolactone (PM 10,000), Eudragit®E100 (PM: 47,000) and solvent organic with sufficient volume for solubilization; 2) Weighing the aqueous phase components including pluronic F-108 triblock copolymer (PM 14,000) and milli-Q-type ultrapure water sufficient to solubilize pluronic F-108 in a separate container; 3) The components of the organic phase are solubilized in acetone, or chloroform or dichloromethane, preferably in acetone and transferred to ultrasonic bath of specific power and frequency for complete solubilization for a period of 15 minutes; 4) The components of the aqueous phase are also transferred to an ultrasonic bath under the same conditions as the organic phase for complete solubilization; 5) The aqueous phase components are diluted 14 to 99 times with milli-Q water; 6) The components of the organic phase are transferred to a falcon tube and disper
  • the suspensions of nanoparticles loaded with red propolis extract (NEU20%, NEU30%, NEU40% and NEU50%), as well as the compositions (NPCL20%, NPCL25%, NPCL30%, NPCL40%, NPCL50%) as well as Compositions (NEUPCL20%, NEUPCL25%, NEUPCL30%, NEUPCL40%) were characterized using pH, mean particle diameter distribution, polydispersion index and zeta potential techniques. For pH determination the polymeric nanoparticle suspensions were placed directly in contact with the pH electrode.
  • compositions were subjected to freeze drying in Terroni® model LD1500 consisting of: 1) vacuum drying chamber containing 3 shelves, 2) a condenser for lowering the temperature to -43 ⁇ 5 Q C, and 3) low vacuum pump.
  • the system pressure was controlled to maintain 30 ° C Hg as indicative of complete drying of the solid polymeric nanoparticles containing propolis extract.
  • the polymeric nanoparticle suspensions were subjected to two main freezing processes which were: A) Slow Freezing, in which the polymeric nanoparticle suspensions were placed in a -20 ° C freezer. Q C for a period of between 48 to 120 hours and immediately transferred to a freeze dryer to perform a drying process for a period of 24 to 48 hours. B) rapid freezing with liquid nitrogen at -196 aid Q C for a period between 10 to 20 minutes. In this process the suspensions of polymeric nanoparticles were placed in 50 mL capacity antibiotic vials, which were packed in a stainless steel metal box before receiving the freezing liquid.
  • cryoprotective agents in this particular freezing process, namely: colloidal silicon dioxide from 0.1 to 10%, as well as sodium starch glycolate solution from 0.1 to 30%. Under these sample preparation conditions prior to the drying process the drying period was reduced by 33 to 50%.
  • Red propolis extract (NPV) loaded solid nanospheres after freeze drying, were characterized by different analytical techniques to demonstrate that the red propolis extract was fully coated by the proposed polymer matrix, and that the nanospheres present in nanometer size even after freeze drying process and that the phenolic compounds present in the red propolis extract were encapsulated in percentages greater than 30%.
  • analytical techniques we mention: A) Infrared Spectroscopy (FTIR-ATR).
  • FTIR-ATR Infrared Spectroscopy
  • the solid propolis extract loaded nanosphere matrix system was subjected to FTIR-ATR analysis in the scan range from 4000 to 400 cm-1 with number of scans from 64 to 128.
  • the equipment used was Thermo Scientific branded with Ommic acquisition. Samples of solid polymeric nanoparticles without propolis extract were also analyzed.
  • DSC Thermal Analysis
  • the solid nanospheres loaded with propolis extract (NPV) were subjected to calorimetric analysis calorimeter Shimadzu model DSC-60 under a nitrogen atmosphere with 50 ml / min flow. At a heating rate of 10 Q C / min. and in the temperature range between 30-400 Q C. An amount of 2.0mg ⁇ 10% was used and placed in aluminum crucible which were hermetically sealed. The equipment was calibrated to indium and zinc standards. Samples of placebo nanospheres (without propolis extract) were also analyzed.
  • Red nanoprobes loaded with red propolis extract were subjected to 2 drying methods: Slow freeze drying followed by lyophilization technique (method A); quick freeze drying followed by lyophilization technique (method B), the latter performed using cryoprotective agents.
  • the solid state NPVs, after drying, were prepared in double carbon tape stubs or directly on the coverslips, submitted to the 10mA gold film metallization system for 7 minutes in a Sanyu Electron Quick Coater SC metallizer. -701 and then analyzed on Shimadzu Electron Scanning Microscope, model SSX-550 Superscan. D) Identification, Dosage and (%) Degree of Encapsulation of flavonoids present in red propolis extract and NPV.
  • the determination (identification and assay) of flavonoids present in red propolis extract and NPV was performed on a Shimadzu UPLC-DAD, consisting of the following modules: a high pressure pump (Model, LC-20ADXR), degasser (model, DGU-20A3R), autoinjector (model, SIL-20AXR), chromatographic column oven, photodiode array (model, SPDM-20A) and fluorescence detector (model RF-20A), a controller (model, CBM-20A) and shimadzu Labsolution software.
  • a high pressure pump Model, LC-20ADXR
  • degasser model, DGU-20A3R
  • autoinjector model, SIL-20AXR
  • chromatographic column oven oven
  • photodiode array model, SPDM-20A
  • fluorescence detector model RF-20A
  • controller model, CBM-20A
  • Flavonoid separation occurred at a stationary phase (C18, 150 x 4.6 mm; 5 ⁇ ), mobile phase consisting of solvent A (ultrapure water) and solvent B (acetonitrile), pumped at a flow rate of 0.3 mL / min. in a gradient elution condition, initially having 70% water and 30% acetonitrile with percentage change from organic to 100% acetonitrile within 40 minutes, followed by an isocratic condition with 100% acetonitrile up to 53 minutes with return to condition 54 minutes, followed by an isocratic condition with 30% acetonitrile up to 60 minutes.
  • solvent A ultrapure water
  • solvent B acetonitrile
  • Analytical standards acquired from sigma-aldrich (formononetin, isoliquiritigenin, daidzein, biochanin A, pinobanksina) and extrasyntesis (liquiritigenin) were exactly weighed 2.0 mg and transferred to 10 mL volumetric flasks to obtain a concentration of 200 ⁇ g / mL.
  • Working solutions were prepared by dilution technique to obtain concentrations of 7.50; 5.00; 2.50; 1.00; 0.50 and 0.15 ⁇ g / mL and obtain calibration curve, which was used to determine the content (dosage) of flavonoids in red propolis extract and NPV.
  • Flavonoid identification was performed using retention times compared to analytical standards using the same analytical conditions as the working day.
  • Red propolis extract 100 mg
  • ml_ 10mg / mL concentration
  • a further dilution step was performed to obtain 1 mg / ml working solution and dilutions to 400 and 250 ⁇ g / mL.
  • NPV containing red propolis extract or NPV without red propolis extract were weighed exactly and corresponding to 1 mg / ml_ of the theoretical concentration of red propolis extract, solubilized with solvent system (acetone: ethanol, 6: 4 v: v.) then was carried dilution to 3 'ug / mL with the same solvent system, in order to determine the maximum amount of flavonoid encapsulated in NPV the NPV containing propolis extract or without extract Propolis (placebo) were also weighed exactly and corresponding to 1 mg / ml_ of the theoretical concentration of red propolis extract, solubilized with solvent system (water: ethanol, 7: 3, v: v), then dilutions were made.
  • Encapsulation Degree (%) (NPEPV marker concentration / EBPV marker concentration) * 100.
  • Solid nanospheres loaded with red propolis extract, placebo solid nanospheres (without red propolis extract) and red propolis extract were evaluated for their antioxidant activities.
  • Stock solutions of 1 mg / ml of NPV compositions containing red propolis extract were prepared, which were accurately weighed to obtain the corresponding 1 mg / ml of red propolis extract in these compositions and solubilized in acetone: ethanol solvent system ( 6: 4, v: v). The same procedure was performed with Red Propolis.
  • NPV stock solution 400 ⁇ _, 50 ⁇ _, 25 ⁇ _ and 13 ⁇ _ aliquots of the NPV stock solution were transferred to 5 mL volumetric flasks and 2 mL of a 3mM DPPH ethanolic solution were added and allowed to stand for 30 minutes in the dark for the reaction to occur. and then the volumetric flask was calibrated with absolute ethanol to obtain concentrations of 80 ⁇ g / ml, 10 ⁇ g / ml, 5 ⁇ g / ml and 2 ⁇ g / ml. The same procedure was performed with red propolis extract and placebo nanospheres (without red propolis extract).
  • the samples of red propolis extract were subjected to UV-vis spectrophotometer reading in wavelength-adjusted mode at 520 nm at the end of the 30 minute period.
  • the NPV and placebo nanospheres showed turbidity at the end of the reaction period and were centrifuged at a rotation speed of 5000 rpm for a period of 5 minutes.
  • the supernatant was subjected to UV-vis spectrophotometer reading under the same conditions. Readings were also taken of the 3 mM DPPH reference solution in ethanol, absolute ethanol solution to zero the equipment and the white solutions (3 mM absolute ethanol + DPPH without addition of red propolis extract or NPV) to calculate% antioxidant activity.
  • Process A Hot Preparation - Obtaining suspension of nanoparticles loaded with red propolis extract (NEU30% or NPCL30% or NEUPCL30%) in aqueous phase; Heating of the aqueous phase containing the suspension of polymeric nanoparticles containing propolis and the oil phase extract containing self-emulsifying cosmetic excipients in a nonionic cream in separate containers in temperature from 75 to 85 Q C, preferably to aqueous phase at 80 Q C and Phase oil 80 Q C; Incorporate the aqueous phase into the oil at the same temperature under stirring at 200 to 400rpm, preferably at 270 to 300rpm, maintaining constant stirring temperature for 10 minutes; Cool with moderate agitation to a temperature of 40 Q C; Add complementary phase (other dermatological actives, sensory modifiers and preservatives) under stirring and allow to cool to room temperature; Check pH and make corrections if necessary to
  • Process B Cold Preparation - Obtaining solid loaded propolis nanospheres obtained by freeze drying using slow freezing or fast freezing; Obtaining nonionic base creams, cream gel, nonionic gel cream, nonionic lotions when prepared with NPCL30% and obtaining cationic creams or cream ointment when obtained with NEU30% and NEUPCL30%; cooling and conditioning of nonionic cream bases at room temperature; Incorporation of the solid polymeric nanospheres (NPCL30%) loaded with red propolis extract into cold "nonionic base creams" by geometric dilution technique under mechanical or manual agitation and without the use of organic or lipid solvents for solubilization of the nanospheres solids loaded with red propolis extract in these base creams.
  • the obtained NPVs can be used in various dermocosmetic compositions, preferably as semi-solid formulations such as: nonionic creams, gel, gel-cream, lotions, nonionic lotions, as well as powders, makeup powders, skin brighteners. , face masks, among others.
  • NPVs can be used in various dermocosmetic compositions, preferably as semi-solid formulations such as: nonionic cream, gel-cream, gel-cream, lotions, nonionic lotions, makeup powders, skin brighteners, masks facials, among others.
  • NPV can also be used in cationic cream formulations as well as cream ointment.
  • compositions proposed herein consist of compositions preferably in the form of nonionic cream, nonionic gel cream, nonionic gel cream, nonionic lotions and also cationic creams, which contain: (i) a nucleus containing red propolis active substance (s) combined with a polymeric nanodevice matrix that may modify or delay the release of active substance (s) contained in the red propolis extract as well as protecting other excipients.
  • oxidative attack cosmetics of phenolic compounds present in propolis extract ii) an intermediate dispersing / stabilizing core layer; iii) an oil / aqueous outer layer responsible for the dilution, dispersion and stabilization processes of the intermediate layer and nucleus in a dermocosmetic composition, as well as promoting the dissolution and permeation of the active components by the skin barriers.
  • Each external may also have other adjuvant active substances with different actions on the skin.
  • the core coating polymer-containing inner layer consists of a synthetic lipophilic polymer or combination of more than one synthetic lipophilic polymer (s).
  • the intermediate layer may also be prepared by conjugating one or more natural hydrophilic polymer (s) to one or more synthetic hydrophilic polymer (s). ).
  • auxiliary surfactant action such as core stabilizing promoters and at the same time promoting core dispersion in the outer layer, they should be in the intermediate layer with varying percentages between 0.05% and 95% according to their emulsifying functionality.
  • the core coating intermediate layer should have percentages between 0.05% and 95%, and preferably between 0.05 and 35% relative to the total weight of the composition.
  • the composition of the outer layer consists of excipients, as well as other dermatological actives with various functionalities, including: emollients, consistency agents, softeners, hydrants, humectants, spreadability, refreshing, film-forming, nourishing, rejuvenating, cell renewal, exfoliators, elasticity promoters, anti-wrinkles, antioxidants, sensory modifiers, preservatives, preservatives, dyes, essences.
  • the outer layer may be composed of 1 or more of these excipients and dermatological actives.
  • outer layer In terms of percentages of pharmaceutical excipients in the outer layer, these may represent between 0.05% and 100% of the total weight.
  • outer layer according to its functionality in the dermocosmetic matrix.
  • the outer layer of the composition should have percentages between 0.01% and 100%, preferably between 1 and 100% of the total weight of the composition.
  • the dermocosmetic composition presented here should be packaged in light-sensitive, matte-looking, light-amber-tinted packaging with optional light photoprotection (optional).
  • Still other dermatological actives used in the composition may be of various types and in different formulations, with varying compositions among the various types of dermatological actives, there are hydrophilic dermatological actives, as well as the lipophilic dermatological actives may be natural or synthetic such as hyaluronic acid®, arnica® glycolic extract, mango butter®, kelpadelie®, rutin® lotus, DMAE bitartrate®, Algisium C®, hydroxyprolisilane C®, Ginkgo biloba® liposomes, biorusol®, Ceramida IMA®, VC- PMG®, lipoic acid®, vitamin E oleosa®, PML AE and AC® liposomes, elastocell®, PML coenzyme Q10® liposomes, sensiline®, structurine®, adenin®, nutriskin®, raffermine®, helioxine®, titanium dioxide micronized®, Neo heliopan E
  • nanospheres loaded with red propolis extract can be used in a wide range of industrial sectors from the cosmetic, cosmeceutical, hygiene and cleaning industries.
  • propolis as an active ingredient in the prevention and treatment of various diseases is already widely described.
  • red propolis has a different composition from other propolis already widely described in the state of the art, which indicates varied biological activities.
  • the dermocosmetic compositions presented herein demonstrate leishmanicidal activity against Leishmania (V.) braziliensis performed by assay of leishmanicidal activity whose IC50 concentration inhibits 50% of the parasite population in in vitro assay. Such activity shows that red propolis extract and nanospheres loaded with red propolis extract can be used against leishmaniasis.
  • red propolis extract-loaded nanospheres and NPV-containing dermocosmetic compositions obtained by the proposed processes may be used for: treatment of the skin and attached organs; skin treatment and rejuvenation, treatment of inflammatory processes of seborrheic origin; postoperative wound treatment, decubitus wound treatment in patients with diabetic foot; treatment of inflammatory processes of the skin in general.
  • suspensions of red propolis extract loaded polymeric nanoparticles as well as solid NPVs obtained by lyophilization will be used for preparation of antioxidant regenerating cream, anti-cream creams, eye area cream.
  • NPV neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-derived neuropeptide-containing the economically viable.
  • NPV obtained through the proposed process can be used, alone or in composition with other products, in various industrial sectors, such as cosmetic, hygiene and cleaning and cosmeceutical in the treatment of leishmaniasis.
  • NPV evaluations obtained using the proposed process demonstrated the obtainment of a nanoparticulate product, with nanometer particle size, adequate encapsulation degree and preserved antioxidant activity.
  • the results obtained are represented in the tables and figures indicated.
  • Table 1 presents the results of mean particle diameter, Polydispersion index, Zeta Potential and pH of Nanoparticle Suspensions containing red propolis extract.
  • compositions Diameter Potential Index Average PH of Zeta Polydispersion (mV)
  • Particle size values ranged from 107.1-177.6 nm with polydispersion index 0.13 to 0.18 for NEU compositions, between 246.7-280.2 nm with polydispersion index 0.1 1 to 0.17 for NPCL compositions between 191.0-221.1 nm with polydispersion index between 0.09 and 0.12 for NEUPCL compositions and all compositions showed unimodal behavior.
  • the zeta potential was between +54.6 to +37.3 mV for the NEU composition, between -3.5 to -12.7 mV for the NPCL composition and between +32.4 to +18.0 mV for the NEUPCL compositions.
  • the compositions have been shown to be stable, dispersed and not prone to aggregation of nanoparticles.
  • the pH values of nanoparticle suspensions containing red propolis extract were similar to nanoparticles suspension without red propolis extract (placebo) with slightly acidic values between 5.20 to 6.00 for NEU compositions, between 5.05 to 4.16 for NPCL compositions, and between 5.65 to 6.58 for NEUPCL compositions.
  • the attached Figure 1 shows Electron Scanning Photomicroscopy of NEU12% (1 A, 1 B), NEU20% (1 C and 1 D) and NPCL30% (1 E and 1 F) without cryoprotectant (1 A, 1 B and 1 D) and with cryoprotectant (1 C, 1 D and 1 F). Photomicrographs with magnification between 3000 times (5 ⁇ scale) and up to 12000 times (1 ⁇ scale) showing nanometric particles.
  • the attached Figure 2 shows DSC thermograms of red propolis extract, polymeric coating matrix and NPV compositions.
  • the DSC thermogram of the red propolis extract showed 4 endothermic events at temperatures of 81, 7 Q C, 92.0 Q C, 107 Q C and 135 Q C referring to wax melting processes and other phenolic components present.
  • the polymeric coatings containing Eudragit®E-100 (NEU), Poli- ⁇ -caprolactone (NPCL) and Eudragit®E-100 (NEU) and Poli-s-caprolactone (NEUPCL) showed endothermic event.
  • the attached Figure 3 shows FTIR-ATR spectra of the extract of red propolis, polymeric coating matrix and NPV compositions.
  • the attached Figure 4 shows the chromatograms (UPLC-DAD) of red propolis extract (4A), coating matrix (4B) (Placebo), composition NEU30% (4C), composition NPCL30% (4D) and composition NEUPCL30% (4E) showing the identification of flavonoids / isoflavonoids present in propolis extract and NPV compositions and absence of flavonoids in the polymeric coating matrix (placebo).
  • UPLC-DAD chromatograms
  • Figures 4A, 4C, 4D and 4E show analytical run chromatograms obtained using chromatographic method developed to detect the presence of flavonoids.
  • Figures 4A, 4C, 4D and 4E show the presence of flavonoids (1) Liquiritigenin (12.53min.), (2) pinobanksin (15.68min.), (3) isoliquiritigenin (17.26min.), (4) formononetine (18.13min.), (5) pinocembrine (23.12min.) and (6) Biochanine A (23.81 min.) at retention times corresponding to and similar to the analytical standards used for chromatographic method development.
  • Table 2 presents the results of Encapsulation Degree of major major flavonoids present in red propolis extract and NPV using UPLC-DAD.
  • composition A B C D E Composition A B C D E
  • A Daidzein; B: Liquiritigenin; C: Isoliquiritigenin; D: Formononetin; E: Biochanina A. * Standard deviation values determined in two chromatographic runs.
  • NEU that presented Eudragit®E-100 was the one that presented the largest losses during washing, centrifugation and drying stage using encapsulation degree freeze drying technique below 60%.
  • higher encapsulation degree was found in the range of 50% to 77%. It was observed that liquiritigenin and daidzein were the flavonoids that presented the greatest losses during the process of obtaining solid NPV.
  • NEU, NPCL and NEUPCL with 30% red propolis extract presented higher content and% encapsulation values (Table 3).
  • Table 3 presents the results of percentages of (%) Encapsulation Degree and (%) Unencapsulated representing the major major flavonoids present in red propolis extract and NPV using UPLC-DAD.
  • NPV showed good antioxidant activity and was concentration dependent. At concentrations of red propolis extract of 80 and 10 ⁇ 9 / ⁇ _, NPVs showed antioxidant activity between 71, 14 to 98.41% and at concentrations of 5 and 2 ⁇ g / ml_, NPV compositions showed percentage antioxidant activity between 58.87 and 97.33% and therefore much higher than the red propolis extract between 25.97 and 40.73%. These results are satisfactory to demonstrate that NPV compositions will act as agents that inhibit oxidative processes and function as biological antioxidants such as skin and appendages, and may function as an anti-aging agent on the skin (Table 4).
  • Table 4 presents the results of antioxidant activity (%) using DPPH method of red propolis extract and NPV.
  • NCEU 70% 100.01 ⁇ 0.1 1 98.41 ⁇ 0.06 79.67 ⁇ 0.17 68.96 ⁇ 0.29
  • the attached Figure 5 shows the IC 50 determination of the Leishmanicidal activity assay against Leishmania (V.) braziliensis using normalized graph (Figure 5A) and some hypothetical biochemical mechanisms of Leishmania (V.) braziliensis cell death by flavonoids, in particular isoliquiritigenin (chalcona) present in red propolis extract and nanospheres loaded with red propolis extract (Figure 5B).
  • the EPV and nanoparticles of the compositions NEU30% and NPCL30% showed proven leishmanicidal activity with IC50 values of 179 ⁇ g / ml_ and 31,16 ⁇ g / ml, respectively.
  • Compositions NCPCL 30% and NPCL40% (47.23 ⁇ g / mL) showed results close to EPV in free form with an IC50 of 38 ⁇ g / mL.
  • Figure 5 is based on research by Torres-Santos and Chen. Torres-Santos et al. (2009) (Torres-Santos EC, Sampaio-Santos M1, Buckner FS, Yokoyama K, Gelb M, Urbina JA, Rossi-Bergmann B (2009) Altered sterol profile induced in Leishmania amazonensis by a natural dihydroxymethoxylated chalcone JAntimicrobial Chemother 63: 469-472) demonstrated that chalcones (dihydro-methoxylated chalcone) alter steroid biosynthesis in L. amazonensis and promote ergosterol accumulation and cholesterol lowering.
  • chalcones dihydro-methoxylated chalcone
  • chalcones destroy the parasite mitochondria ultrastructure, inhibit mitochondrial respiration and the activity of mitochondrial dehydrogenase (SDH) enzymes, succinate dehydrogenase (SDH), cytochrome succinate and reductase, NADH dehydrogenase, especially NADH cytochrome a, fumarate reductase (FDH) activity present only in the Leishmania parasite.
  • SDH mitochondrial dehydrogenase
  • SDH succinate dehydrogenase
  • cytochrome succinate and reductase cytochrome succinate and reductase
  • NADH dehydrogenase especially NADH cytochrome a, fumarate reductase (FDH) activity present only in the Leishmania parasite.
  • Attached Figure 6 shows photos of non-ionic creams containing NPCL30% loaded with red propolis extract to achieve final concentration in cosmetic cream of 2.0% (A), 1.5% (B), 1.0 % (C) and 0.5% (D) of the red propolis extract.
  • Solid state NPV nanospheres were either directly incorporated into the nonionic cream or using the suspensions of polymeric nanoparticles (B).

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Abstract

The present invention discloses red propolis extract-loaded nanospheres (RPNs), composed of standardized hydroalcoholic extract of red propolis originating from red propolis in its natural form, and of polymeric matrices having a function of nanoencapsulation, dispersion and stabilization, and preventing auto-oxidation of creams. The RPNs in suspension or in solid form are considered to be intermediate products in the production of dermocosmetic compositions. The invention also relates to the processes of characterization of the nanoscale red propolis extract-loaded nanospheres, and also to the identification of isoflavonoids and antioxidant activity and a leishmanicidal action. In addition, the present embodiment of the invention discloses processes for obtaining the dermocosmetic compositions containing the RPNs, preferably in the form of non-ionic creams, a cationic cream form also being possible. Lastly, uses are disclosed for the dermocosmetic compositions containing red propolis extract-loaded nanospheres in the treatment of leishmaniasis, as healing agent and antioxidant.

Description

Relatório Descritivo de Patente de Invenção  Patent Invention Descriptive Report
NANOESFERAS CARREGADAS COM EXTRATO DE PRÓPOLIS VERMELHA, PROCESSO DE OBTENÇÃO DE NANOESFERAS, COMPOSIÇÕES  LOADED NANOPHESES WITH RED PROPOLIS EXTRACT, NANOPHESES PROCESSING, COMPOSITIONS
DERMOCOSMÉTICAS CONTENDO AS MESMAS E USOS  DERMOCOSMETICS CONTAINING THE SAME AND USES
Campo da Invenção Field of the Invention
[0001] A presente invenção pertence ao campo de produtos naturais com utilidade na saúde pública em estética e beleza, mais especificamente aqueles dos sistemas de nanopartículas carregadas com princípios ativos do extrato de própolis vermelha com atividades biológicas, destinados a uma administração na via dérmica e nas mucosas. São propostos nanoesferas carregadas com extrato de própolis vermelha (NPV), processos de obtenção de NPV, e composições dermocosméticas contendo NPV e usos. A produção de NPV está ligada à utilização de extrato hidroalcoolico padronizado de própolis vermelha, princípio ativo das NPV, e de sistema de matrizes de polímeros farmacêuticos orgânicos com função encapsulante, dispersante e estabilizante destas nanoesferas em suspensão, que serão nanoprecipitadas e liofilizadas para obter nanoesferas poliméricas contendo extrato de própolis vermelha no estado sólido que serão utilizadas para produção de composições dermocosméticas. Por fim, as composições dermocosméticas contendo as NPEPV apresentam aplicação em setores dermocosméticos e cosmecêuticos, em tratamento de estética, beleza, em cirurgias reparadoras, devido a sua atividade antioxidante, anti-inflamatória, anticancerígena, cicatrizante, bem como por sua ação leishmanicida.  [0001] The present invention pertains to the field of natural public health utility products in aesthetics and beauty, more specifically those of the active ingredient loaded nanoparticle systems of the biologically active red propolis extract, intended for dermal administration and in the mucous membranes. Red propolis extract (NPV) loaded nanospheres, processes for obtaining NPV, and dermocosmetic compositions containing NPV and uses are proposed. NPV production is linked to the use of standardized red propolis hydroalcoholic extract, active ingredient of NPV, and matrix system of organic pharmaceutical polymers with encapsulating, dispersing and stabilizing function of these suspended nanospheres, which will be nanoprecipitated and lyophilized to obtain nanospheres. solid propolis extract containing polymeric compounds which will be used for the production of dermocosmetic compositions. Finally, NPEPV-containing dermocosmetic compositions have application in dermocosmetic and cosmeceutical sectors, in aesthetic treatment, beauty, in reparative surgeries, due to their antioxidant, anti-inflammatory, anticancer and healing activity, as well as their leishmanicidal action.
Antecedentes da Invenção Background of the Invention
[0002] Os constituintes da própolis vêm sendo identificados no mundo inteiro (Pietta, P.; Gardana, C; Scaglianti, M.; Simonetti, P., Journal of Pharmaceutical and Biomedical Analysis, V. 45, p. 390-399, 2007). Os flavonoides, ácidos fenólicos e terpenos são as principais substâncias encontradas e utilizadas para rastrear a qualidade e, em alguns casos, para demonstrar a autenticidade da própolis de algumas regiões geográficas (Volpi, N.; Bergonzini, G., Journal of Pharmaceutical and Biomedical Analysis, V. 42, p. 354-361 , 2006). Dentre os flavonoides e ácidos fenólicos mais comumente utilizados como marcadores, podem ser citados: quercetina, canferol, naringenina, crisina, pinocembrina, galangina (flavonoides); ácido gálico, ácido caféico, ácido p-coumárico, ácido ferúlico, ácido cinâmicos e derivados (ácido fenólicos), clerodanos (diterpenóides) (Rosalen, P.L.; Castro, M. L; Cury, J.A., Química Nova, V. 30, N. 07, 1512-1517, 2007); (Yao, L; Jiang, Y.; Singanusong, R., Datta, N.; Raymont, K., Food Chemistry, V. 86, p.169-177, 2004); (Weston, R. J., Mitchell, K. R., Allen, K. L, Food Research International, V. 37, p. 166-174, 2004). Propolis constituents have been identified worldwide (Pietta, P.; Gardana, C.; Scaglianti, M.; Simonetti, P., Journal of Pharmaceutical and Biomedical Analysis, V. 45, p. 390-399, 2007). Flavonoids, phenolic acids and terpenes are the main substances found and used to track quality and, in some cases, to demonstrate the authenticity of propolis in some geographical regions (Volpi, N.; Bergonzini, G., Journal of Pharmaceutical and Biomedical Analysis, V. 42, p. 354-361, 2006). Flavonoids and phenolic acids most commonly used as markers include: quercetin, canferol, naringenin, chrysin, pinocembrine, galangin (flavonoids); gallic acid, caffeic acid, p-coumaric acid, ferulic acid, cinnamic acids and derivatives (phenolic acid), clerodanes (diterpenoids) (Rosalen, PL; Castro, M.L; Cury, JA, Química Nova, V. 30, N 07, 1512-1517, 2007); (Yao, L.; Jiang, Y .; Singanusong, R., Datta, N.; Raymont, K., Food Chemistry, V. 86, p.169-177, 2004); (Weston, RJ, Mitchell, KR, Allen, K.L. Food Research International, V. 37, pp. 166-174, 2004).
[0003] O uso popular cada vez crescente de própolis e seus derivados com ação antimicrobiana (Sforcin, J.M; Fernandes Jr., A.; Lopes, C. A. M.; Bankova, V. And Funari, S. R. C, J. Ethnopharmacology, V. 73, p. 243-249, 1998), anti-inflamatória (Khayyal, M.T.; el-Ghazaly, M.A.; el-Khatib, A.S., Drug Experimental and Clinicai Research, V. 19, p. 197-203, 1993), antiviral (Vynograd, N.; Vynograd, I.; Sosnowisky, Z., Phytomedicine, V. 7, p. 1 -6, 2000), anticarcinogênica (Bazo, A. P.; Rodrigues, M. A. M.; Sforcin, J.M.; Camargo, J.L.V., Ribeiro, J.L.; Salvadori, D. M.F., Teratogenis, Carcinogenis and Mutagenis, V. 22, p. 183-194, 2002) e imunomodulatória (Sforcin, J. M., Kanemo, R. Funari, S. R. C, Journal of Venemous Animal and Toxins, V. 8, p. 19-29, 2002); (Sá-Nunes, A.; Faccioli, L.H., Sforcin, J. M., Journal of Ethnopharmacology, V.83, p. 93-97, 2003) vem demonstrando o grande poder terapêutico da própolis em substituição aos medicamentos sintéticos convencionais.  The ever increasing popular use of propolis and its derivatives with antimicrobial action (Sforcin, JM; Fernandes Jr., A.; Lopes, CAM; Bankova, V. And Funari, SR C, J. Ethnopharmacology, V. 73 243-249, 1998), anti-inflammatory (Khayyal, MT; el-Ghazaly, MA; el-Khatib, AS, Drug Experimental and Clinical Research, V. 19, p. 197-203, 1993), antiviral (Vynograd, N .; Vynograd, I.; Sosnowisky, Z., Phytomedicine, V. 7, pp. 1-6, 2000), anticarcinogenic (Bazo, AP; Rodrigues, MAM; Sforcin, JM; Camargo, JLV, Ribeiro , JL; Salvadori, DMF, Teratogenis, Carcinogenis and Mutagenis, V. 22, pp. 183-194, 2002) and immunomodulatory (Sforcin, JM, Kanemo, R. Funari, SR C, Journal of Venemous Animal and Toxins, V. 8, pp. 19-29, 2002); (Sa-Nunes, A.; Faccioli, L.H., Sforcin, J.M., Journal of Ethnopharmacology, V.83, pp. 93-97, 2003) has been demonstrating the great therapeutic power of propolis as a substitute for conventional synthetic drugs.
[0004] A caracterização de todas estas atividades biológicas associadas à tendência de utilização de produtos naturais tem resultado num aumento da demanda de própolis e produtos contendo própolis, como extratos, cremes, pomadas, comprimidos, cápsulas, ou pós (H. Menezes, M. Bacci Jr., S. D. Oliveira, F. C. Pagnocca, Antibacterial properties of própolis and products containingpropolis from Brazil, Apidologie, V. 28, p. 71 -76, 1997). The characterization of all these biological activities associated with the tendency to use natural products has resulted in an increased demand for propolis and propolis-containing products such as extracts, creams, ointments, tablets, capsules, or powders (H. Menezes, M Bacci Jr., SD Oliveira, FC Pagnocca, Antibacterial properties of propolis and products. containingpropolis from Brazil, Apidologie, V. 28, p. 71-76, 1997).
[0005] Em se tratando da própolis vermelha, esta foi classificada como o 13° subtipo de própolis brasileira encontrada nas regiões de mangues, lagoas, rios e praias do nordeste brasileiro entre os estados da Sergipe, Alagoas, Pernambuco e Paraíba pelo pesquisador (Andreas Daugsch, Cleber S. Moraes, Patrícia Fort and Yong K. Park, Brazilian Red Própolis— Chemical Composition and Botanical Origin, eCAM 2008;5(4)435^141 doi:10.1093/ecam/nem057). A principal origem botânica da própolis vermelha se deve a planta Dalbergia ecastophyllum, localmente conhecida como Rabo-de-Bugio, que apresenta um exsudado resinoso vermelho liberado da sua seiva. As far as red propolis is concerned, it was classified as the 13th subtype of Brazilian propolis found in the mangroves, lagoons, rivers and beaches of northeastern Brazil between the states of Sergipe, Alagoas, Pernambuco and Paraíba by the researcher (Andreas Daugsch, Cleber S. Moraes, Patricia Fort and Yong K. Park, Brazilian Red Propolis — Chemical Composition and Botanical Origin, eCAM 2008; 5 (4) 435-141 doi: 10.1093 / ecam / nem057). The main botanical origin of red propolis is the Dalbergia ecastophyllum plant, locally known as the Howler's Tail, which has a red resinous exudate released from its sap.
[0006] Pesquisadores nacionais e internacionais com expertise em própolis vêm identificando os constituintes da própolis vermelha. Foram identificados 1 1 isoflavonóides e 1 chalcona da própolis vermelha cubana e dentre estas substâncias podemos citar: Formononetina (Biochanina B), Bichanina A, vestitol, orto-metil vestitol, medicarpina, homopterocarpina, outros derivados pterocarpanos, liquiritigenina e isoliquiritigenina (chalcona) (Anna Piccinelli e col. J. Agric. Food Chem. 2005, 53, 9010-9016). Foram identificadas também outras subclasses de flavonoides, incluindo novos tipos de flavonoides da própolis vermelha da região nordeste e que podemos citar: rutina, quercetina, luteolina, pinocembrina e biochanina A, formononetina, daidzeina, liquiritigenina, pinobanksina, dalbergina (neoflavonóides) (Andreas Daugsch, Cleber S. Moraes, Patrícia Fort and Yong K. Park, Brazilian Red Própolis— Chemical Composition and Botanical Origin, eCAM, Evidence-based complementary and alternative medicine, 2008;5(4)435-441 doi:10.1093/ecam/nem057).  [0006] National and international researchers with propolis expertise have been identifying the constituents of red propolis. We identified 1 isoflavonoids and 1 chalcone of Cuban red propolis and among these substances we can mention: Formononetina (Biochanina B), Bichanina A, vestitol, ortho-methyl vestitol, medicarpine, homopterocarpine, other pterocarpane derivatives, liquiritigenin and isoliquiritigenin (chalcona) ( Anna Piccinelli et al J. Agric. Food Chem. 2005, 53, 9010-9016). Other flavonoid subclasses have also been identified, including new types of flavonoids from northeastern red propolis that we can mention: rutin, quercetin, luteoline, pinocembrin and biochanin A, formononetin, daidzein, liquiritigenin, pinobanksin, dalbergin (neoflavonoids) (Andreas Daugsch) , Cleber S. Moraes, Patricia Fort and Yong K. Park, Brazilian Red Propolis - Chemical Composition and Botanical Origin, eCAM, Evidence-based complementary and alternative medicine, 2008; 5 (4) 435-441 doi: 10.1093 / ecam / nem057 ).
[0007] Pesquisadores estudando a própolis vermelha da região dos manguezais de Marechal Deodoro-Alagoas, identificaram o flavonóide crisina e quercetina, novos isoflavonóides na própolis vermelha de Alagoas, o vestitol, 2'4'dihidroximetoxi flavana, benzofenonas isopreniladas, bem como ácido ferúlico e ésteres fenólicos metoxieugenol, metileugenol, guiacol, ésteres dimetílicos de ácido butanenodióico, ésteres metílicos de ácido hexadecanóico (Bruno B. Silva, Pedro L. Rosalen, Jaime A. Cury, Masaharu Ikegaki, Vinícius C. Souza, Alessandro Esteves and Severino M. Alencar Chemical Composition and Botanical Origin of Red Propolis,a New Type of Brazilian Propolis, eCAM, evidence based complementary and alternative medicine 2008;5(3)313-316, doi:10.1093/ecam/nem059). [0007] Researchers studying propolis in the region of Deodoro, Alagoas Marechal Mangrove identified chrysin flavonoid and quercetin, new isoflavones in propolis of Alagoas, the vestitol, 2 '4' dihidroximetoxi flavana, isopreniladas benzophenones and ferulic acid and phenolic esters methoxyheugenol, methylheugenol, guiacol, esters butanedioic acid dimethyls, hexadecanoic acid methyl esters (Bruno B. Silva, Pedro L. Rosalen, Jaime A. Cury, Masaharu Ikegaki, Vinicius C. Souza, Alessandro Esteves and Severino M. Alencar Chemical Composition and Botanical Origin of Red Propolis, a New Type of Brazilian Propolis, eCAM, evidence based complementary and alternative medicine 2008; 5 (3) 313-316, doi: 10.1093 / ecam / nem059).
[0008] Adicionalmente, pesquisadores identificaram 14 substâncias na própolis vermelha dos manguezais de Alagoas, dentre eles triterpenos (cicloartenol, lupeol, elemicina, α-amirina e β-amirina), ácidos fenólicos (anetol, eugenol e metileugenol), isoflavonoides (isosativan, medicarpina) (Boryana Trusheva, Milena Popova, Vassya Bankova, Svetiana Simova, Maria Cristina Marcucci, Patrícia Laguna Miorin, Flavia da Rocha Pasin and Iva Tsvetkova, Bioactive Constituents of Brazilian Red Propolis, eCAM, evidence-based complementary and alternative medicine 2006;3(2)249-254, doi:10.1093/ecam/nel006).  In addition, researchers have identified 14 substances in the Alagoas mangrove red propolis, including triterpenes (cycloartenol, lupeol, elemicin, α-amyrin and β-amirine), phenolic acids (anethole, eugenol and metileugenol), isoflavonoids (isosativan, medicarpina) (Boryana Trusheva, Milena Popova, Vassya Bankova, Svetiana Simova, Maria Cristina Marcucci, Patricia Laguna Miorin, Flavia da Pasin and Iva Tsvetkova, Bioactive Constituents of Brazilian Red Propolis, eCAM, evidence-based complementary and alternative medicine 2006; 3 (2) 249-254, doi: 10,1093 / ecam / level 006).
[0009] Com vistas à descoberta de moléculas e seus alvos terapêuticos para cura das doenças, a própolis vermelha tem sido submetida a diversos estudos detalhados sobre suas atividades biológicas. Pesquisas de identificação e elucidação estrutural de novas substâncias continuam sendo desenvolvidas na identificação de novas substâncias presentes no extrato de própolis vermelha. Foram identificados uma nova chalcona, 3, 4, 2',3'tetrahidroxchalcona e um novo flavonóide C-glicosilado, a narigenina-8-C- hexoside, além do (3S)-vestitol e (3S)-7-O-metilvestitol (Adne A Righi, Thiago R Alves, Giuseppina Negri, Lucas M Marques, Henrique Breyer and Antonio Saiatino, Brazilian red propolis: unreported substances, antioxidant and antimicrobial actívities, J Sei Food Agric 201 1 ; 91 : 2383-2370, DOI 10.1002/jsfa.4468). Pesquisas biológicas com o extrato etanólico de própolis vermelha e substâncias isoladas, o vestitol e o neovestitol, demonstraram atividade antiinflamatória na concentração de 10mg/Kg e atividade antimicrobiana em concentações entre 6 a 100μg/mL (Bruno Bueno-Silva, Severino M. Alencar, Hyun Koo, Masaharu Ikegaki, Gil V. J. Silva, Marcelo H. Napimoga, and Pedro L. Rosalen, Anti-lnflammatory and Antimicrobial Evaluation of Neovestitol and Vestitol Isolated from Brazilian Red Propolis, J. Agric. Food Chem. 2013, V. 61 , p. 4546-4550, dx.doi.org/10.1021/jf305468f). [0009] With a view to the discovery of molecules and their therapeutic targets for the cure of diseases, red propolis has been subjected to several detailed studies on its biological activities. Research into the identification and structural elucidation of new substances continues to be developed in the identification of new substances present in red propolis extract. A new chalcone, 3, 4, 2 ' , 3 ' tetrahydroxchalcona and a new C-glycosylated flavonoid, narigenin-8-C-hexoside, as well as (3S) -vestitol and (3S) -7-O-methylvestitol have been identified. (Adne A Righi, Thiago R Alves, Giuseppina Negri, Lucas M Marques, Henry Breyer and Antonio Saiatino, Brazilian red propolis: unreported substances, antioxidant and antimicrobial activities, J Sei Food Agric 201 1; 91: 2383-2370, DOI 10.1002 / jsfa.4468). Biological researches with ethanolic extract of red propolis and isolated substances, vestitol and neovestitol, demonstrated antiinflammatory activity at 10mg / kg concentration and antimicrobial activity at concentrations between 6 and 100μg / mL (Bruno Bueno-Silva, Severino M. Alencar, Hyun Koo, Masaharu Ikegaki, Gil VJ Silva, Marcelo H. Napimoga, and Pedro L. Rosalen, Anti-Inflammatory and Antimicrobial Evaluation of Neovestitol and Vestitol Isolated from Brazilian Red Propolis, J. Agric. Food Chem. 2013, V. 61, p. 4546-4550, dx.doi.org/10.1021/jf305468f).
[0010] OS extratos de própolis vermelha enriquecidos com xantochimol e formononetina vêm apresentando atividades antitumoral in vitro contra diferentes linhagens de células como melanoma usando linhagem celular B16F10, ATCC CRL6475 (Estela Maria Novak, Martha Silveira e Costa Silva,Maria Cristina Marcucci, Alexandra Christine Helena Frankland Sawaya, Begoha Giménez-Cassina López, Maria Angela Henriques Zanella Fortes, Ricardo Rodrigues Giorgi, Kamila Tamie Marumo, Rosângela Felipe Rodrigues, Durvanei Augusto Maria. Antitumoural activity of Brazilian red propolis fraction enriched with xanthochymol and formononetin: An in vitro and in vivo study, Journal of Functional Foods, 1 1 , (2014), 91 -102). Xanthochimol and formononetin enriched red propolis extracts have been showing in vitro antitumor activity against different cell lines such as melanoma using B16F10 cell line, ATCC CRL6475 (Maria Novak, Martha Silveira and Costa Silva, Maria Cristina Marcucci, Alexandra Christine Helena Frankland Sawaya, Begoha Giménez-Cassina Lopez, Maria Angela Henriques Zanella Fortes, Ricardo Rodrigues Giorgi, Kamila Tamie Marumo, Rosangela Felipe Rodrigues, Durvanei Augusto Maria Antitumoural activity of Brazilian red propolis fraction enriched with xanthochymol and formononetin vivo study, Journal of Functional Foods, 11, (2014), 91-102).
[0011] Extrato etanólico de própolis vermelha vem apresentando atividade contra células de câncer das linhagens de glioblastoma (SF-295), ovário (Ovcar-8) e de cólon (HCT-1 16) (Izabel Cristina Gomes de Mendonça, Isabel Cristina Celerino de Moraes Porto, Ticiano Gomes do Nascimento, Naiana Soares de Souza, José Marcos dos Santos Oliveira, Rodolfo Elleson dos Santos Arruda, Kristiana Cerqueira Mousinho, Aldenir Feitosa dos Santos, Irinaldo Diniz Basílio-Júnior, Abhishek Parolia, Francisco Stefânio Barreto, Brazilian red propolis: phytochemical screening, antioxidant activity and effect against câncer cells, BMC Complementary and Alternative Medicine (2015) 15:357.DOI 10.1 186/s12906-015-0888-9).  Red propolis ethanolic extract has been showing activity against cancer cells of the glioblastoma (SF-295), ovary (Ovcar-8) and colon (HCT-1 16) strains (Izabel Cristina Gomes de Mendonça, Isabel Cristina Celerino Porto, Ticiano Gomes do Nascimento, Naiana Soares de Souza, Jose Marcos dos Santos Oliveira, Rodolfo Elleson dos Santos Arruda, Kristiana Cerqueira Mousinho, Aldenir Feitosa dos Santos, Irinaldo Diniz Basilio Junior, Abhishek Parolia, Francisco Stefânio Barreto, Brazilian red propolis: phytochemical screening, antioxidant activity and effect against cancer cells, BMC Complementary and Alternative Medicine (2015) 15: 357.DOI 10.1 186 / s12906-015-0888-9).
[0012] Estudos de identificação proteômica dos marcadores celulares de câncer de laringe também demonstraram atividade do extrato de própolis vermelha contra linhagens de células laringeal epidermóide frente as cepas ATCC Hep-2 (Caroline Olivieri da Silva Frozza, Tanara da Silva Ribeiro, Gabriela Gambato, Caroline Menti, Sidnei Moura, Paulo Marcos Pinto, Charley Christian Staats, Francine Ferreira Padilha, Karine Rech Begnini, Priscila Marques Moura de Leon, Sibele Borsuk, Lucielli Savegnago, Odir Dellagostin, Tiago Collares, Fabiana Kõmmling Seixas, João Antonio Pêgas Henriques, Mariana Roesch-Ely, Proteomic analysis identifies differentially expressed proteins after red propolis treatment in Hep-2 cells, Food and Chemical Toxicology 63 (2014) 195-204). Proteomic identification studies of laryngeal cancer cell markers have also demonstrated red propolis extract activity against epidermoid laryngeal cell lines against ATCC Hep-2 strains (Caroline Olivieri da Silva Frozza, Tanara da Silva Ribeiro, Gabriela Gambato, Caroline Menti, Sidnei Moura, Paulo Marcos Pinto, Charley Christian Staats, Francine Ferreira Padilha, Karine Rech Begnini, Priscila Marques Moura de Leon, Sibele Borsuk, Lucielli Savegnago, Odir Dellagostin, Tiago Collares, Fabiana Kömmling Seixas, Joao Antonio Pêgas Henriques, Mariana Roesch-Ely, Proteomic analysis identifies differently expressed proteins after red propolis treatment in Hep-2 cells, Food and Chemical Toxicology 63 (2014) 195-204).
[0013] Em todo o mundo existem pesquisas para curar doenças negligenciáveis como a leishmaniose e os extratos de própolis também vem demonstrando atividade contra estes parasitas. Duran et al. (201 1 ) (Duran, N., Muz, M., Culha, G., Duran, G., Ozer, B. 201 1 . GC-MS analysis and antileishmanial activities of two Turkish propolis types. Parasitology research, 108, 95-105,) mostraram atividade leishmanicida para 2 tipos de extratos de própolis da Turquia (própolis Hatay e própolis Bursa) e demonstrou um IC50 de 250μg/mL e 500μg/mL, respectivamente. Outro estudo relevante por Duran et al. (2008) (Duran, G., Duran, N., Culha, G., Ozcan, B., Oztas, H., Ozer, B. 2008. In vitro antileishmanial activity of Adana propolis samples on Leishmania trópica: a preliminary study. Parasitology Research, 102, 1217-1225) revelou bons resultados para Adana Própolis contra leishmania em concentrações de 250μg/mL. Outro tipo de extrato de própolis turca (própolis Kayseri) estudada por Ozbilge et al. (2010) (Ozbilge, H., Kaya, E.G., Albayrak, S., Silici, S. 201 0. Anti-leishmanial activities of ethanolic extract of Kayseri propolis. African Journal of Microbiology Research. 4, 556-560.) demonstraram atividade leishmanicida contra a Leishmania trópica com IC50 de 32μg/mL. Estudo comparativo dos extratos de própolis verde Brasileira e extrato de própolis búlgara realizadas por Machado et al. (2007) mostraram atividade contra leishmanicidas 4 espécies diferentes de Leishmania chagasi (amazonensis braziliensis e Major). Extrato de própolis verde brasileira mostrou IC50 perto de 49μg/mL contra espécies braziliensis chagasi e Major, enquanto o extrato de própolis búlgara apresentou atividade leishmanicida para as espécies amazonensis, chagasi e Major com IC50 entre 2,8 μg/mL e 41 ^g/mL. Extrato de própolis vermelha brasileira foi avaliado em macrófagos infectados com Leishmania amazonensis, e demonstrou que a concentração de 25 ug/mL é capaz de aumentar a actividade redutora de MTT sendo activos contra parasitas intracelulares presentes em macrófagos (Ayres, D.C., Marcucci, M.C., Giorgio, S. 2007. Effects of Brazilian propolis on Leishmania amazonensis. 39 Memórias Instituto Oswaldo Cruz, 102, 215-220.). There is research around the world to cure negligible diseases such as leishmaniasis and propolis extracts have also been showing activity against these parasites. Duran et al. (2011) (Duran, N., Muz, M., Culha, G., Duran, G., Ozer, B. 201. GC-MS analysis and antileishmanial activities of two Turkish propolis types. Parasitology research, 108, 95-105,) showed leishmanicidal activity for 2 types of Turkish propolis extracts (Hatay propolis and Bursa propolis) and demonstrated an IC50 of 250μg / mL and 500μg / mL, respectively. Another relevant study by Duran et al. (2008) (Duran, G., Duran, N., Culha, G., Ozcan, B., Oztas, H., Ozer, B. 2008. In vitro antileishmanial activity of Adana propolis samples on tropic Leishmania: a preliminary study Parasitology Research, 102, 1217-1225) has shown good results for Adana Propolis against leishmania at concentrations of 250μg / mL. Another type of Turkish propolis extract (Kayseri propolis) studied by Ozbilge et al. (2010) (Ozbilge, H., Kaya, EG, Albayrak, S., Silici, S. 201. Anti-leishmanial activities of ethanolic extract of Kayseri propolis. African Journal of Microbiology Research. 4,556-560.) leishmanicidal activity against tropic Leishmania with IC50 of 32μg / mL. Comparative study of Brazilian green propolis extracts and Bulgarian propolis extract by Machado et al. (2007) showed activity against leishmanicides 4 different species of Leishmania chagasi (amazonensis braziliensis and Major). Brazilian green propolis extract showed IC50 close to 49μg / mL against braziliensis chagasi and Major species, while Bulgarian propolis extract showed leishmanicidal activity for amazonensis, chagasi and Major species with IC50 between 2.8 μg / mL and 41 ^ g / mL. Brazilian red propolis extract was evaluated in macrophages infected with Leishmania amazonensis, and demonstrated that the concentration of 25 µg / mL is capable of increasing MTT reducing activity by being active against intracellular parasites present in macrophages (Ayres, DC, Marcucci, MC, Giorgio, S. 2007. Effects of Brazilian propolis on Leishmania amazonensis 39 Memories Oswaldo Cruz Institute, 102, 215-220.).
[0014] A presença de vários flavonoides e ácidos fenólicos em extratos e tinturas de propolis mostram que estas substâncias agem sinergisticamente em ação citostática/citotóxica, anti-inflamatória, cicatrizante, antimicrobiana e antioxidante. Desta forma, o coquetel de substâncias combinadas, mesmo que em baixas concentrações, irá promover uma potente ação contra os agentes patogênicos. O desenvolvimento e produção de fitoterápicos e opoterápicos vêm sendo regulado no país com as resoluções que estabelecem metodologias de controle de qualidade desses produtos. Estudos de padronização do fitoterápico e opoterápicos preconizam ensaios de modo a garantir a qualidade destas classes de medicamentos desde a coleta, screening fitoquímico, determinação qualitativa e quantitativa de marcadores fitoquímicos, teste de autenticidade, processamento, produção, estudo de estabilidade, validação de metodologias analíticas, embalagens e controle de qualidade de produto.  The presence of various flavonoids and phenolic acids in propolis extracts and tinctures show that these substances act synergistically on cytostatic / cytotoxic, anti-inflammatory, healing, antimicrobial and antioxidant action. Thus, the cocktail of combined substances, even at low concentrations, will promote a potent action against pathogens. The development and production of phytotherapics and opotherapics have been regulated in the country with the resolutions that establish quality control methodologies of these products. Phytotherapeutic and opotherapeutic standardization studies advocate trials to ensure the quality of these drug classes from collection, phytochemical screening, qualitative and quantitative determination of phytochemical markers, authenticity testing, processing, production, stability study, validation of analytical methodologies. , packaging and product quality control.
[0015] Devido às suas características diferenciadas em termos de composição química e múltiplas atividades biológicas, bem como por apresentar características de produção e manejo padronizado nas áreas de manguezais de Alagoas-Brasil, respeitando uma política de preservação e conservação ambiental das áreas de mata atlântica e manguezais, a produção de propolis vermelha in natura e extrato hidroalcoolico faz parte de uma cadeia produtiva de produtos opoterápicos que promove o desenvolvimento sustentável da região dos mangues e lagoas sendo contemplada com o selo de indicação geográfica com a denominação de origem. Due to its different characteristics in terms of chemical composition and multiple biological activities, as well as presenting production and standardized management characteristics in the mangrove areas of Alagoas-Brazil, respecting a policy of preservation and environmental conservation of the Atlantic Forest areas. and mangroves, the production of fresh red propolis and hydroalcoholic extract is part of a production chain of opotherapeutic products that promotes the sustainable development of the mangroves and lakes region and is awarded the geographical indication seal with the designation of origin.
[0016] Desenvolvimento de novos sistemas de entrega do fármaco ou substâncias ativas sobre determinados alvos terapêuticos é uma necessidade para se obter o sucesso terapêutico ou que demonstre benefício fisiológico à saúde na prevenção de doenças ou cura das mesmas ou ainda com ação de estética e beleza, e é tão importante quanto a descoberta das moléculas com determinada ação terapêutica. Desta forma, na presente invenção foram planejadas diferentes composições dermocosméticas na forma semissólida de uso tópico ou nas mucosas, preferencialmente tópico cutâneo e de liberação modificada. Development of new drug delivery systems or active substances on certain therapeutic targets is a necessity to achieve therapeutic success or to demonstrate physiological benefit to the patient. health in preventing diseases or curing them or with aesthetic and beauty action, and is as important as the discovery of molecules with a certain therapeutic action. Thus, in the present invention different dermocosmetic compositions have been devised in semi-solid topical or mucosal form, preferably cutaneous and modified release topical.
[0017] No desenvolvimento de novos sistemas de liberação de extratos ativos contendo várias substâncias ativas com atividade polimultifuncional nos dermocosméticos faz-se necessário ter o conhecimento da pele como órgão com função de barreira contra agentes químicos e físicos, das vias de permeação e fatores de penetração na pele, bem como dos principais excipientes cosméticos responsáveis pela permeação destas substâncias ativas na pele e órgãos anexos. A pele funciona como um grande órgão que recobre uma superfície superior a 20 000 cm2 com propriedades e funções variadas, pois além proteger o corpo da invasão de microrganimos, protege contra raios UV, produz melanina e imunoglobulinas, além de controlar a temperatura corporal, pressão sanguínea, bem como de apresentar função sensorial. O conhecimento anatómico da pele faz-se necessário na escolha dos sistemas de encapsulação de extrato ativo, bem como de excipientes cosméticos que irão compor uma composição dermocosmética. A pele possui diversas camadas histológicas, sendo dividida em estrato córneo, epiderme, derme e tecido adiposo subcutâneo. O estrato córneo e folículos pilosos e glândulas (sudorípara e sebácea) são considerados as primeiras barreiras que limita a velocidade de entrada das substâncias ativas que compõe um creme cosmético (Leon Lachman, Herbert A. Lieberman, Joseph L Kanig, Teoria e Prática na Indústria Farmacêutica, Vol. II, capítulo 18, páginas 907-953, 2001 ). In the development of new active extract release systems containing various active substances with polymultifunctional activity in dermocosmetics, it is necessary to have knowledge of the skin as an organ with barrier function against chemical and physical agents, permeation pathways and factors of skin penetration as well as major cosmetic excipients responsible for the permeation of these active substances in the skin and attached organs. The skin acts as a large organ that covers a surface of over 20,000 cm 2 with varying properties and functions, as it protects the body from microorganism invasion, protects against UV rays, produces melanin and immunoglobulins, and controls body temperature, blood pressure as well as having sensory function. Anatomical knowledge of the skin is required when choosing active extract encapsulation systems as well as cosmetic excipients that will make up a dermocosmetic composition. The skin has several histological layers, being divided into stratum corneum, epidermis, dermis and subcutaneous adipose tissue. The stratum corneum and hair follicles and glands (sweat and sebaceous) are considered the first barriers that limit the entry speed of the active substances that make up a cosmetic cream (Leon Lachman, Herbert A. Lieberman, Joseph L Kanig, Theory and Practice in Industry Pharmaceuticals, Vol. II, Chapter 18, pages 907-953, 2001).
[0018] Nos dermocosméticos, são de extrema importância a escolha dos agentes encapsulantes do extrato ativo e excipientes utilizados em cosmetologia com propriedades multifuncional, isto é, excipientes com diversas funções como: facilitadores da permeação cutânea, ação fotoprotetora, ação cosmética específica, estabilizantes do sistema lipófilo/hidrófilo, controle da viscosidade e sensorial, além de serem biocompatíveis e biodegradáveis. Os excipientes cosméticos são essenciais numa composição dermocosméticas, pois apresentam características que asseguram estabilidade à composição, facilitam a administração, promovem a liberação das substâncias ativas da matriz dérmica promovendo desta forma a biodisponibilidade das substâncias ativas nas diferentes camadas da pele e consequentemente a ação farmacológica adequada sem toxicidade, bem com a aceitabilidade do consumidor do ponto de vista sensorial e estético. Para o desenvolvimento de sistemas semissólidos com aplicação em cosmetologia é necessário ter um conjunto de excipientes que irão liberar adequadamente o fármaco ou substâncias ativas de uma matriz dermocosmética e dentre eles estão os excipientes: emulgentes, emolientes, umectantes, bases absorventes de água, bases hidrófilas, ceras, hidrocarbonetos, substâncias oleaginosas, álcoois, ácidos gordos, esteres, polióis, pó insolúvel, essências, conservantes, antissépticos. Alguns excipientes utilizados em composições cosméticas podem alterar a estrutura interna do estrato córneo alterando a função de barreira e aumentando a velocidade de permeação de substâncias ativas pelo estrato córneo, dentre eles podemos citar: água, propilenoglicol, ácido oléico, ureia, DMSO, laurocapranos, pirrolidonas, e demais tensoativos(Leon Lachman, Herbert A. Lieberman, Joseph L Kanig, Teoria e Prática na Indústria Farmacêutica, Vol. II, capítulo 18, páginas 907-953, 2001 ). In dermocosmetics, the choice of active extract encapsulating agents and excipients used in cosmetology with multifunctional properties is extremely important, ie excipients with various functions such as: skin permeation facilitators, photoprotective action, specific cosmetics, lipophilic / hydrophilic system stabilizers, viscosity and sensory control, as well as being biocompatible and biodegradable. Cosmetic excipients are essential in a dermocosmetic composition as they have characteristics that ensure stability to the composition, facilitate administration, promote the release of active substances from the dermal matrix thus promoting the bioavailability of active substances in different layers of the skin and consequently the appropriate pharmacological action. without toxicity, as well as consumer acceptability from a sensory and aesthetic point of view. For the development of semi-solid systems with cosmetology application it is necessary to have a set of excipients that will adequately release the drug or active substances from a dermocosmetic matrix and among them are the excipients: emulgents, emollients, humectants, water absorbent bases, hydrophilic bases. , waxes, hydrocarbons, oilseeds, alcohols, fatty acids, esters, polyols, insoluble powder, essences, preservatives, antiseptics. Some excipients used in cosmetic compositions may alter the internal structure of the stratum corneum by altering the barrier function and increasing the permeation rate of active substances by the stratum corneum. These include water, propylene glycol, oleic acid, urea, DMSO, laurocaprans, pyrrolidones, and other surfactants (Leon Lachman, Herbert A. Lieberman, Joseph L Kanig, Theory and Practice in the Pharmaceutical Industry, Vol. II, Chapter 18, pages 907-953, 2001).
[0019] Do ponto de vista do processo e dos custos de produção, incompatibilidades farmacotécnicas sejam elas químicas ou físicas, entre as substâncias ativas e excipientes cosméticos devem ser evitadas. Alguns excipientes cosméticos podem gerar influências negativas para a composição dermocosmética. As composições cosméticas devido a presença de excipientes lipídicos, ou pela presença de substâncias ativas lipídicas, estão sujeitas a reações de auto-oxidação com posterior decomposição, sendo um inconveniente para estas formulações, resultando em aroma e aspecto desagradáveis e reações de rancificação. Portanto, a escolha dos excipientes deve ser bem definida para evitar problemas de incompatibilidade fármaco- excipiente ou excipiente-excipiente, reduzindo a estabilidade da(s) substância(s) ativa(s), na composição, bem como problemas de toxicidade. Algumas substâncias ativas podem ser altamente reativas numa composição dermocosmética e produzir reações químicas das mais diversas reduzindo a estabilidade destas composições semissólidas. Dentre as reações químicas comumente previstas estão as reações de hidrólise de ésteres, amidas, imidas, lactona; reações de oxidação devido a presença de alta concentração de oxigénio na composição e auto-oxidação; reações de isomerização; reações de fotodegradação iniciadas pela ação da luz causando a diminuição da potência das substâncias ativas (Katsunori Yoshida, Tomoko Sekine, Fumiaki Matsuzaki, Toshio Yanaki*, and Michihiro Yamaguchi. Stability of Vitamin A in Oil-ln-Water- In-Oil-Type Multiple Emulsions. JAOCS, Vol. 76, no. 2 (1999) PP.6); reações de degradação com formação de substâncias tóxicas como formaldeído na composição cosmética e gerando possíveis efeitos tóxicos como dermatites atópicas em consumidores destes produtos (Margareta Bergh, Kerstin Magnusson, J. Lars G. Nilsson and Ann-Therese Karlberg. Formation of formaldehyde and peroxides by air oxidation of high purity polyoxyethylene surfactants. Contact Dermatitis, 1998, 39, 14-20). From the point of view of process and production costs, pharmaceutical and chemical incompatibilities, whether chemical or physical, between active substances and cosmetic excipients should be avoided. Some cosmetic excipients may generate negative influences for dermocosmetic composition. Cosmetic compositions due to the presence of lipid excipients, or the presence of lipid active substances, are subject to self-oxidation reactions with subsequent decomposition, which is a drawback for these formulations, resulting in aroma and appearance. unpleasant and rancid reactions. Therefore, the choice of excipients should be well defined to avoid problems of drug-excipient or excipient-excipient incompatibility, reducing the stability of the active substance (s) in the composition as well as toxicity problems. Some active substances may be highly reactive in a dermocosmetic composition and produce various chemical reactions reducing the stability of these semi-solid compositions. Commonly anticipated chemical reactions include hydrolysis reactions of esters, amides, imides, lactone; oxidation reactions due to the presence of high oxygen concentration in the composition and self-oxidation; isomerization reactions; Photodegradation reactions initiated by the action of light causing the potency of the active substances to decrease (Katsunori Yoshida, Tomoko Sekine, Fumiaki Matsuzaki, Toshio Yanaki * , and Michihiro Yamaguchi. Stability of Vitamin A in Oil-ln-Water-In-Oil-Type Multiple Emulsions, JAOCS, Vol. 76, No. 2 (1999) PP.6); degradation reactions with formation of toxic substances as formaldehyde in the cosmetic composition and generating possible toxic effects as atopic dermatitis in consumers of these products (Margareta Bergh, Kerstin Magnusson, J. Lars G. Nilsson and Ann-Therese Karlberg. Formation of formaldehyde and peroxides by air oxidation of high purity polyoxyethylene surfactants (Contact Dermatitis, 1998, 39, 14-20).
[0020] Em geral extratos de própolis apresentam em sua composição diversas substâncias ativas de diferentes classes fitoquímicas, a saber: flavonoides, ácidos fenólicos, terpenos, clerodanos, xantonas, benzofenonas dentre outras, que podem em contato com diferentes excipientes dermocosméticos promoverem reações químicas das mais diversas reduzindo a potência, a estabilidade, além de resultar em produtos com aspecto desagradável e ainda resultar em efeitos tóxicos. Neste sentido, faz necessário desenvolver técnicas de micro ou nanoencapsulação de substâncias ativas de origem natural.  In general propolis extracts have in their composition several active substances of different phytochemical classes, namely: flavonoids, phenolic acids, terpenes, clerodanes, xanthones, benzophenones among others, which may in contact with different dermocosmetic excipients promote chemical reactions of the propolis. more diverse reducing power, stability, and result in unpleasant looking products and also result in toxic effects. In this sense, it is necessary to develop micro or nanoencapsulation techniques of active substances of natural origin.
[0021] Técnicas de micro e nanoencapsulação são utilizadas pelas indústrias farmacêutica, alimentícia e cosmética com o intuito de solucionar gargalos tecnológicos dentre eles: proteger a(s) substância(s) ativa(s) contra agentes extrínsecos (umidade, luz, oxigénio) que podem causar reações de hidrólise, fotodegradação, oxidação; prevenir a perda de substâncias voláteis; aumentar o prazo de validade do produto; promover liberação controlada de substâncias ativas; bem como melhorar características específicas de entrega de substâncias ativas em tecidos alvos. Micro and nanoencapsulation techniques are used by pharmaceutical, food and cosmetic industries in order to solve technological bottlenecks among them: protecting the active substance (s) against extrinsic agents (moisture, light, oxygen) that may cause hydrolysis, photodegradation, oxidation reactions; prevent loss of volatile substances; increase the shelf life of the product; promote controlled release of active substances; as well as improving specific delivery characteristics of active substances in target tissues.
[0022] As nanopartículas, em geral, são obtidas por diversas técnicas de preparação que dentre elas podemos citar as técnicas de formação de nanoemulsões, e dentre elas podemos citar: método de dupla emulsificação, método de emulsificação-coacervação, método de revestimento com polímero, método de revestimento camada por camada, método emulsificação-difusão e método da nanoprecipitação(Mora-Huertas, C. E. Fessi, H. Elaissari, A. Polymer-based nanocapsules for drug delivery. International Journal of Pharmaceutics 385 (2010) 1 13-142).  Nanoparticles, in general, are obtained by various preparation techniques which among them we can mention the techniques of nanoemulsion formation, and among them we can mention: double emulsification method, emulsification-coacervation method, polymer coating method , layer-by-layer coating method, emulsification-diffusion method, and nanoprecipitation method (Mora-Huertas, CE Fessi, H. Elaissari, A. Polymer-based nanocapsules for drug delivery. International Journal of Pharmaceutics 385 (2010) 1 13-142 ).
[0023] Porém estes métodos de obtenção de nanopartículas em suspensão, se não preparados de forma adequada podem apresentar desvantagens como risco de contaminação microbiológica por estar em meio aquoso, degradação do polímero por hidrólise, instabilidade físico-química devido à agregação das partículas ou diminuição da atividade biológica das substâncias ativas incorporadas. Algumas técnicas de preparação de nanopartículas evitam contaminação microbiana, problemas de degradação do polímero e possíveis instabilidades; então o uso de processos de secagem de nanopartículas é um dos recursos para garantir estabilidade das substâncias por tempos prolongados.  However, these methods of obtaining suspended nanoparticles, if not properly prepared, may present disadvantages such as risk of microbiological contamination by being in aqueous medium, degradation of polymer by hydrolysis, physicochemical instability due to particle aggregation or decreased of the biological activity of the incorporated active substances. Some nanoparticle preparation techniques prevent microbial contamination, polymer degradation problems and possible instabilities; So, the use of nanoparticle drying processes is one of the resources to guarantee the stability of the substances for long time.
[0024] Existem técnicas de secagem de nanopartículas, como as técnicas de evaporação de solvente por rotaevaporador, secagem por spray- dryer, liofilização e outras. Um método de escolha para obtenção de microencapsulado e nanoencapsulados muito utilizado é o método de secagem por liofilização. A liofilização é bastante empregada na secagem de substâncias termolábeis, como material contendo substâncias proteicas, microrganismos como bactérias lácticas usadas na produção de fermentos, fungos comestíveis, dentre outros. É, também, empregada como método de encapsulação de material ativos sensíveis à temperatura e alta pressão. A secagem de nanopartículas apresenta vantagens de aumentar prazo de validade de substâncias ativas, evita degradação de substâncias em meio aquosos e em suspensão, facilitar processo de redispersão e reincorporação em sistemas semissólidos. There are nanoparticle drying techniques, such as solvent evaporation by spin-evaporator, spray-drying, lyophilization and others. One method of choice for obtaining widely used microencapsulated and nanoencapsulated is the freeze drying method. Freeze drying is widely used in drying substances thermolabile, as a material containing protein substances, microorganisms such as lactic bacteria used in the production of yeast, edible fungi, among others. It is also employed as a method of encapsulating temperature sensitive and high pressure active materials. Nanoparticle drying has the advantages of extending shelf life of active substances, prevents degradation of substances in aqueous and suspended media, facilitates redispersion process and reincorporation into semi-solid systems.
[0025] Até o presente momento existem alguns documentos no estado da técnica com potencial de inovação para microencapsulação de própolis e poucos documentos para nanoencapsulação de própolis, porém até o momento não foi identificado documentos no estado da técnica com nanoencapsulação da própolis vermelha para fins dermocosméticos. Os documentos a seguir irão citá-los.  To date there are some state-of-the-art documents with potential for innovation for propolis microencapsulation and few documents for propolis nanoencapsulation, but so far no documents have been identified with dermocosmetic red propolis nanoencapsulation. . The following documents will cite them.
[0026] Alguns documentos no estado da técnica demonstram a utilização da liofilização para obtenção de microencapsulados. A patente de invenção KR1020030063053 produz-se grânulos de uma mistura de 10 a 30% de própolis, 40 a 70% de lactose, 0,1 a 1 ,0% de vitamina e 10 a 30% de mel desidratado por meio de liofilização. O documento chinês CN171 1992 obtém microcapsulas de própolis usando na composição excipiente de alto custo, a beta-ciclodextrina seguido por processo de liofilização. Já o documento JP2006028174 produz grânulos de própolis usando uma mistura de aminoácidos na proporção de 50% (extrato de própolis: mistura de aminoácidos). Microencapsulados de própolis contendo um amido modificado (Octenil-succinato de amido) e goma arábica foram obtidos pela técnica de Spray-Dryer (Silva F. C, Thomazini M., Alencar S. M. and Favaro-Trindade C. S., XVMth International Conference on Bioencapsulation, Groningen, Netherlands ; September 24-26, 2009). A obtenção de microencapsulados de própolis para diversos fins é descrito no estado da técnica. Por exemplo, a patente de invenção CN102920652 prepara microencapsulados de própolis com outros biopolímeros: a quitosana, hidroxipropil betaciclodextrina e glicerofosfato de sódio para tratamento de patologias periodontais. Patente de invenção CN102772596 utiliza multimistura de extrato de planta, própolis e 5% de quitina para obter preparações usando a técnica de spray-dryer com aplicação no tratamento da obesidade. Patente de invenção CN102641256 obtém microencapsulados de própolis com uréia, gelatina, parafina, e solvente tóxico (hexano) para preparo de microencapsulados com tamanho de partícula grande entre 70 a 150μιη. O documento CN102429141 utiliza sistema gelatina/pectina e própolis para preparação de microcápsulas de liberação lenta usando a liofilização. Some prior art documents demonstrate the use of lyophilization to obtain microencapsulates. KR1020030063053 produces granules from a mixture of 10 to 30% propolis, 40 to 70% lactose, 0.1 to 1.0% vitamin and 10 to 30% dehydrated honey by lyophilization. Chinese document CN171 1992 obtains propolis microcapsules using in the high cost excipient composition beta-cyclodextrin followed by lyophilization process. JP2006028174 produces propolis granules using a 50% amino acid mixture (propolis extract: amino acid mixture). Propolis microencapsulates containing a modified starch (starch octenyl succinate) and gum arabic were obtained by Spray-Dryer technique (Silva F. C, Thomazini M., Alencar SM and Favaro-Trinity CS, XVMth International Conference on Bioencapsulation, Groningen , Netherlands; September 24-26, 2009). Obtaining propolis microencapsulates for various purposes is described in the prior art. For example, CN102920652 prepares propolis microencapsulates with other biopolymers: chitosan, hydroxypropyl betacyclodextrin and sodium glycerophosphate for treatment of periodontal disorders. CN102772596 uses multimixture of plant extract, propolis and 5% chitin to obtain preparations using the spray-dryer technique for obesity treatment. CN102641256 obtains microencapsulates of propolis with urea, gelatin, paraffin, and toxic solvent (hexane) for preparation of microencapsulates with large particle size between 70 to 150μιη. CN102429141 uses gelatin / pectin and propolis system for preparation of slow release microcapsules using lyophilization.
[0027] Processo de obtenção de componentes da própolis no estado seco utilizando gelatina contendo solução de própolis da cidade de Maringá (Paraná) foram preparadas por secagem por spray-dryer. A otimização das condições de secagem por pulverização e as proporções de gelatina e manitol foram investigados (M.L. Bruschi, M.L.C. Cardoso, M.B. Lucchesi, M.P.D. Gremião, International Journal of Pharmaceutics (2003) 264, 45-55). É fundamental se destacar que, a utilização de diferentes tipos de excipientes permite diferentes mecanismos de liberação das substâncias ativas da matriz farmacêutica (microencapsulados), além de diferentes ações fisiológicas. O pedido de patente brasileiro PI05001757 utiliza apenas dois excipientes (gelatina e manitol) e demonstra apenas processo de preparação de uma matriz intermediária contendo extratos de própolis não divulgando a procedência da própolis utilizada. Os pedidos de patente WO 2014/186851 e BR 10 2012 013590-6 realizados pelo nosso grupo de pesquisa e inovação com medicamentos opoterápicos, em particular com própolis vermelha, faz uso de gelatina, amido pré-gelatinizado e dióxido de silício coloidal para preparação de microencapsulados de própolis vermelha brasileira com liberação gastroresistente. O pedido de patente francês FR 725623 tem como objetivo um sistema galênico multi-microparticulado de liberação prolongada de diferentes princípios ativos. Essas microcápsulas são formadas por um núcleo de princípio ativo, revestido com uma cobertura à base por exemplo de etilcelulose, de polivinilpirrolidona, de Óleo de rícino e de estearato de magnésio. A patente européia EP 548356 reivindica um comprimido multiparticulado de desagregação rápida que compreende uma substância ativa sob a forma de microcristais ou de microgrânulos providos de uma cobertura. Patente de invenção WO 2014/166994 A1 desenvolve um sistema misto de liberação do ingrediente ativo e compreende um sistema nano-micro particulado que apresenta ingredientes ativos no interior da nanopartícula, bem como no interior da micropartícula com aplicação em composições de uso oral. [0027] Process for obtaining propolis components in the dry state using gelatin containing propolis solution from the city of Maringá (Paraná) were prepared by spray-dryer drying. Optimization of spray drying conditions and proportions of gelatin and mannitol were investigated (ML Bruschi, MLC Cardoso, MB Lucchesi, MPD Gremião, International Journal of Pharmaceutics (2003) 264, 45-55). It is important to highlight that the use of different types of excipients allows different mechanisms of release of the active substances of the pharmaceutical matrix (microencapsulated), besides different physiological actions. Brazilian patent application PI05001757 uses only two excipients (gelatin and mannitol) and only demonstrates the process of preparing an intermediate matrix containing propolis extracts without disclosing the origin of the propolis used. Patent applications WO 2014/186851 and BR 10 2012 013590-6 filed by our research and innovation group with opotherapeutic drugs, in particular with red propolis, make use of gelatin, pregelatinized starch and colloidal silicon dioxide for preparation of microencapsulates of Brazilian red propolis with gastroresistant release. French patent application FR 725623 is for a multi-microparticulate extended release galenic system of different active ingredients. These microcapsules are formed by a nucleus active ingredient, coated with a coating based for example on ethylcellulose, polyvinylpyrrolidone, castor oil and magnesium stearate. European patent EP 548356 claims a rapidly disintegrating multiparticulate tablet comprising an active substance in the form of microcrystals or microgranules provided with a coating. WO 2014/166994 A1 develops a mixed active ingredient release system and comprises a nano-micro particulate system which has active ingredients within the nanoparticle as well as within the microparticle for use in oral compositions.
[0028] Poucos produtos e processos nanotecnológicos de preparação de nanopartíclas utilizando própolis têm sido desenvolvidos no mundo inteiro, por diferentes grupos de pesquisas, de diferentes modos e para diferentes finalidades (nutracêutica, terapêutica bem como cosmecêutica). Uma formulação de nanopartículas polimérica de própolis (própolis nanoalimento) foi desenvolvida utilizando agregados micelares de copolímeros reticulados e aleatória de N-isopropilacrilamida (NIPAAm) com N-vinil-2-pirrolidona (VP) e poli (etilenoglicol) monoacrilato (PEG-A) (Kim, Dong-Myung, Lee, Gee-Dong, Aum, Seung-Hyun, Kim, Ho-Jun, Biological & Pharmaceutical Bulletin, (2008) 31 , 1704-1710). Patente de invenção americana US 20130295181 e patente EP2633862 da mesma família de patente desenvolve nanocápsulas de própolis a base de álcool polivinílico (PVA), tamanho de partículas submicromético (<1000nm) compreendido entre 220nm a 438nm com diferentes aplicabilidades e diferentes áreas: agronómica, farmacêutica e cosmética, etc. Família de patente de invenção WO/201 1 /001 181 e CA2765920 utiliza extrato de própolis para obter uma matriz carreadora sólida de substâncias ativas, especificamente desenvolve um filme sólido contendo própolis. Few nanotechnological products and processes for preparing nanoparticles using propolis have been developed worldwide by different research groups in different ways and for different purposes (nutraceutical, therapeutic as well as cosmeceutical). A polymeric propolis nanoparticle formulation (nanofood propolis) was developed using cross-linked micellar aggregates of N-isopropylacrylamide (NIPAAm) with N-vinyl-2-pyrrolidone (VP) and poly (ethylene glycol) monoacrylate (PEG-A) (Kim, Dong-Myung, Lee, Gee-Dong, Aum, Seung-Hyun, Kim, Ho-Jun, Biological & Pharmaceutical Bulletin, (2008) 31, 1704-1710). US 20130295181 and EP2633862 of the same patent family develops polyvinyl alcohol (PVA) propolis nanocapsules, submicrometic particle size (<1000nm) ranging from 220nm to 438nm with different applicability and different areas: agronomic, pharmaceutical and cosmetics, etc. WO / 2011/001 181 and CA2765920 utilize propolis extract to obtain a solid carrier matrix of active substances, specifically developing a propolis-containing solid film.
[0029] Alguns exemplos mostram a aplicabilidade específica em área de cosméticos usando nanopartículas revestidas contendo substâncias hidrofílicas e/ou oleofílicas em composições dermocosméticas, bem como em composições orais. Patente de invenção WO2015/022471 A1 utiliza nanocápsulas de revestimento para substâncias oleofílicas ativas presentes em filtro solares UVA/UVB. O documento KR20100078349 revela preparações de nano emulsões de própolis utilizando excipiente β-ciclodextrina. O documento de patente WO 2015/003155 A1 mostra aplicabilidade de nanofibras biocompatíveis contendo quitosana, mel, ácido acético, gelatina, colágeno, alginatos, BSA e outros agentes formadores de redes nanométricas reticuladas e substâncias ativas naturais ou sintéticas contra diversas patologias como bactérias, hipertensão arterial, cicatrização de feridas, câncer. Documento de patente PCT/US2013/075714 trata de composições contendo pó anidro contendo sistema particulado submicrométrico líquido (< 1000nm) utilizado para preparação de emulsões, suspensões, soluções e pós para fins cosmético ou farmacêutico. Este sistema consiste de um núcleo hidrofílico contendo um revestimento hidrofóbico que irá proteger todos os ingredientes ativos em seu núcleo. Documento de patente chinês CN103520069 utiliza mel e própolis em composições e produtos de beleza para unhas incluindo composições tipo gel, cremes e adesivos. Cápsulas de gelatina mole foram desenvolvidas no documento de patente para manter a beleza e juventude, bem como regular a imunidade, colesterol e senescência. Documento de patente chinês CN1031 160399 desenvolve sabonete com extratos naturais, própolis e ingredientes base para fabricação de sabonete para o tratamento multifuncional de limpeza, remoção de bactérias, redução de alergias e antisséptico. Patente de invenção chinês CN101461846 desenvolve um gel contendo extrato de plantas e própolis contra herpes zoster. Documento de patente japonês JP2008247830 utiliza extrato de própolis e extrato de amaranto em composição cosmética para inibir manchas de pele, pigmentações, manchas senis. Documento de patente de invenção CN101 181 198 utiliza ácido kojico, fibroina, própolis e outros ingredientes na preparação de um creme de beleza nutritivo para remoção de manchas de pele. Patente de invenção utiliza mel, pó de pérolas, própolis, glicerina para produzir um creme multifuncional para massagem e de beleza reduzindo processos inflamatórios da pele como a acne. Documento de patente de invenção JP2006016373 com propriedade multifuncional de redução de peso, redução capilar e tratamento de beleza utiliza própolis, geleia real, extratos de plantas, e colágeno. Patente de invenção KR1020030093890 utiliza extrato de própolis e extrato de chá verde e outros extratos de planta para produzir um sabonete com ação polimultifuncional antibacteriana, antisséptica, dermatites atópicas e ação odorizante. Patente de invenção japonesa JP2003055138 utiliza mel e extrato de própolis do alecrim brasileira para produzir um tipo de emplastro com ação antibacteriana e antifúngica, bem como para tratamento de beleza. Patente de invenção chinesa CN1244386 utiliza própolis e extrato de perilla para produzir partículas entre 0,01 a 10μιη, em composições usadas no tratamento e prevenção da cárie, inflamação periodontais, agente antibacteriano. Some examples show the specific applicability in cosmetics using coated nanoparticles containing hydrophilic and / or oleophilic substances in dermocosmetic compositions as well as in oral compositions. WO2015 / 022471 A1 uses coating nanocapsules for active oleophilic substances present in UVA / UVB sunscreens. KR20100078349 discloses propolis nano emulsion preparations using β-cyclodextrin excipient. WO 2015/003155 A1 shows applicability of biocompatible nanofibers containing chitosan, honey, acetic acid, gelatin, collagen, alginates, BSA and other crosslinked nanometric network forming agents and natural or synthetic active substances against various pathologies such as bacteria, hypertension artery, wound healing, cancer. PCT / US2013 / 075714 deals with compositions containing anhydrous powder containing liquid submicron particulate system (<1000nm) used for the preparation of emulsions, suspensions, solutions and powders for cosmetic or pharmaceutical purposes. This system consists of a hydrophilic core containing a hydrophobic coating that will protect all active ingredients in its core. Chinese Patent Document CN103520069 utilizes honey and propolis in nail beauty products and compositions including gel-like compositions, creams and adhesives. Soft gelatin capsules have been developed in the patent document to maintain beauty and youth as well as regulate immunity, cholesterol and senescence. Chinese Patent Document CN1031 160399 develops soap with natural extracts, propolis and base ingredients for soap making for multifunctional treatment of cleaning, bacterial removal, allergy reduction and antiseptic. Chinese patent CN101461846 develops a gel containing plant extract and herpes zoster propolis. Japanese Patent Document JP2008247830 utilizes propolis extract and amaranth extract in cosmetic composition to inhibit skin blemishes, pigmentation, age spots. CN101 181 198 utilizes kojic acid, fibroin, propolis and other ingredients in the preparation of a nourishing beauty cream for removing skin blemishes. Patent utilizes honey, pearl powder, propolis, glycerin to produce a multifunctional massage and beauty cream reducing inflammatory skin processes such as acne. JP2006016373 with multifunctional property of weight reduction, hair reduction and beauty treatment utilizes propolis, royal jelly, plant extracts, and collagen. KR1020030093890 uses propolis extract and green tea extract and other plant extracts to produce an antibacterial, antiseptic, multi-functional soap, atopic dermatitis and odorant action soap. Japanese Patent JP2003055138 uses honey and propolis extract from Brazilian rosemary to produce a type of plaster with antibacterial and antifungal action, as well as for beauty treatment. Chinese patent CN1244386 uses propolis and perilla extract to produce particles between 0.01 to 10μιη, in compositions used in the treatment and prevention of caries, periodontal inflammation, antibacterial agent.
[0030] No processo de nanoencapsulação de fármacos e substâncias ativas vem sendo utilizados alguns polímeros com propriedades nanoencapsulantes em composições ditas nanoesferas contendo um núcleo sólido ou nanocápsulas contendo um núcleo oleoso ou líquido. Uma combinação de polímeros nanoencapsulantes também vem sendo utilizados para formar as chamadas matrizes poliméricas sólidas (nanoesferas) ou nanocápsulas de núcleo líquido revestido por uma parede sólida. O termo "nanoencapsulados" também pode ser utilizado de forma mais abrangente sem definição do tipo de nanopartícula polimérica (nanoesfera ou nanocáspula). O uso de sistemas poliméricos ou também chamadas de matrizes poliméricas vem sendo encontrado na literatura científica, podendo realizar a combinação de um ou mais dos seguintes polímeros: Span, álcool polivinílico, polietilenoglicol, polioxipropileno, Tween, pluronic, polioxietileno, policaprolactona, ceramida, ácido poliláctico/poliglicólico, alfa-tocoferol polietilenoglicol succinato , cloreto de trimetil quitosana, dentre outros (M. Wulff- Perez, A. Torcello-Gomez, MJ. Galvez-Ruiz, A. Martin-Rodnguez, Stability of emulsions for parenteral feeding: Preparation and characterization of o/w nanoemulsions with natural oils and Pluronic F68 as surfactant, Food Hydrocolloids 23 (2009) 1096-1 102.; Hearan Suh, Byeongmoon Jeong, Ramesh Rathi, Sung Wan Kim, Regulation of smooth muscle cell proliferation using paclitaxel-loaded poly(ethylene oxide)-poly(lactide/ glycolide) nanospheres, J Biomed Mater Res. 1998 Nov;42(2):331 -8.; Régis Coco, Laurence Plapied, Vincent Pourcelle, Christine Jérôme, David J. Brayden, Yves-Jacques Schneider, Véronique Préat, Drug delivery to inflamed colon by nanoparticles: Comparison of different strategies, International Journal of Pharmaceutics 440 (2013) 3-12). In the nanocapsulation process of drugs and active substances some polymers with nanoencapsulant properties have been used in compositions of said nanospheres containing a solid core or nanocapsules containing an oily or liquid core. A combination of nanoencapsulant polymers has also been used to form so-called solid polymeric matrices (nanospheres) or solid-walled liquid core nanocapsules. The term "nanoencapsulated" can also be used more broadly without defining the type of polymeric nanoparticle (nanosphere or nanocapsule). The use of polymeric systems or also called polymeric matrices has been found in the scientific literature and can combine one or more of the following polymers: Span, polyvinyl alcohol, polyethylene glycol, polyoxypropylene, Tween, pluronic, polyoxyethylene, polycaprolactone, ceramide, acid polylactic / polyglycolic, alpha-tocopherol polyethylene glycol succinate, trimethyl chitosan chloride, among others (M. Wulff-Perez, A. Torcello-Gomez, MJ. Galvez-Ruiz, A. Martin-Rodnguez, Stability of emulsions for parenteral feeding: Preparation and characterization of o / w nanoemulsions with natural oils and Pluronic F68 as surfactant, Food Hydrocolloids 23 (2009) 1096-1102; Hearan Suh, Byeongmoon Jeong, Ramesh Rathi, Sung Wan Kim, Regulation of smooth muscle cell proliferation using paclitaxel-loaded poly (ethylene oxide) -poly (lactide / glycolide) nanospheres, J Biomed Mater Res. 1998 Nov; 42 (2): 331-8; Regis Coco, Laurence Plapied, Vincent Pourcelle, Christine Jérôme, David J. Brayden, Yves-Jacques Schneider, Veronique Préat, Comparison of different strategies, International Journal of Pharmaceuticals 440 (2013) 3-12) .
[0031] Uso de matrizes de revestimento polimérico garantem vantagens ao formulador dentre elas, estabilidade ao sistema matricial nanoparticulado, evita problemas de degradação ou reações de hidrólise, fotodegradação, dentre outras, e ainda garante processo padronizado de liberação das substâncias ativas. Até o momento algumas pesquisas mostram o uso de matrizes poliméricas no processo de micro ou nanoencapsulação de fármacos com atividade farmacológica contra câncer (tamoxifen, paclitaxel), usando extrato de plantas, extrato de própolis, bem como fitofármacos (rutina) (Juliana S. Almeida, Fernanda Lima, Simoni Da Ros, Luis O. S. Bulhões, Leandro M. de Carvalho, Ruy C. R. Beck, Nanostructured Systems Containing Rutin: In Vitro Antioxidant Activity and Photostability Studies, Nanoscale Res Lett (2010) 5:1603-1610.; Jugminder S. Chawla, Mansoor M. Amiji, Biodegradable poly(o- caprolactone) nanoparticles for tumor targeted delivery of tamoxifen, International Journal of Pharmaceutics 249 (2002) 127-138; Ezequiel Bernabeu, Gustavo Helguera, Maria J. Legaspi, Lorena Gonzalez,Christian Hocht, Carlos Taira, Diego A. Chiappetta, Paclitaxel-loaded PCL-TPGS nanoparticles: In vitro and in vivo performance compared with Abraxane®, Colloids and Surfaces B: Biointerfaces 1 13 (2014) 43- 50.; Alexandru Luca, Betul Cilek, Vasif Hasirci, Serpil Sahin, Gulum Sumnu, Storage and Baking Stability of Encapsulated Sour Cherry Phenolic Compounds Prepared from Micro- and Nano-Suspensions, Food Bioprocess Technol (2014) 7:204-21 1 ).  Use of polymeric coating matrices guarantees advantages to the formulator among them, stability to the nanoparticulate matrix system, avoids degradation problems or hydrolysis reactions, photodegradation, among others, and also ensures standardized process of release of active substances. To date some research has shown the use of polymeric matrices in the process of micro or nanoencapsulation of drugs with pharmacological activity against cancer (tamoxifen, paclitaxel), using plant extract, propolis extract as well as phytopharmaceuticals (rutin) (Juliana S. Almeida , Fernanda Lima, Simoni Da Ros, Luis OS Bulhões, Leandro M. de Carvalho, Ruy CR Beck, Nanostructured Systems Containing Rutin: In Vitro Antioxidant Activity and Photostability Studies, Nanoscale Res Lett (2010) 5: 1603-1610 .; Jugminder S Chawla, Mansoor M. Amiji, Biodegradable poly (o-caprolactone) nanoparticles for targeted tumor delivery of tamoxifen, International Journal of Pharmaceutics 249 (2002) 127-138; Ezequiel Bernabeu, Gustavo Helguera, Maria J. Legaspi, Lorena Gonzalez, Christian Hocht, Carlos Taira, Diego A. Chiappetta, Paclitaxel-loaded PCL-TPGS nanoparticles: In vitro and in vivo performance compared to Abraxane®, Colloids and Surfaces B: Biointerfaces 1 13 (2014) 43- 50 Alexandru Luca, Betul Cilek, Vasif Hasirci, Serpil Sahin, Gulum Sumnu, Storage and Baking Stability of Encapsulated Sour Cherry Phenolic Compounds Prepared from Micro- and Nano-Suspensions, Food Bioprocess Technol (2014) 7: 204-21 1).
[0032] Até o momento foi detectado um sistema polimérico contendo matriz polimérica policaprolactona e álcool polivinílico (PCL-PVA) e um tipo particular de própolis da região sudeste do Brasil (Mogi das Cruzes-SP), com processo de preparação pela técnica de emulsificação, seguido de evaporação. Porém as partículas obtidas apresentavam-se como microparticulados com tamanho de partícula entre 5 a 10μιη (N. Duran, P. D. Marcato, C. M. S. Buffo, M. M. M. De Azevedo, E. Esposito, Poly(e-caprolactone)/propolis extract: microencapsulation and antibacterial activity evaluation, Pharmazie 62 (2007) 4, 287-290). Família de patente US20130295181 e EP2633862 desenvolve nanocápsulas de própolis a base de álcool polivinílico (PVA) com tamanho compreendido entre 220nm a 438nm com diferentes aplicabilidades incluindo áreas agronómica, farmacêutica e cosmética. To date a polymeric system containing polymeric matrix polycaprolactone and polyvinyl alcohol (PCL-PVA) and a particular type of propolis from southeastern Brazil (Mogi das Cruzes-SP), with preparation process by emulsification technique, followed by evaporation. However the particles obtained were microparticulate with particle size between 5 and 10μιη (N. Duran, PD Marcato, CMS Buffo, MMM De Azevedo, E. Esposito, Poly (e-caprolactone) / propolis extract: microencapsulation and antibacterial activity evaluation, Pharmazie 62 (2007) 4, 287-290). US20130295181 and EP2633862 develops nanocapsules of polyvinyl alcohol (PVA) -based propolis ranging in size from 220nm to 438nm with different applicability including agronomic, pharmaceutical and cosmetic areas.
[0033] Nenhum dos documentos de patente descritos revela a obtenção de nanopartículas poliméricas contendo extrato de própolis vermelha (NPEPV) usando sistema polimérico PCL-pluronic (F-108) através de método de nanoprecipitação seguido por centrifugação e secagem por liofilização para obtenção de nanopartículas poliméricas sólidas com aplicação na preparação de cremes dermocosméticos. Desta forma, as modalidades da invenção descritas neste documento apresentam vantagens consideráveis frente ao estado da técnica.  None of the disclosed patent documents disclose obtaining polymeric nanoparticles containing red propolis extract (NPEPV) using PCL-pluronic polymeric system (F-108) by nanoprecipitation method followed by centrifugation and lyophilization drying to obtain nanoparticles. solid polymers with application in the preparation of dermocosmetic creams. Accordingly, the embodiments of the invention described herein have considerable advantages over the state of the art.
[0034] A invenção proposta no presente documento busca o preenchimento de uma lacuna tecnológica em termos da aplicação da própolis vermelha como elemento ativo de composições dermocosméticas de liberação modificada para o tratamento e prevenção de doenças, propondo-se uma forma de liberação modificada das nanopartículas poliméricas contendo extrato de própolis vermelha (NPEPV). Desta forma, propõem-se nanopartículas poliméricas contendo extrato de própolis vermelha de liberação modificada, processos para obtenção de NPEPV, composições dermocosméticas com NPEPV, processos de preparação das composições dermocosméticas com NPEPV e usos destes composições dermocosméticos com ação antioxidante, anti-inflamatória, citostática/citotóxica, cicatrizante de feridas no pós-operatório, feridas de decúbito, tratamentos de estética e beleza, higiene e limpeza. [0034] The invention proposed herein seeks to fill a technological gap in the application of red propolis as an active element of modified release dermocosmetic compositions for the treatment and prevention of diseases by proposing a modified release form of nanoparticles. propolis extract containing polymeric compounds (NPEPV). Thus, polymeric nanoparticles containing modified release red propolis extract, processes for obtaining NPEPV, dermocosmetic compositions with NPEPV, preparation processes for dermocosmetic compositions with NPEPV and uses of these antioxidant, anti-inflammatory, cytostatic dermocosmetic compositions are proposed. / cytotoxic, wound healing in the postoperative period, decubitus wounds, beauty and beauty treatments, hygiene and cleanliness.
[0035] Cumpre salientar que, a utilização da própolis vermelha é característica determinante na inovação, em termos das novas tecnologias em saúde com uso de própolis e opoterápicos, visto que, a própolis vermelha apresenta composição característica, que, terá função primordial na atividade das NPEPV e demais composições dermocosméticas, para prevenção e tratamento de doenças, estética e beleza. It should be noted that the use of red propolis is a determining feature in innovation, in terms of new health technologies with the use of propolis and opotherapics, since red propolis has a characteristic composition, which will play a primary role in the activity of NPEPV and other dermocosmetic compositions for disease prevention and treatment, aesthetics and beauty.
[0036] O desenvolvimento de método analítico de identificação e quantificação de isoflavonóides no extrato e nas nanopartículas poliméricas contendo extrato de própolis vermelha por cromatografia líquida de alta eficiência acoplada ao detector de arranjo de diodo também surge como elemento inovador deste documento.  [0036] The development of analytical method of identification and quantification of isoflavonoids in the extract and polymeric nanoparticles containing red propolis extract by high performance liquid chromatography coupled to the diode array detector also emerges as an innovative element of this document.
[0037] Ainda, como diferencial essencial frente ao estado da técnica, as nanopartículas poliméricas contendo própolis vermelha e composições dermocosméticas propostas na presente invenção, utilizam compostos encapsulantes, estabilizantes e dispersantes do núcleo, na matriz intermediária que irão promover a liberação controlada, bem como evitar processos oxidativos entre os compostos fenólicos pressentes na própolis vermelha com demais excipientes cosméticos presentes na matriz externa, além de uma matriz externa óleo/aquosa polimultifuncional com atividades acessórias que contêm excipientes cosméticos biocompatíveis, biodegradáveis, atóxicos e estáveis com característica reológica para semissólidos cosméticos/cosmecêuticos de liberação modificada. Desta forma, a composição aqui revelada pode ser usada para solucionar problemas técnicos semi-industriais e industriais nas áreas dermocosmética, bem como algumas aplicações de uso terapêutico.  Also, as an essential differential compared to the state of the art, the red propolis-containing polymeric nanoparticles and dermocosmetic compositions proposed in the present invention utilize encapsulating, stabilizing and dispersing compounds of the core in the intermediate matrix which will promote controlled release as well as avoid oxidative processes between phenolic compounds present in red propolis with other cosmetic excipients present in the external matrix, as well as a polymultifunctional external oil / aqueous matrix with accessory activities that contain biocompatible, biodegradable, non-toxic and stable cosmetic excipients with rheological characteristic for cosmetic semisolids / modified release cosmeceuticals. Thus, the composition disclosed herein can be used to solve semi-industrial and industrial technical problems in the dermocosmetic areas, as well as some therapeutic applications.
Sumário da Invenção Summary of the Invention
[0038] A presente invenção apresenta nanoesferas carregadas com extrato de própolis vermelha (NPV), composto por extrato padronizado de própolis vermelha proveniente da matéria-prima própolis vermelha, e três sistemas matriciais nanopolimérica composto por polímeros de revestimento e dispersante/estabilizante. Estes sistemas nanoencapsulantes é composto por sistema binário ou sistema ternário, com propriedades biocompatível, biodegradável e promotora de liberação modificada do princípio ativo, protege as composições dermocosméticas de ataque oxidativo das substâncias fenólicas presentes no extrato ativo de própolis vermelha, além de apresentar atividade leishmanicida. A invenção trata também do processo de obtenção de suspensões de nanopartículas poliméricas que são nanoencapsuladas em sistema de liberação modificada, por meio da técnica de nanoprecipitação seguido por centrifugação e liofilização para produzir nanoesferas no estado sólido carregadas com extrato de própolis vermelha. Adicionalmente, apresenta-se nesta modalidade de invenção em termos de composições semissólidas contendo as NPV, preferencialmente, na forma de creme não- iônico, creme-gel não-iônico, gel-creme não-iônico, loções não-iônicas, podendo também preparar cremes catiônicos. A composição revelada compreende: i) um núcleo contendo substância(s) ativa(s) de própolis vermelha combinado com uma matriz polimérica de nanorevestimento que pode modificar ou retardar a liberação das substância(s) ativa(s) contidas no extrato de própolis vermelha; (ii) uma camada intermediária dispersante/ estabilizante do núcleo; (iii) uma camada externa óleo/aquosa responsável pelos processos diluição, dispersão e estabilização de camada intermediária e núcleo numa composição dermocosmética, bem como por ser promotor da dissolução e permeação dos componentes ativos pelas barreiras da pele. Ainda, a invenção trata do processo de preparação da composição dermocosméticas, a partir da preparação de suspensões de nanopartículas poliméricas contendo própolis vermelha, bem como a partir das nanopartículas poliméricas no estado sólido contendo extrato de própolis vermelha. Por fim, são apresentados usos das NPV e das composições dermocosméticas contendo as NPV com ação antioxidante e leishmanicida. Breve Descrição das Figuras The present invention features nanobeads loaded with red propolis extract (NPV), composed of standardized extract of red propolis from the red propolis raw material, and three nanopolymer matrix systems composed of coating and dispersing / stabilizing polymers. These nanoencapsulant systems are composed of binary or ternary systems, with biocompatible, biodegradable and modified release properties promoting the active principle, protect dermocosmetic compositions from oxidative attack of phenolic substances present in the active extract of red propolis, besides presenting leishmanicidal activity. The invention also relates to the process of obtaining polymeric nanoparticle suspensions which are nanoencapsulated in a modified release system by the nanoprecipitation technique followed by centrifugation and lyophilization to produce solid state nanospheres loaded with red propolis extract. Additionally, it is presented in this embodiment of the invention in terms of semisolid compositions containing the NPV, preferably in the form of nonionic cream, nonionic cream gel, nonionic gel cream, nonionic lotions, and may also prepare cationic creams. The disclosed composition comprises: i) a core containing red propolis active substance (s) combined with a polymeric nanoclay matrix that may modify or delay the release of the active substance (s) contained in the red propolis extract. ; (ii) a core dispersing / stabilizing intermediate layer; (iii) an oil / aqueous outer layer responsible for the dilution, dispersion and stabilization processes of the intermediate layer and core in a dermocosmetic composition, as well as promoting dissolution and permeation of the active components by skin barriers. Further, the invention addresses the process of preparing the dermocosmetic composition from preparing suspensions of red propolis-containing polymeric nanoparticles, as well as from solid state polymeric nanoparticles containing red propolis extract. Finally, uses of NPV and dermocosmetic compositions containing NPV with antioxidant and leishmanicidal action are presented. Brief Description of the Figures
[0039] A modalidade da invenção, juntamente com vantagens adicionais da mesma podem ser melhor explanadas e compreendidas mediante referência as figuras em anexo e a seguinte descrição:  [0039] The embodiment of the invention, together with further advantages thereof, may be further explained and understood by reference to the attached figures and the following description:
[0040] A Figura 1 anexa apresenta Fotomicroscopias de Varredura Eletrônica das NPV - NCEU20% (A e B), NCEU 12% (C e D) e NCPCL30% (E e F) usando crioprotetores dióxido de silício coloidal (A, B e E) e glicolato de amido sódico (C, D e F). Fotomicrografias com ampliação entre 3000 vezes (escala 5μιη) e ampliação de até 12000 vezes (escala 1 μιη) mostrando partículas em escala nanométrica.  The attached Figure 1 shows NPV Electron Scanning Photomicroscopy - NCEU20% (A and B), NCEU 12% (C and D) and NCPCL30% (E and F) using colloidal silicon dioxide cryoprotectants (A, B and E) and sodium starch glycolate (C, D and F). Photomicrographs with magnification between 3000 times (5μιη scale) and magnification up to 12000 times (1μμη scale) showing nanoscale particles.
[0041] A Figura 2 anexa apresenta termogramas de DSC das NPV. Composições NEU (A), composições NPCL (B) e composições NEUPCL (C).  The accompanying Figure 2 shows NPV DSC thermograms. NEU (A) compositions, NPCL (B) compositions and NEUPCL (C) compositions.
[0042] A Figura 3 anexa apresenta espectros FTIR-ATR das NPV. Extrato de própolis vermelha, composição placebo e composição NEU 30% (A e B). Extrato de própolis vermelha, composição placebo e composição NPCL 30% (C e D). Extrato de própolis vermelha, composição placebo e composição NEUPCL 30% (E e F). The attached Figure 3 shows NPV FTIR-ATR spectra. Red propolis extract, placebo composition and 30% NEU composition (A and B). Red propolis extract, placebo composition and NPCL composition 30% (C and D). Red propolis extract, placebo composition and composition 30% NEUPCL (E and F).
[0043] A Figura 4 anexa apresenta os cromatogramas (UPLC-DAD) do extrato de própolis vermelha (A), Placebo NEU (B), composição NEU30% (C), composição NPCL 30% (D) e composição NEUPCLC 30% (E) que identifica flavonoides/isoflavonóide presente no extrato de própolis vermelha e composições e ausência de flavonóides no placebo.  The attached Figure 4 shows the chromatograms (UPLC-DAD) of red propolis extract (A), Placebo NEU (B), NEU30% composition (C), NPCL 30% composition (D) and NEUPCLC 30% composition ( E) which identifies flavonoids / isoflavonoid present in red propolis extract and compositions and absence of flavonoids in placebo.
[0044] A Figura 5 anexa apresenta gráfico normalizado de determinação da IC50 em ensaio leishmanicida in vitro contra Leishmania (V.) braziliensis (A). Ilustração esquemática de alguns mecanismos bioquímicos hipotéticos de inibição celular da Leishmania (V.) braziliensis pelos flavonóides presentes no extrato de própolis vermelha de Alagoas bem como nas nanoesferas carregadas com extrato de própolis vermelha (B).  The accompanying Figure 5 shows a standardized IC50 determination plot in an in vitro leishmanicidal assay against Leishmania (V.) braziliensis (A). Schematic illustration of some hypothetical biochemical mechanisms of cellular inhibition of Leishmania (V.) braziliensis by flavonoids present in the red propolis extract from Alagoas as well as in nanospheres loaded with red propolis extract (B).
[0045] A Figura 6 anexa apresenta fotos de cremes não-iônicos contendo NPCL30% carregadas com extrato de própolis vermelha para obter concentração final no creme cosmético de 2,0% (A), 1 ,5% (B), 1 ,0% (C) e 0,5% (D) do extrato de própolis vermelha. As nanoesferas NPV no estado sólido foram diretamente incorporadas ao creme não-iônico ou usando as suspensões de nanopartículas poliméricas (B). The attached Figure 6 shows photos of non-ionic creams containing NPCL30% loaded with red propolis extract to obtain final concentration in the cosmetic cream of 2.0% (A), 1.5% (B), 1.0% (C) and 0.5% (D) of the red propolis extract. Solid state NPV nanospheres were either directly incorporated into the nonionic cream or using polymeric nanoparticle suspensions (B).
Descrição Detalhada da Invenção Detailed Description of the Invention
[0046] Os processos e composições descritos na presente invenção podem ser melhores detalhados e compreendidos mediante referência às figuras presentes neste documento e a seguinte descrição:  The processes and compositions described in the present invention may be further detailed and understood by reference to the figures herein and the following description:
- Nanoesferas carregadas com Extrato de Própolis Vermelha (NPV) - Nanospheres loaded with Red Propolis Extract (NPV)
[0047] As nanopartículas (sólidas ou em suspensão) apresentadas no presente documento apresentam concentrações de flavonoides, isoflavonóides, chalconas e ácidos fenólicos diferenciados devido à especificidade da própolis vermelha. Adicionalmente, as nanopartículas em questão apresentam importante característica de proteger o sistema matricial semissólido (creme cosmético) da ação pró-oxidante dos flavonoides e compostos fenólicos presentes no extrato de própolis vermelha, visto que cremes cosméticos preparados diretamente com o extrato de própolis vermelha sofrem escurecimento (cor marrom) em até 10 dias, devido reações oxidantes de compostos fenólicos frente aos excipientes cosméticos presente neste sistema matricial de liberação dérmica. Vale ressaltar também que o revestimento polimérico proposto apresenta uma segunda funcionalidade importante, de liberação modificada dos compostos ativos, retardando a liberação dos flavonoides/isoflavonóides, para uma ação de maior duração, além de manutenção dos princípios ativos por mais tempo com atividade biológica nas camadas da pele, sendo importante em tratamentos contra inflamação da pele, câncer, ação leishmanicida, cicatrização de feridas em leishmanioses, além de promover ação antienvelhecimento devida as suas propriedades antioxidantes. The nanoparticles (solid or in suspension) presented herein have differentiated flavonoid, isoflavonoid, chalcone and phenolic acid concentrations due to the specificity of red propolis. In addition, the nanoparticles in question have an important feature of protecting the semi-solid matrix system (cosmetic cream) from the pro-oxidant action of flavonoids and phenolic compounds present in red propolis extract, since cosmetic creams prepared directly with red propolis extract are darkened. (brown color) within 10 days, due to oxidizing reactions of phenolic compounds against the cosmetic excipients present in this matrix dermal release system. It is also noteworthy that the proposed polymeric coating has a second important functionality, modified release of active compounds, delaying the release of flavonoids / isoflavonoids, for a longer action, and maintenance of active principles longer with biological activity in the layers. It is important in treatments against skin inflammation, cancer, leishmanicidal action, wound healing in leishmaniasis, besides promoting anti-aging action due to its antioxidant properties.
[0048] As modalidades de invenção de nanoesferas carregadas com extrato de própolis vermelha apresentadas nesta invenção também pode ser aplicadas para produção de cápsulas de própolis vermelha. [0049] As NPV, além do extrato com os princípios ativos de interesse, obtidos da própolis vermelha, apresentam em sua composição os excipientes farmacêuticos com diferentes funcionalidades sendo eles: composto ativo (extrato de própolis vermelha), matriz polimérica de revestimento com função nanoencapsulante, emulsificante, dispersante, estabilizante e antioxidante. [0048] The embodiments of the red propolis extract loaded nanospheres presented in this invention may also be applied to the production of red propolis capsules. NPV, in addition to the extract with the active principles of interest, obtained from red propolis, have in their composition pharmaceutical excipients with different functionalities: active compound (extract of red propolis), polymeric coating matrix with nanoencapsulant function. , emulsifier, dispersant, stabilizer and antioxidant.
[0050] Uma modalidade da presente invenção, uma nanopartícula polimérica NEU em diferentes percentagens de extrato de própolis vermelha, apresenta-se como uma composição de: extrato padronizado de própolis vermelha proveniente da matéria-prima própolis vermelha em proporção entre 5 a 80%, preferivelmente entre 10 e 55%; agente encapsulante em proporção entre 50 a 70% e agente dispersante/estabilizante em proporção entre 15 e 30%. One embodiment of the present invention, a NEU polymeric nanoparticle in different percentages of red propolis extract, is presented as a composition of: standardized red propolis extract from the red propolis raw material in a proportion of 5 to 80%, preferably between 10 and 55%; 50 to 70% encapsulating agent and 15 to 30% dispersing / stabilizing agent.
[0051] Outra modalidade da invenção proposta, uma nanopartícula polimérica NPCL em diferentes percentagens de extrato de própolis vermelha, apresenta-se com a seguinte composição: extrato hidroalcoólico padronizado de própolis vermelha proveniente da matéria-prima própolis vermelha em proporção entre 10 a 80%, preferivelmente entre 15 e 55%; agente encapsulante em proporção entre 40 a 90% e um agente dispersante/estabilizante em proporção entre 1 e 20%.  Another embodiment of the proposed invention, an NPCL polymeric nanoparticle in different percentages of red propolis extract, has the following composition: standardized hydroalcoholic extract of red propolis from the red propolis raw material in a ratio of 10 to 80%. preferably between 15 and 55%; 40 to 90% encapsulating agent and 1 to 20% dispersing / stabilizing agent.
[0052] Outra modalidade da invenção proposta, uma nanopartícula polimérica NEUPCL em deferentes percentagens de extrato de própolis vermelha, apresenta-se com a seguinte composição: extrato hidroalcoólico padronizado de própolis vermelha proveniente da matéria-prima própolis vermelha em proporção entre 10 a 95%, preferivelmente entre 15 e 50%; agentes encapsulantes em proporção entre 30 a 80% e um agente dispersante/estabilizante em proporção entre 1 a 20%.  Another embodiment of the proposed invention, a NEUPCL polymeric nanoparticle in different percentages of red propolis extract, has the following composition: standardized hydroalcoholic extract of red propolis from the red propolis raw material in a proportion of 10 to 95%. preferably between 15 and 50%; encapsulating agents in a ratio of 30 to 80% and a dispersing / stabilizing agent in a ratio of 1 to 20%.
[0053] Nas modalidades da invenção compostas por NEU, todos as NEU20%, NEU25%, NEU30%, NEU40% e NEU50%, o sistema matricial polimérico é caracterizado por ser um sistema binário que promove a nanoencapsulação da nanopartícula, além da estabilização e dispersão após nanoprecipitação em meio aquoso, composto por Eudragit® E100, também conhecido como o poli-(metacrilato de butila-co(2-dimetilaminoetila) (metacrilato de metila) (P.M.: 47.000) e pluronic F-108 copolímero tribloco (P.M.: 14000). Nas modalidades de invenção em NEU20%, NEU25%, NEU30%, NEU40% e NEU50%, o sistema dispersante/estabilizante também pode ser caracterizado por ser composto por Eudragit®E100 (P.M.: 47.000) e pluronic F-68 copolímero tribloco (P.M.: 14.000). In the embodiments of the invention composed of NEU, all NEU20%, NEU25%, NEU30%, NEU40% and NEU50%, the polymeric matrix system is characterized by being a binary system that promotes nanoparticle nanoencapsulation, in addition to stabilization and dispersion after aqueous nanoprecipitation, composed of Eudragit® E100, also known as poly (butyl-co (2-dimethylaminoethyl methacrylate) (methyl methacrylate) (PM: 47,000) and pluronic F-108 triblock copolymer (PM: 14000) In the embodiments of NEU20%, NEU25%, NEU30%, NEU40% and NEU50%, the dispersing / stabilizing system can also be characterized by being composed of Eudragit®E100 (PM: 47,000) and pluronic F-68 triblock copolymer ( PM: 14,000).
[0054] As nanoesferas poliméricas carregadas com extrato de própolis vermelha (NEU20%, NEU25%, NEU30%, NEU40% e NEU50%), por consistir de excipientes biocompatíveis e biodegradáveis, além de conferir liberação modificada ao nanoencapsulado, ela também reduz o ataque oxidativo dos compostos fenólicos presentes no extrato própolis vermelha aos excipientes lipídicos, os quais compõe a composição de formas semissólidas como: cremes catiônicos e pomada-creme para fins dermocosméticas, bem como em formas farmacêuticas sólidas e injetáveis.  The polymeric nanospheres loaded with red propolis extract (NEU20%, NEU25%, NEU30%, NEU40% and NEU50%), consisting of biocompatible and biodegradable excipients, as well as conferring modified release to the nanoencapsulated, also reduces attack. oxidative of phenolic compounds present in the red propolis extract to lipid excipients, which make up the composition of semisolid forms such as cationic creams and cream ointment for dermocosmetic purposes, as well as solid and injectable pharmaceutical forms.
[0055] Nas modalidades da invenção compostas por NEUPCL, todos as NEUPCL20%, NEUPCL25%, NEUPCL30% e NEUPCL40%, o sistema matricial polimérico é caracterizado por ser um sistema ternário que promove a nanoencapsulação da nanopartícula, além da estabilização e dispersão após nanoprecipitação em meio aquoso, composto por poli-s-caprolactona ou PCL, Eudragit® E100, também conhecido como o poli-(metacrilato de butila-co(2- dimetilaminoetila) (metacrilato de metila) (P.M.: 47.000) e pluronic F-108 copolímero tribloco (P.M.: 14000). Nesta modalidade de invenção em NEUPCL20%, NEUPCL25%, NEUPCL30% e NEUPCL40%, o sistema dispersante/estabilizante também pode ser caracterizado por ser composto por poli-s-caprolactona (P.M.: 10.000), Eudragit®E100 (P.M.: 47.000) e pluronic F- 68 copolímero tribloco (P.M.: 14.000).  In the embodiments of the invention composed of NEUPCL, all NEUPCL20%, NEUPCL25%, NEUPCL30% and NEUPCL40%, the polymeric matrix system is characterized by being a ternary system that promotes nanoparticle nanoencapsulation as well as stabilization and dispersion after nanoprecipitation. in aqueous medium consisting of poly-s-caprolactone or PCL, Eudragit® E100, also known as poly (butyl-co (2-dimethylaminoethyl) methacrylate (methyl methacrylate) (PM: 47,000) and pluronic F-108 triblock copolymer (PM: 14000) In this embodiment of the invention in NEUPCL20%, NEUPCL25%, NEUPCL30% and NEUPCL40%, the dispersing / stabilizing system can also be characterized by being composed of poly-s-caprolactone (PM: 10,000), Eudragit ®E100 (PM: 47,000) and pluronic F-68 triblock copolymer (PM: 14,000).
[0056] Ainda, é importante destacar que, as nanoesferas poliméricas de própolis vermelha contendo PCL e Eudragit®E100 propostos no presente documento apresentam-se inovadores por apresentarem tamanho de partículas manométricos numa modalidade de invenção e também apresentarem-se aqui apresentadas e em relação a outros sistemas microencapsulados de própolis, já citados anteriormente, além de serem usadas para resolver o problema de cremes catiônicos e pomada-creme contendo extrato de própolis vermelha, bem como em formas farmacêuticas sólidas e injetáveis. It is also important to highlight that the PCL and Eudragit®E100-containing red propolis polymeric nanospheres proposed herein are innovative because they have particle size pressure gauge in one embodiment of the invention and also presented herein and in connection with other previously mentioned microencapsulated propolis systems, in addition to being used to solve the problem of cationic creams and cream ointment containing red propolis extract as well as in solid and injectable pharmaceutical forms.
[0057] Nas modalidades da invenção compostas por NPCL, todos as NPCL20%, NPCL25%, NPCL30%, NPCL40% e NPCL50%, o sistema matricial polimérico é caracterizado por ser um sistema binário que promove a nanoencapsulação da nanopartícula, além da estabilização e dispersão após nanoprecipitação em meio aquoso, composto por poli-s-caprolactona ou PCL, (P.M.: 10.000) e pluronic F-108 copolímero tribloco (P.M.: 14.000). Nesta modalidade de invenção em NPCL20%, NPCL25%, NPCL30%, NPCL40% e NPCL50%, o sistema dispersante/estabilizante também pode ser caracterizado por ser composto por poli-s-caprolactona (P.M.: 10.000) e pluronic F-68 copolímero tribloco (P.M.: 14.000). As composições aqui apresentadas são usadas para resolver o problema de cremes não-iônicos carregadas com extrato de própolis vermelha, bem como poder serem usadas em formas farmacêuticas sólidas e injetáveis. In the embodiments of the invention composed of NPCL, all NPCL20%, NPCL25%, NPCL30%, NPCL40% and NPCL50%, the polymeric matrix system is characterized by being a binary system that promotes nanoparticle nanoencapsulation, in addition to stabilization and dispersion after nanoprecipitation in aqueous medium composed of poly-s-caprolactone or PCL (PM: 10,000) and pluronic F-108 triblock copolymer (PM: 14,000). In this embodiment of the invention in NPCL20%, NPCL25%, NPCL30%, NPCL40% and NPCL50%, the dispersing / stabilizing system can also be characterized by being composed of poly-s-caprolactone (PM: 10,000) and pluronic F-68 triblock copolymer. (PM: 14,000). The compositions presented herein are used to solve the problem of non-ionic creams loaded with red propolis extract as well as being used in solid and injectable pharmaceutical forms.
- Processo de obtenção de NPV - Process of obtaining NPV
[0058] As preparações das nanopartículas poliméricas contendo extrato de própolis vermelha são obtidas através do extrato hidroalcoólico superconcentrado e padronizado de própolis vermelha com percentagem residual de solvente entre 0,01 a 10%, preferencialmente entre 0,01 a 3% usando o processo de maceração e álcool etílico entre 75 a 85 QGL por período de tempo entre 48 a 72 horas de extração e concentração do extrato em rotaevaporador usando temperatura de 45°C e pressão de 650 immHg. Preparations of the polymeric nanoparticles containing red propolis extract are obtained by superconcentrated and standardized red propolis hydroalcoholic extract with residual solvent percentage from 0.01 to 10%, preferably from 0.01 to 3% using the process. maceration and ethyl alcohol at 75 to 85 Q GL for a time period between 48 to 72 hours of extraction and extract concentration in rotary evaporator using a temperature of 45 ° C and a pressure of 650 immHg.
[0059] Uma massa sólida vermelho-escura, com rendimento entre 50 a 70%, será obtida com percentagem de solvente entre 0,01 a 35%, sendo denominado extrato de própolis vermelha. O extrato, preferivelmente, deverá apresenta percentagem de solvente entre 0,01 a 3%. - Preparação do sistema de nanoesferas carregadas com extrato de própolis vermelha (NPV) A dark red solid mass, with a yield between 50 and 70%, will be obtained with a solvent percentage between 0.01 and 35%, being called red propolis extract. Preferably the extract should have a solvent percentage of 0.01 to 3%. - Preparation of the Red Propolis Extract (NPV) loaded nanosphere system
[0060] As etapas de obtenção das NPV incluem: 1 ) Pesagem dos componentes da fase orgânica incluindo o extrato padronizado de própolis vermelha, polímero de nanorevestimento poli-s-caprolactona (P.M 10.000), Eudragit®E100 (P.M.: 47.000) e solvente orgânico com volume suficiente para solubilização; 2) Pesagem dos componentes da fase aquosa incluindo o pluronic F-108 copolímero tribloco (P.M. 14.000) e água ultrapura do tipo milli-Q suficiente para solubilização do pluronic F-108, em recipiente separado; 3) Os componentes da fase orgânica são solubilizados em acetona, ou clorofórmio ou diclorometano, preferencialmente em acetona e transferidos para banho ultrassônico com potência e frequência específicas para a completa solubilização por um período de 15 minutos; 4) Os componentes da fase aquosa são também transferidos para banho ultrassônico nas mesmas condições da fase orgânica para a completa solubilização; 5) Os componentes da fase aquosa são diluídos para um volume entre 14 a 99 vezes com água milli-Q; 6) Os componentes da fase orgânica são transferidos para um tubo falcon e dispersos com a fase aquosa com fator de diluição entre 15 a 100 vezes, por exemplo, (1 parte do volume da fase orgânica: e 99 partes do volume da fase aquosa, v:v) para obter uma suspensão de nanopartículas poliméricas contendo extrato de própolis vermelha; 7) a concentração de massa sólida do extrato de própolis vermelha, do polímero de nanorevestimento e do pluronic F-108 com função dispersante compreende uma quantidade de massa sólida entre 0,01 10 a 0,706% que são dispersos no seio da água ultrapura tipo milli-Q (quantidade de massa sólida dos componentes das fases orgânica e aquosa dispersos em água milli-Q); 8) Neste momento ocorre o fenómeno de nanoprecipitação com obtenção de uma suspensão de nanopartículas poliméricas contendo extrato de própolis vermelha; 9) O processo segue com a centrifugação da suspensão de nanopartículas poliméricas contendo extrato de própolis vermelha usando velocidade de centrifugação entre 4000 a 6000rpm por período de 15 minutos e remoção do sobrenadante; 10) seguido de etapa de lavagem com água milli - Q(opcional) das nanopartículas poliméricas nanoprecipitadas; 1 1 ) seguido por novo processo de centrifugação, com descarte do sobrenadante; 12) Após etapa de centrifugação e descarte do sobrenadante, as nanopartículas poliméricas nanoprecipitadas pelo processo de centrifugação são reunidas e concentradas em volume entre 1 a 10 mL, preferencialmente entre 3 a 6 mL para iniciarem processo caracterização físico-química e processo de secagem por liofilização. NPV steps include: 1) Weighing of organic phase components including standardized red propolis extract, poly-s-caprolactone (PM 10,000), Eudragit®E100 (PM: 47,000) and solvent organic with sufficient volume for solubilization; 2) Weighing the aqueous phase components including pluronic F-108 triblock copolymer (PM 14,000) and milli-Q-type ultrapure water sufficient to solubilize pluronic F-108 in a separate container; 3) The components of the organic phase are solubilized in acetone, or chloroform or dichloromethane, preferably in acetone and transferred to ultrasonic bath of specific power and frequency for complete solubilization for a period of 15 minutes; 4) The components of the aqueous phase are also transferred to an ultrasonic bath under the same conditions as the organic phase for complete solubilization; 5) The aqueous phase components are diluted 14 to 99 times with milli-Q water; 6) The components of the organic phase are transferred to a falcon tube and dispersed with the aqueous phase with dilution factor between 15 to 100 times, for example (1 part volume of the organic phase: and 99 parts volume of the aqueous phase, v: v) to obtain a suspension of polymeric nanoparticles containing red propolis extract; (7) The solid mass concentration of the red propolis extract, nanoclay polymer and dispersing pluronic F-108 comprises an amount of solid mass of 0.01 10 to 0.706% which is dispersed within milli-ultrapure water. Q (solid mass amount of components of the organic and aqueous phases dispersed in milli-Q water); 8) At this moment the phenomenon of nanoprecipitation occurs with obtaining a suspension of polymeric nanoparticles containing red propolis extract; 9) The process proceeds with centrifugation of the suspension of polymeric nanoparticles containing red propolis extract using spin speed from 4000 to 6000rpm for 15 minutes and removal of supernatant; 10) followed by milli - Q water washing step (optional) of the nanoprecipitated polymer nanoparticles; 1 1) followed by a new centrifugation process, discarding the supernatant; 12) After centrifugation and supernatant disposal step, the nanoprecipitated polymeric nanoparticles by centrifugation process are pooled and concentrated in volume between 1 to 10 mL, preferably between 3 to 6 mL to initiate physicochemical characterization process and freeze drying process .
- Caracterização Físico-Química das Suspensões de Nanopartículas Poliméricas contendo extrato de própolis vermelha (NPEPV)  - Physicochemical Characterization of Suspensions of Polymeric Nanoparticles containing Red Propolis Extract (NPEPV)
[0061] As suspensões de nanopartículas carregadas com extrato de própolis vermelha (NEU20%, NEU30%, NEU40% e NEU50%), além das composições (NPCL20%, NPCL25%, NPCL30%, NPCL40%, NPCL50%), bem como as composições (NEUPCL20%, NEUPCL25%, NEUPCL30%, NEUPCL40%) foram caracterizadas usando técnicas de pH, distribuição do diâmetro médio de partículas, índice de polidispersão e potencial zeta. Para determinação do pH as suspensões de nanopartículas poliméricas foram colocadas diretamente em contato com o eletrodo de pH. Para determinação dos demais parâmetros físico-químicos aqui citados, a solução foi diluída 4 vezes com água Milli-Q e analisadas em equipamento Zetasizer, Nano ZS da Malvern. Os resultados foram expressos como a média de 2 ciclos de 20 scans (Tabela 1 ).  The suspensions of nanoparticles loaded with red propolis extract (NEU20%, NEU30%, NEU40% and NEU50%), as well as the compositions (NPCL20%, NPCL25%, NPCL30%, NPCL40%, NPCL50%) as well as Compositions (NEUPCL20%, NEUPCL25%, NEUPCL30%, NEUPCL40%) were characterized using pH, mean particle diameter distribution, polydispersion index and zeta potential techniques. For pH determination the polymeric nanoparticle suspensions were placed directly in contact with the pH electrode. In order to determine the other physicochemical parameters mentioned here, the solution was diluted 4 times with Milli-Q water and analyzed in Malvern's Zetasizer, Nano ZS equipment. Results were expressed as the average of 2 cycles of 20 scans (Table 1).
- Secagem das suspensões de nanopartículas poliméricas contendo extrato de própolis vermelha  - Drying of polymeric nanoparticle suspensions containing red propolis extract
[0062] As suspensões de nanopartículas carregadas com extrato de própolis vermelha (NEU20%, NEU30%, NEU40% e NEU50%), além das composições (NPCL20%, NPCL25%, NPCL30%, NPCL40%, NPCL50%), bem como as composições (NEUPCL20%, NEUPCL25%, NEUPCL30%, NEUPCL40%) foram submetidas a secagem por liofilização em equipamento Terroni®, modelo LD1500 constituído de: 1 )câmara de secagem a vácuo contendo 3 prateleiras, 2) um condensador para abaixamento da temperatura para -43 ± 5QC e, 3) bomba de baixo vácuo. A pressão do sistema foi controlada para manter 30C^Hg como indicativo de completa secagem das nanopartículas poliméricas sólidas contendo extrato de própolis. The suspensions of nanoparticles loaded with red propolis extract (NEU20%, NEU30%, NEU40% and NEU50%), as well as compositions (NPCL20%, NPCL25%, NPCL30%, NPCL40%, NPCL50%) as well as compositions (NEUPCL20%, NEUPCL25%, NEUPCL30%, NEUPCL40%) were subjected to freeze drying in Terroni® model LD1500 consisting of: 1) vacuum drying chamber containing 3 shelves, 2) a condenser for lowering the temperature to -43 ± 5 Q C, and 3) low vacuum pump. The system pressure was controlled to maintain 30 ° C Hg as indicative of complete drying of the solid polymeric nanoparticles containing propolis extract.
[0063] É importante destacar que, antes do processo de secagem por liofilização as suspensões de nanopartículas poliméricas foram submetidas a dois principais processos de congelamento que foram: A) Congelamento Lento, no qual as suspensões de nanopartículas poliméricas foram colocadas em freezer a -20QC por período entre 48 a 120 horas e imediatamente transferidas para liofilizador para realizar processo de secagem por período entre 24 e 48 horas. B) Congelamento rápido com auxílio de nitrogénio líquido a -196QC por período entre 10 a 20 minutos. Neste processo as suspensões de nanopartículas poliméricas foram colocadas em frascos do tipo antibiótico com capacidade para 50 mL, as quais foram acondicionadas em caixa metálica tipo aço inox antes do recebimento do líquido congelante. Vale destacar também o uso de agentes crioprotetores neste processo particular de congelamento, sendo eles: dióxido de silício coloidal entre 0,1 a 10%, bem como solução glicolato de amido sódico entre 0,1 a 30%. Nestas condições de preparo de amostra previamente ao processo de secagem o período de secagem foi reduzido entre 33 a 50%. Importantly, prior to the freeze drying process the polymeric nanoparticle suspensions were subjected to two main freezing processes which were: A) Slow Freezing, in which the polymeric nanoparticle suspensions were placed in a -20 ° C freezer. Q C for a period of between 48 to 120 hours and immediately transferred to a freeze dryer to perform a drying process for a period of 24 to 48 hours. B) rapid freezing with liquid nitrogen at -196 aid Q C for a period between 10 to 20 minutes. In this process the suspensions of polymeric nanoparticles were placed in 50 mL capacity antibiotic vials, which were packed in a stainless steel metal box before receiving the freezing liquid. Also noteworthy is the use of cryoprotective agents in this particular freezing process, namely: colloidal silicon dioxide from 0.1 to 10%, as well as sodium starch glycolate solution from 0.1 to 30%. Under these sample preparation conditions prior to the drying process the drying period was reduced by 33 to 50%.
- Caracterização físico-química das nanopartículas poliméricas sólidas contendo extrato de própolis vermelha  - Physicochemical characterization of solid polymeric nanoparticles containing red propolis extract
[0064] As nanoesferas sólidas carregadas com extrato de própolis vermelha (NPV), após processo de secagem por liofilização, foram caracterizadas através de diferentes técnicas analíticas para demonstrar que o extrato de própolis vermelha foi totalmente revestido pela matriz polimérica proposta, e que as nanoesferas apresentam-se em tamanho nanométrico mesmo após processo de secagem por liofilização e que os compostos fenólicos presentes no extrato de própolis vermelha foram encapsulados em percentagens superiores a 30%. Dentre as técnicas analíticas, citamos: A) Espectroscopia de infravermelho (FTIR-ATR). O sistema matricial de nanoesferas sólidas carregado com extrato de própolis vermelha foram submetidos à análise FTIR-ATR na faixa de varredura entre 4000 a 400 cm-1 com número de scans entre 64 a 128. O equipamento utilizado foi da marca Thermo Scientific com software de aquisição Ommic. Amostras de nanopartículas poliméricas sólidas sem extrato de própolis também foram analisadas. B) Análise Térmica (DSC). As nanoesferas sólidas carregadas com extrato de própolis vermelha (NPV) foram submetidos a análise calorimétrica em calorímetro Shimadzu, modelo DSC-60 sob atmosfera de nitrogénio com fluxo de 50mL/min., numa razão de aquecimento de 10QC/min. e na faixa de temperatura entre 30-400QC. Uma quantidade de 2,0mg ± 10% foi utilizada e colocadas em cadinho de alumínio que foram hermeticamente selados. O equipamento foi calibrado com padrões de índio e zinco. Amostras de nanoesferas placebo (sem extrato de própolis) também foram analisadas. C) Análise da Morfologia e Diâmetro Médio das Partículas das NPEPV usando Microscopia de Varredura Eletrônica (MEV). As nanoesferas sólidas carregadas com extrato de própolis vermelha (NPV) foram submetidas aos 2 métodos de secagem: Secagem pelo congelamento lento, seguido de técnica de liofilização (método A); secagem pelo congelamento rápido, seguido de técnica de liofilização(método B), este último realizado com o uso de agentes crioprotetores. As NPV no estado sólido, após secagem, foram preparadas em stubs com fita dupla de carbono ou diretamente nas lamínulas, submetidas ao sistema de metalização com filme de ouro sob corrente de 10 mA durante 7 minutos em um metalizador Sanyu Electron, modelo Quick Coater SC-701 e em seguidas analisadas em Microscópio de Varredura Eletrônico da Shimadzu, modelo SSX-550 Superscan. D) Identificação, Doseamento e (%) Grau de Encapsulação de flavonóides presentes no extrato de própolis vermelha e nas NPV. A determinação (identificação e doseamento) dos flavonóides presentes no extrato de própolis vermelha e nas NPV foi realizada num equipamento UPLC-DAD da Shimadzu, que consiste nos seguintes módulos: uma bomba de alta pressão (Modelo, LC-20ADXR), desgaseificador (modelo, DGU-20A3R), autoinjetor (modelo, SIL-20AXR), forno para coluna cromatográfica, detectores de arranjo de fotodiodo (modelo, SPDM-20A) e detector de fluorescência (modelo RF-20A), uma controladora (modelo, CBM-20A) e um software Labsolution da shimadzu. A separação dos flavonoides ocorreu numa fase estacionária (C18, 150 x 4,6 mm; 5μιη), fase móvel que consistia de solvente A (água ultrapura) e solvente B (acetonitrila), bombeadas num fluxo de 0,3 mL/min. numa condição eluição gradiente, tendo no início 70% de água e 30% de acetonitrila com variação da percentagem de orgânico para 100% de acetonitrila em 40 minutos, seguido por uma condição isocrática com 100% de acetonitrila até 53 minutos, com retorno à condição inicial em 54 minutos, seguido de condição isocrática com 30% de acetonitrila até 60 minutos. Red propolis extract (NPV) loaded solid nanospheres, after freeze drying, were characterized by different analytical techniques to demonstrate that the red propolis extract was fully coated by the proposed polymer matrix, and that the nanospheres present in nanometer size even after freeze drying process and that the phenolic compounds present in the red propolis extract were encapsulated in percentages greater than 30%. Among the analytical techniques, we mention: A) Infrared Spectroscopy (FTIR-ATR). The solid propolis extract loaded nanosphere matrix system was subjected to FTIR-ATR analysis in the scan range from 4000 to 400 cm-1 with number of scans from 64 to 128. The equipment used was Thermo Scientific branded with Ommic acquisition. Samples of solid polymeric nanoparticles without propolis extract were also analyzed. B) Thermal Analysis (DSC). The solid nanospheres loaded with propolis extract (NPV) were subjected to calorimetric analysis calorimeter Shimadzu model DSC-60 under a nitrogen atmosphere with 50 ml / min flow. At a heating rate of 10 Q C / min. and in the temperature range between 30-400 Q C. An amount of 2.0mg ± 10% was used and placed in aluminum crucible which were hermetically sealed. The equipment was calibrated to indium and zinc standards. Samples of placebo nanospheres (without propolis extract) were also analyzed. C) Analysis of NPEPV Morphology and Particle Diameter using Electron Scanning Microscopy (SEM). Red nanoprobes loaded with red propolis extract (NPV) were subjected to 2 drying methods: Slow freeze drying followed by lyophilization technique (method A); quick freeze drying followed by lyophilization technique (method B), the latter performed using cryoprotective agents. The solid state NPVs, after drying, were prepared in double carbon tape stubs or directly on the coverslips, submitted to the 10mA gold film metallization system for 7 minutes in a Sanyu Electron Quick Coater SC metallizer. -701 and then analyzed on Shimadzu Electron Scanning Microscope, model SSX-550 Superscan. D) Identification, Dosage and (%) Degree of Encapsulation of flavonoids present in red propolis extract and NPV. The determination (identification and assay) of flavonoids present in red propolis extract and NPV was performed on a Shimadzu UPLC-DAD, consisting of the following modules: a high pressure pump (Model, LC-20ADXR), degasser (model, DGU-20A3R), autoinjector (model, SIL-20AXR), chromatographic column oven, photodiode array (model, SPDM-20A) and fluorescence detector (model RF-20A), a controller (model, CBM-20A) and shimadzu Labsolution software. Flavonoid separation occurred at a stationary phase (C18, 150 x 4.6 mm; 5μιη), mobile phase consisting of solvent A (ultrapure water) and solvent B (acetonitrile), pumped at a flow rate of 0.3 mL / min. in a gradient elution condition, initially having 70% water and 30% acetonitrile with percentage change from organic to 100% acetonitrile within 40 minutes, followed by an isocratic condition with 100% acetonitrile up to 53 minutes with return to condition 54 minutes, followed by an isocratic condition with 30% acetonitrile up to 60 minutes.
[0065] Padrões analíticos adquiridos da sigma-aldrich (formononetina, isoliquiritigenina, daidzeína, Biochanina A, pinobanksina) e Extrasyntesis (Liquiritigenina) foram exatamente pesados 2,0 mg e transferidos para balões volumétricos de 10 mL para obter concentração de 200μg/mL. Soluções de trabalho foram preparadas por técnica de diluição para obter concentrações de 7,50; 5,00; 2,50; 1 ,00; 0,50 e 0,15μg/mL e obter curva de calibração, a qual foi utilizada para determinação do teor (doseamento) de flavonoides no extrato de própolis vermelha e nas NPV. A identificação dos flavonóide foi realizada usando tempos de retenção comparativos com os padrões analíticos usando as mesmas condições analíticas do dia de trabalho. O estudo de grau de encapsulação também foi realizado nestas mesmas condições analíticas e com os 5 marcadores (Liquiritigenina, pinobanksina, formononetina, isoliquiritigenina, Biochanina A) presente no extrato de própolis vermelha, os quais foram determinados nas NPV e no extrato de própolis vermelha seguindo estas condições de separação. O extrato de própolis vermelha (100 mg) no estado sólido (<3% de solvente) foi solubilizado em etanol absoluto com auxílio de banho ultrassônico (5 minutos) e transferido para balão volumétrico de 10 ml_ para obter concentração de 10mg/mL. Uma nova etapa de diluição foi realizada para obter solução de trabalho de 1 mg/ml_ e realizar diluições para 400 e 250μg/mL. Estas soluções foram filtradas em unidades filtrantes de 0,45μιη e injetadas (2μΙ_) no sistema UPLC-DAD. As NPV contendo extrato de própolis vermelha ou NPV sem extrato de própolis vermelha (Composição Placebo) foram pesados exatamente e correspondente a 1 mg/ml_ da concentração teórica de extrato de própolis vermelha, solubilizados com sistema de solvente (acetona:etanol, 6:4, v:v), em seguida foi realizada a diluição para 3"^g/ml_ com o mesmo sistema de solvente, a fim de verificar a quantidade máxima de flavonóide encapsulada nas NPV. As NPV contendo extrato de própolis vermelha ou sem extrato de própolis (placebo) também foram pesados exatamente e correspondente a uma 1 mg/ml_ da concentração teórica de extrato de própolis vermelha, solubilizados com sistema de solvente (água:etanol, 7:3, v:v), em seguida foram realizadas as diluições para 3"^g/ml_ com o mesmo sistema de solvente (água:etanol, 7:3, v:v), a fim de verificar a quantidade de flavonóide não encapsulada nas NPV, mas presentes na porção externa das nanopartículas poliméricas (NPV). A determinação do grau de encapsulação se deu pela seguinte fórmula: Grau de Encapsulação (%) = (concentração do marcador na NPEPV/Concentração do marcador no EBPV)*100. Analytical standards acquired from sigma-aldrich (formononetin, isoliquiritigenin, daidzein, biochanin A, pinobanksina) and extrasyntesis (liquiritigenin) were exactly weighed 2.0 mg and transferred to 10 mL volumetric flasks to obtain a concentration of 200μg / mL. Working solutions were prepared by dilution technique to obtain concentrations of 7.50; 5.00; 2.50; 1.00; 0.50 and 0.15μg / mL and obtain calibration curve, which was used to determine the content (dosage) of flavonoids in red propolis extract and NPV. Flavonoid identification was performed using retention times compared to analytical standards using the same analytical conditions as the working day. The encapsulation degree study was also performed under these same analytical conditions and with the 5 markers (Liquiritigenin, pinobanksin, formononetin, isoliquiritigenin, Biochanina A) present in the red propolis extract, which were determined in the NPV and the red propolis extract following these separation conditions. Red propolis extract (100 mg) in solid state (<3% solvent) was solubilized in absolute ethanol with the aid of an ultrasonic bath (5 minutes) and transferred to a 10-liter volumetric flask. ml_ to obtain 10mg / mL concentration. A further dilution step was performed to obtain 1 mg / ml working solution and dilutions to 400 and 250μg / mL. These solutions were filtered on 0.45 μιη filter units and injected (2μΙ_) into the UPLC-DAD system. NPV containing red propolis extract or NPV without red propolis extract (Placebo Composition) were weighed exactly and corresponding to 1 mg / ml_ of the theoretical concentration of red propolis extract, solubilized with solvent system (acetone: ethanol, 6: 4 v: v.) then was carried dilution to 3 'ug / mL with the same solvent system, in order to determine the maximum amount of flavonoid encapsulated in NPV the NPV containing propolis extract or without extract Propolis (placebo) were also weighed exactly and corresponding to 1 mg / ml_ of the theoretical concentration of red propolis extract, solubilized with solvent system (water: ethanol, 7: 3, v: v), then dilutions were made. to 3 "ug / mL with the same solvent system (water: ethanol, 7: 3, v: v) in order to check the amount of flavonoid not encapsulated in NPV, but present in the outer portion of the polymeric nanoparticles (NPV ). The degree of encapsulation was determined by the following formula: Encapsulation Degree (%) = (NPEPV marker concentration / EBPV marker concentration) * 100.
- Caracterização da Atividade Antioxidante das NPV e Extrato de Própolis Vermelha usando DPPH  - Characterization of NPV Antioxidant Activity and Red Propolis Extract using DPPH
[0066] As nanoesferas sólidas carregadas com extrato de própolis vermelha, nanoesferas sólidas placebo (sem extrato de própolis vermelha) e o extrato de própolis vermelha foram avaliados quanto as suas atividades antioxidantes. Preparou-se soluções estoques de 1 mg/ml_ das composições NPV contendo extrato de própolis vermelha, as quais foram exatamente pesadas para obter o correspondente de 1 mg/ml_ de extrato de própolis vermelha nestas composições e solubilizadas em sistema de solvente acetona:etanol (6:4, v:v). Mesmo procedimento foi realizado com extrato de própolis vermelha. Alíquotas de 400μΙ_, 50μΙ_, 25μΙ_ e 13μΙ_ da solução estoque das NPV foram transferidas para balões volumétricos de 5 mL e adicionado 2 mL de uma solução etanólica de DPPH a 3mM, sendo deixadas em repouso por 30 minutos no escuro para que ocorresse a reação, e em seguida, o balão volumétrico foi aferido com etanol absoluto para obter concentrações de 80^g/ml_, 10,C^g/ml_, 5,C^g/ml_ e 2^g/ml_. Mesmo procedimento foi realizado com o extrato de própolis vermelha e com as nanoesferas placebo (sem extrato de própolis vermelha). Solid nanospheres loaded with red propolis extract, placebo solid nanospheres (without red propolis extract) and red propolis extract were evaluated for their antioxidant activities. Stock solutions of 1 mg / ml of NPV compositions containing red propolis extract were prepared, which were accurately weighed to obtain the corresponding 1 mg / ml of red propolis extract in these compositions and solubilized in acetone: ethanol solvent system ( 6: 4, v: v). The same procedure was performed with Red Propolis. 400μΙ_, 50μΙ_, 25μΙ_ and 13μΙ_ aliquots of the NPV stock solution were transferred to 5 mL volumetric flasks and 2 mL of a 3mM DPPH ethanolic solution were added and allowed to stand for 30 minutes in the dark for the reaction to occur. and then the volumetric flask was calibrated with absolute ethanol to obtain concentrations of 80 µg / ml, 10 µg / ml, 5 µg / ml and 2 µg / ml. The same procedure was performed with red propolis extract and placebo nanospheres (without red propolis extract).
[0067] As amostras de extrato de própolis vermelha foram submetidas a leitura em espectrofotometro do UV-vis em modo fotométrico ajustado para comprimento de onda de 520 nm ao final do período de 30 minutos. As NPV e nanoesferas placebo apresentaram turvação ao final do período reacional, sendo submetidas a uma centrifugação com velocidade de rotação de 5000rpm por período de 5 minutos. O sobrenadante foi submetido a leitura no espectrofotometro UV-vis nas mesmas condições. Também foram realizados leituras da solução de referência de DPPH 3 mM em etanol, solução de etanol absoluto para zerar o equipamento e das soluções branco (etanol absoluto + DPPH a 3 mM sem adição de extrato de própolis vermelha ou NPV) para realizar cálculo % da atividade antioxidante. O cálculo de % da atividade antioxidante foi dado pela seguinte fórmula: (%) Atividade antioxidante = 100- (((A amostra - A branco)x100)/A controle). Onde: A amostra = Absorbâncias das amostras NPV contendo solução de DPPH, A branco = Absorbâncias das amostras NPV sem a adição da solução de DPPH, A controle = Absorbância da solução de referência de DPPH em etanol.  The samples of red propolis extract were subjected to UV-vis spectrophotometer reading in wavelength-adjusted mode at 520 nm at the end of the 30 minute period. The NPV and placebo nanospheres showed turbidity at the end of the reaction period and were centrifuged at a rotation speed of 5000 rpm for a period of 5 minutes. The supernatant was subjected to UV-vis spectrophotometer reading under the same conditions. Readings were also taken of the 3 mM DPPH reference solution in ethanol, absolute ethanol solution to zero the equipment and the white solutions (3 mM absolute ethanol + DPPH without addition of red propolis extract or NPV) to calculate% antioxidant activity. The calculation of% antioxidant activity was given by the following formula: (%) Antioxidant activity = 100- (((The sample - A blank) x100) / A control). Where: Sample = Absorbances of NPV samples containing DPPH solution, A white = Absorbances of NPV samples without addition of DPPH solution, Control = Absorbance of DPPH reference solution in ethanol.
- Processo de preparação de composições dermocosméticas contendo NPV  - Process for the preparation of NPV-containing dermocosmetic compositions
[0068] As etapas de obtenção de composições dermocosméticas contendo NPV podem ser identificadas pelos seguintes Procesos principais: 1 ) Processo A: Preparação à quente - Obtenção de suspensão de nanopartículas carregadas com extrato de própolis vermelha (NEU30% ou NPCL30% ou NEUPCL30%) em fase aquosa; Aquecimento da fase aquosa contendo a suspensão de nanopartículas poliméricas contendo extrato de própolis e da fase oleosa contendo excipientes cosméticos autoemulsionantes de um creme não-iônico em recipientes separados em temperatura entre 75 a 85QC, preferivelmente a fase aquosa a 80QC e fase oleosa a 80QC; Incorporar a fase aquosa sobre a oleosa na mesma temperatura sob agitação entre 200 a 400rpm, preferivelmente entre 270 a 300rpm, mantendo temperatura e agitação constante por 10 minutos; Resfriar com agitação moderada para temperatura de 40QC; Adicionar fase complementar (outros ativos dermatológicos, modificadores sensoriais e conservantes) sob agitação e deixar resfriar até temperatura ambiente; Verificar pH e fazer correções se necessárias para 5,5 a 6,5. 2) Processo B: Preparação a frio - Obtenção de nanoesferas sólidas carrgadas com extrato de própolis vermelha obtidas por liofilização usando congelamento lento ou congelamento rápido; Obtenção de cremes bases não- iônicos, creme-gel, gel-creme não-iônico, loções não-iônicas quando preparados com NPCL30% e obtenção de cremes catiônicos ou creme- pomada quando obtidos com NEU30% e NEUPCL30%; resfriamento e acondicionamento dos creme bases não-iônicos a temperatura ambiente; Incorporação das nanoesferas poliméricas sólidas (NPCL30%) carregados com extrato de própolis vermelha em "cremes bases não-iônicos" a frio através de técnica de diluição geométrica sob agitação mecânica ou manual e sem a utilização de solventes orgânicos ou lipídicos para a solubilização das nanoesferas sólidas carregadas com extrato de própolis vermelha nestes cremes bases. Ou, Incorporação das nanoesferas poliméricas sólidas (NEU30% ou NEUPCL30%) carregados com extrato de própolis vermelha em "cremes bases catiônicos" a frio através de técnica de diluição geométrica sob agitação mecânica ou manual e sem a utilização de solventes orgânicos ou lipídicos para a solubilização das nanoesferas sólidas carregadas com extrato de própolis vermelha nestes "cremes catiônicos" ou "creme-pomada". [0068] The steps for obtaining NPV-containing dermocosmetic compositions can be identified by the following main processes: 1) Process A: Hot Preparation - Obtaining suspension of nanoparticles loaded with red propolis extract (NEU30% or NPCL30% or NEUPCL30%) in aqueous phase; Heating of the aqueous phase containing the suspension of polymeric nanoparticles containing propolis and the oil phase extract containing self-emulsifying cosmetic excipients in a nonionic cream in separate containers in temperature from 75 to 85 Q C, preferably to aqueous phase at 80 Q C and Phase oil 80 Q C; Incorporate the aqueous phase into the oil at the same temperature under stirring at 200 to 400rpm, preferably at 270 to 300rpm, maintaining constant stirring temperature for 10 minutes; Cool with moderate agitation to a temperature of 40 Q C; Add complementary phase (other dermatological actives, sensory modifiers and preservatives) under stirring and allow to cool to room temperature; Check pH and make corrections if necessary to 5.5 to 6.5. 2) Process B: Cold Preparation - Obtaining solid loaded propolis nanospheres obtained by freeze drying using slow freezing or fast freezing; Obtaining nonionic base creams, cream gel, nonionic gel cream, nonionic lotions when prepared with NPCL30% and obtaining cationic creams or cream ointment when obtained with NEU30% and NEUPCL30%; cooling and conditioning of nonionic cream bases at room temperature; Incorporation of the solid polymeric nanospheres (NPCL30%) loaded with red propolis extract into cold "nonionic base creams" by geometric dilution technique under mechanical or manual agitation and without the use of organic or lipid solvents for solubilization of the nanospheres solids loaded with red propolis extract in these base creams. Or, Incorporation of solid polymeric nanospheres (NEU30% or NEUPCL30%) loaded with red propolis extract into cold "cationic base creams" by geometric dilution technique under mechanical or manual agitation and without the use of organic or lipid solvents for solubilization of solid nanospheres loaded with red propolis extract in these "cationic creams" or "cream-ointments".
[0069] Os processos A e B descritos foram comparados com a preparação em cremes catiônicos contendo suspensões de nanopartículas poliméricas, bem como com preparação de "cremes catiônicos" contendo extrato de própolis vermelha solúvel em solvente orgânico e sem o processo tecnológico de nanoencapsulamento, os quais mostravam incompatibilidades físicas e químicas após 10 dias de preparadas com escurecimento da composição para cor marrom. No entanto, os dois processos de obtenção de cremes dermocosméticos contendo as NPV, as composições se mantiveram compatíveis e estáveis durante período maior que 180 dias. The described processes A and B were compared with the preparation in cationic creams containing polymeric nanoparticle suspensions, as well as preparation of "cationic creams" containing organic solvent soluble red propolis extract and without the technological process of nanocapsulation, which showed physical and chemical incompatibilities after 10 days of preparation with darkening from composition to brown color. However, the two processes for obtaining dermocosmetic creams containing NPV, the compositions remained compatible and stable for a period longer than 180 days.
[0070] As NPV obtidos podem ser utilizados em diversas composições dermocosméticas, preferencialmente como formulações semissólidas, como: cremes não-iônicos, gel, gel-creme, loções, loções não-iônicas, também como pós, pós para maquiagem, clareadores de pele, máscaras faciais, dentre outras. The obtained NPVs can be used in various dermocosmetic compositions, preferably as semi-solid formulations such as: nonionic creams, gel, gel-cream, lotions, nonionic lotions, as well as powders, makeup powders, skin brighteners. , face masks, among others.
- Composições Dermocosméticas contendo NPV  - Dermocosmetic Compositions Containing NPV
[0071] As NPV obtidas podem ser utilizadas em diversas composições dermocosméticas, preferencialmente como formulações semissólidas, como: creme não-iônico, creme-gel, gel-creme, loções, loções não-iônicas, pós para maquiagem, clareadores de pele, máscaras faciais, dentre outras. As NPV também podem ser usadas em formulações de cremes catiônicos e também pomada-creme.  The obtained NPVs can be used in various dermocosmetic compositions, preferably as semi-solid formulations such as: nonionic cream, gel-cream, gel-cream, lotions, nonionic lotions, makeup powders, skin brighteners, masks facials, among others. NPV can also be used in cationic cream formulations as well as cream ointment.
[0072] As composições farmacêuticas, propostas no presente documento, consistem em composições preferencialmente na forma de creme não-iônico, creme-gel não-iônico, gel-creme não-iônico, loções não-iônicas e também cremes catiônicos, que apresentam: i) um núcleo contendo substância(s) ativa(s) de própolis vermelha combinado com matriz polimérica de nanorevestimento que pode modificar ou retardar a liberação de substância(s) ativa(s) contidas no extrato de própolis vermelha, bem como proteger demais excipientes cosméticos do ataque oxidativo de compostos fenólicos presente no extrato de própolis; ii) uma camada intermediária dispersante / estabilizante do núcleo; iii) uma camada externa óleo/aquosa responsável pelos processos de diluição, dispersão, e estabilização da camada intermediária e núcleo numa composição dermocosmética, bem como por ser promotora da dissolução e permeação dos componentes ativos pelas barreiras da pele. Esta cada externa também poderá apresentar outras substâncias ativas adjuvantes com diferentes ações na pele. [0072] The pharmaceutical compositions proposed herein consist of compositions preferably in the form of nonionic cream, nonionic gel cream, nonionic gel cream, nonionic lotions and also cationic creams, which contain: (i) a nucleus containing red propolis active substance (s) combined with a polymeric nanodevice matrix that may modify or delay the release of active substance (s) contained in the red propolis extract as well as protecting other excipients. oxidative attack cosmetics of phenolic compounds present in propolis extract; ii) an intermediate dispersing / stabilizing core layer; iii) an oil / aqueous outer layer responsible for the dilution, dispersion and stabilization processes of the intermediate layer and nucleus in a dermocosmetic composition, as well as promoting the dissolution and permeation of the active components by the skin barriers. Each external may also have other adjuvant active substances with different actions on the skin.
[0073] A camada interna contendo polímero de revestimento do núcleo consiste de um polímero lipofílico sintético ou combinação de mais de um polímero(s) lipofílio(s) sintético(s).  The core coating polymer-containing inner layer consists of a synthetic lipophilic polymer or combination of more than one synthetic lipophilic polymer (s).
[0074] Em uma modalidade distinta da invenção, a camada intermediária pode ser preparada também pela conjugação entre um ou mais polímero(s) hidrofílico(s) natura(is) e um ou mais polímero(s) hidrofílico(s) sintético(s). Com ação surfactante auxiliar, como os agentes promotores de estabilização do núcleo e ao mesmo tempo promovendo a dispersão do núcleo na camada externa, estes deverão constar na camada intermediária com percentagens variáveis entre 0,05% e 95% de acordo com a sua funcionalidade emulsificante de dispersar agentes lipofílicos (núcleo) no seio da solução aquosa (camada externa). Em termos de proporção da camada intermediária na composição dermocosmética final, a camada intermediária de revestimento do núcleo deve apresentar percentagens entre 0,05% e 95%, e preferencialmente entre 0,05 e 35% em relação ao peso total da composição.  In a distinct embodiment of the invention, the intermediate layer may also be prepared by conjugating one or more natural hydrophilic polymer (s) to one or more synthetic hydrophilic polymer (s). ). With auxiliary surfactant action, such as core stabilizing promoters and at the same time promoting core dispersion in the outer layer, they should be in the intermediate layer with varying percentages between 0.05% and 95% according to their emulsifying functionality. disperse lipophilic agents (nucleus) into the aqueous solution (outer layer). In terms of the proportion of the intermediate layer in the final dermocosmetic composition, the core coating intermediate layer should have percentages between 0.05% and 95%, and preferably between 0.05 and 35% relative to the total weight of the composition.
[0075] Já a composição da camada externa consiste de excipientes, além de outros ativos dermatológicos com diversas funcionalidades, dentre eles citamos: emolientes, agentes de consistência, amaciantes, hidrantes, umectantes, espalhabilidade, refrescante, formador de filme, nutritivo, rejuvenecedores, renovação celular, esfoliantes, promotores de elasticidade, antirrugas, antioxidantes, modificadores sensoriais, preservantes, conservantes, corantes, essências. A camada externa pode ser composta de 1 ou mais destes excipientes e ativos dermatológicos. The composition of the outer layer consists of excipients, as well as other dermatological actives with various functionalities, including: emollients, consistency agents, softeners, hydrants, humectants, spreadability, refreshing, film-forming, nourishing, rejuvenating, cell renewal, exfoliators, elasticity promoters, anti-wrinkles, antioxidants, sensory modifiers, preservatives, preservatives, dyes, essences. The outer layer may be composed of 1 or more of these excipients and dermatological actives.
[0076] Em termos de porcentagens dos excipientes farmacêuticos na camada externa, estes podem representar entre 0,05% e 100% do peso total da camada externa, de acordo com a sua funcionalidade na matriz dermocosmética. Em termos de proporção da camada externa na composição dermocosmética final, a camada externa da composição deve apresentar percentagens entre 0,01 % e 100%, preferencialmente entre 1 e 100% em relação ao peso total da composição. In terms of percentages of pharmaceutical excipients in the outer layer, these may represent between 0.05% and 100% of the total weight. outer layer according to its functionality in the dermocosmetic matrix. In terms of the proportion of the outer layer in the final dermocosmetic composition, the outer layer of the composition should have percentages between 0.01% and 100%, preferably between 1 and 100% of the total weight of the composition.
[0077] A composição dermocosmética aqui apresentada deve ser acondicionadas em embalagens para substâncias sensíveis à luz, com aspecto fosco e tonalidades entre o âmbar ao escuro e com fotoproteção interna à luz (opcional).  The dermocosmetic composition presented here should be packaged in light-sensitive, matte-looking, light-amber-tinted packaging with optional light photoprotection (optional).
[0078] Ainda, outros ativos dermatológicos utilizados na composição podem ser de diversos tipos e em formulações diversas, com composições variáveis entre os diversos tipos de ativos dermatológicos, têm-se ativos dermatológicos hidrofílicos, bem como os ativos dermatológicos lipofílicos podendo ser naturais ou sintéticos como ácido hialurônico®, extrato glicólico de arnica®, manteiga de manga®, kelpadelie®, lótus rutina®, DMAE bitartarato®, Algisium C®, hydroxyprolisilane C®, lipossomas de Ginkgo biloba®, biorusol®, Ceramida IMA®, VC-PMG®, ácido-lipóico®, vitamina E oleosa®, lipossomas PML AE e AC®, elastocell®, lipossomas PML coenzima Q10®, sensiline®, structurine®, Adenin®, nutriskin®, raffermine®, helioxine®, dióxido de titânio micronizado®, Neo heliopan E 1000®, Neo Helliopan OS®, Neo heliopan BB®, Neo heliopan MA®, óleo de macadâmia, pentacare®, remoduline®, dermatan sulfato®, óleo de girassol®, fucogel®, lipossomas de colágenos®, extrato glicólico de pêssego®, extrato glicólico de oliva®, extrato glicólico de damasco®, maturine®, alistin®, algisium C®, nanosferas de ácido salicílico®, DSBC®, extrato glicólico de maracujá®, pentaglycan®, glucan E-20®, óleo de girassol®, extrato glicólico de melissa®, extrato glicólico de melissa®, fucogel®, extrato glicólico de hipérico®, Iris Iso®, extrato de algas®, GPS trealose vetorizada®, PABC hidratante®, ácido retinóico®, hidroquinona®, VC- IP®, skin whitening complex®, vitamina F hidrossolúvel®, cafeisilane C®, Bioex antilipêmico®, filtro UVA/UVB hidrossolúvel®, benzofena-3®, metoxicinamato de etil-hexila®, octocrileno®, metoxicinamato de isoamila®, salicilato de octila®, antranilato de metila®, microesponjas de retinol®, prosoja, densiskin®, soybean oil®, Coup Déclat®, biorusol II SCA®, essência de cereja®, Trealose®, phytospingosine®, matrixil®, argireline®, tensine®, elastinol®, ascobosilane C®, whitespheres®, DMAE fluid®, alantoína®, genisteina tópica®, nutripeptídeos®, liftline®, kinetin L®, cerealmilk Premium®, biomimetric LRF complex®, sílica shells®, sebonormine®, Net FS®, physiogenyl®, fomblin HC/R®, óleo de copaíba®, manteiga de cupuaçu®, Sk influx®, hidroviton 24®, palmifruit fitoconcentral®, clerilys W®, pelemol IN 2®, lactil®, Clariskin II®, metil parabeno, propilparabeno, propilenoglicol, BHT, ultraderme nanovitamina A®, phenogard MP®, cerearet 20®, álcool cetearílico, óleo mineral, álcool de lanolina e vaselina, imidazolidiniluréia, base automeulsionante e emoliente para cremes e loções O/A. Still other dermatological actives used in the composition may be of various types and in different formulations, with varying compositions among the various types of dermatological actives, there are hydrophilic dermatological actives, as well as the lipophilic dermatological actives may be natural or synthetic such as hyaluronic acid®, arnica® glycolic extract, mango butter®, kelpadelie®, rutin® lotus, DMAE bitartrate®, Algisium C®, hydroxyprolisilane C®, Ginkgo biloba® liposomes, biorusol®, Ceramida IMA®, VC- PMG®, lipoic acid®, vitamin E oleosa®, PML AE and AC® liposomes, elastocell®, PML coenzyme Q10® liposomes, sensiline®, structurine®, adenin®, nutriskin®, raffermine®, helioxine®, titanium dioxide micronized®, Neo heliopan E 1000®, Neo Helliopan OS®, Neo heliopan BB®, Neo heliopan MA®, macadamia oil, pentacare®, remoduline®, dermatan sulfato®, sunflower oil®, fucogel®, colonic liposomes agenos®, peach glycolic extract®, olive glycolic extract®, apricot glycolic extract, maturine®, alistin®, algisium C®, salicylic acid nanospheres®, DSBC®, passion fruit® glycolic extract, pentaglycan®, glucan E-20®, sunflower oil®, melissa® glycolic extract, melissa® glycolic extract, fucogel®, hypercylic glycolic extract®, Iris Iso®, algae extract®, GPS vectorized trehalose®, PABC hydrating®, acid retinoic®, hydroquinone®, VC-IP®, skin whitening complex®, water soluble vitamin F, cafeisilane C®, antilipemic bioex®, water soluble UVA / UVB filter, benzofena-3®, methoxycinnamate -hexila®, octocrylene®, isoamila® methoxycinnamate, octila® salicylate, methyl® anthranilate, retinol® microspheres, prosoja, densiskin®, soybean oil®, Coup Déclat®, biorusol II SCA®, cherry essence ®, Trealose®, phytospingosine®, matrixil®, argireline®, tensine®, elastinol®, ascobosilane C®, whitespheres®, DMAE fluid®, allantoin®, topical genistein®, nutripeptides®, liftline®, kinetin L®, cerealmilk Premium ®, biomimetric LRF complex®, silica shells®, sebonormine®, Net FS®, physiogenyl®, fomblin HC / R®, copaiba® oil, cupuaçu® butter, Sk influx®, hydroviton 24®, fitoconcentral® palmifruit, clerilys W®, pelemol IN 2®, lactil®, Clariskin II®, methyl paraben, propylparaben, propylene glycol, BHT, ultraviolet nanovitamin A®, phenogard MP®, cerearet 20®, cetearyl alcohol, mineral oil, lanolin and vaseline alcohol, imidazolidinylurea , self-propelling and emollient base for O / A creams and lotions.
- Usos de NPV em composições dermocosméticas contendo os mesmos - Uses of NPV in dermocosmetic compositions containing them
[0079] Diante de atividades biológicas do extrato de própolis vermelha, as nanoesferas carregadas com extrato de própolis vermelha podem ser utilizados em uma ampla gama de setores industriais dos ramos cosmético, cosmecêutico, higiene e limpeza. Given the biological activities of red propolis extract, nanospheres loaded with red propolis extract can be used in a wide range of industrial sectors from the cosmetic, cosmeceutical, hygiene and cleaning industries.
[0080] A utilização da própolis como princípio ativo na prevenção e tratamento de diversas doenças já é amplamente descrito. Entretanto, a própolis vermelha apresenta composição diferenciada das demais própolis já amplamente descritas no estado da técnica, o que indica atividades biológicas variadas.  The use of propolis as an active ingredient in the prevention and treatment of various diseases is already widely described. However, red propolis has a different composition from other propolis already widely described in the state of the art, which indicates varied biological activities.
[0081] As composições dermocosméticas aqui apresentadas demonstram atividade leishmanicida contra Leishmania (V.) braziliensis realizado através de ensaio de atividade leishmanicida cuja IC50 concentração que inibe 50% da população do parasita em ensaio in vitro. Tal atividade mostra que o extrato de própolis vermelha e as nanoesferas carregadas com extrato de própolis vermelha pode ser utilizada contra leishmanioses.  The dermocosmetic compositions presented herein demonstrate leishmanicidal activity against Leishmania (V.) braziliensis performed by assay of leishmanicidal activity whose IC50 concentration inhibits 50% of the parasite population in in vitro assay. Such activity shows that red propolis extract and nanospheres loaded with red propolis extract can be used against leishmaniasis.
[0082] Devido à sua característica biológica comprovadamente antioxidante, as nanoesferas carregadas com extrato de própolis vermelha e composições dermocosméticas contendo NPV obtidos pelos processos propostos podem ser utilizadas para: tratamento da pele e órgãos anexos; tratamento e rejuvenecimento da pele, tratamento de processos inflamatórios de origem seborréica; tratamento de feridas em pós-operatórios, tratamento de feridas de decúbitos em pacientes com pé diabético; tratamento de processos inflamatórios da pele de maneira geral. Preferencialmente, para a preparação de creme não-iônicos contendo NPV, as suspensões de nanopartículas poliméricas carregadas com extrato de própolis vermelha, bem como as NPV sólidas obtidas por liofilização serão utilizadas para preparação de creme regenerador antioxidante, cremes antiedemas, creme para áreas dos olhos, creme rejuvenescedor, creme despigmentante, creme com branqueamento da pele, creme para flacidez da área dos olhos, creme anticelulite, creme de massagem, creme anti-sinais Oil-Free, creme para peles ressecadas, creme para pré-cirurgia, creme Pós-Cirurgia, creme antienvelhecimento, incluindo para peles sensíveis, pele cansada, pele madura, creme para bolsas dos olhos; Loção fotoprotetora FPS entre 5 a 60, loção aceleradora do bronzeamento FPS 10, Loção Pós-Sol hidratante, loção hidratante para o corpo, loção Pós Peeling, loção despigmentante, Loção anti-rugas com retinol, Gel-Creme Pós- Sol com formononetina, Gel-Creme Pós-sol com concentrado de isoflavonas, Gel antienvelhecimento com ação tipo-Botox, Creme-Gel nutritivo, Loção Pós- Banho, Gel para peles oleoasas, Gel-Creme Antiacne e oleosidade, Creme regenerador para as mãos, Creme barreira para as mãos, Loção para as mãos com filtro solar. Tais usos para o produto aqui propostos, em termos de aplicação de nanopartículas poliméricas contendo extrato de própolis vermelha, apresentam-se como um diferencial frente ao já exposto pelo estado da técnica. Due to its proven biological characteristics antioxidant, red propolis extract-loaded nanospheres and NPV-containing dermocosmetic compositions obtained by the proposed processes may be used for: treatment of the skin and attached organs; skin treatment and rejuvenation, treatment of inflammatory processes of seborrheic origin; postoperative wound treatment, decubitus wound treatment in patients with diabetic foot; treatment of inflammatory processes of the skin in general. Preferably for the preparation of non-ionic cream containing NPV, suspensions of red propolis extract loaded polymeric nanoparticles as well as solid NPVs obtained by lyophilization will be used for preparation of antioxidant regenerating cream, anti-cream creams, eye area cream. , rejuvenating cream, depigmenting cream, skin whitening cream, eye area sagging cream, anti-cellulite cream, massage cream, Oil-Free Anti-Signal Cream, dry skin cream, pre-surgery cream, Post-Cream Surgery, anti-aging cream, including for sensitive skin, tired skin, mature skin, eye bag cream; FPS 10 to 60 SPF Protective Lotion, FPS 10 Tanning Accelerating Lotion, Moisturizing Post Lotion, Moisturizing Body Lotion, Post Peeling Lotion, Depigmenting Lotion, Retinol Anti-Wrinkle Lotion, Formononetin After-Cream Gel, Isoflavone Concentrate Gel-Cream, Botox-type Anti-Aging Gel, Nourishing Gel-Cream, After-Bath Lotion, Oily Skin Gel, Anti-acne and Oily Gel Cream, Regenerating Hand Cream, Barrier Cream hands, Hand lotion with sunscreen. Such uses for the product proposed here, in terms of application of polymeric nanoparticles containing red propolis extract, are a differential compared to that already exposed by the state of the art.
[0083] Assim, as modalidades da invenção descritas no presente documento apresentam-se como um avanço no estado da técnica já que, permitem a produção de NPV e composições dermocosméticas contendo as mesmas de maneira economicamente viável. Desta forma, as NPV obtidos através do processo proposto podem ser utilizados, isolada ou em composição com outros produtos, em diversos setores industriais, como cosmético, higiene e limpeza e cosmecêutico no tratamento das leishmanioses. Thus, the embodiments of the invention described herein present an advance in the state of the art as they allow the production of NPV and dermocosmetic compositions containing the economically viable. Thus, NPV obtained through the proposed process can be used, alone or in composition with other products, in various industrial sectors, such as cosmetic, hygiene and cleaning and cosmeceutical in the treatment of leishmaniasis.
EXEMPLOS EXAMPLES
[0084] As avaliações das NPV obtidos utilizando o processo proposto demonstraram a obtenção de um produto nanoparticulado, com tamanho de partículas nanométricas, grau de encapsulação adequado e atividade antioxidante preservada. Os resultados obtidos estão representados nas tabelas e figuras indicadas.  [0084] NPV evaluations obtained using the proposed process demonstrated the obtainment of a nanoparticulate product, with nanometer particle size, adequate encapsulation degree and preserved antioxidant activity. The results obtained are represented in the tables and figures indicated.
[0085] A Tabela 1 apresenta os resultados de diâmetro médio das partículas, índice de Polidispersão, Potencial Zeta e pH das Suspensões de Nanopartículas contendo extrato de própolis vermelha. Table 1 presents the results of mean particle diameter, Polydispersion index, Zeta Potential and pH of Nanoparticle Suspensions containing red propolis extract.
Tabela 1 Table 1
Composições Diâmetro índice de Potencial PH médio das Polidispersão Zeta (mV)  Compositions Diameter Potential Index Average PH of Zeta Polydispersion (mV)
partículas (PDI)  particles (PDI)
(nm)  (nm)
NEUPIacebo 177,6±0,7 0,13 +54,6±0,80 6,0±0,05 NEUPIacebo 177.6 ± 0.13 0.13 + 54.6 ± 0.80 6.0 ± 0.05
NEU12% 107,1 ±0,7 0,18 +37,3±1 ,30 5,98±0,1NEU12% 107.1 ± 0. 0.18 + 37.3 ± 1.30 5.98 ± 0.1
NEU20% 1 17,4±0,9 0,15 +40,4±0,60 5,88±0,2NEU20% 1 17.4 ± 0.9 0.15 + 40.4 ± 0.60 5.88 ± 0.2
NEU25% 140,4±1 ,7 0,14 +44,8±0,50 5,36±0,2NEU25% 140.4 ± 1.7 0.14 + 44.8 ± 0.50 5.36 ± 0.2
NEU30% 151 ,9±2,3 0,17 +37,9±0,90 5,9±0,07NEU30% 151.9 ± 2.3 0.17 + 37.9 ± 0.90 5.9 ± 0.07
NEU50% - - - 5,6±0,10NEU50% - - - 5.6 ± 0.10
NEU70% - - - 5,2±0,10NEU70% - - - 5.2 ± 0.10
NPCLPIacebo 279,6±1 ,4 0,17 -33,5±6,10 5,05±0,1NPCLPIacebo 279.6 ± 1.4 0.17 -3.5 ± 6.10 5.05 ± 0.1
NPCL25% 262,2±7,1 0,13 -18,7±0,51 4,53±0,1NPCL25% 262.2 ± 7.1 0.13 -18.7 ± 0.51 4.53 ± 0.1
NPCL30% 280,2±6,7 0,17 -26,8±4,60 4,64±0,1NPCL30% 280.2 ± 6.7 0.17 -26.8 ± 4.60 4.64 ± 0.1
NPCL50% 208,5±4,8 0,12 -12,7±5,30 4,54±0,2NPCL50% 208.5 ± 4.8 0.12 -12.7 ± 5.30 4.54 ± 0.2
NPCL70% 246,7±7,0 0,1 1 -19,2±4,60 4,16±0,1NPCL70% 246.7 ± 7.0 0.1 1 -19.2 ± 4.60 4.16 ± 0.1
NEUPCLPIaceb 221 ,1 ±1 ,2 0,10 +32,4±0,80 5,78±0,1NEUPCLPIaceb 221.1 ± 1.2, 0.10 + 32.4 ± 0.80 5.78 ± 0.1
0 0
NEUPCL20% 200,8±1 ,6 0,12 +25,0±0,60 6,58±0,1 NEUPCL20% 200.8 ± 1.6 0.12 + 25.0 ± 0.60 6.58 ± 0.1
NEUPCL30% 191 ,0±1 ,8 0,09 +29,6±0,60 6,30±0,1NEUPCL 30% 191.0 ± 1.8, 0.09 + 29.6 ± 0.60 6.30 ± 0.1
NEUPCL40% 195,8±3,4 0,1 1 +18,0±1 ,50 5,65±0,1NEUPCL40% 195.8 ± 3.4 0.11 + 18.0 ± 1.50 5.65 ± 0.1
*Valores referentes ; à média de duas leituras para diâmetro médio das partículas e PDI; Valores referentes à média da determinação de três valores do potencial zeta ± desvio padrão; Valores referentes à média da determinação de três valores de pH ± desvio padrão. * Referring values; the mean of two readings for mean particle diameter and PDI; Values referring to the mean of determining three values of zeta potential ± standard deviation; Values referring to the mean of the determination of three pH values ± standard deviation.
[0086] Valores de tamanho de partículas variaram entre 107,1 -177, 6nm com índice de polidispersão entre 0,13 a 0,18 para as composições NEU, entre 246, 7-280, 2nm com índice de polidispersão entre 0,1 1 a 0,17 para as composições NPCL, entre 191 , 0-221 , 1 nm com índice de polidispersão entre 0,09 e 0,12 para as composições NEUPCL e todas as composições mostraram um comportamento unimodal. O potencial zeta se mostrou entre +54,6 a +37,3 mV, para a composição NEU, entre -33,5 a -12,7 mV para a composição NPCL e entre +32,4 a +18,0 mV para composições NEUPCL. As composições demonstraram-se estáveis, dispersas e sem tendência a agregação das nanopartículas. Os valores de pH das suspensões de nanopartículas contendo extrato de própolis vermelha mostraram-se semelhantes à suspensão de nanopartículas sem extrato de própolis vermelha (placebo) com valores levemente ácidos entre 5,20 a 6,00 para composições NEU, entre 5,05 a 4,16 para composições NPCL, e entre 5,65 a 6,58 para as composições NEUPCL. Particle size values ranged from 107.1-177.6 nm with polydispersion index 0.13 to 0.18 for NEU compositions, between 246.7-280.2 nm with polydispersion index 0.1 1 to 0.17 for NPCL compositions between 191.0-221.1 nm with polydispersion index between 0.09 and 0.12 for NEUPCL compositions and all compositions showed unimodal behavior. The zeta potential was between +54.6 to +37.3 mV for the NEU composition, between -3.5 to -12.7 mV for the NPCL composition and between +32.4 to +18.0 mV for the NEUPCL compositions. The compositions have been shown to be stable, dispersed and not prone to aggregation of nanoparticles. The pH values of nanoparticle suspensions containing red propolis extract were similar to nanoparticles suspension without red propolis extract (placebo) with slightly acidic values between 5.20 to 6.00 for NEU compositions, between 5.05 to 4.16 for NPCL compositions, and between 5.65 to 6.58 for NEUPCL compositions.
[0087] A Figura 1 anexa apresenta Fotomicroscopia de Varredura Eletrônica das NEU12% (1 A, 1 B), NEU20% (1 C e 1 D) e NPCL30% (1 E e 1 F) sem crioprotetor (1 A, 1 B e 1 D) e com crioprotetor (1 C, 1 D e 1 F). Fotomicrografias com ampliação entre 3000 vezes (escala 5μιη) e ampliação de até 12000 vezes (escala 1 μιη) mostrando partículas nanométricas. The attached Figure 1 shows Electron Scanning Photomicroscopy of NEU12% (1 A, 1 B), NEU20% (1 C and 1 D) and NPCL30% (1 E and 1 F) without cryoprotectant (1 A, 1 B and 1 D) and with cryoprotectant (1 C, 1 D and 1 F). Photomicrographs with magnification between 3000 times (5μιη scale) and up to 12000 times (1 μιη scale) showing nanometric particles.
[0088] A Figura 2 anexa apresenta termogramas de DSC do extrato de própolis vermelha, da matriz de revestimento polimérico e composições de NPV. The attached Figure 2 shows DSC thermograms of red propolis extract, polymeric coating matrix and NPV compositions.
[0089] O termograma de DSC do extrato de própolis vermelha apresentou 4 eventos endotérmicos nas temperaturas de 81 ,7QC, 92,0QC, 107QC e 135QC referente a processos de fusão de ceras e demais componentes fenólicos presentes no extrato de própolis vermelha, enquanto os revestimentos poliméricos contendo Eudragit®E-100 (NEU), Poli-ε- caprolactona (NPCL) e Eudragit®E-100 (NEU) e Poli-s-caprolactona (NEUPCL) apresentaram evento endotérmico de fusão próximo a temperatura de 60QC e que foi semelhante as composições das NPV na faixa de temperatura entre 48 a 59QC com pico em 52°C e sem eventos endotérmicos adicionais na faixa de temperatura do extrato de própolis vermelha, o que mostra pelas propriedades térmicas que o extrato de própolis vermelha foi encapsulado. The DSC thermogram of the red propolis extract showed 4 endothermic events at temperatures of 81, 7 Q C, 92.0 Q C, 107 Q C and 135 Q C referring to wax melting processes and other phenolic components present. in the red propolis extract, while the polymeric coatings containing Eudragit®E-100 (NEU), Poli-ε-caprolactone (NPCL) and Eudragit®E-100 (NEU) and Poli-s-caprolactone (NEUPCL) showed endothermic event. melting near a temperature of 60 Q C and which was similar to the compositions of NPV in the temperature range of 48 to 59 W C with a peak at 52 ° C without additional endothermal events at propolis extract temperature range, which shows by the thermal properties that the red propolis extract was encapsulated.
[0090] A Figura 3 anexa apresenta espectros FTIR-ATR do extrato de própolis vermelha, da matriz de revestimento polimérico e composições de NPV. The attached Figure 3 shows FTIR-ATR spectra of the extract of red propolis, polymeric coating matrix and NPV compositions.
[0091] O Espectro FTIR-ATR do extrato de própolis vermelha apresentou estiramentos característicos de compostos fenólicos em 3336cm-1 (Hidroxila fenólica), estiramentos em 1617cm-1 , 1496cm-1 e 1450cm-1 (deformação axial C=C de anéis aromáticos em compostos fenólicos) e estiramentos em 1045 cm-1 referente a deformação axial de C-O de éter aromático e estiramentos em 877cm-1 referentes a deformação angular fora do plano de C-H de aromáticos. Os espectros FTIR-ATR de matriz de revestimento polimérico e composições NPCL apresentaram estiramento similares em 2863cm-1 , 1400-1340cm-1 referente a deformações axiais e angulares, respectivamente de CH2 e deformação axial e angular de C-(C-O)-C em 1 171 cm-1 , além de deformação axial de carbonila de cetona alifática (C=O) em 1725cm-1 , e desaparecimento dos estiramentos característicos do extrato de própolis vermelha comprovando por técnica espectroscópica no infravermelho a encapsulação do extrato de própolis vermelha durante processo de obtenção das NPCL.  The FTIR-ATR Spectrum of the red propolis extract showed characteristic stretches of phenolic compounds at 3336cm-1 (phenolic hydroxyl), stretches at 1617cm-1, 1496cm-1 and 1450cm-1 (axial deformation C = C of aromatic rings phenolic compounds) and 1045 cm-1 stretches for axial CO deformation of aromatic ether and 877cm-1 stretches for off-plane angular deformation of aromatics. The polymeric coating matrix FTIR-ATR spectra and NPCL compositions showed similar stretching at 2863cm-1, 1400-1340cm-1 for axial and angular deformations, respectively of CH2 and axial and angular deformation of C- (CO) -C in 1 171 cm-1, besides axial deformation of aliphatic ketone carbonyl (C = O) at 1725cm-1, and disappearance of the characteristic strains of red propolis extract, confirming by infrared spectroscopic technique the encapsulation of red propolis extract during the process. obtaining the NPCL.
[0092] Para as composições apresentando Eudragit®E-100 (NEU e NEUPCL) foi observado estiramentos característicos de Eudragit-E-100 dentre eles uma deformação axial simétrica C-H de CH2 e CH3 entre 2970-2850 cm- 1 , uma estiramento intenso (forte) em 1740 cm-1 (1750-1735 cm-1 ) decorrente da deformação axial do grupamento C = O de éster, duas banda de absorção de baixa intensidade em 1464 cm-1 e 1378 cm-1 referente à deformação angular C-H de CH2 e CH3 e duas bandas em 1210 - 1 190 cm-1 característica de deformação axial média-forte de C-O-C e C-(C=O)-C-O- C de éster. Deformação axial fraca-fraca em 1035 cm-1 , referente a ligação C-N de amina terciária de baixa intensidade. Nas composições NEU e NEUPCL carregadas com extrato de própolis vermelha foram observados supressão das bandas de absorção ou estiramentos característicos de hidroxilas fenólicas na região de 3400 a 3250 cm-1 e do estiramento característico de anel aromático na região de 1600 a 1450 cm-1 nas composições contendo estas matrizes poliméricas. For the compositions presenting Eudragit®E-100 (NEU and NEUPCL) characteristic Eudragit-E-100 stretches were observed among them a symmetrical axial CH deformation of CH2 and CH3 between 2970-2850 cm-1, an intense stretching ( 1740 cm-1 (1750-1735 cm-1) due to the axial deformation of the C = O ester group, two low intensity absorption bands at 1464 cm-1 and 1378 cm-1 relative to the angular deformation CH of CH2 and CH3 and two bands at 1210 - 1 190 cm -1 feature medium-strong axial deformation of COC and ester C- (C = O) -CO-C. Weak-weak axial deformation at 1035 cm -1, relative to CN binding of low intensity tertiary amine. In the NEU and NEUPCL compositions loaded with red propolis extract, suppression of the absorption bands or characteristic stretches of phenolic hydroxyls in the region of 3400 to 3250 cm-1 and the characteristic aromatic ring stretch in the region was observed. 1600 to 1450 cm -1 in compositions containing these polymeric matrices.
[0093] A Figura 4 anexa apresenta os cromatogramas (UPLC-DAD) do extrato de própolis vermelha (4A), da matriz de revestimento (4B)(Placebo), composição NEU30% (4C), composição NPCL30% (4D) e composição NEUPCL30% (4E) mostrando a identificação de flavonoides/isoflavonóide presente no extrato de própolis e composições NPV e ausência de flavonoides na matriz de revestimento polimérico (placebo). The attached Figure 4 shows the chromatograms (UPLC-DAD) of red propolis extract (4A), coating matrix (4B) (Placebo), composition NEU30% (4C), composition NPCL30% (4D) and composition NEUPCL30% (4E) showing the identification of flavonoids / isoflavonoids present in propolis extract and NPV compositions and absence of flavonoids in the polymeric coating matrix (placebo).
[0094] As figuras 4A, 4C, 4D e 4E mostram cromatogramas de corridas analíticas obtidas usando método cromatográfico desenvolvido para detectar a presença de flavonoides. As figuras 4A, 4C, 4D e 4E mostram a presença dos flavonoides (1 ) Liquiritigenina (12,53min.), (2) pinobanksina (15,68min.), (3) isoliquiritigenina (17,26min.), (4) formononetina (18,13min.), (5) pinocembrina (23,12min.) e (6) Biochanina A (23,81 min.) nos tempos de retenção correspondentes e similares aos padrões analíticos utilizados para desenvolvimento de método cromatográfico.  Figures 4A, 4C, 4D and 4E show analytical run chromatograms obtained using chromatographic method developed to detect the presence of flavonoids. Figures 4A, 4C, 4D and 4E show the presence of flavonoids (1) Liquiritigenin (12.53min.), (2) pinobanksin (15.68min.), (3) isoliquiritigenin (17.26min.), (4) formononetine (18.13min.), (5) pinocembrine (23.12min.) and (6) Biochanine A (23.81 min.) at retention times corresponding to and similar to the analytical standards used for chromatographic method development.
[0095] A Tabela 2 apresenta os resultados de Grau de Encapsulação dos principais flavonoides majoritários presentes no extrato de própolis vermelha e nas NPV usando UPLC-DAD. Table 2 presents the results of Encapsulation Degree of major major flavonoids present in red propolis extract and NPV using UPLC-DAD.
Tabela 2 Table 2
(%) Grau de encapsulação dos flavonóides majoritários usando UPLC-DAD* (%) Encapsulation degree of major flavonoids using UPLC-DAD *
Composição A B C D EComposition A B C D E
NEU12% 30,29±2,69 32,12±1 ,13 42,70±0,14 33,21 ±0,06 60,16±0,16NEU12% 30.29 ± 2.69 32.12 ± 1.13 42.70 ± 0.14 33.21 ± 0.06 60.16 ± 0.16
NEU20% 20,51 ±0,57 25,71 ±0,35 35,70±0,42 27,85±0,74 59,39±0,32NEU20% 20.51 ± 0.57 25.71 ± 0.35 35.70 ± 0.42 27.85 ± 0.74 59.39 ± 0.32
NEU25% 15,52±0,12 20,98±0,13 28,31 ±0,28 23,12±0,29 52,57±5,48NEU25% 15.52 ± 0.12 20.98 ± 0.13 28.31 ± 0.28 23.12 ± 0.29 52.57 ± 5.48
NEU30% 37,54±1 ,70 47,55±1 ,17 62,98±0,03 51 ,07±0,02 77,58±1 ,33NEU30% 37.54 ± 1.70 47.55 ± 1.17 62.98 ± 0.03 51.07 ± 0.02 77.58 ± 1.33
NEU50% 24,32±0,09 28,05±0,61 37,39±0,1 1 32,54±0,05 64,78±0,37NEU50% 24.32 ± 0.09 28.05 ± 0.61 37.39 ± 0.1 1 32.54 ± 0.05 64.78 ± 0.37
NEU70% 26,34±0,87 29,97±0,03 48,74±0,08 40,97±0,01 72,04±0,80NEU70% 26.34 ± 0.87 29.97 ± 0.03 48.74 ± 0.08 40.97 ± 0.01 72.04 ± 0.80
NPCL25% 18,41 ±0,72 31 ,19+1 ,1 1 81 ,87±1 ,34 53,39±0,85 74,54±0,26NPCL25% 18.41 ± 0.72 31, 19 + 1,111 81, 87 ± 1, 34 53.39 ± 0.85 74.54 ± 0.26
NPCL30% 30,71 ±1 ,20 39,78±0,21 1 1 1 ,86±0,31 88,48±0,02 1 12,79±0,7NPCL30% 30.71 ± 1.20 39.78 ± 0.21 1 1 1, 86 ± 0.31 88.48 ± 0.02 1 12.79 ± 0.7
NPCL40% 27,16±0,36 41 ,35±0,67 94,01 ±0,47 78,67±0,18 92,53±0,86NPCL40% 27.16 ± 0.36 41, 35 ± 0.67 94.01 ± 0.47 78.67 ± 0.18 92.53 ± 0.86
NPCL70% 27,36±4,45 30,93±0,83 69,94±1 ,23 60,58±1 ,08 74,48±0,10NPCL70% 27.36 ± 4.45 30.93 ± 0.83 69.94 ± 1.23 60.58 ± 1.08 74.48 ± 0.10
NEUPCL20% 27,22±1 ,12 43,25±0,35 102,96±1 ,5 77,26±0,64 92,52±1 ,08NEUPCL20% 27.22 ± 1.12 12 43.25 ± 0.35 102.96 ± 1.5 77.26 ± 0.64 92.52 ± 1.08
NEUPCL30% 29,88±0,02 57,63±5,97 106,71 ±1 ,2 88,67±1 ,63 94,87±2,04NEUPCL 30% 29.88 ± 0.02 57.63 ± 5.97 106.71 ± 1, 2 88.67 ± 1.63 94.87 ± 2.04
NEUPCL40% 29,80±1 ,01 37,31 ±0,56 97,92±0,44 78,02±1 ,73 93,54±0,77NEUPCL40% 29.80 ± 1.01 37.31 ± 0.56 97.92 ± 0.44 78.02 ± 1.73 93.54 ± 0.77
A:Daidzeína; B: Liquiritigenina; C: Isoliquiritigenina; D: Formononetina; E: Biochanina A. * Valores de desvio padrão determinado em duas corridas cromatográficas. A: Daidzein; B: Liquiritigenin; C: Isoliquiritigenin; D: Formononetin; E: Biochanina A. * Standard deviation values determined in two chromatographic runs.
[0096] Dentre as composições estudadas NEU que apresentou Eudragit®E-100 foi a que apresentou as maiores perdas durante etapa de lavagem, centrifugação e secagem usando técnica de liofilização com grau de encapsulação abaixo de 60%. Para as composições NPCL e NEU PCL observou-se grau de encapsulação maiores na faixa compreendida entre 50% a 77%. Observou-se que a liquiritigenina e daidzeína foram os flavonoides que apresentaram as maiores perdas durante processo de obtenção das NPV sólidas. Dentre as composições estudadas, NEU, NPCL e NEUPCL com 30% de extrato de própolis vermelha apresentaram maiores valores de teor e % de encapsulação (Tabela 3).  Among the studied compositions NEU that presented Eudragit®E-100 was the one that presented the largest losses during washing, centrifugation and drying stage using encapsulation degree freeze drying technique below 60%. For NPCL and NEU PCL compositions, higher encapsulation degree was found in the range of 50% to 77%. It was observed that liquiritigenin and daidzein were the flavonoids that presented the greatest losses during the process of obtaining solid NPV. Among the studied compositions, NEU, NPCL and NEUPCL with 30% red propolis extract presented higher content and% encapsulation values (Table 3).
[0097] A Tabela 3 apresenta os resultados de percentagens de (%)Grau de Encapsulação e (%)Não-encapsulados representando os principais flavonoides majoritários presentes no extrato de própolis vermelha e nas NPV usando UPLC-DAD. Table 3 presents the results of percentages of (%) Encapsulation Degree and (%) Unencapsulated representing the major major flavonoids present in red propolis extract and NPV using UPLC-DAD.
Tabela 3  Table 3
(%) Grau de encapsulação usando UPLC-DAD  (%) Encapsulation degree using UPLC-DAD
Composição %GE Médio de flavonoides Composition% GE Flavonoid Medium
NEU12% 39,69  NEU12% 39.69
NEU20% 33,83  NEU20% 33.83
NEU25% 28,04  NEU25% 28.04
NEU30% 55,34  NEU30% 55.34
NEU50% 37,42  NEU50% 37.42
NEU70% 43,61  NEU70% 43.61
NPCL25% 51 ,88  NPCL25% 51.88
NPCL30% 76,72  NPCL30% 76.72
NPCL40% 66,74  NPCL40% 66.74
NPCL70% 52,66  NPCL70% 52.66
NEUPCL20% 68,64  NEUPCL20% 68.64
NEUPCL30% 75,43  NEUPCL30% 75.43
NEUPCL40% 67,32  NEUPCL40% 67.32
GE: Grau de encapsulação;  GE: degree of encapsulation;
[0098] As NPV apresentaram boa atividade antioxidante e foi concentração dependente. Nas concentrações de extrato de própolis vermelha de 80 e 10μ9/ιτιΙ_, as NPVs mostraram atividade antioxidante entre 71 ,14 a 98,41 % e nas concentrações de 5 e 2^g/ml_, as composições de NPV apresentaram percentagem de atividade antioxidante entre 58,87 e 97,33% e, portanto, muito superior ao extrato de própolis vermelha entre 25,97 e 40,73%. Esses resultados são satisfatórios para demonstrar que as composições de NPV irão atuar como agentes que inibem processos oxidativos e funcionando como agentes antioxidantes em nível biológico como pele e órgãos anexos, podendo funcionar como agente antienvelhecimento na pele(Tabela 4).  NPV showed good antioxidant activity and was concentration dependent. At concentrations of red propolis extract of 80 and 10μ9 / ιτιΙ_, NPVs showed antioxidant activity between 71, 14 to 98.41% and at concentrations of 5 and 2 ^ g / ml_, NPV compositions showed percentage antioxidant activity between 58.87 and 97.33% and therefore much higher than the red propolis extract between 25.97 and 40.73%. These results are satisfactory to demonstrate that NPV compositions will act as agents that inhibit oxidative processes and function as biological antioxidants such as skin and appendages, and may function as an anti-aging agent on the skin (Table 4).
[0099] A Tabela 4 apresenta os resultados de atividade antioxidante (%) usando método DPPH do extrato de própolis vermelha e das NPV. Tabela 4 Table 4 presents the results of antioxidant activity (%) using DPPH method of red propolis extract and NPV. Table 4
% de Atividade Antioxidante  % Antioxidant Activity
Composição 80ng/ml_ 10μg/mL 5μg/mL  Composition 80ng / ml_ 10μg / mL 5μg / mL
EPV 98,06±0,18 79,00±0,13 40,73±0,03 25,97±0,04 EPV 98.06 ± 0.18 79.00 ± 0.13 40.73 ± 0.03 25.97 ± 0.04
NCEU 12% 97,30±0,17 71 ,14±0,06 65,89±0,06 58,87±0,1 1NCEU 12% 97.30 ± 0.17 71, 14 ± 0.06 65.89 ± 0.06 58.87 ± 0.1 1
NCEU 20% 97,30±0,42 78,34±0,06 72,17±0,1 1 67,07±0,19NCEU 20% 97.30 ± 0.42 78.34 ± 0.06 72.17 ± 0.1 1 67.07 ± 0.19
NCEU 25% 95,90±0,17 76,68±0,22 69,77±0,23 66,70±0,46NCEU 25% 95.90 ± 0.17 76.68 ± 0.22 69.77 ± 0.23 66.70 ± 0.46
NCEU 30% 95,87±0,1 1 96,12±0,22 77,38±0,22 69,70±0,06NCEU 30% 95.87 ± 0.1 1 96.12 ± 0.22 77.38 ± 0.22 69.70 ± 0.06
NCEU 50% 98,37±0,06 97,33±0,25 75,42±0,32 66,1 1 ±0,26NCEU 50% 98.37 ± 0.06 97.33 ± 0.25 75.42 ± 0.32 66.1 1 ± 0.26
NCEU 70% 100,01 ±0,1 1 98,41 ±0,06 79,67±0,17 68,96±0,29NCEU 70% 100.01 ± 0.1 1 98.41 ± 0.06 79.67 ± 0.17 68.96 ± 0.29
NCPCL 20% 99,58±0,55 93,12±0,87 86,07±0,51 80,12±0,06NCPCL 20% 99.58 ± 0.55 93.12 ± 0.87 86.07 ± 0.51 80.12 ± 0.06
NCPCL 30% 97,39±0,36 84,61 ±0,14 97,39±0,44 86,18±0,34NCPCL 30% 97.39 ± 0.36 84.61 ± 0.14 97.39 ± 0.44 86.18 ± 0.34
NCPCL 40% 94,48±0,37 78,47±0,1 1 89,52±0,46 86,09±0,36NCPCL 40% 94.48 ± 0.37 78.47 ± 0.1 1 89.52 ± 0.46 86.09 ± 0.36
NCPCL 70% 94,48±0,05 90,08±0,1 1 84,1 1 ±0,17 76,50±0,67NCPCL 70% 94.48 ± 0.05 90.08 ± 0.1 1 84.1 1 ± 0.17 76.50 ± 0.67
NCEUP 20% 100,83±0,46 91 ,94±0,67 92,30±0,80 74,31 ±0,34NCEUP 20% 100.83 ± 0.46 91, 94 ± 0.67 92.30 ± 0.80 74.31 ± 0.34
NCEUP 30% 101 ,80±0,97 84,1 1 ±0,05 78,52±0,96 72,87±0,1 1NCEUP 30% 101, 80 ± 0.97 84.1 1 ± 0.05 78.52 ± 0.96 72.87 ± 0.1 1
NCEUP 40% 101 ,75±0,60 79,82±0,19 79,79±0,28 75,33±0,13NCEUP 40% 101.75 ± 0.60 79.82 ± 0.19 79.79 ± 0.28 75.33 ± 0.13
*Valores referentes à média da determinação de três valores de pH ± desvio padrão. * Mean values for the determination of three pH values ± standard deviation.
[0100] Observa-se que as formulações apresentaram percentual de atividade antioxidante mais elevado que o extrato na forma livre, evidenciando que essas composições podem atuar por mais tempo ou em menor concentração que o extrato bioativo de própolis vermelha aumentando o poder antioxidante dos compostos fenólicos. A exacerbação da atividade antioxidante nas formulações deve-se provavelmente, ao efeito de liberação controlada destas matrizes de revestimento destes sistemas matriciais.  [0100] It is observed that the formulations presented higher antioxidant activity percentage than the free form extract, evidencing that these compositions can act for a longer or lower concentration than the red propolis bioactive extract increasing the antioxidant power of the phenolic compounds. . The exacerbation of antioxidant activity in the formulations is probably due to the controlled release effect of these coating matrices of these matrix systems.
[0101] A Figura 5 anexa apresenta a determinação da IC50 do ensaio de atividade leishmanicida contra Leishmania (V.) braziliensis usando gráfico normalizado (figura 5A) e alguns mecanismos bioquímicos hipotéticos de morte celular para Leishmania (V.) braziliensis pelos flavonoides, em especial isoliquiritigenina (chalcona), presentes no extrato de própolis vermelha e nas nanoesferas carregadas com extrato de própolis vermelha (figura 5B). O EPV e as nanopartículas das composições NEU30% e NPCL30% apresentaram atividade leishmanicida comprovadas com valores de IC50 de 179^g/ml_ e 31 ,16 μg/mL, respectivamente. As composições NCPCL 30% e NPCL40% (47,23 μg/mL) apresentaram resultados próximo ao EPV na forma livre com IC50 de 38^g/ml_. The attached Figure 5 shows the IC 50 determination of the Leishmanicidal activity assay against Leishmania (V.) braziliensis using normalized graph (Figure 5A) and some hypothetical biochemical mechanisms of Leishmania (V.) braziliensis cell death by flavonoids, in particular isoliquiritigenin (chalcona) present in red propolis extract and nanospheres loaded with red propolis extract (Figure 5B). The EPV and nanoparticles of the compositions NEU30% and NPCL30% showed proven leishmanicidal activity with IC50 values of 179 µg / ml_ and 31,16 µg / ml, respectively. Compositions NCPCL 30% and NPCL40% (47.23 μg / mL) showed results close to EPV in free form with an IC50 of 38 µg / mL.
[0102] A figura 5 é baseada nas pesquisas de Torres-Santos e Chen. Torres-Santos et a/.(2009)(Torres-Santos EC, Sampaio-Santos Ml, Buckner FS, Yokoyama K, Gelb M, Urbina JA, Rossi-Bergmann B (2009) Altered sterol profile induced in Leishmania amazonensis by a natural dihydroxymethoxylated chalcone. JAntimicrobial Chemother 63:469-472) demonstraram que as chalconas (chalcona di-hidro-metoxilado) alteram a biossíntese de esteroides na L. amazonensis e promovem o acúmulo de ergosterol e diminuição do colesterol. Esta alteração resulta no aumento da fluidez da membrana e do efeito tóxico no parasito com valores de IC50 igual a 5,5 μΜ. Estudo realizado por Chen et al. (2001 ) (Chen M, Zhai L, Christensen SB, Theander TG, Kharazmi A (2001 ) Inhibition of fumarate reductase in leishmania major and I. donovani by chalcones. Antimicrobial Agents Chemother 45(7):2023-2029) mostrou que chalconas (licochalcona A em estudo específico) destroem a ultraestrutura da mitocôndria do parasito, inibem a respiração mitocondrial e a atividade das enzimas desidrogenase mitocondrial, que são succinato desidrogenase (SDH), succinato citocromo e redutase, NADH desidrogenase, NADH citocromo e redutase, especialmente a atividade da enzima fumarato redutase (FDH) presentes somente no parasita Leishmania. [0102] Figure 5 is based on research by Torres-Santos and Chen. Torres-Santos et al. (2009) (Torres-Santos EC, Sampaio-Santos M1, Buckner FS, Yokoyama K, Gelb M, Urbina JA, Rossi-Bergmann B (2009) Altered sterol profile induced in Leishmania amazonensis by a natural dihydroxymethoxylated chalcone JAntimicrobial Chemother 63: 469-472) demonstrated that chalcones (dihydro-methoxylated chalcone) alter steroid biosynthesis in L. amazonensis and promote ergosterol accumulation and cholesterol lowering. This change results in increased membrane fluidity and toxic effect on the parasite with IC 50 values of 5.5 μΜ. Study by Chen et al. (2001) (Chen M, Zhai L, Christensen SB, Theander TG, Kharazmi A (2001) Inhibition of fumarate reductase in Leishmania major and I. donovani by chalcones. Antimicrobial Agents Chemother 45 (7): 2023-2029) showed that chalcones (lycochalcona A in specific study) destroy the parasite mitochondria ultrastructure, inhibit mitochondrial respiration and the activity of mitochondrial dehydrogenase (SDH) enzymes, succinate dehydrogenase (SDH), cytochrome succinate and reductase, NADH dehydrogenase, especially NADH cytochrome a, fumarate reductase (FDH) activity present only in the Leishmania parasite.
[0103] A Figura 6 anexa apresenta fotos de cremes não-iônicos contendo NPCL30% carregadas com extrato de própolis vermelha para obter concentração final no creme cosmético de 2,0% (A), 1 ,5% (B), 1 ,0% (C) e 0,5% (D) do extrato de própolis vermelha. As nanoesferas NPV no estado sólido foram diretamente incorporadas ao creme não-iônico ou usando as suspensões de nanopartículas poliméricas (B). Attached Figure 6 shows photos of non-ionic creams containing NPCL30% loaded with red propolis extract to achieve final concentration in cosmetic cream of 2.0% (A), 1.5% (B), 1.0 % (C) and 0.5% (D) of the red propolis extract. Solid state NPV nanospheres were either directly incorporated into the nonionic cream or using the suspensions of polymeric nanoparticles (B).

Claims

Reivindicações Claims
1 . Nanoesferas carregadas com extrato de própolis vermelha caracterizado por compreender princípios ativos derivados da própolis vermelha.  1 . Nanospheres loaded with red propolis extract characterized by comprising active ingredients derived from red propolis.
2. Nanoesferas carregadas com extrato de própolis caracterizado por os princípios ativos serem derivados de extratos de própolis vermelha e apresentar porcentagem final de extrato de própolis vermelha entre 14 e 69%.  2. Nanospheres loaded with propolis extract characterized in that the active ingredients are derived from red propolis extracts and have a final percentage of red propolis extract between 14 and 69%.
3. Nanoesferas carregadas com extrato de própolis vermelha, de acordo com as reivindicações 1 e 2, caracterizado pelo fato de compreender:  Red propolis extract loaded nanospheres according to Claims 1 and 2, characterized in that they comprise:
a) extrato padronizado de própolis vermelha proveniente da própolis vermelha in natura dos manguezais de Alagoas em proporção entre 5 a 100%; b) encapsulante de nanorevestimento em proporção entre 5 e 95%, preferencialmente entre 30 e 86%;  a) standardized extract of red propolis from the fresh red propolis of the mangroves of Alagoas in a proportion of 5 to 100%; b) nanocoat encapsulant in a proportion from 5 to 95%, preferably from 30 to 86%;
c) dispersante com função estabilizante em proporção entre 5 e 95%, preferencialmente entre 15 e 35%.  c) dispersant with stabilizing function in a ratio of 5 to 95%, preferably between 15 and 35%.
4. Nanoesferas carregadas com extrato de própolis vermelha, de acordo com a reivindicação 3, caracterizado por compreender:  Red propolis extract loaded nanospheres according to Claim 3, characterized in that they comprise:
a) sistema encapsulante de nanorevestimento composto por sistema binário, composto por a) poli-s-caprolactona (P.M 10.000) e pluronic F-108 (P.M. 14.000), ou b) poli-s-caprolactona (P.M. 10.000) e Pluronic F-68 para obter composições NPCL;  a) binary coating encapsulant system composed of a) poly-s-caprolactone (PM 10,000) and pluronic F-108 (PM 14,000), or b) poly-s-caprolactone (PM 10,000) and Pluronic F- 68 to obtain NPCL compositions;
b) sistema encapsulante de nanorevetimento composto por sistema binário, composto por eudragit E-100 (P.M 14.000) e pluronic F-108 (P.M. 14.000) para obter composições NEU;  b) binary encapsulant nanorevetment system composed of eudragit E-100 (P.M 14,000) and pluronic F-108 (P.M. 14,000) to obtain NEU compositions;
c) sistema encapsulante de nanorevestimento composto por sistema ternário, composto por poli-s-caprolactona (P.M 10.000), eudragit E-100 e pluronic F-108 (P.M. 14.000) para obter composições NEUPCL.  c) nanoparticulate encapsulating system composed of ternary system composed of poly-s-caprolactone (P.M 10,000), eudragit E-100 and pluronic F-108 (P.M. 14,000) to obtain NEUPCL compositions.
5. Nanoesferas carregadas com extrato de própolis vermelha, de acordo com as reivindicações 1 a 4, caracterizado por apresentar propriedades de liberação controlada. Red propolis extract loaded nanospheres according to Claims 1 to 4, characterized in that they have controlled release properties.
6. Nanoesferas carregadas com extrato de própolis vermelha, de acordo com as reivindicações 1 a 4, caracterizado por apresentar propriedades de proteção da composição dermocosmética de incompatibilidades químicas entre os compostos fenólicos presentes no extrato de própolis e demais excipientes cosméticos. Red propolis extract loaded nanospheres according to Claims 1 to 4, characterized in that they have protective properties of the dermocosmetic composition of chemical incompatibilities between the phenolic compounds present in the propolis extract and other cosmetic excipients.
7. Nanoesferas carregadas com extrato de própolis vermelha, de acordo com as reivindicações 1 a 4, caracterizado por apresentar propriedades de fotoproteção dos compostos fenólicos presentes no extrato de própolis.  Red propolis extract loaded nanospheres according to any of claims 1 to 4, characterized in that they have photoprotection properties of the phenolic compounds present in the propolis extract.
8. Nanoesferas carregadas com extrato de própolis vermelha, de acordo com as reivindicações 1 a 4, caracterizado por apresentar propriedades de biocompatibilidade e biodegradabilidade sem apresentar qualquer risco quando aplicado topicamente sobre a pele.  Red propolis extract loaded nanospheres according to any one of claims 1 to 4, characterized in that it has biocompatibility and biodegradability properties without any risk when applied topically to the skin.
9. Nanoesferas carregadas com extrato de própolis vermelha, de acordo com as reivindicações de 1 a 8, caracterizado por conter, entre seus componentes, compostos derivados de própolis vermelha, preferencialmente extrato padronizado de própolis vermelha, para uso no tratamento dermocosmético com ação leishmanicida para tratamento de leishmanioses cutâneas.  Red propolis extract-loaded nanospheres according to claims 1 to 8, characterized in that they contain, among their components, compounds derived from red propolis, preferably standardized red propolis extract, for use in dermocosmetic treatment with leishmanicidal action. treatment of cutaneous leishmaniasis.
10. Nanoesferas carregadas com extrato de própolis vermelha, de acordo com a reivindicação 9, caracterizado por conter, entre seus componentes, compostos derivados de própolis vermelha, preferencialmente extrato padronizado de própolis vermelha, para uso no tratamento dermocosmético com ação leishmanicida para tratamento de leishmanioses e, especificamente, causados pelo género Leishmania brazilisensis.  Red propolis extract loaded nanospheres according to Claim 9, characterized in that it contains, among its components, compounds derived from red propolis, preferably standardized red propolis extract, for use in the treatment of leishmaniasis for dermocosmetic treatment of leishmaniasis. and specifically caused by the genus Leishmania brazilisensis.
1 1 . Nanoesferas carregadas com extrato de própolis vermelha, de acordo com as reivindicações 9 e 10, caracterizado por conter, entre seus componentes, compostos derivados de própolis vermelha, preferencialmente extrato padronizado de própolis vermelha, para uso no tratamento dermocosmético com ação antioxidante e anti-inflamatória para tratamento de leishmanioses cutâneas causadas pela Leishmania brazilisensis. 1 1. Red propolis extract loaded nanospheres according to claims 9 and 10, characterized in that they contain, among their components, compounds derived from red propolis, preferably standardized red propolis extract, for use in dermocosmetic treatment with antioxidant and anti-inflammatory action. for treatment of cutaneous leishmaniasis caused by Leishmania brazilisensis.
12. Nanoesferas carregadas com extrato de própolis vermelha, de acordo com as reivindicações de 1 a 8, caracterizado por conter, entre seus componentes, compostos derivados de própolis vermelha, preferencialmente extrato padronizado de própolis vermelha, para uso no tratamento dermocosmético com ação antioxidante e hidratante em processos inflamatórios eritematosos, causados, preferencialmente, por ação de excesso de exposição de luz solar ou luz artificial. Red propolis extract-loaded nanospheres according to any of claims 1 to 8, characterized in that they contain, among their components, compounds derived from red propolis, preferably standard red propolis extract, for use in the dermocosmetic treatment with antioxidant action. moisturizing in erythematous inflammatory processes, caused preferably by the action of overexposure of sunlight or artificial light.
13. Nanoesferas carregadas com extrato de própolis vermelha, de acordo com as reivindicações de 1 a 8, caracterizado por conter, entre seus componentes, compostos derivados de própolis vermelha, preferencialmente extrato padronizado de própolis vermelha, para uso no tratamento dermocosmético com ação antioxidante e hidratante em processos inflamatórios de dermatite atópica.  Red propolis extract loaded nanospheres according to any one of claims 1 to 8, characterized in that they contain, among their components, compounds derived from red propolis, preferably standard red propolis extract, for use in the dermocosmetic treatment with antioxidant action. moisturizer in inflammatory processes of atopic dermatitis.
14. Nanoesferas carregadas com extrato de própolis vermelha, de acordo com as reivindicações de 1 a 8, caracterizado por conter, entre seus componentes, compostos derivados de própolis vermelha, preferencialmente extrato padronizado de própolis vermelha, para uso no tratamento dermocosmético com ação antioxidante e antirrugas em processos de envelhecimento natural da pele causados, preferencialmente, por exposição da pele as variações climáticas ou exposição da pele a agentes químicos, físicos, bem como deficiências bioquímicas ou nutricionais.  Red propolis extract-loaded nanospheres according to claims 1 to 8, characterized in that they contain, among their components, compounds derived from red propolis, preferably standardized red propolis extract, for use in the dermocosmetic treatment with antioxidant action. anti-wrinkles in processes of natural aging of the skin caused, preferably, by exposure of the skin to climatic variations or exposure of the skin to chemical, physical as well as biochemical or nutritional deficiencies.
15. Nanoesferas carregadas com extrato de própolis vermelha, de acordo com as reivindicações de 1 a 8, caracterizado por conter, entre seus componentes, compostos derivados de própolis vermelha, preferencialmente extrato padronizado de própolis vermelha, para uso no tratamento dermocosmético com ação antioxidante retardando o envelhecimento precoce da pele e demais efeitos causados por ação de agentes extrínsecos.  Red propolis extract-loaded nanospheres according to Claims 1 to 8, characterized in that they contain, among their components, compounds derived from red propolis, preferably standardized red propolis extract, for use in the dermocosmetic treatment with retarding antioxidant action. premature skin aging and other effects caused by the action of extrinsic agents.
16. Processo de obtenção de nanoesferas carregadas com extrato de própolis vermelha, caracterizado por compreender as seguintes etapas:  Process for obtaining nanopheres loaded with red propolis extract, comprising the following steps:
i) Preparação do extrato padronizado de própolis vermelha a partir da própolis vermelha in natura; (i) Preparation of the standardized red propolis extract from the fresh red propolis;
ii) Preparação de suspensões de nanoesferas carregadas com extrato de própolis vermelha por solubilização dos componentes da fase orgânica e componentes da fase aquosa em volumes e concentrações específicas, em recipientes separados, usando banho ultrassônico;  (ii) Preparation of suspensions of red propolis extract-loaded nanospheres by solubilizing the organic phase components and aqueous phase components in specific volumes and concentrations in separate containers using an ultrasonic bath;
iii) Preparação das suspensões de nanoesferas carregadas com extrato de própolis vermelha por técnica de nanoprecipitação dos componentes da fase orgânica sobre a fase aquosa usando proporções e concentrações específicas dos componentes da fase orgânica e da fase aquosa, conforme descrito em ii);  (iii) Preparation of red propolis extract-loaded nanosphere suspensions by nanoprecipitation of the organic phase components over the aqueous phase using specific proportions and concentrations of the organic phase and aqueous phase components, as described in ii);
iv) Preparação das nanoesferas carregadas com extrato de própolis vermelha por centrifugação, usando velocidade de rotação e tempo específicos seguido de técnica de concentração das suspensões de nanoesferas carregadas com extrato de própolis vermelha;  iv) Preparation of the red propolis extract loaded nanospheres by centrifugation using specific rotation speed and time followed by concentration technique of the red propolis extract loaded nanosphere suspensions;
v) Secagem das nanoesferas carregadas com extrato de própolis vermelha com obtidas em iv) usando técnicas de congelamento rápido, seguido de secagem por liofilização usando condições de baixa pressão;  v) Drying the red propolis extract-loaded nanospheres as obtained in iv) using rapid freezing techniques, followed by freeze drying using low pressure conditions;
vi) Caracterização das suspensões de nanoesferas carregadas com extrato de própolis vermelha usando técnicas de: distribuição do diâmetro médio das partículas, índice de polidispersão, potencial zeta e pH;  vi) Characterization of nanoprobes suspensions loaded with red propolis extract using techniques of: mean particle diameter distribution, polydispersion index, zeta potential and pH;
vii) Caracterização das nanoesferas sólidas carregadas com extrato de própolis vermelha usando técnicas de: DSC, FTIR, MEV, UPLC-DAD, atividade antioxidante;  vii) Characterization of solid nanospheres loaded with red propolis extract using: DSC, FTIR, SEM, UPLC-DAD, antioxidant activity;
viii) Caracterização biológica das nanoesferas sólidas carregadas com extrato de própolis vermelha usando método in vitro de atividade leishmanicida contra Leishmania braziliensis.  viii) Biological characterization of solid nanospheres loaded with red propolis extract using in vitro method of leishmanicidal activity against Leishmania braziliensis.
17. Processo de obtenção de nanoesferas carregadas com extrato de própolis vermelha, de acordo com a reivindicação 16, caracterizado pelo fato de, na etapa iii), o extrato bruto é solubilizado juntamente com agente encapsulante poli-s-caprolactona responsável pela nanoencapsulação do extrato de própolis vermelha para obter composições NPCL. Process for obtaining red propolis extract loaded nanospheres according to claim 16, characterized in that, in step iii), the crude extract is solubilized together with the poly-s-caprolactone encapsulating agent responsible for the nanoencapsulation of red propolis extract to obtain NPCL compositions.
18. Processo de obtenção de nanoesferas carregadas com extrato de própolis vermelha, de acordo com a reivindicação 16, caracterizado pelo fato de, na etapa iii), o extrato bruto é solubilizado juntamente com agente encapsulante Eudragit E-100 responsável pela nanoencapsulação do extrato de própolis vermelha para obter composições NEU.  Process for obtaining red propolis extract loaded nanospheres according to claim 16, characterized in that, in step iii), the crude extract is solubilized together with the Eudragit E-100 encapsulating agent responsible for the nanoencapsulation of the extract. red propolis to get NEU compositions.
19. Processo de obtenção de nanoesferas carregadas com extrato de própolis vermelha, de acordo com a reivindicação 16, caracterizado pelo fato de, na etapa iii), o extrato bruto é solubilizado juntamente com agentes encapsulantes poli-s-caprolactona e Eudragit E-100 responsáveis pela nanoencapsulação do extrato de própolis vermelha para obter composições NEUPCL.  Process for obtaining red propolis extract loaded nanospheres according to claim 16, characterized in that, in step iii), the crude extract is solubilized together with poly-s-caprolactone and Eudragit E-100 encapsulating agents. responsible for nanoencapsulation of red propolis extract to obtain NEUPCL compositions.
20. Processo de obtenção de nanoesferas carregadas com extrato de própolis vermelha, de acordo com a reivindicação 16, caracterizado pelo fato de, na etapa iii), são adicionados a fase aquosa agente dispersante/estabilizante pluronic F-108 ou pluronic F-68, preferencialmente, pluronic F-108 copolímero tribloco responsável pela nanodispersão e estabilização das nanopartículas poliméricas em suspensão.  Process for obtaining red propolis extract loaded nanospheres according to Claim 16, characterized in that, in step iii), the dispersing / stabilizing agent pluronic F-108 or pluronic F-68 is added to the aqueous phase. preferably pluronic F-108 triblock copolymer responsible for nanodispersion and stabilization of polymeric nanoparticles in suspension.
21 . Processo de obtenção de nanoesferas carregadas com extrato de própolis vermelha, de acordo com a reivindicação 16, caracterizado pelo fato de, na etapa iii) a adição dos componentes da composição com proporção de sólidos entre 5% a 80% do extrato padronizado de própolis vermelha, entre 10% a 70% de agente encapsulante, e entre 10% a 35% de agente dispersante.  21 Process for obtaining nanopheres loaded with red propolis extract according to claim 16, characterized in that, in step iii) the addition of the components of the composition with a proportion of solids between 5% to 80% of the standardized red propolis extract from 10% to 70% encapsulating agent and from 10% to 35% dispersing agent.
22. Processo de obtenção de nanoesferas carregadas com extrato de própolis vermelha, de acordo com a reivindicação 16, caracterizado pelo fato de, na etapa v), a secagem das nanoesferas no estado sólido podem ocorrer na presença de crioprotetores aerosil entre 0,1 a 10%, bem como glicolato de amido sódico entre 0,1 a 30%.  Process for obtaining red propolis extract loaded nanospheres according to Claim 16, characterized in that, in step v), solid-state drying of the nanospheres may occur in the presence of aerosil cryoprotectants between 0.1 to 10% as well as sodium starch glycolate between 0.1 to 30%.
23. Processo de obtenção suspensões de nanopartículas carregadas com extrato de própolis vermelha, de acordo com a reivindicação 16, caracterizado pelo fato de, na etapa vi) obter um diâmetro médio das partículas entre 107 a 177 nm, índice de polidispersão entre 0,130 a 0,180, e potencial zeta entre +37,3 a +54,6mV para as composições ditas NEU. 23. Process for obtaining charged nanoparticle suspensions Red propolis extract according to Claim 16, characterized in that, in step vi) an average particle diameter of 107 to 177 nm is obtained, a polydispersion index of 0.130 to 0.180 and a zeta potential of +37.3. at + 54.6mV for the so-called NEU compositions.
24. Processo de obtenção suspensões de nanopartículas carregadas com extrato de própolis vermelha, de acordo com a reivindicação 16, caracterizado pelo fato de, na etapa vi) obter um diâmetro médio das partículas entre 191 a 221 ,1 nm, índice de polidispersão entre 0,090 a 0,120, e potencial zeta entre +18,0 a +32,4mV para as composições ditas NEUPCL;  Process for obtaining nanoparticle suspensions loaded with red propolis extract according to claim 16, characterized in that, in step vi) an average particle diameter between 191 and 221.1 nm, polydispersion index between 0.090 is obtained. at 0.120, and zeta potential between +18.0 to + 32.4mV for said NEUPCL compositions;
25. Processo de obtenção de nanoesferas carregadas com extrato de própolis vermelha, de acordo com a reivindicação 16, caracterizado pelo fato de, na etapa vii) comprova-se o revestimento das nanopartículas poliméricas contendo extrato de própolis vermelha usando DSC, FTIR e MEV.  Process for obtaining red propolis extract loaded nanospheres according to claim 16, characterized in that the coating of the red propolis extract-containing polymeric nanoparticles using DSC, FTIR and MEV is demonstrated in step vii).
26. Processo de obtenção de nanoesferas carregadas com extrato de própolis vermelha, de acordo com a reivindicação 16, caracterizado pelo fato de, na etapa vii) comprova-se o grau de encapsulação das nanoesferas carregadas com extrato de própolis vermelha usando UPLC-DAD.  Process for obtaining red propolis extract loaded nanospheres according to Claim 16, characterized in that, in step vii) the degree of encapsulation of the red propolis extract loaded nanospheres using UPLC-DAD is verified.
27. Processo de obtenção suspensões de nanopartículas carregadas com extrato de própolis vermelha, de acordo com a reivindicação 16, caracterizado pelo fato de, na etapa viii) as nanoesferas carregadas com extrato de própolis vermelha comprova-se atividade leishmaniciada para as composições NEU e NPCL com valores de IC50 de 179^g/ml_ e 31 ,16 μg/mL, respectivamente.  Process for obtaining suspensions of red propolis extract loaded nanoparticles according to claim 16, characterized in that, in step viii) the nanopheres loaded with red propolis extract is shown to have leishmanicidal activity for the NEU and NPCL compositions. with IC 50 values of 179 µg / ml and 31,16 µg / ml respectively.
28. Composição, caracterizada por compreender nanoesferas carregadas com extrato de própolis vermelha como princípio ativo, isoladamente ou em conjunto com outros ativos.  Composition, characterized in that it comprises nanospheres loaded with red propolis extract as active ingredient, alone or in conjunction with other actives.
29. Composição, de acordo com a reivindicação 28, caracterizada por ser, preferencialmente formulações semissólidas, como creme não-iônicos, gel, gel-creme, loções, loções não-iônicas, cremes cationicos, pomada-creme, pós, pós para maquiagem, clareadores de pele, máscaras faciais; Composition according to Claim 28, characterized in that it is preferably semi-solid formulations such as non-ionic cream, gel, gel-cream, lotions, nonionic lotions, cationic creams, cream-ointment, powders, makeup powders. , skin whiteners, face masks;
30. Composição, de acordo com a reivindicação 28, caracterizada por compreender (i) um núcleo contendo substância(s) ativa(s) de própolis vermelha combinado com agente de revestimento; (ii) uma camada intermediária de dispersante e estabilizante de as substância(s) ativa(s) contidas no núcleo; (iii) uma camada externa óleo-aquosa responsável pelos processos dissolução e permeação dos constituintes ativos do extrato de própolis vermelha pelas barreiras da pele. Composition according to Claim 28, characterized in that it comprises (i) a core containing red propolis active substance (s) combined with a coating agent; (ii) an intermediate layer of dispersant and stabilizer of the active substance (s) contained in the core; (iii) an oil-aqueous outer layer responsible for the dissolution and permeation processes of the active constituents of the red propolis extract through the skin barriers.
31 . Composição, de acordo com a reivindicação 28, caracterizada por apresentar característica polimultifuncional de liberação modificada, antioxidante, ação leishmanicida e fotoproteção.  31 Composition according to Claim 28, characterized in that it has a polymultifunctional modified release characteristic, antioxidant, leishmanicidal action and photoprotection.
32. Composição, de acordo com a reivindicação 28, caracterizada por apresentar um núcleo i) com função de proteção da composição contra auto- oxidação e fotodegradação dos constituintes presentes na composição dermocosmética.  Composition according to Claim 28, characterized in that it has a core (i) with a function of protecting the composition against auto-oxidation and photodegradation of the constituents present in the dermocosmetic composition.
33. Composição, de acordo com a reivindicação 28, caracterizada por compreender um núcleo (i) composto pelas substâncias ativas da própolis vermelha, derivadas do extrato padronizado da própolis vermelha e matriz de revestimento polimérico biocompatível e biodegradável e, apresentar proporção entre 0,01 % e 10%, preferencialmente entre 0,01 e 5%, em relação ao peso total da composição.  Composition according to Claim 28, characterized in that it comprises a core (i) composed of the active substances of red propolis derived from the standardized extract of red propolis and biocompatible and biodegradable polymeric coating matrix and having a ratio of 0.01 % to 10%, preferably 0.01 to 5%, based on the total weight of the composition.
34. Composição, de acordo com a reivindicação 28, caracterizada por compreender uma camada intermediária (ii) composta por excipientes cosméticos, preferencialmente agentes dispersantes e estabilizantes do núcleo, na porcentagem entre 0,01 % e 95% do peso total da camada intermediária, de acordo com a funcionalidade do excipiente cosmética na matriz dermocosmética e, apresentar proporção entre 0,01 % e 100%, preferencialmente entre 0,01 e 75%, em relação ao peso total da composição.  Composition according to Claim 28, characterized in that it comprises an intermediate layer (ii) composed of cosmetic excipients, preferably core dispersing and stabilizing agents, in the percentage between 0.01% and 95% of the total weight of the intermediate layer. according to the functionality of the cosmetic excipient in the dermocosmetic matrix and having a ratio of 0.01% to 100%, preferably 0.01 to 75%, relative to the total weight of the composition.
35. Composição, de acordo com a reivindicação 28, caracterizada por compreender uma camada externa (iii) com excipientes cosméticos, com diversas funcionalidades, preferencialmente pela diluição, dispersão e estabilização da camada intermediária e do núcleo na composição, bem como pela dissolução e permeação dos constituintes ativos do extrato de própolis vermelha pelas barreiras da pele, na porcentagem entre 0,01 % e 100% do peso total da camada externa, de acordo com a funcionalidade do excipiente farmacêutico na matriz farmacêutica e, apresentar proporção entre 0,01 % e 100%, preferencialmente entre 0,01 e 99,9%, em relação ao peso total da composição. Composition according to Claim 28, characterized in that it comprises an outer layer (iii) with cosmetic excipients, having various functionalities, preferably by dilution, dispersion and dispersion. stabilization of the intermediate layer and core in the composition, as well as the dissolution and permeation of the active constituents of the red propolis extract by the skin barriers, in the percentage between 0.01% and 100% of the total weight of the outer layer, according to functionality of the pharmaceutical excipient in the pharmaceutical matrix and have a ratio of 0.01% to 100%, preferably 0.01 to 99.9%, of the total weight of the composition.
36. Composição, de acordo com a reivindicação 28, caracterizada por compreender na camada externa (iii) excipientes cosméticos, com diversas funcionalidades, preferencialmente emolientes, agentes de consistência, amacientes, hidrantes, umectantes, promotores de espalhabilidade, refrescantes, formadores de filme, nutritivos, rejuvenecedores, promotores de renovação celular, esfoliantes, promotores de elasticidade, agentes de ação antirrugas, agentes de ação antioxidante, modificadores sensoriais, preservantes, conservantes, odorizantes e, apresentar proporção entre 0,01 % e 100%, preferencialmente entre 0,01 e 99,9%, em relação ao peso total da composição.  Composition according to Claim 28, characterized in that it comprises in the outer layer (iii) cosmetic excipients having various functionalities, preferably emollients, consistency agents, softeners, hydrants, humectants, spreadability promoters, refreshers, film-forming agents. nutrients, rejuvenators, cell renewal promoters, exfoliating agents, elasticity promoters, anti-wrinkle action agents, antioxidant action agents, sensory modifiers, preservatives, preservatives, odorants, and have a ratio between 0.01% and 100%, preferably between 0, 01 and 99.9%, relative to the total weight of the composition.
37. Composição, de acordo com a reivindicação 28, caracterizada por compreender uma camada externa com excipientes cosméticos como emolientes, agentes de consistência, amaciente, hidrantes, umectantes, espalhabilidade, refrescante, formador de filme, nutritivo, rejuvenecedores, renovação celular, esfoliantes, promotores de elasticidade, antirrugas, antioxidantes, modificadores sensoriais, preservantes, conservantes, odorizantes e, apresentar proporção entre 0,01 % e 100%, preferencialmente entre 0,01 e 99,9%, em relação ao peso total da composição.  Composition according to Claim 28, characterized in that it comprises an outer layer with cosmetic excipients such as emollients, consistency agents, softener, hydrants, humectants, spreadability, refreshing, film-forming, nourishing, rejuvenating, cell renewal, exfoliating, elasticity promoters, anti-wrinkles, antioxidants, sensory modifiers, preservatives, preservatives, odorants and, have a ratio between 0.01% and 100%, preferably between 0.01 and 99.9%, in relation to the total weight of the composition.
38. Composição, de acordo com a reivindicação 28, caracterizada por compreender uma camada externa com e outros ativos dermatológicos hidrofílicos e lipofílicos podendo ser naturais ou sintéticos e, apresentar proporção entre 0,01 % e 100%, preferencialmente entre 0,01 e 99,9%, em relação ao peso total da composição. Composition according to Claim 28, characterized in that it comprises an outer layer with and other hydrophilic and lipophilic dermatological actives, which may be natural or synthetic, and have a ratio between 0.01% and 100%, preferably between 0.01 and 99%. , 9%, relative to the total weight of the composition.
39. Uso de composições combinadas com nanoesferas carregadas com extrato de própolis vermelha, de acordo com as reivindicações 9, 10, 1 1 e 27, caracterizado por ser isoladamente um agente leishmanicida ou como adjuvante, para tratar a leishmaniose cutânea causada pelo parasita Leishmania sp. Use of compositions with red propolis extract loaded nanospheres according to claims 9, 10, 11 and 27, characterized in that it is either a leishmanicidal agent alone or as an adjuvant to treat cutaneous leishmaniasis caused by the parasite Leishmania sp. .
40. Uso de composições combinadas com nanoesferas carregadas com extrato de própolis vermelha, de acordo com as reivindicações 9, 10, 1 1 e 27, caracterizado por ser isoladamente um agente leishmanicida ou como adjuvante, para combater lesões cutâneas causados pelo Leishmania (V.) braziliensis.  Use of compositions with red propolis extract loaded nanospheres according to claims 9, 10, 11 and 27, characterized in that it is either a leishmanicidal agent or as an adjuvant to combat skin lesions caused by Leishmania (V. ) braziliensis.
41 . Uso de composições combinadas com nanopartículas poliméricas contendo extrato de própolis vermelha, de acordo com as reivindicações 28 a 38, caracterizado por ser isoladamente ou como agente adjuvante, para tratamento dermocosmético com ação antioxidante, cicatrizante de feridas causadas pela leishmaniose cutânea.  41 Use of compositions containing red propolis extract-containing polymeric nanoparticles according to any one of claims 28 to 38, characterized in that it is isolated or as an adjuvant for dermocosmetic treatment with wound healing caused by cutaneous leishmaniasis.
42. Uso de composições combinadas com nanopartículas poliméricas contendo extrato de própolis vermelha, de acordo com as reivindicações 28 a 38, caracterizado por ser isoladamente ou como agente adjuvante, para tratamento dermocosmético com ação antioxidante, cicatrizante em tratamento pré-cirúrgicos e pós-cirúrgicos de feridas.  Use of compositions containing red propolis extract-containing polymeric nanoparticles according to any one of claims 28 to 38, characterized in that it is alone or as an adjuvant for antioxidant dermocosmetic treatment, healing in pre-surgical and post-surgical treatment of wounds.
43. Uso de composições combinadas com nanopartículas poliméricas contendo extrato de própolis vermelha, de acordo com as reivindicações 28 a 38, caracterizado por ser isoladamente ou como agente adjuvante, para tratamento dermocosmético com ação antioxidante, nutritiva e estética da pele causadas por deficiências bioquímicas ou nutricional.  Use of compositions containing red propolis extract-containing polymeric nanoparticles according to any one of claims 28 to 38, characterized in that it is alone or as an adjuvant for dermocosmetic treatment with antioxidant, nourishing and aesthetic action of the skin caused by biochemical or nutritional
44. Uso de composições combinadas com nanopartículas poliméricas contendo extrato de própolis vermelha, de acordo com as reivindicações 28 a 38, caracterizado por ser isoladamente um agente antioxidante ou como adjuvante, para combater processos de antienvelhecimento da pele e órgãos anexos. Use of combined compositions with red propolis extract-containing polymeric nanoparticles according to any one of claims 28 to 38, characterized in that it is an antioxidant agent alone or as an adjuvant to combat antiaging processes of the skin and adjoining organs.
45. Uso de composições combinadas com nanopartículas poliméricas contendo extrato de própolis vermelha, de acordo com as reivindicações 28 a 38, caracterizado por ser isoladamente ou com um agente adjuvante, para tratamento dermocosmético de processos de renovação celular cutânea, antiinflamatório e hidratante em processos inflamatórios eritematosos causados por ação de excesso de exposição de luz solar ou luz artificial. Use of compositions containing red propolis extract-containing polymeric nanoparticles according to any one of claims 28 to 38, characterized in that it is alone or with an adjuvant for dermocosmetic treatment of skin, antiinflammatory and hydrating cell renewal processes in inflammatory processes. erythematosus caused by overexposure to sunlight or artificial light.
46. Uso de composições combinadas com nanopartículas poliméricas contendo extrato de própolis vermelha, de acordo com as reivindicações 28 a 38, caracterizado por ser isoladamente ou como agente adjuvante, para tratamento dermocosmético com ação antioxidante e antirrugas em processos de envelhecimento natural da pele causados por exposição da pele às variações climáticas ou exposição da pele a agentes químicos e físicos.  Use of compositions containing red propolis extract-containing polymeric nanoparticles according to any one of claims 28 to 38, characterized in that it is alone or as an adjuvant for antioxidant and anti-wrinkle dermocosmetic treatment in natural skin aging processes caused by skin exposure to climatic variations or skin exposure to chemical and physical agents.
47. Uso de composições combinadas com nanopartículas poliméricas contendo extrato de própolis vermelha, de acordo com as reivindicações 28 a 38, caracterizado por ser isoladamente ou como agente adjuvante, para tratamento dermocosmético com ação antioxidante e na lipoxidação de gorduras localizadas e celulites.  Use of combined compositions with red propolis extract-containing polymeric nanoparticles according to any one of claims 28 to 38, characterized in that it is alone or as an adjuvant for dermocosmetic treatment with antioxidant action and in the lipoxidation of localized fats and cellulite.
48. Uso de composições combinadas com nanopartículas poliméricas contendo extrato de própolis vermelha, de acordo com as reivindicações 28 a 38, caracterizado por ser isoladamente ou como agente adjuvante, para tratamento dermocosmético com ação antiacne e para tratamento de peles com produção excessiva de oleosidade.  Use of the combined compositions with red propolis extract-containing polymeric nanoparticles according to any one of claims 28 to 38, characterized in that it is alone or as an adjuvant for anti-acne dermocosmetic treatment and for the treatment of excessively oiled skin.
49. Uso de composições combinadas com nanopartículas poliméricas contendo extrato de própolis vermelha, de acordo com as reivindicações 28 a 38, caracterizado por ser isoladamente ou como agente adjuvante, para tratamento dermocosmético e estético de clareamento da pele.  Use of combined compositions with red propolis extract-containing polymeric nanoparticles according to any one of claims 28 to 38, characterized in that it is alone or as an adjuvant for dermocosmetic and aesthetic skin whitening treatment.
50. Uso de composições combinadas com nanopartículas poliméricas contendo extrato de própolis vermelha, de acordo com as reivindicações 28 a 38, caracterizado por ser isoladamente ou como agente adjuvante, para tratamento dermocosmético e estético de hipopigmentação da pele e tratamento do vitiligo. Use of combined compositions with red propolis extract-containing polymeric nanoparticles according to any one of claims 28 to 38, characterized in that it is alone or as an adjuvant for the dermocosmetic and aesthetic treatment of skin hypopigmentation and Vitiligo treatment.
PCT/BR2017/050088 2016-08-04 2017-04-13 Red propolis extract-loaded nanospheres, method for obtaining nanospheres, dermocosmetic compositions containing same, and uses WO2018023182A1 (en)

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